Transplant recipients developing donor-specific HLA class II (HLA-II) Abs are at higher risk for Ab-mediated rejection (AMR) and transplant vasculopathy. To understand how HLA-II Abs cause AMR and transplant vasculopathy, we determined the signaling events triggered in vascular endothelial cells (EC) following Ab ligation of HLA-II molecules. HLA-II expression in EC was induced by adenoviral vector expression of CIITA or by pretreatment with TNF-α/IFN-γ. Ab ligation of class II stimulated EC proliferation and migration. Class II Ab also induced activation of key signaling nodes Src, focal adhesion kinase, PI3K, and ERK that regulated downstream targets of the mammalian target of rapamycin (mTOR) pathway Akt, p70 ribosomal S6 kinase, and S6 ribosomal protein. Pharmacological inhibitors and small interfering RNA showed the protein kinases Src, focal adhesion kinase, PI3K/Akt, and MEK/ERK regulate class II Ab-stimulated cell proliferation and migration. Treatment with rapalogs for 2 h did not affect HLA-II Ab-induced phosphorylation of ERK; instead, mTOR complex (mTORC)1 targets were dependent on activation of ERK. Importantly, suppression of mTORC2 for 24 h with rapamycin or everolimus or treatment with mTOR active-site inhibitors enhanced HLA-II Ab-stimulated phosphorylation of ERK. Furthermore, knockdown of Rictor with small interfering RNA caused overactivation of ERK while abolishing phosphorylation of Akt Ser473 induced by class II Ab. These data are different from HLA class I Ab-induced activation of ERK, which is mTORC2-dependent. Our results identify a complex signaling network triggered by HLA-II Ab in EC and indicate that combined ERK and mTORC2 inhibitors may be required to achieve optimal efficacy in controlling HLA-II Ab-mediated AMR.

Solid organ transplant recipients developing donor-specific HLA Abs (DSA) are at a higher risk for acute and chronic Ab-mediated rejection (AMR) and graft loss (13). Acute AMR is estimated to affect 10–15% of allografts (1, 46), whereas chronic AMR occurs in as many as 50% of allografts by 10 y after transplant (79). Notably, chronic Ab-mediated allograft injury shares common histologic features across all transplanted organs and manifests as an insidious vascular disease known as transplant vasculopathy (TV). The affected vessels of the donor organ exhibit neointimal growth and perivascular fibrosis (1015). Cell proliferation and angiogenic processes appear to be a central mechanism for the formation of these vascular lesions (16, 17).

Although strong clinical evidence supports the association between HLA DSA, chronic AMR, and TV, the exact mechanisms whereby DSA cause neointimal hyperplasia and fibrosis are largely unknown. Vascular endothelial cell (EC) injury mediated by complement-fixing DSA was thought to mediate chronic AMR and graft failure (18, 19). However, recent studies indicate that DSA can contribute to alterations in EC function through complement-independent mechanisms by transducing intracellular signals (2024). Studies by our group and others have shown that crosslinking of HLA class I molecules with Ab on the surface of EC promotes diverse biological functions, including cellular proliferation and survival in clinically relevant in vivo and in vitro models of AMR (25, 26). Engagement of class I molecules by HLA Abs stimulates phosphorylation of protein kinases Src, focal adhesion kinase (FAK), and paxillin and assembly of focal adhesions and activation of the PI3K/protein kinase B/Akt pathway (2729). The activation of PI3K and Akt leads to upregulation of antiapoptotic Bcl-2 and Bcl-xL protein expression in EC (27). Ligation of class I molecules on EC results in cell proliferation (28, 3032) via activation of the mammalian target of rapamycin (mTOR) complex (mTORC)1 and downstream signal targets, including p70 ribosomal S6 kinase (S6K) and S6 ribosomal protein (S6RP) (31, 33, 34), as well as the mTORC2 signaling targets Akt and ERK (31, 33, 35).

HLA class II (HLA-II) molecules, in addition to their classical role in Ag presentation, have been reported to regulate various cellular processes, including proliferation, maturation, cytokine production, and apoptosis, in macrophages, B cells, and dendritic cells (36, 37). These functions of HLA-II have been shown to engage various intracellular signaling events, in APCs through agonistic actions after engagement by TCRs, including activation of protein kinases Src, Syk, protein kinase C, MAPK p38, and ERK (36, 38). Allograft recipients may form Abs against any mismatched HLA Ags carried by the donor, but DSA to HLA-II molecules vastly predominate, particularly in the late posttransplant period (3943). However, despite the strong correlation between DSA to HLA-II and poor graft outcome across solid organs, very little is known about the intracellular signaling in graft vascular cells activated by HLA-II Ab binding and how they contribute to allograft injury and the process of TV.

Under physiological conditions, most human vascular EC do not express HLA-II molecules, and vascular EC in culture rapidly lose HLA-II expression. Inflammatory insults, occurring during the process of transplantation including surgical trauma, and ischemia/reperfusion injury, as well as rejection, produce proinflammatory cytokines such as TNF-α, IL-1β, and IFN-γ. In turn, cytokines such as IFN-γ activate the HLA CIITA, turn on transcription, and induce HLA-II molecule expression on EC (44, 45).

In this study, we sought to elucidate the role of HLA-II DSA in intracellular signal transduction, cell proliferation, and migration in vascular EC, the angiogenic processes thought to drive TV. To overcome historical limitations of studying HLA-II in human EC, we constructed and transfected an adenovirus-based vector encoding CIITA (Ad-CIITA) into primary human aortic EC or pretreated EC with cytokines TNF-α and IFN-γ to induce HLA-II expression. Ab ligation of HLA-II molecules on EC triggered a network of intracellular signals, including activation of protein kinases Src, FAK, and PI3K/Akt, and the mTOR signaling cascade, including mTOR, S6K, S6RP, and MAPK ERK. HLA-II Abs also stimulated angiogenic responses in EC, including proliferation and migration. Studies using pharmacological inhibitors and small interfering RNA (siRNA) demonstrated that FAK/Src, PI3K, PDK1/Akt, and ERK function as upstream signaling elements regulating downstream targets of the mTOR pathway. Disruption of signaling events elicited through Src/FAK, ERK, or mTOR prevented class II–mediated EC proliferation and migration. Importantly, pharmacological or siRNA suppression of mTORC2 blocked AKT at Ser473 and led to hyperphosphorylation of ERK in response to Ab ligation of HLA-II molecules in EC. These results identify a novel feedback loop in EC stimulated with HLA-II Abs and underscore a major functional difference in the signaling networks that modify EC function through HLA class I and class II molecules (33). Our results identify mechanisms of HLA-II Ab-mediated vascular injury and suggest that novel combinations of mTORC2 and MEK inhibitors may be clinically useful to treat AMR and TV.

Cell culture reagents were from Invitrogen Life Technologies (Carlsbad, CA). Purified mouse mAbs against HLA-II (clone ID no. F002-6C6G1, IgG2a), DR (clone ID no. FM5203, IgG1), DQ (clone ID no. SPVL3, IgG2a), and DP (clone ID: BRA, IgG2b) were provided by Dr. J.-h. Lee (One Omega, Canoga Park, CA). Plasmid pcDNA3 myc CIITA (plasmid no. 14650) was a gift from M. Peterlin (plasmid no. 14650; Addgene, Cambridge, MA). Plasmid pENTR 4, plasmid pAd/PL-DEST Gateway, DH5 competent cells, the HEK293A cell line, Gateway LR Clonase II enzyme mix, Lipofectamine 2000, and Opti-MEM I medium were from Invitrogen. Adeno-XTM rapid titer kits were from Clontech (Mountain View, CA). Rapamycin, everolimus, and dasatinib were purchased from Sigma-Aldrich (St. Louis, MO). PP242 was from Chemdea (Ridgewood, NJ). PP2, PP3, LY294002, U0126, and U0124 were from Calbiochem (La Jolla, CA). A66 and anti–integrin αvβ3 Ab (mouse IgG [mIgG]1) were obtained from R&D Systems (Minneapolis, MN). Anti-CD105 Ab (clone 43A3, mIgG1) was from BioLegend (San Diego, CA). MK-2206, GDC-0068, and PD0325901 were obtained from Selleckchem (Houston, TX). Polyclonal Ab against phospho-Src (Tyr418), phospho-FAK (Tyr576), and phospho-FAK (Tyr577) were obtained from Invitrogen/BioSource International (Camarillo, CA). Rabbit polyclonal Ab against phospho-mTOR (Ser2448), phospho-S6K (Thr389), phospho-S6K (Thr421/Ser424), phospho-S6RP (Ser235/236), phospho-S6RP (Ser240/300), phospho-PI3K p85 (Tyr458)/p55 (Tyr199), phospho-Akt (Thr308), phospho-Akt (Ser473), phospho–c-Raf (Ser259), phospho-ERK (Thr202/Tyr204), mTOR, S6K, S6RP, Akt, MEK, ERK, and β-actin Ab were from Cell Signaling Technology (Beverly, MA). Anti-mTOR, Rictor, and Raptor mAb were from Millipore/Upstate (San Diego, CA). Anti-mTOR (A300-504A), Raptor (A300-506A), and Rictor (A300-459A) rabbit Ab were from Bethyl Laboratories (Montgomery, TX). The rabbit polyclonal Ab against c-Src (SRC2), β-tubulin (H-235), goat anti-rabbit HRP, goat anti-mouse HRP Ab, and protein A/G plus–agarose were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-Vinculin (clone Hvin-1) and mitomycin C (M4287) were from Sigma-Aldrich. FITC-conjugated donkey anti-rabbit IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Vybrant CFDA SE cell tracer kit (V-12883) was purchased from Molecular Probes (Eugene, OR). The BD Pharmingen BrdU flow kit was from BD Biosciences (San Jose, CA).

Primary human aortic EC were isolated from the aortic rings of explanted donor hearts, as described previously (46), or commercial EC (lot no. EC5555) were obtained from Lonza/Clonetics (Walkersville, MD) and cultured in M199 medium (Mediatech, Manassas, VA) supplemented with 20% (v/v) FBS (HyClone), penicillin-streptomycin (100 U/ml and 100 μg/ml, respectively; both from Invitrogen), sodium pyruvate (1 mmol/l), heparin (90 μg/ml; Sigma-Aldrich), and EC growth supplement (20 μg/ml; BD Biosciences). Cells from passage 3 to 8 were used at a confluence of 80%. Prior to use in experiments, cells were grown for 6 h in medium M199 containing 0.2% FBS.

The DNA fragment encoding CIITA, a transcription factor regulating HLA-II expression, was subcloned from plasmid pcDNA3 myc CIITA into the adenovirus-based vector pAd/PL-DEST. Recombinant Ad-CIITA was then produced, amplified, and tittered. Primary cultures of human aortic EC were infected with Ad-CIITA. Expression of HLA-II in EC was determined by flow cytometric analysis. CIITA-expressing EC were stimulated with HLA-II Abs, and protein phosphorylation of distinguished signaling pathways was examined by Western blot.

Primary cultures of human aortic EC were infected with Ad-CIITA at a multiplicity of infection (MOI) of 2 or were pretreated with 200 U of TNF-α and 500 U of IFN-γ for 2 d. Expression of HLA-II Ags on EC was determined by flow cytometric analysis using anti-human HLA-II Abs. MOI is the ratio of infectious agents, virus particles, to infection targets, EC. As virus particles increased, the ratio of infected cells with at least one viral particle also increased. MOI was defined as follows: MOI of 1, percentage of infected cells is 63.2%; MOI of 2, percentage of infected cells is 86.5%; MOI of 3, percentage of infected cells is 95.0%; and MOI of 5, percentage of infected cells is 99.3% (47).

The human mTOR siRNA duplexes (5′-CCA AAG UGC UGC AGU ACU AUU-3′ and 5′-UAG UAC UGC AGC ACU UUG GUU-3′), human Raptor siRNA duplexes (5′-GGA CAA CGG CCA CAA GUA C dTdT-3′ and 5′-GUA CUU GUG GCC GUU GUC C dTdT-3′), human Rictor siRNA duplexes (5′-ACU UGU GAA GAA UCG UAU C dTdT-3′ and 5′-GUA ACG AUU CUU CAC AAG U dTdT-3′), human FAK siRNA duplexes (5′-GGU UCA AGC UGG AUU AUU U-3′ and 5′-AAA UAA UCC AGC UUG AAC C-3′), human c-Src siRNA SMARTpool, human MEK siRNA SMARTpool, and control nontargeting siRNA duplexes (5′-UAG CGA CUA AAC ACA UCA AUU-3′ and 5′-AAU UGA UGU GUU UAG UCG CUA-3′) were synthesized by Dharmacon (Lafayette, CO). Transfection of siRNA for EC was previously described (28, 31). Briefly, EC were plated at a density of 70% confluency in 35-mm dishes in FBS-free medium M199 and transfected with siRNA using Mirus TransIT-TKO transfection reagents. For each transfection, 6 μl of the Mirus transfection reagent was mixed with 200 μl of serum-free medium Opti-MEMI in a 5-ml tube and incubated for 5 min at room temperature. Following the incubation, 100 nM siRNA was added into the mixture and incubated for 5 min at room temperature. Fresh complete medium was added to the cells 5 h after transfection, and experiments were conducted 48 h posttransfection. Immunoblotting with anti-mTOR, Raptor, Rictor, or FAK Ab was performed to monitor the efficiency of siRNA knockdown. Anti-Vinculin, β-tubulin, or β-actin Ab was used to confirm equal loading of cellular proteins in lysates.

Cell lysates for Western blot were prepared as previously described (29). Briefly, EC were seeded in 35-mm dishes coated with 0.1% gelatin. Serum and growth factor–starved cells were treated with anti–HLA-II Ab or control IgG at 37°C, washed twice with ice-cold PBS containing 1 mM sodium orthovanadate, and lysed in buffer (containing 20 mM Tris [pH 7.9], 137 mM NaCl, 5 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 10 mM NaF, 1 mM PMSF, 1 mM sodium orthovanadate, 10 μg/ml aprotinin, and 10 μg/ml leupeptin) for 10 min on ice. The cell lysates were centrifuged at 15,000 × g for 10 min at 4°C, and the supernatants were collected and precleared for 2 h with 15 μl of protein A/G plus–agarose at 4°C. The total protein content was measured using the Bradford protein assay method (Pierce) using BSA as standard. Proteins in whole-cell lysates were heated for 5 min at 95°C in 6× SDS sample buffer, electrophoresed on SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. The membranes were blocked using 5% nonfat dry milk in TBS (pH 7.4) containing 0.05% Tween 20 (TBST) for 1 h at room temperature, and incubated with appropriate primary Ab overnight at 4°C. The blots were washed with TBST followed by incubation in HRP-conjugated secondary Ab for 1 h at room temperature. The blots were subsequently washed with TBST and developed with ECL (Amersham). The phosphorylated protein bands were scanned using the Epson Perfection V700 photo scanner and were quantified using ImageJ software (National Institutes of Health, Bethesda, MD).

EC were labeled with CFSE (Molecular Probes) according to the manufacturer’s protocol. Briefly, EC were grown in 35-mm culture dishes coated with 0.1% gelatin up to 70% confluence. The cells were starved in M199 medium without FBS for 6 h, labeled with 2 μM CFSE at 37°C for 15 min, and washed twice in warm M199. CFSE-labeled cells were stimulated with anti–HLA-II Ab for 48 h in M199 with 2% FBS. Cells were detached using 0.125% trypsin/0.05% EDTA, washed with paraformaldehyde (PFA; 2.5% FBS, 0.1% sodium azide, in PBS), and analyzed by flow cytometry. Analysis was performed on 10,000 cells per sample using a FACSCalibur (Becton Dickinson, Mountain View, CA). Freshly CFSE-labeled EC and CFSE-labeled EC grown in complete medium were used as negative and positive controls for staining, respectively. Data were analyzed using CellQuestPro (Becton Dickinson) and ModFit LT software (Verity Software House, Topsham, ME). Cell proliferation was calculated using the Proliferation Wizard Model (Verity Software House). The proliferation index is the sum of the cells in all generations divided by the computed number of original parent cells present at the start of the experiment (33). Our previous studies verified that EC do not exhibit dye extrusion up to 72 h after CFSE labeling (33, 48).

DNA synthesis was measured by BrdU incorporation using BD Pharmingen BrdU flow kits (BD Biosciences) according to the manufacturer’s protocol. Briefly, EC grown to 70% confluence in 35-mm culture dishes coated with 0.1% gelatin in M199 medium containing 2.0% FBS were treated with anti–HLA-II Ab or isotype control IgG for 48 h. During the final 2 h of incubation, 10 μM BrdU was added to the cell culture. The cells were detached with Accutase (Innovative Cell Technologies, San Diego, CA), fixed, and permeabilized at room temperature. DNA was denatured by incubation for 60 min at 37°C with 50 μl of DNase. The cells were incubated with 20 μl of FITC–anti-BrdU Ab for 20 min at room temperature, and then total DNA was stained with 7-aminoactinomycin D. Thereafter, the cells were analyzed for simultaneous green (FL1) and red (FL3) fluorescence emission on a FACSCalibur flow cytometer (Becton Dickinson).

An in vitro wound healing assay was performed as previously described (33, 48). Briefly, EC were grown in 35-mm culture dishes coated with 0.1% gelatin up to confluence. Starved cells were pretreated with 10 μg/ml mitomycin C for 2 h to inhibit cell proliferation. A scratch wound was created with a sterile 200-μl pipette tip. The dishes were rinsed with M199 to remove detached cells. Wounded cells were treated with anti–HLA-II mAb for 16 h. The cells were fixed with 10% neutral buffered formalin, stained with Wright–Giemsa (Sigma-Aldrich), and wound closure was monitored by microscopy. The cell number between two initiated front edges was counted. Migration rate was analyzed by calculating the cell number between two initiated front edges of class II–stimulated EC divided by the cell number between two initiated front edges of control EC.

The migration of EC was measured in a transwell insert system (8 μm pore size; Corning). EC were grown in 24-well plates coated with 0.1% gelatin and were infected with Ad-CIITA or pretreated with TNF-α/IFN-γ for 2 d. Cells were trypsinized, resuspended, and 30,000 cells were seeded on the upper chamber of the insert and stimulated with anti–HLA-II Ab in M199 plus 2% FBS. After 16 h, cells on the upper surface of the membrane were removed, and the migrated cells were fixed with methanol and stained with crystal violet. Three fields per insert were photographed with a ×10 objective lens and were counted (33).

Each experiment was repeated three times independently. Unless otherwise noted, data are presented as mean ± SEM. Differences in protein phosphorylation, cell proliferation, or cell migration were calculated using the Student t test or the one-way ANOVA with Fisher least significant difference (LSD). All p values were two-sided, and <0.05 was considered significant.

HLA-II molecules are not constitutively expressed on primary EC in culture, but they can be upregulated by inflammatory cytokines such as TNF-α and IFN-γ (44). However, treatment of EC with cytokines induces intracellular signaling cascades and phenotypic changes that may interfere and/or confound interpretation of the signaling pathways and crosstalk mechanisms induced by engagement of HLA-II. To overcome this limitation, we engineered an adenoviral vector to express the CIITA, a master regulator of HLA-II gene expression in primary EC (Supplemental Fig. 1A). Prior to initiation of experiments, we optimized the pretreatment time and concentration of cytokines TNF-α/IFN-γ and Ad-CIITA to achieve HLA-II expression. We determined that an MOI of 2 Ad-CIITA achieved high levels of HLA-II expression with low background. Pretreatment of EC with a combination of 200 U of TNF-α and 500 U of IFN-γ for 48 h induced expression of CIITA and HLA-II (Supplemental Fig. 1B). Infection of EC with Ad-CIITA (Supplemental Fig. 1C) or pretreatment of EC with TNF-α and IFN-γ (Supplemental Fig. 1D) induced CIITA protein production, but not on LacZ-transfected or unstimulated EC (Supplemental Fig. 1E, 1F). The bar graph shows that median fluorescence intensity of HLA-II molecules increased on Ad-CIITA–transfected EC and cytokine-pretreated EC as Ab binding to EC increased, with saturation at ∼1 μg/ml (Supplemental Fig. 1G, 1H). Expression of HLA-DR was the highest, followed by nearly equivalent levels of DQ and DP (Supplemental Fig. 1I, 1J).

Having established conditions to generate EC-expressing HLA-II, we determined whether Ab-mediated ligation of HLA-II stimulates proliferation and migration. Prior to initiation of experiments, we performed dose response experiments and established 1 μg/ml HLA-II Ab to elicit the highest degree of EC proliferation and migration (data not shown). This concentration was then used in subsequent experiments. EC from three different donors were infected with Ad-CIITA or pretreated with TNF-α/IFN-γ for 48 h to upregulate HLA-II expression, and then treated with HLA-II Ab for 48 h. Significantly, more DNA synthesis was seen in Ad-CIITA–infected EC exposed to HLA-II Abs compared with isotope control mIgG (Fig. 1A, 1B). In contrast, ligation of LacZ-infected EC with anti–HLA-II Ab failed to stimulate cell proliferation (Fig. 1A, 1B), consistent with lack of HLA-II expression (Supplemental Fig. 1E, 1G). Likewise, ligation of HLA-II on TNF-α/IFN-γ–pretreated EC with a saturating dose of 1 μg/ml Ab stimulated a 2.7-fold increase in cell proliferation compared with isotype control mIgG (Fig. 1A, 1C). Similar results were observed when cell proliferation was assayed by CFSE labeling in Ad-CIITA–transfected or TNF-α/IFN-γ–pretreated EC in response to ligation of HLA-II (Fig. 1D–F). Specifically, more EC underwent several rounds of division, as measured by dilution of CFSE, a 1.5-fold increase in cell proliferation in Ad-CIITA–infected EC, or a 2.7-fold increase in TNF-α/IFN-γ–pretreated EC, in the presence of HLA-II Abs compared with mIgG (Fig. 1D–F).

FIGURE 1.

HLA-II Ab stimulates EC proliferation and migration. EC were infected with Ad-CIITA or pretreated with TNF-α/IFN-γ in 35-mm dishes coated with 0.1% gelatin for 48 h. (A) EC were stimulated with 1 μg/ml HLA-II Ab for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry. DNA synthesis S phase was gated, and proliferation index (PI) is presented as fold increase in the percentage of cells positive for BrdU normalized to untreated control. (D) EC were labeled with CFSE, stimulated with 1 μg/ml HLA-II Ab for 48 h, and harvested. EC proliferation was measured by flow cytometry and analyzed by ModFit LT software. EC proliferation was calculated using the Proliferation Wizard Model. The proliferation index is the sum of the cells in all generations divided by the computed number of original parent cells present at the start of the experiment. (G) EC were pretreated with 10 μg/ml mitomycin C for 2 h to inhibit cell proliferation before being assayed for their ability to migrate. A scratch wound was created with a sterile 200-μl pipette tip. Wounded cells were stimulated with 1 μg/ml anti–HLA-II Ab for 16 h. Representative microscopy fields are shown. The cell number between two initiated front edges was counted; migration rate was analyzed by calculating the cell number between two front edges of class II–stimulated EC divided by the cell number between two front edges of control EC. (J) Cell migration was measured in a transwell insert system. EC infected with Ad-CIITA or pretreated with TNF-α/IFN-γ were seeded to the upper chamber of the insert and stimulated with 1 μg/ml class II Ab. After incubation for 16 h at 37°C, the cells on the upper surface of the insert membrane were removed with a cotton swab, the migrated cells on the reverse side of the insert membrane were fixed with methanol and stained with crystal violet, three middle fields per insert were photographed with a ×10 objective lens, and migrated cells were counted. Fluorescence microscopy images are presented. Original magnification, ×10. (B, C, E, and F) The bar graphs show the mean ± SEM fold change of the proliferation index. (H, I, K, and L) The bar graph shows the mean ± SEM number of migrated cells. Data represent a minimum of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 were analyzed by one-way ANOVA with a Fisher LSD test.

FIGURE 1.

HLA-II Ab stimulates EC proliferation and migration. EC were infected with Ad-CIITA or pretreated with TNF-α/IFN-γ in 35-mm dishes coated with 0.1% gelatin for 48 h. (A) EC were stimulated with 1 μg/ml HLA-II Ab for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry. DNA synthesis S phase was gated, and proliferation index (PI) is presented as fold increase in the percentage of cells positive for BrdU normalized to untreated control. (D) EC were labeled with CFSE, stimulated with 1 μg/ml HLA-II Ab for 48 h, and harvested. EC proliferation was measured by flow cytometry and analyzed by ModFit LT software. EC proliferation was calculated using the Proliferation Wizard Model. The proliferation index is the sum of the cells in all generations divided by the computed number of original parent cells present at the start of the experiment. (G) EC were pretreated with 10 μg/ml mitomycin C for 2 h to inhibit cell proliferation before being assayed for their ability to migrate. A scratch wound was created with a sterile 200-μl pipette tip. Wounded cells were stimulated with 1 μg/ml anti–HLA-II Ab for 16 h. Representative microscopy fields are shown. The cell number between two initiated front edges was counted; migration rate was analyzed by calculating the cell number between two front edges of class II–stimulated EC divided by the cell number between two front edges of control EC. (J) Cell migration was measured in a transwell insert system. EC infected with Ad-CIITA or pretreated with TNF-α/IFN-γ were seeded to the upper chamber of the insert and stimulated with 1 μg/ml class II Ab. After incubation for 16 h at 37°C, the cells on the upper surface of the insert membrane were removed with a cotton swab, the migrated cells on the reverse side of the insert membrane were fixed with methanol and stained with crystal violet, three middle fields per insert were photographed with a ×10 objective lens, and migrated cells were counted. Fluorescence microscopy images are presented. Original magnification, ×10. (B, C, E, and F) The bar graphs show the mean ± SEM fold change of the proliferation index. (H, I, K, and L) The bar graph shows the mean ± SEM number of migrated cells. Data represent a minimum of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 were analyzed by one-way ANOVA with a Fisher LSD test.

Close modal

To determine whether HLA-II signaling stimulates EC migration, primary aortic EC from three different donors were infected with Ad-CIITA or pretreated with TNF-α/IFN-γ for 48 h, then treated with anti–HLA-II Ab for 16 h. Ab ligation of HLA-II on Ad-CIITA–infected EC stimulated a 1.9-fold increase in cell migration (wound healing assay) compared with mIgG-treated EC (Fig. 1G, 1H). Ligation of HLA-II on LacZ-infected EC with anti–HLA-II Ab failed to stimulate detectable EC migration (Fig. 1G, 1H). Ligation of HLA-II on TNF-α/IFN-γ–pretreated EC with Ab stimulated a 4.7-fold increase in cell migration compared with isotype control mIgG (Fig. 1G, 1I). These results were corroborated by using the transwell migration assay. Ligation of HLA-II on Ad-CIITA–infected EC stimulated a 1.9-fold increase in cell migration (Fig. 1J, 1K). Again, no effect of HLA-II Ab was seen in LacZ-infected EC. Furthermore, ligation of HLA-II on TNF-α/IFN-γ–pretreated EC with Ab stimulated a 2.9-fold increase in cell migration compared with isotope control mIgG (Fig. 1J, 1L). EC binding Abs not directed against HLA, anti-CD105 Ab, or anti–integrin αvβ3 Ab did not significantly stimulate cell migration in wound healing and transwell migration assays (Supplemental Fig. 4C–F). These results demonstrate that ligation of HLA-II with Abs stimulates proliferation and migration in primary human EC.

We next sought to identify the intracellular signaling pathways that lead to HLA-II–induced EC proliferation and migration. To characterize HLA-II Ab-triggered activation of intracellular signal transduction networks, we initially established the time and concentration of HLA-II Ab necessary to elicit optimal Ab-stimulated activation of signal transduction pathways in Ad-CIITA–infected and TNF-α/IFN-γ–pretreated EC (Supplemental Figs. 2, 3). Stimulation of Ad-CIITA–infected EC or TNF-α/IFN-γ–pretreated EC with HLA-II Ab at various concentrations (Supplemental Fig. 2) and times (Supplemental Fig. 3) induced a marked increase in the phosphorylation of Src Tyr418, FAK Tyr577, mTOR Ser2448, and S6K Thr389, a site directly targeted by mTORC1, S6RP Ser240/244, a site directly downstream to S6K, Akt Thr308, a site targeted by PDK1 in response to PI3K, Akt Ser473, targeted by mTORC2, and ERK Thr202/Tyr204 compared with mIgG. No increased phosphorylation of any of these targets was detected in LacZ-infected EC (Supplemental Figs. 2, 3). EC-binding non-HLA Abs against CD105 and integrin αvβ3 also did not significantly increase phosphorylation of these signal molecules (Supplemental Fig. 4A, 4B). Given that treatment for 15 min using HLA-II Ab at a concentration of 0.1 μg/ml produced optimal responses with increased phosphorylation of all signaling molecules tested, these conditions were adopted in subsequent experiments. The results shown in Supplemental Figs. 2 and 3 prompted us to use multiple inhibitors and siRNAs to unravel the cause/effect relationships and regulatory feedback loops in this set of signaling events.

The Akt/mTOR network is a key signaling pathway in the regulation of cell metabolism, migration, survival, and proliferation (49) that plays a critical role in HLA class I Ab-induced EC activation, survival, proliferation, and migration (27, 31, 33, 34). However, the function of this pathway in HLA-II Ab-induced EC signaling, proliferation, and migration is unknown. Consequently, we explored the in-depth relationship between mTOR complexes (mTORC1 and mTORC2) and their downstream targets, including S6K, S6RP, and Akt, following ligation of HLA-II molecules with Abs.

Pretreatment of EC with rapamycin for 2 h, which inhibits mTORC1 but does not interfere with mTORC2 signaling, prevented HLA-II–induced phosphorylation of mTOR Ser2448, S6K Thr389, and S6RP Ser240/244, but did not have any detectable effect on the phosphorylation of Akt Ser473 or ERK Thr202/Tyr204 in either Ad-CIITA–infected or TNF-α/IFN-γ–pretreated EC (Fig. 2A, 2B). In contrast, long-term treatment (24 h) with rapamycin, which blocks both mTORC1 and mTORC2 in EC (33), additionally abolished phosphorylation of Akt Ser473, in line with the notion that this residue is targeted by mTORC2 (Fig. 2A, 2B). Interestingly, long-term exposure to rapamycin caused hyperphosphorylation of ERK in Ad-CIITA–infected (Fig. 2A, 2C) or TNF-α/IFN-γ–pretreated EC (Fig. 2B, 2D), and did not affect phosphorylation of c-Raf-1 Ser259. Importantly, either short-term or long-term treatment with rapamycin of Ad-CIITA–infected or TNF-α/IFN-γ–pretreated EC did not affect HLA-II Ab-stimulated phosphorylation of Src Tyr418, FAK Tyr576, p85 PI3K Tyr458, or Akt Thr308 (Fig. 2), implying that Src, FAK, and PI3K/Akt function upstream of the mTOR complexes in the signaling network was triggered by engagement of HLA-II molecules in EC.

FIGURE 2.

mTOR regulates HLA-II Ab-stimulated activation of intracellular signal networks, cell proliferation and migration. EC were infected with Ad-LacZ (A, C, and I) or Ad-CIITA or (B, DF, and J) pretreated with TNF-α/IFN-γ for 48 h. (A, B, E, and F) Starved EC were pretreated with 30 nM rapamycin for 2 or 24 h; or (E and F) with 10 nM everolimus for 2 or 24 h; or (I and J) with 30 nM rapamycin or 10 nM everolimus for 24 h; or (E, F, I, and J) with 1 μM PP242 for 2 h. (A, B, E, and F) Cells were stimulated with 0.1 μg/ml HLA-II Ab or control mIgG for 15 min. Proteins in the precleared cell lysates were separated by 6–15% SDS-PAGE followed by immunoblotting with (A and B) anti–phospho-Src Tyr418, FAK Tyr576, p85 PI3K Tyr458, or Akt Thr308, (A, B, E, and F) Akt Ser473, mTOR Ser2448, S6K Thr389, S6RP Ser240/244, c-Raf-1 Ser259, or ERK1/2 Thr202/Tyr204 Abs. The membrane was reprobed with anti-vinculin, S6K, S6RP, Akt, ERK or (A and B) Src, FAK, PI3K, mTOR, or β-actin total Abs to confirm equal loading of proteins. (C, D, G, and H) The bar graphs show phosphorylated protein bands shown in (A), (B), (E), and (F) were quantified by densitometry scan analysis, and results are expressed as the mean ± SEM percentage of maximal increase in phosphorylation above control values. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were analyzed by one-way ANOVA with a Fisher LSD test. (I and J) EC were detached with 0.125% trypsin/0.05% EDTA, washed with PBS containing 2.5% FBS and 0.1% sodium azide (PFA), and incubated with 1 μg/ml anti–HLA-II mAbs for 30 min at 4°C. EC were washed three times with PFA and incubated with FITC-conjugated goat anti-mouse F(ab′)2 fragment for 30 min at 4°C. The fluorescence intensity was measured by LSRFortessa flow cytometry using the FACSDiva program (Becton Dickinson). The bar graphs show the mean ± SEM of median channel fluorescence values of HLA-II expression on EC and were analyzed by one-way ANOVA with a Fisher LSD test. Data represent at least three independent experiments.

FIGURE 2.

mTOR regulates HLA-II Ab-stimulated activation of intracellular signal networks, cell proliferation and migration. EC were infected with Ad-LacZ (A, C, and I) or Ad-CIITA or (B, DF, and J) pretreated with TNF-α/IFN-γ for 48 h. (A, B, E, and F) Starved EC were pretreated with 30 nM rapamycin for 2 or 24 h; or (E and F) with 10 nM everolimus for 2 or 24 h; or (I and J) with 30 nM rapamycin or 10 nM everolimus for 24 h; or (E, F, I, and J) with 1 μM PP242 for 2 h. (A, B, E, and F) Cells were stimulated with 0.1 μg/ml HLA-II Ab or control mIgG for 15 min. Proteins in the precleared cell lysates were separated by 6–15% SDS-PAGE followed by immunoblotting with (A and B) anti–phospho-Src Tyr418, FAK Tyr576, p85 PI3K Tyr458, or Akt Thr308, (A, B, E, and F) Akt Ser473, mTOR Ser2448, S6K Thr389, S6RP Ser240/244, c-Raf-1 Ser259, or ERK1/2 Thr202/Tyr204 Abs. The membrane was reprobed with anti-vinculin, S6K, S6RP, Akt, ERK or (A and B) Src, FAK, PI3K, mTOR, or β-actin total Abs to confirm equal loading of proteins. (C, D, G, and H) The bar graphs show phosphorylated protein bands shown in (A), (B), (E), and (F) were quantified by densitometry scan analysis, and results are expressed as the mean ± SEM percentage of maximal increase in phosphorylation above control values. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were analyzed by one-way ANOVA with a Fisher LSD test. (I and J) EC were detached with 0.125% trypsin/0.05% EDTA, washed with PBS containing 2.5% FBS and 0.1% sodium azide (PFA), and incubated with 1 μg/ml anti–HLA-II mAbs for 30 min at 4°C. EC were washed three times with PFA and incubated with FITC-conjugated goat anti-mouse F(ab′)2 fragment for 30 min at 4°C. The fluorescence intensity was measured by LSRFortessa flow cytometry using the FACSDiva program (Becton Dickinson). The bar graphs show the mean ± SEM of median channel fluorescence values of HLA-II expression on EC and were analyzed by one-way ANOVA with a Fisher LSD test. Data represent at least three independent experiments.

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Rapamycin and everolimus are allosteric inhibitors of mTOR that act via FKBP-12 (33), whereas the compound PP242 acts at the catalytic site of mTOR, thereby inhibiting both mTORC1 and mTORC2 (50). Exposure to everolimus or PP242 prevented the phosphorylation of mTOR Ser2448, S6K Thr389, and S6RP Ser240/244 (Fig. 2E), indicating that allosteric or active-site inhibitors of mTOR blocked the mTORC1/S6K signal cascade. Long-term treatment with allosteric inhibitors or exposure to PP242 abolished phosphorylation of Akt Ser473 (Fig. 2F, 2G), in line with the notion that these interventions blocked mTORC2. In agreement with data shown in Fig. 2A–D, long-term treatment with rapamycin or everolimus or exposure to PP242 caused overactivation of ERK (Fig. 2F, 2H) with no effects on HLA-II expression (Fig. 2I, 2J), and did not affect phosphorylation of c-Raf-1 Ser259 (Fig. 2F). These results imply that mTORC2 mediates a novel negative feedback loop, which reduces the activation of ERK in HLA-II–stimulated EC cells.

We next determined whether inhibition of mTOR in these cells prevents HLA-II Ab-stimulated cell proliferation and migration. Treatment with everolimus, rapamycin, or PP242 blocked HLA-II Ab-stimulated cell proliferation in either Ad-CIITA–infected EC (Fig. 3A) or TNF-α/IFN-γ–pretreated EC (Fig. 3B).

FIGURE 3.

mTOR regulates HLA-II Ab-stimulated EC proliferation and migration. EC were infected with Ad-LacZ or Ad-CIITA (A, C, and E) or pretreated with TNF-α/IFN-γ for 48 h (B, D, and F). (A and B) Cells were stimulated with 1 μg/ml HLA-II Ab or control mIgG for 48 h. Cells were incorporated with BrdU for 2 h and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and the proliferation index (PI) is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. (C and D) Cells were pretreated with 10 μg/ml mitomycin C for 2 h to inhibit cell proliferation before being assayed for their ability to migrate. A scratch wound was created with a sterile 200-μl pipette tip. Wounded cells were stimulated with 1 μg/ml anti–HLA-II Ab for 16 h. The cell number between two initiated front edges was counted; migration rate was analyzed by calculating the cell number between two initiated front edges of class II–stimulated EC divided by the cell number between two initiated front edges of control EC. (E and F) Cell migration was measured in a transwell insert system. EC infected with Ad-CIITA or pretreated with TNF-α/IFN-γ were added to the upper chamber of the insert and stimulated with 1 μg/ml class II Ab. After incubation for 16 h at 37°C, the cells on the upper surface of the insert membrane were removed with a cotton swab, the migrated cells on the bottom of the insert membrane were fixed with methanol and stained with crystal violet, three middle fields per insert were photographed with a ×10 objective lens, and migrated cells were counted. The bar graphs show the mean ± SEM fold change of (A and B) proliferation index or (C–F) migrated cells. Data represent at least three independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001 were analyzed by one-way ANOVA with a Fisher LSD test.

FIGURE 3.

mTOR regulates HLA-II Ab-stimulated EC proliferation and migration. EC were infected with Ad-LacZ or Ad-CIITA (A, C, and E) or pretreated with TNF-α/IFN-γ for 48 h (B, D, and F). (A and B) Cells were stimulated with 1 μg/ml HLA-II Ab or control mIgG for 48 h. Cells were incorporated with BrdU for 2 h and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and the proliferation index (PI) is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. (C and D) Cells were pretreated with 10 μg/ml mitomycin C for 2 h to inhibit cell proliferation before being assayed for their ability to migrate. A scratch wound was created with a sterile 200-μl pipette tip. Wounded cells were stimulated with 1 μg/ml anti–HLA-II Ab for 16 h. The cell number between two initiated front edges was counted; migration rate was analyzed by calculating the cell number between two initiated front edges of class II–stimulated EC divided by the cell number between two initiated front edges of control EC. (E and F) Cell migration was measured in a transwell insert system. EC infected with Ad-CIITA or pretreated with TNF-α/IFN-γ were added to the upper chamber of the insert and stimulated with 1 μg/ml class II Ab. After incubation for 16 h at 37°C, the cells on the upper surface of the insert membrane were removed with a cotton swab, the migrated cells on the bottom of the insert membrane were fixed with methanol and stained with crystal violet, three middle fields per insert were photographed with a ×10 objective lens, and migrated cells were counted. The bar graphs show the mean ± SEM fold change of (A and B) proliferation index or (C–F) migrated cells. Data represent at least three independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001 were analyzed by one-way ANOVA with a Fisher LSD test.

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Similarly, pretreatment with these pharmacological agents inhibited HLA-II Ab-stimulated cell migration in Ad-CIITA–infected EC (Fig. 3C) or TNF-α/IFN-γ–pretreated EC (Fig. 3D). These results were corroborated using a transwell migration assay (Fig. 3E, 3F). Collectively, these results indicate that mTORC1 and mTORC2 play a critical role in HLA-II Ab-stimulated EC proliferation and migration.

To corroborate our findings with pharmacological inhibitors of mTOR in HLA-II Ab-stimulated EC proliferation and signaling, we next determined the effect of siRNA-mediated knockdown of mTOR, Raptor (mTORC1), or Rictor (mTORC2) protein expression on cell proliferation. Initially, we characterized the efficiency of siRNA knockdown. As shown in Fig. 4A, transfection of EC with siRNA targeting mTOR siRNA markedly reduced mTOR protein expression but did not alter the expression of Raptor, Rictor, S6K, Akt, ERK, Vinculin, β-tubulin, and β-actin in the same cell lysates. Furthermore, transfection of EC with nontargeting control siRNA had no effect on mTOR expression (Fig. 4A). Transfection of EC with siRNAs targeting Raptor and Rictor also caused a marked decrease in the expression of these proteins (Fig. 4A). Importantly, siRNA-mediated knockdown of mTOR, Raptor, or Rictor did not reduce HLA-II expression level on Ad-CIITA–infected EC (Fig. 4B) and TNF-α/IFN-γ–pretreated EC (Fig. 4C) measured by flow cytometry and completely blocked HLA-II Ab-stimulated cell proliferation (Fig. 4D, 4E), supporting the notion that mTORC1 and mTORC2 play key roles in EC proliferation triggered by engagement of HLA-II.

FIGURE 4.

Effects of mTOR, Raptor, and Rictor siRNA knockdown on HLA-II Ab-stimulated EC proliferation and activation of signal transduction networks. (AE) EC were transfected with 100 nM mTOR or (A–G) Raptor, Rictor, or control siRNA for 48 h. (A, F, and G) Protein in precleared cell lysates were separated by 6 ∼ 15% SDS-PAGE followed by immunoblotting with anti-mTOR, Raptor, Rictor, S6K, Akt, ERK, Vinculin, β-tubulin, and β-actin. (B, D, and F) EC were infected with Ad-LacZ or Ad-CIITA or (C, E, and G) pretreated with TNF-α/IFN-γ for 48 h. (B and C) EC were detached with 0.125% trypsin/0.05% EDTA, washed with PFA, and incubated with 1 μg/ml HLA-II Ab for 30 min at 4°C. EC were washed three times with PFA and incubated with FITC-conjugated goat anti-mouse F(ab′)2 fragment for 30 min at 4°C. The fluorescence intensity was measured on an LSRFortessa flow cytometer and calculated using the FACSDiva program (Becton Dickinson). The bar graphs show the mean ± SEM of median channel fluorescence values of HLA-II expression on EC and were analyzed by one-way ANOVA with a Fisher LSD test. (D and E) Transfected EC were stimulated with 1 μg/ml HLA-II Ab or mIgG for 48 h. EC were incorporated with BrdU for 2 h and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and proliferation index (PI) is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. The bar graphs show the mean ± SEM fold change of proliferation index. (F and G) Transfected EC were stimulated with 0.1 μg/ml HLA-II Ab for 15 min. Treatment of EC with mIgG serves as negative control. Proteins in the precleared cell lysates were separated by 6 ∼ 15% SDS-PAGE followed by immunoblotting with Abs to Raptor, Rictor for knockdown efficiency, anti–phospho-Src Tyr418, FAK Tyr576, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, S6K Thr389, S6RP Ser240/244, c-Raf-1 Ser259, or ERK Thr202/Tyr204. The membranes were reprobed with anti-Src, FAK, PI3K, Akt, S6K, S6RP, ERK, or Vinculin Abs to confirm equal loading of proteins. (H and I) Phosphorylated protein bands shown in (F and G) were quantified by densitometry scan analysis and results are expressed as the mean ± SEM percentage of maximal increase in phosphorylation above control values. ***p < 0.001, ****p < 0.0001 were analyzed by one-way ANOVA with a Fisher LSD test. Data represent at least three independent experiments.

FIGURE 4.

Effects of mTOR, Raptor, and Rictor siRNA knockdown on HLA-II Ab-stimulated EC proliferation and activation of signal transduction networks. (AE) EC were transfected with 100 nM mTOR or (A–G) Raptor, Rictor, or control siRNA for 48 h. (A, F, and G) Protein in precleared cell lysates were separated by 6 ∼ 15% SDS-PAGE followed by immunoblotting with anti-mTOR, Raptor, Rictor, S6K, Akt, ERK, Vinculin, β-tubulin, and β-actin. (B, D, and F) EC were infected with Ad-LacZ or Ad-CIITA or (C, E, and G) pretreated with TNF-α/IFN-γ for 48 h. (B and C) EC were detached with 0.125% trypsin/0.05% EDTA, washed with PFA, and incubated with 1 μg/ml HLA-II Ab for 30 min at 4°C. EC were washed three times with PFA and incubated with FITC-conjugated goat anti-mouse F(ab′)2 fragment for 30 min at 4°C. The fluorescence intensity was measured on an LSRFortessa flow cytometer and calculated using the FACSDiva program (Becton Dickinson). The bar graphs show the mean ± SEM of median channel fluorescence values of HLA-II expression on EC and were analyzed by one-way ANOVA with a Fisher LSD test. (D and E) Transfected EC were stimulated with 1 μg/ml HLA-II Ab or mIgG for 48 h. EC were incorporated with BrdU for 2 h and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and proliferation index (PI) is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. The bar graphs show the mean ± SEM fold change of proliferation index. (F and G) Transfected EC were stimulated with 0.1 μg/ml HLA-II Ab for 15 min. Treatment of EC with mIgG serves as negative control. Proteins in the precleared cell lysates were separated by 6 ∼ 15% SDS-PAGE followed by immunoblotting with Abs to Raptor, Rictor for knockdown efficiency, anti–phospho-Src Tyr418, FAK Tyr576, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, S6K Thr389, S6RP Ser240/244, c-Raf-1 Ser259, or ERK Thr202/Tyr204. The membranes were reprobed with anti-Src, FAK, PI3K, Akt, S6K, S6RP, ERK, or Vinculin Abs to confirm equal loading of proteins. (H and I) Phosphorylated protein bands shown in (F and G) were quantified by densitometry scan analysis and results are expressed as the mean ± SEM percentage of maximal increase in phosphorylation above control values. ***p < 0.001, ****p < 0.0001 were analyzed by one-way ANOVA with a Fisher LSD test. Data represent at least three independent experiments.

Close modal

To further characterize the mechanisms by which mTOR complexes regulate HLA-II Ab-stimulated activation of signal transduction pathways, we determined the effect of siRNA knockdown of Raptor and Rictor on HLA-II Ab-induced signaling. Similar to short-term exposure to rapamycin (Fig. 2A, 2B, 2E), knockdown of Raptor with siRNA blocked HLA-II Ab-induced phosphorylation of S6K Thr389 and S6RP Ser240/244 (Fig. 4F, 4G). Interestingly, transfection of Ad-CIITA–infected EC and TNF-α/IFN-γ–pretreated EC with Raptor siRNA to dissect mTORC1 caused hyperactivation of Akt Ser473 (Fig. 4F–I), suggesting that mTORC1 mediates a negative feedback loop, reducing the activity of mTORC2. Conversely, siRNA-mediated knockdown of Rictor to block mTORC2 abolished HLA-II Ab-mediated phosphorylation of Akt Ser473 and caused overactivation of ERK (Fig. 4F–I), and it did not affect phosphorylation of c-Raf-1 Ser259 (Fig. 4F, 4G), thus confirming our results with long-term treatment with allosteric inhibitors or a catalytic inhibitor of mTOR (Fig. 2A–H). As expected, transfection of EC with Rictor siRNA did not inhibit mTORC1-mediated phosphorylation of S6K Thr389 and S6RP Ser240/244 (Fig. 4F, 4G). It is noteworthy that knockdown of Raptor or Rictor did not affect HLA-II Ab-stimulated phosphorylation of Src Tyr418, FAK Tyr576, p85 PI3K Tyr458, or Akt Thr308, indicating that Src, FAK, PI3K, and PDK1/Akt function upstream of the mTOR signaling pathway.

As PI3K/Akt is a major pathway leading to mTOR activation, we determined whether HLA-II Ab-stimulated mTOR activation also proceeds through PI3K/Akt in human EC. Pretreatment of starved Ad-CIITA–infected and TNF-α/IFN-γ–pretreated EC with either the dual PI3K/mTOR inhibitor LY294002 or the compound A66, a specific and potent inhibitor of the p110α catalytic subunit of PI3K (51, 52), suppressed HLA-II Ab-stimulated phosphorylation of p85 PI3K Tyr458, Akt Thr308, and Ser473 and also abolished phosphorylation of mTOR Ser2448, S6K Thr389, and S6RP Ser240/244 (Fig. 5A, 5B). Exposure of EC to either LY294002 or A66 did not affect HLA-II Ab-induced phosphorylation of Src Tyr418 or FAK Tyr576 (Fig. 5A, 5B), implying that the FAK/Src kinases function upstream or in parallel to PI3K/Akt/mTOR in EC.

FIGURE 5.

PI3K regulates HLA-II Ab-mediated signal transduction pathways, cell proliferation, and migration. EC were (A, C, E, G, I, and K) infected with Ad-LacZ or Ad-CIITA or (B, D, F, H, J, and L) pretreated with TNF-α/IFN-γ for 48 h. (A–D and G–L) Starved cells were pretreated with 20 μM LY294002 or with 10 μM A66, or (E and F) with 1 μM MK-2206 or with 1 μM GDC-0068 for 60 min. (A, B, E, and F) Pretreated EC were stimulated with 0.1 μg/ml HLA-II Ab or mIgG control for 15 min. Proteins in the precleared cell lysates were separated by 6 ∼ 15% SDS-PAGE followed by immunoblotting with anti–phospho-Src Tyr418, FAK Tyr576, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, mTOR Ser2448, S6K Thr389, S6RP Ser240/244, MEK Ser217/221, c-Raf-1 Ser259, or ERK Thr202/Tyr204. The membranes were reprobed with anti-Src, FAK, PI3K, Akt, mTOR, S6K, S6RP, ERK, or β-actin Abs to confirm equal loading of proteins. (C and D) Phosphorylated protein bands shown in (A) and (B) were quantified by densitometry scan analysis and results are expressed as the mean ± SEM percentage of maximal increase in phosphorylation above control values. **p < 0.01, ***p < 0.001, ****p < 0.0001 were analyzed by one-way ANOVA with a Fisher LSD test. (G and H) Starved cells were stimulated with 1 μg/ml HLA-II Ab for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and proliferation index is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. The bar graphs show the mean ± SEM fold change of proliferation index. (I and J) Cells were pretreated with 10 μg/ml mitomycin C for 2 h to inhibit cell proliferation before being assayed for their ability to migrate. A scratch wound was created with a sterile 200-μl pipette tip. Wounded cells were stimulated with 1 μg/ml anti–HLA-II Ab or mIg for 16 h. The cell number between two initiated front edges was counted; migration rate was analyzed by calculating the cell number between two initiated front edges of class II–stimulated EC divided by the cell number between two initiated front edges of negative control EC. (K and L) Cell migration was measured in a transwell insert system. EC (K) infected with Ad-CIITA or (L) pretreated with TNF-α/IFN-γ were seeded to the upper chamber of the insert and stimulated with 1 μg/ml HLA-II Ab or mIgG for 16 h at 37°C. After incubation, the cells on the upper surface of the insert membrane were removed with a cotton swab, the migrated cells on the bottom of the insert membrane were fixed with methanol and stained with crystal violet, three middle fields per insert were photographed with a ×10 objective lens, and migrated cells were counted. (G and H) The bar graphs show the mean ± SEM fold change of proliferation index. (I–L) The bar graph shows the mean ± SEM number of migrated cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were analyzed by one-way ANOVA with a Fisher LSD test. Data represent at least three independent experiments.

FIGURE 5.

PI3K regulates HLA-II Ab-mediated signal transduction pathways, cell proliferation, and migration. EC were (A, C, E, G, I, and K) infected with Ad-LacZ or Ad-CIITA or (B, D, F, H, J, and L) pretreated with TNF-α/IFN-γ for 48 h. (A–D and G–L) Starved cells were pretreated with 20 μM LY294002 or with 10 μM A66, or (E and F) with 1 μM MK-2206 or with 1 μM GDC-0068 for 60 min. (A, B, E, and F) Pretreated EC were stimulated with 0.1 μg/ml HLA-II Ab or mIgG control for 15 min. Proteins in the precleared cell lysates were separated by 6 ∼ 15% SDS-PAGE followed by immunoblotting with anti–phospho-Src Tyr418, FAK Tyr576, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, mTOR Ser2448, S6K Thr389, S6RP Ser240/244, MEK Ser217/221, c-Raf-1 Ser259, or ERK Thr202/Tyr204. The membranes were reprobed with anti-Src, FAK, PI3K, Akt, mTOR, S6K, S6RP, ERK, or β-actin Abs to confirm equal loading of proteins. (C and D) Phosphorylated protein bands shown in (A) and (B) were quantified by densitometry scan analysis and results are expressed as the mean ± SEM percentage of maximal increase in phosphorylation above control values. **p < 0.01, ***p < 0.001, ****p < 0.0001 were analyzed by one-way ANOVA with a Fisher LSD test. (G and H) Starved cells were stimulated with 1 μg/ml HLA-II Ab for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and proliferation index is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. The bar graphs show the mean ± SEM fold change of proliferation index. (I and J) Cells were pretreated with 10 μg/ml mitomycin C for 2 h to inhibit cell proliferation before being assayed for their ability to migrate. A scratch wound was created with a sterile 200-μl pipette tip. Wounded cells were stimulated with 1 μg/ml anti–HLA-II Ab or mIg for 16 h. The cell number between two initiated front edges was counted; migration rate was analyzed by calculating the cell number between two initiated front edges of class II–stimulated EC divided by the cell number between two initiated front edges of negative control EC. (K and L) Cell migration was measured in a transwell insert system. EC (K) infected with Ad-CIITA or (L) pretreated with TNF-α/IFN-γ were seeded to the upper chamber of the insert and stimulated with 1 μg/ml HLA-II Ab or mIgG for 16 h at 37°C. After incubation, the cells on the upper surface of the insert membrane were removed with a cotton swab, the migrated cells on the bottom of the insert membrane were fixed with methanol and stained with crystal violet, three middle fields per insert were photographed with a ×10 objective lens, and migrated cells were counted. (G and H) The bar graphs show the mean ± SEM fold change of proliferation index. (I–L) The bar graph shows the mean ± SEM number of migrated cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were analyzed by one-way ANOVA with a Fisher LSD test. Data represent at least three independent experiments.

Close modal

Interestingly, treatment with LY294002 enhanced ERK phosphorylation induced by Ab-mediated ligation of HLA-II in either Ad-CIITA–infected EC (Fig. 5A, 5C) or TNF-α/IFN-γ–pretreated EC (Fig. 5B, 5D) and did not affect phosphorylation of c-Raf-1 Ser259 (Fig. 5A, 5B). These results reinforce the notion (posited above) that mTORC2 mediates negative feedback on ERK activation.

Akt may inhibit RAF-1 by directly phosphorylating c-Raf-1 at Ser259 (53). However, this negative crosstalk is controversial (54, 55). Therefore, we determined whether ERK overactivation induced by mTORC2 and PI3K inhibition is mediated by reduction of c-Raf-1 phosphorylation at Ser259. Starved Ad-CIITA–infected EC and TNF-α/IFN-γ EC were pretreated with allosteric (MK-2206) or active site (GDC-0068) inhibitors of Akt. Inhibition of Akt blocked PI3K/Akt targeting molecules S6K Thr389 and S6RP Ser240/244, but it did not affect constitutive phosphorylation of c-Raf-1 at Ser259. Moreover, hyperphosphorylation of ERK at Thr202/Tyr204 was not observed under Akt inhibition (Fig. 5E, 5F). Our results indicate that PI3K inhibition with LY294002 suppresses a negative feedback loop via mTORC2, thereby leading to enhanced MEK/ERK pathway activity in EC.

Next, we determined whether PI3K was also required for class II Ab-induced cell proliferation and migration, as it would be expected in the light of our previous results with mTOR inhibitors and knockdown of Raptor and Rictor. To test this prediction, starved Ad-CIITA–infected EC and TNF-α/IFN-γ–pretreated EC were exposed to LY294002 or A66 and then stimulated with anti–HLA-II Ab. Cell proliferation was measured by BrdU incorporation, and cell migration was measured by wound healing and Transwell migration assays. Pretreatment with LY294002 or A66 completely prevented class II Ab-stimulated cell proliferation in Ad-CIITA–infected EC (Fig. 5G) and TNF-α/IFN-γ–pretreated EC (Fig. 5H). Pretreatment with LY294002 also blocked class II Ab-mediated EC migration in Ad-CIITA–infected EC (Fig. 5I, 5K) and TNF-α/IFN-γ–pretreated EC (Fig. 5J, 5L) by wound healing or transwell migration assay. These data indicate that PI3K regulates class II Ab-stimulated mTOR and thereby leads to cell proliferation and migration.

Thus far, our analysis of HLA-II signaling shows that multiple pharmacological inhibitors or transfection of siRNAs targeting Raptor and Rictor do not affect the activation of Src (scored by phosphorylation at Tyr418) or FAK (at Tyr576/577). These findings imply that these kinases are likely to function as upstream elements in the signaling cascade triggered by engagement of HLA-II in EC. To explore this hypothesis, we next evaluated the impact of Src inhibition on phosphorylation of FAK, PI3K, mTOR, S6K, S6RP, Akt, and ERK. Exposure of Ad-CIITA–infected or TNF-α/IFN-γ–pretreated EC to the Src inhibitor PP2 prevented class II Ab-stimulated phosphorylation of Src Tyr418, FAK Tyr576/577, p85 PI3KTyr458, Akt Thr308, and Akt Ser473 and also abolished phosphorylation of mTOR Ser2448, S6K Thr389, and S6RP Ser240/244 with no effect on HLA-II expression level (Fig. 6C, 6D). Importantly, the Src inhibitor also prevented phosphorylation of ERK at Thr202/Tyr204 in both Ad-CIITA–infected and TNF-α/IFN-γ–pretreated EC (Fig. 6A, 6B).

FIGURE 6.

Src regulates HLA-II Ab-stimulated activation of intracellular signal networks, cell proliferation, and migration. EC were (A, C, E, G, I, and K) infected with Ad-LacZ or Ad-CIITA or (B, D, F, H, J, and L) pretreated with TNF-α/IFN-γ for 48 h. (A–D and G–L) Starved cells were pretreated with 12.5 μM PP2, or (A, B, G, and H) with 1 μM dasatinib, or (I–L) with PP2 inactive analog PP3 for 30 min. (E and F) Starved EC were transfected with 100 nM c-Src or control siRNA for 48 h. (A, B, E, and F) EC were stimulated with 0.1 μg/ml HLA-II Ab or mIgG control for 15 min. Treatment of EC with mIgG serves as negative control. Proteins in the precleared cell lysates were separated by 6 ∼ 15% SDS-PAGE followed by immunoblotting with anti–phospho-Src Tyr418, FAK Tyr576, FAK Tyr577, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, mTOR Ser2448, S6K Thr389, S6RP Ser240/244, or ERK Thr202/Tyr204. The membranes were reprobed with anti-Src, FAK, PI3K, Akt, mTOR, S6K, S6RP, ERK, or β-actin Abs to confirm equal loading of proteins. (C and D) EC were detached, incubated with 1 μg/ml HLA-II Ab for 30 min at 4°C, washed three times with PFA, and the cells were incubated with FITC-conjugated goat anti-mouse F(ab′)2 fragment for 30 min at 4°C. The fluorescence intensity was measured by LSRFortessa flow cytometry and calculated using the FACSDiva program. The bar graphs show the mean ± SEM of median channel fluorescence values of HLA-II expression on EC and were analyzed by one-way ANOVA with a Fisher LSD test. (G and H) Starved cells were stimulated with 1 μg/ml HLA-II Ab for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and proliferation index (PI) is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. (I and J) Cells were pretreated with 10 μg/ml mitomycin C for 2 h to inhibit cell proliferation before being assayed for their ability to migrate. A scratch wound was created with a sterile 200-μl pipette tip. Wounded cells were stimulated with 1 μg/ml anti–HLA-II Ab for 16 h. The cell number between two initiated front edges was counted; migration rate was analyzed by calculating the cell number between two initiated front edges of class II–stimulated EC divided by the cell number between two initiated front edges of negative control EC. (K and L) Cell migration was measured in a transwell insert system. EC infected with Ad-CIITA or pretreated with TNF-α/IFN-γ were seeded to the upper chamber of the insert and stimulated with 1 μg/ml HLA-II Ab for 16 h at 37°C. After incubation, the cells on the upper surface of the insert membrane were removed with a cotton swab, the migrated cells on the bottom of the insert membrane were fixed with methanol and stained with crystal violet, three middle fields per insert were photographed with a ×10 objective lens, and migrated cells were counted. (G and H) The bar graphs show the mean ± SEM fold change of proliferation index. (I–L) The bar graph shows the mean ± SEM number of migrated cells. Data represent at least three independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001 were analyzed by one-way ANOVA with a Fisher LSD test.

FIGURE 6.

Src regulates HLA-II Ab-stimulated activation of intracellular signal networks, cell proliferation, and migration. EC were (A, C, E, G, I, and K) infected with Ad-LacZ or Ad-CIITA or (B, D, F, H, J, and L) pretreated with TNF-α/IFN-γ for 48 h. (A–D and G–L) Starved cells were pretreated with 12.5 μM PP2, or (A, B, G, and H) with 1 μM dasatinib, or (I–L) with PP2 inactive analog PP3 for 30 min. (E and F) Starved EC were transfected with 100 nM c-Src or control siRNA for 48 h. (A, B, E, and F) EC were stimulated with 0.1 μg/ml HLA-II Ab or mIgG control for 15 min. Treatment of EC with mIgG serves as negative control. Proteins in the precleared cell lysates were separated by 6 ∼ 15% SDS-PAGE followed by immunoblotting with anti–phospho-Src Tyr418, FAK Tyr576, FAK Tyr577, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, mTOR Ser2448, S6K Thr389, S6RP Ser240/244, or ERK Thr202/Tyr204. The membranes were reprobed with anti-Src, FAK, PI3K, Akt, mTOR, S6K, S6RP, ERK, or β-actin Abs to confirm equal loading of proteins. (C and D) EC were detached, incubated with 1 μg/ml HLA-II Ab for 30 min at 4°C, washed three times with PFA, and the cells were incubated with FITC-conjugated goat anti-mouse F(ab′)2 fragment for 30 min at 4°C. The fluorescence intensity was measured by LSRFortessa flow cytometry and calculated using the FACSDiva program. The bar graphs show the mean ± SEM of median channel fluorescence values of HLA-II expression on EC and were analyzed by one-way ANOVA with a Fisher LSD test. (G and H) Starved cells were stimulated with 1 μg/ml HLA-II Ab for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and proliferation index (PI) is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. (I and J) Cells were pretreated with 10 μg/ml mitomycin C for 2 h to inhibit cell proliferation before being assayed for their ability to migrate. A scratch wound was created with a sterile 200-μl pipette tip. Wounded cells were stimulated with 1 μg/ml anti–HLA-II Ab for 16 h. The cell number between two initiated front edges was counted; migration rate was analyzed by calculating the cell number between two initiated front edges of class II–stimulated EC divided by the cell number between two initiated front edges of negative control EC. (K and L) Cell migration was measured in a transwell insert system. EC infected with Ad-CIITA or pretreated with TNF-α/IFN-γ were seeded to the upper chamber of the insert and stimulated with 1 μg/ml HLA-II Ab for 16 h at 37°C. After incubation, the cells on the upper surface of the insert membrane were removed with a cotton swab, the migrated cells on the bottom of the insert membrane were fixed with methanol and stained with crystal violet, three middle fields per insert were photographed with a ×10 objective lens, and migrated cells were counted. (G and H) The bar graphs show the mean ± SEM fold change of proliferation index. (I–L) The bar graph shows the mean ± SEM number of migrated cells. Data represent at least three independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001 were analyzed by one-way ANOVA with a Fisher LSD test.

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To substantiate that Src family kinases are required for triggering the signaling network characterized in this study in response to HLA-II ligation, we next tested dasatinib, a second-generation tyrosine kinase inhibitor, including the Src family, recently used in clinical trials in solid tumors and leukemia (56, 57). Dasatinib, similar to PP2, abolished HLA-II Ab-stimulated phosphorylation of Src Tyr418, FAK Tyr576/577, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, mTOR Ser2448, S6K Thr389, and S6RP Ser240/244 and ERK Thr202/Tyr204 in both Ad-CIITA–infected and TNF-α/IFN-γ–pretreated EC (Fig. 6A, 6B).

There are nine members of Src family, including c-Src, Fyn, and Yes, expressed in EC (29). Different members of the Src family may play a different role in HLA-II Ab-triggered signal networks. To confirm our observation using pharmacological inhibitors of Src, we next determined the effect of c-Src siRNA on HLA-II Ab-induced Src activation and its downstream targets. First, we determined the efficiency of c-Src siRNA on c-Src expression. As shown in Fig. 6E and 6F, c-Src expression was markedly decreased by siRNA knockdown whereas transfection of EC with nontargeted siRNA had no effect on c-Src protein expression (Fig. 6E, lanes 1–4, 6F, lanes 1 and 2). To verify c-Src siRNA specificity, we determined the expression of several cytoskeletal proteins in the same cell lysates of EC transfected with c-Src siRNA. Transfection of EC with c-Src siRNA or control siRNA did not affect expression of vinculin and β-actin (Fig. 6E, 6F). Furthermore, transfection of Ad-CIITA–infected and TNF-α/IFN-γ–pretreated EC with c-Src siRNA prevented HLA-II Ab-stimulated phosphorylation of FAK Tyr576/577, p85 PI3KTyr458, Akt Thr308, and Akt Ser473 and abolished phosphorylation of mTOR Ser2448, S6K Thr389, and S6RP Ser240/244 (Fig. 6E, 6F). Importantly, the c-Src siRNA also prevented phosphorylation of ERK at Thr202/Tyr204 in both Ad-CIITA–infected and TNF-α/IFN-γ–pretreated EC (Fig. 6E, 6F).

In view of these results, we anticipated that Src should be essential for EC proliferation and migration induced by Ab-mediated ligation of HLA-II. Pretreatment with PP2 or dasatinib completely inhibited class II Ab-stimulated cell proliferation in both Ad-CIITA–infected and TNF-α/IFN-γ–pretreated EC (Fig. 6G, 6H). Furthermore, pretreatment of EC with PP2 abolished HLA-II Ab-stimulated cell migration as determined by wound healing and transwell migration assays (Fig. 6I–L). In contrast, the PP2 inactive analog PP3 did not inhibit class II–stimulated cell migration in either Ad-CIITA–infected EC or TNF-α/IFN-γ–pretreated EC (Fig. 6I–L). Our results are consistent with the notion that Src activation is one of the earliest events in the HLA-II Ab-triggered signal transduction network elucidated in this study and required for EC proliferation and migration.

The interaction between FAK and Src plays a critical role in the activation of these kinases in response to a variety of stimuli, but the role of FAK in Src activation in EC remains unknown. Consequently, we next determined the role of FAK in HLA-II Ab-induced Src activation and its downstream targets. Initially, we determined the efficiency of FAK siRNA on FAK expression. As shown in Fig. 7A–C, FAK expression was markedly decreased by siRNA knockdown whereas transfection of EC with nontargeted siRNA had no effect on FAK protein expression (Fig. 7A, lanes 1–4, 7B, lanes 1 and 2, 7C, lane 1). To verify FAK siRNA specificity, we determined the expression of several cytoskeletal proteins in the same cell lysates of EC transfected with FAK siRNA. Transfection of EC with FAK siRNA or control siRNA did not affect expression of vinculin, β-actin (Fig. 7A–C), and β-tubulin (Fig. 7C) and also did not affect HLA-II expression level on Ad-CIITA–infected and TNF-α/IFN-γ–pretreated EC (Fig. 7D, 7E). To determine whether FAK is associated with Src in HLA-II Ab-stimulated activation of Src, we examined the effect of FAK siRNA on HLA-II–induced phosphorylation of Src at Tyr418 in the catalytic domain of Src. Transfection of Ad-CIITA–infected and TNF-α/IFN-γ–pretreated EC with FAK siRNA inhibited class II Ab-induced phosphorylation of Src at Tyr418 (Fig. 7A, 7B). In contrast, transfection of EC with non–target control siRNA had no effect on class II–mediated activation of Src.

FIGURE 7.

Effect of FAK siRNA transfection on HLA-II Ab-stimulated activation of signal transduction networks and cell proliferation. (A, D, and F) EC were infected with Ad-LacZ or Ad-CIITA or (B, E, and G) pretreated with TNF-α/IFN-γ for 48 h. (A–G) Starved EC were transfected with 100 nM FAK or control siRNA for 48 h. (A and B) Transfected EC were stimulated with 0.1 μg/ml HLA-II Ab or control mIgG for 15 min. (A–C) Proteins in the precleared cell lysates were separated by 6 ∼ 15% SDS-PAGE followed by immunoblotting with anti-FAK Ab for siRNA knockdown efficiency, or (A and B) with anti–phospho-Src Tyr418, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, mTOR Ser2448, S6K Thr389, S6RP Ser240/244, or ERK Thr202/Tyr204. The membranes were reprobed with anti-Vinculin, Src, PI3K, Akt, mTOR, S6K, S6RP, ERK, or β-actin Abs to confirm equal loading of proteins. (C) The membrane was immunoblotted with anti-Vinculin, β-tubulin, or β-actin Ab for FAK siRNA knockdown specificity. (D and E) EC were detached, incubated with 1 μg/ml HLA-II Ab for 30 min at 4°C, washed three times, and incubated with FITC-conjugated goat anti-mouse F(ab′)2 fragment for 30 min at 4°C. The fluorescence intensity was measured on an LSRFortessa flow cytometer and calculated using the FACSDiva program. The bar graphs show the mean ± SEM of median channel fluorescence values of HLA-II expression on EC and were analyzed by one-way ANOVA with a Fisher LSD test. (F and G) Starved cells were stimulated with 1 μg/ml HLA-II Ab for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and proliferation index is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. The bar graphs show the mean ± SEM fold change of proliferation index. Data represent at least three independent experiments. *p < 0.05, **p < 0.01 were analyzed by one-way ANOVA with a Fisher LSD test.

FIGURE 7.

Effect of FAK siRNA transfection on HLA-II Ab-stimulated activation of signal transduction networks and cell proliferation. (A, D, and F) EC were infected with Ad-LacZ or Ad-CIITA or (B, E, and G) pretreated with TNF-α/IFN-γ for 48 h. (A–G) Starved EC were transfected with 100 nM FAK or control siRNA for 48 h. (A and B) Transfected EC were stimulated with 0.1 μg/ml HLA-II Ab or control mIgG for 15 min. (A–C) Proteins in the precleared cell lysates were separated by 6 ∼ 15% SDS-PAGE followed by immunoblotting with anti-FAK Ab for siRNA knockdown efficiency, or (A and B) with anti–phospho-Src Tyr418, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, mTOR Ser2448, S6K Thr389, S6RP Ser240/244, or ERK Thr202/Tyr204. The membranes were reprobed with anti-Vinculin, Src, PI3K, Akt, mTOR, S6K, S6RP, ERK, or β-actin Abs to confirm equal loading of proteins. (C) The membrane was immunoblotted with anti-Vinculin, β-tubulin, or β-actin Ab for FAK siRNA knockdown specificity. (D and E) EC were detached, incubated with 1 μg/ml HLA-II Ab for 30 min at 4°C, washed three times, and incubated with FITC-conjugated goat anti-mouse F(ab′)2 fragment for 30 min at 4°C. The fluorescence intensity was measured on an LSRFortessa flow cytometer and calculated using the FACSDiva program. The bar graphs show the mean ± SEM of median channel fluorescence values of HLA-II expression on EC and were analyzed by one-way ANOVA with a Fisher LSD test. (F and G) Starved cells were stimulated with 1 μg/ml HLA-II Ab for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and proliferation index is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. The bar graphs show the mean ± SEM fold change of proliferation index. Data represent at least three independent experiments. *p < 0.05, **p < 0.01 were analyzed by one-way ANOVA with a Fisher LSD test.

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Our results demonstrating that FAK plays a critical role in class II Ab-stimulated Src activation raised the possibility that FAK is required for the activation of the whole signaling network triggered by engagement of HLA-II. In line with this possibility, transfection of Ad-CIITA–infected and TNF-α/IFN-γ–pretreated EC with FAK siRNA abolished HLA-II Ab-induced phosphorylation of PI3K, Akt, mTOR, S6K, S6RP, and ERK (Fig. 7A, 7B). Additionally, siRNA-mediated knockdown of FAK blocked HLA-II Ab-stimulated proliferation in Ad-CIITA–infected EC (Fig. 7F) and in TNF-α/IFN-γ–pretreated EC (Fig. 7G). In contrast, transfection of EC with non–target control siRNA had no effect on HLA-II Ab-mediated cell proliferation (Fig. 7F, 7G). Collectively, our results imply that the activation of the FAK/Src complex is one of the earliest events in the HLA-II Ab-activated intracellular signal network.

Our results implicated FAK/Src in the activation of ERK, but the role of ERK in HLA-II Ab-triggered activation of intracellular signal transduction network was not known. To clarify the role of ERK in HLA-II signaling, starved Ad-CIITA–infected EC or TNF-α/IFN-γ–pretreated EC were pretreated with MEK inhibitors UO126 or PD0325901 followed by stimulation with class II Ab. Pretreatment of EC with either UO126 or PD0325901 had no effect on HLA-II Ab-induced phosphorylation of Src Tyr418, FAK Tyr576/577, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, and mTOR Ser2448 but prevented phosphorylation of S6K Thr389, S6K Thr421/Ser424, S6RP Ser240, and ERK Thr202/Tyr204 triggered by HLA-II crosslinking with class II Ab in Ad-CIITA–infected EC and TNF-α/IFN-γ–pretreated EC (Fig. 8A, 8B). These results indicate that ERK is required for full mTORC1 activation, as phosphorylation of S6K and S6RP was reduced when ERK was inactivated.

FIGURE 8.

ERK1/2 regulates HLA-II Ab-induced activation of intracellular signal transduction networks, cell proliferation, and migration. EC were (A, C, E, G, and I) infected with Ad-LacZ or Ad-CIITA or (B, D, F, H, and J) pretreated with TNF-α/IFN-γ for 48 h. (A and B) Starved cells were pretreated with 5 μM UO126, or (E–J) with 1 μM UO126, or (A and B) with 5 μM PD0325901, or (E and F) with 1 μM PD0325901, or (G–J) with 1 μM UO124 for 60 min, or (C and D) starved EC were transfected with 100 nM of MEK or control siRNA for 48 h. (A–D) Pretreated EC were stimulated with 0.1 μg/ml HLA-II Ab for 15 min. Treatment of EC with mIgG serves as a negative control. Proteins in the precleared cell lysates were separated by 6 ∼ 15% SDS-PAGE followed by immunoblotting with anti–phospho-Src Tyr418, FAK Tyr576, FAK Tyr577, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, mTOR Ser2448, S6K Thr389, S6K Thr421/424, S6RP Ser240/244, or ERK Thr202/Tyr204. The membranes were reprobed with anti-Src, FAK, PI3K, Akt, mTOR, S6K, S6RP, ERK, or β-actin Abs to confirm equal loading of proteins. (E and F) Cells were stimulated with 1 μg/ml HLA-II Ab for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry. DNA synthesis S phase was gated, and proliferation index (PI) is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. (G and H) Cells were pretreated with 10 μg/ml mitomycin C for 2 h to inhibit cell proliferation before being assayed for their ability to migrate. A scratch wound was created with a sterile 200-μl pipette tip. Wounded cells were stimulated with 1 μg/ml anti–HLA-II Ab for 16 h. The cell number between two initiated front edges was counted; migration rate was analyzed by calculating the cell number between two initiated front edges of HLA-II Ab-stimulated EC divided by the cell number between two initiated front edges of negative control EC. (I and J) Cell migration was measured in a transwell insert system. EC infected with Ad-LacZ or Ad-CIITA or pretreated with TNF-α/IFN-γ were seeded to the upper chamber of the insert, pretreated with UO126 or UO124 for 60 min, and stimulated with 1 μg/ml HLA-II Ab for 16 h at 37°C. After incubation, the cells on the upper surface of the insert membrane were removed with a cotton swab, the migrated cells on the bottom of the insert membrane were fixed with methanol and stained with crystal violet, three middle fields per insert were photographed with a ×10 objective lens, and migrated cells were counted. The bar graphs show the mean ± SEM fold change of (C and D) proliferation index and (E–H) migrated cells. Data represent at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were analyzed by one-way ANOVA with a Fisher LSD test.

FIGURE 8.

ERK1/2 regulates HLA-II Ab-induced activation of intracellular signal transduction networks, cell proliferation, and migration. EC were (A, C, E, G, and I) infected with Ad-LacZ or Ad-CIITA or (B, D, F, H, and J) pretreated with TNF-α/IFN-γ for 48 h. (A and B) Starved cells were pretreated with 5 μM UO126, or (E–J) with 1 μM UO126, or (A and B) with 5 μM PD0325901, or (E and F) with 1 μM PD0325901, or (G–J) with 1 μM UO124 for 60 min, or (C and D) starved EC were transfected with 100 nM of MEK or control siRNA for 48 h. (A–D) Pretreated EC were stimulated with 0.1 μg/ml HLA-II Ab for 15 min. Treatment of EC with mIgG serves as a negative control. Proteins in the precleared cell lysates were separated by 6 ∼ 15% SDS-PAGE followed by immunoblotting with anti–phospho-Src Tyr418, FAK Tyr576, FAK Tyr577, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, mTOR Ser2448, S6K Thr389, S6K Thr421/424, S6RP Ser240/244, or ERK Thr202/Tyr204. The membranes were reprobed with anti-Src, FAK, PI3K, Akt, mTOR, S6K, S6RP, ERK, or β-actin Abs to confirm equal loading of proteins. (E and F) Cells were stimulated with 1 μg/ml HLA-II Ab for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry. DNA synthesis S phase was gated, and proliferation index (PI) is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. (G and H) Cells were pretreated with 10 μg/ml mitomycin C for 2 h to inhibit cell proliferation before being assayed for their ability to migrate. A scratch wound was created with a sterile 200-μl pipette tip. Wounded cells were stimulated with 1 μg/ml anti–HLA-II Ab for 16 h. The cell number between two initiated front edges was counted; migration rate was analyzed by calculating the cell number between two initiated front edges of HLA-II Ab-stimulated EC divided by the cell number between two initiated front edges of negative control EC. (I and J) Cell migration was measured in a transwell insert system. EC infected with Ad-LacZ or Ad-CIITA or pretreated with TNF-α/IFN-γ were seeded to the upper chamber of the insert, pretreated with UO126 or UO124 for 60 min, and stimulated with 1 μg/ml HLA-II Ab for 16 h at 37°C. After incubation, the cells on the upper surface of the insert membrane were removed with a cotton swab, the migrated cells on the bottom of the insert membrane were fixed with methanol and stained with crystal violet, three middle fields per insert were photographed with a ×10 objective lens, and migrated cells were counted. The bar graphs show the mean ± SEM fold change of (C and D) proliferation index and (E–H) migrated cells. Data represent at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were analyzed by one-way ANOVA with a Fisher LSD test.

Close modal

To confirm our observation using pharmacological inhibitors of MEK, we next determined the effect of MEK siRNA on HLA-II Ab-induced ERK activation and its targets. For this, we determined the efficiency of MEK siRNA on MEK expression first. As shown in Fig. 8C and 8D, MEK expression was markedly reduced by siRNA knockdown whereas transfection of EC with nontargeted siRNA had no effect on MEK protein expression (Fig. 8C, lanes 1–4, 8D, lanes 1 and 2). To verify MEK siRNA specificity, we determined the expression of several cytoskeletal proteins in the same cell lysates of EC transfected with MEK siRNA. Transfection of EC with MEK siRNA or control siRNA did not affect expression of vinculin and β-actin (Fig. 8C, 8D). Transfection of starved Ad-CIITA–infected and TNF-α/IFN-γ–pretreated EC with MEK siRNA had no effect on HLA-II Ab-induced phosphorylation of Src Tyr418, FAK Tyr576/577, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, and mTOR Ser2448 but abrogated phosphorylation of S6K Thr389, S6K Thr421/Ser424, S6RP Ser240/244, and ERK at Thr202/Tyr204 in both Ad-CIITA–infected and TNF-α/IFN-γ–pretreated EC (Fig. 8C, 8D).

Next we determined the impact of ERK inhibition on EC proliferation and migration. Pretreatment of starved Ad-CIITA–infected and TNF-α/IFN-γ–pretreated EC with UO126 or PD0325901 inhibited HLA-II Ab-stimulated EC proliferation (Fig. 8E, 8F) and EC migration, as determined by wound healing (Fig. 8G, 8H) or transwell migration assays (Fig. 8I, 8J). In contrast, UO124, an inactive analog of UO126, had no effect on HLA-II Ab-induced cell migration (Fig. 8G–J). These data indicated that the ERK signaling contributes to HLA-II Ab-stimulated mTORC1 activation, proliferation, and migration of EC stimulated by Ab-mediated ligation of HAL II.

Because long-term treatment with rapamycin or everolimus, exposure to PP242, or siRNA knockdown of Rictor inhibited Akt phosphorylation on Ser473 but induced concomitant overactivation of ERK, we proposed that mTORC2 mediates negative feedback on ERK activation. To elucidate this mechanism, we next determined whether EC exposure to a MEK inhibitor prevents overactivation of ERK in response to mTORC2 inhibition. Treatment with UO126 abrogated hyperphosphorylation of ERK induced by rapamycin 24 h treatment (Fig. 9A, 9B) but did not prevent phosphorylation of Akt Ser473.

FIGURE 9.

Treatment of EC with MEK inhibitor abrogates mTORC2-mediated overactivation of ERK mediated in response to HLA-II Ab. EC were (C) infected with Ad-LacZ or Ad-CIITA or (A, B, and D) pretreated with TNF-α/IFN-γ for 48 h. Starved cells were pretreated with 30 nM rapamycin for 24 h and/or 1 μM UO126 for 60 min. (A and B) Cells were stimulated with 0.1 μg/ml HLA-II Ab for 15 min. Treatment of EC with mIgG serves as negative control. Proteins in the precleared cell lysates were separated by 6 ∼ 15% SDS-PAGE followed by immunoblotting with anti–phospho-Akt Ser473 or ERK1/2 Thr202/Tyr204 Abs. The membranes were reprobed with anti-Akt or ERK total Abs to confirm equal loading of proteins. (B) Phosphorylated protein bands shown in (A) were quantified by densitometry scan analysis and results are expressed as the mean ± SEM percentage of maximal increase in phosphorylation above control values. **p < 0.01, ***p < 0.001, ****p < 0.0001 were analyzed by one way ANOVA with a Fisher LSD test. (C and D) Cells were stimulated with 1 μg/ml HLA-II Ab for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry. DNA synthesis S phase was gated, and proliferation index is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. The bar graphs show the mean ± SEM fold change of proliferation index. Data represent at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 were analyzed by one-way ANOVA with a Fisher LSD test. (E) Scheme representing HLA-II Ab-triggered activation of intracellular signal networks in EC leading to cell proliferation and migration. EC were infected with Ad-CIITA or pretreated with TNF-α/IFN-γ for 48 h to upregulate HLA-II molecule expression. Ligation of HLA-II molecules with Ab stimulates phosphorylation of protein tyrosine kinases, including Src Tyr418, FAK Tyr576 and Tyr577. Dissecting the upstream/downstream interactions using siRNA and pharmacological inhibitors, we show that Src is the earliest protein kinase in the HLA-II Ab-induced signaling cascade. Activated FAK/Src signaling complex mediates phosphorylation of p85 PI3K Tyr458, Akt Thr308, and Akt Ser473. The PI3K/Akt cascade stimulates phosphorylation of mTOR and assembly of mTORC1 and mTORC2. mTORC1 induces phosphorylation of S6K Thr389 and Thr421/Ser424. mTORC1/S6K signal axis phosphorylates S6RP at Ser240/244. mTORC2 mediates phosphorylation of Akt at Ser473. In parallel to the PI3K/Akt/mTOR signal pathways, activated FAK/Src complex also stimulates activation of the MEK/ERK pathway. These signal networks, solid lines, regulate HLA Ab-stimulated cell proliferation and migration. Long-term treatment with rapamycin, everolimus, an active-site inhibitor or knockdown Rictor with siRNA mediates mTORC2 negatively feedbacks to enhance phosphorylation of ERK (negative feedback loop 1). Solid line with arrow presents stimulatory signal transduction pathways. The dotted line presents the negative feedback loop.

FIGURE 9.

Treatment of EC with MEK inhibitor abrogates mTORC2-mediated overactivation of ERK mediated in response to HLA-II Ab. EC were (C) infected with Ad-LacZ or Ad-CIITA or (A, B, and D) pretreated with TNF-α/IFN-γ for 48 h. Starved cells were pretreated with 30 nM rapamycin for 24 h and/or 1 μM UO126 for 60 min. (A and B) Cells were stimulated with 0.1 μg/ml HLA-II Ab for 15 min. Treatment of EC with mIgG serves as negative control. Proteins in the precleared cell lysates were separated by 6 ∼ 15% SDS-PAGE followed by immunoblotting with anti–phospho-Akt Ser473 or ERK1/2 Thr202/Tyr204 Abs. The membranes were reprobed with anti-Akt or ERK total Abs to confirm equal loading of proteins. (B) Phosphorylated protein bands shown in (A) were quantified by densitometry scan analysis and results are expressed as the mean ± SEM percentage of maximal increase in phosphorylation above control values. **p < 0.01, ***p < 0.001, ****p < 0.0001 were analyzed by one way ANOVA with a Fisher LSD test. (C and D) Cells were stimulated with 1 μg/ml HLA-II Ab for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry. DNA synthesis S phase was gated, and proliferation index is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. The bar graphs show the mean ± SEM fold change of proliferation index. Data represent at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 were analyzed by one-way ANOVA with a Fisher LSD test. (E) Scheme representing HLA-II Ab-triggered activation of intracellular signal networks in EC leading to cell proliferation and migration. EC were infected with Ad-CIITA or pretreated with TNF-α/IFN-γ for 48 h to upregulate HLA-II molecule expression. Ligation of HLA-II molecules with Ab stimulates phosphorylation of protein tyrosine kinases, including Src Tyr418, FAK Tyr576 and Tyr577. Dissecting the upstream/downstream interactions using siRNA and pharmacological inhibitors, we show that Src is the earliest protein kinase in the HLA-II Ab-induced signaling cascade. Activated FAK/Src signaling complex mediates phosphorylation of p85 PI3K Tyr458, Akt Thr308, and Akt Ser473. The PI3K/Akt cascade stimulates phosphorylation of mTOR and assembly of mTORC1 and mTORC2. mTORC1 induces phosphorylation of S6K Thr389 and Thr421/Ser424. mTORC1/S6K signal axis phosphorylates S6RP at Ser240/244. mTORC2 mediates phosphorylation of Akt at Ser473. In parallel to the PI3K/Akt/mTOR signal pathways, activated FAK/Src complex also stimulates activation of the MEK/ERK pathway. These signal networks, solid lines, regulate HLA Ab-stimulated cell proliferation and migration. Long-term treatment with rapamycin, everolimus, an active-site inhibitor or knockdown Rictor with siRNA mediates mTORC2 negatively feedbacks to enhance phosphorylation of ERK (negative feedback loop 1). Solid line with arrow presents stimulatory signal transduction pathways. The dotted line presents the negative feedback loop.

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To further determine whether the overactivation of the MEK/ERK pathway counterbalances the EC growth-suppressing effect of the mTORC2 inhibitor, we examined the proliferation (BrdU incorporation assay) of Ad-CIITA–infected EC and TNF-α/IFN-γ–pretreated EC treated with rapamycin and without or with UO126. Pretreatment with either rapamycin or UO126 inhibited HLA-II Ab-stimulated cell proliferation, but the combination of rapamycin and UO126 produced a further inhibitory effect on EC proliferation (Fig. 9C, 9D). These results indicate that cotargeting mTORC2 and MEK/ERK pathways mediates a profound inhibition of EC proliferation. Similar results were obtained when PD0325901 was used to inhibit MEK instead of UO126. Pretreatment of EC with PD0325901 also abolished overactivation of ERK in response to rapamycin 24 h treatment, by which inhibits mTORC2. Similar results were seen in proliferation in Ad-CIITA–infected EC and TNF-α/IFN-γ–pretreated EC (data not shown). Our results show that overactivation of ERK induced by suppression of mTORC2 can be abrogated by administration of a MEK inhibitor in EC, a conclusion with potential translational implications.

EC line the inner surface of blood vessels and serve as the interface between circulating blood and surrounding tissue. After solid organ transplantation, the endothelium in the transplanted organ forms a vast network that dynamically regulates vascular barrier functions. Physiologically, human artery EC do not express HLA-II Ags. During the process of transplantation, surgical trauma and ischemia/reperfusion injury induce an inflammatory process leading to TNF-α, IL-1β, and IFN-γ production by EC (44). MHC-II promoters share a common set of cis-acting elements, including the W/S, X1, X2, and Y boxes (S-X-Y module), which provide the appropriate interaction surface for the recruitment of CIITA. Upon IFN-γ induction, CIITA is produced and induces or enhances the association of transcription factors with the promoter (45). Subsequently, these inflammatory cytokines stimulate HLA-II Ag expression on EC.

Owing to the technical limitations of studying HLA-II–mediated functions in cultured EC, studies of the effects of HLA-II Abs on EC have been limited and very little is known about the signaling cascades triggered by these Abs in these cells. Recently, Taflin et al. (58) described a model in which HLA-DR expression was induced in cultured EC through lentiviral delivery and was sufficient to drive activation of memory CD4+ T cells. However, the forced expression of classical HLA-II molecules without accessory HLA-DM, HLA-DO, and invariant chain expression, which are necessary for proper Ag presentation, may yield artificial CLIP-loaded HLA-DR at the cell surface. Moreover, this model did not permit investigation of other classical HLA-II molecules, HLA-DQ and HLA-DP, which are clinically relevant in transplantation. In this study, we describe a new model of HLA-II expression in human primary EC utilizing adenoviral vector-mediated expression of CIITA, a transcription factor that promotes expression of both classical and nonclassical HLA-II molecules, as well as the invariant chain and cathepsin S (59). Adenovirus was chosen for higher transfection efficiency in primary EC. Importantly, this approach drives expression of endogenous alleles of HLA-DR, DQ, and DP. In parallel, we used EC treated with IFN-γ and TNF-α to mimic inflammatory conditions under which EC might physiologically and pathologically upregulate HLA-II Ags. Similar to the Ad-CIITA model, this parallel treatment of EC with cytokines also induced CIITA production and expression of HLA-DR, DQ, and DP on EC. Using these two model systems together with multiple chemical inhibitors and siRNA-mediated knockdown of signal transducers, we explored in depth the organization of the signaling network elicited by Ab-mediated ligation of class II molecules in primary EC that leads to proliferation and migration.

Transplant vasculopathy is caused by proliferation of both EC and smooth muscle cells, as evident from proliferating cell nuclear Ag staining in chronic lesions (60, 61). Angiogenic changes resulting from EC activation, proliferation, and migration, characterized by new microvessel formation under the hyperplastic neointima, occur in cardiac transplant recipients with TV (10). In this study, we found that ligation of HLA-II molecules with Ab in Ad-CIITA–infected or TNF-α/IFN-γ–pretreated EC stimulates the proliferation and migration of these cells, as demonstrated using different assays. Activated EC may also stimulate smooth muscle cells through paracrine effects promoting fibroproliferation, such as through production of VEGF (62).

Studies using pharmacological inhibitors and siRNAs to knock down the expression of signal transducers demonstrate that the protein kinases Src/FAK, PI3K/Akt, mTOR, and ERK play critical roles in mediating class II Ab-stimulated EC proliferation and migration. Pretreatment of EC with either structurally unrelated Src inhibitors or siRNA-mediated knockdown of c-Src and FAK blocked HLA-II Ab-stimulated activation of all signaling molecules detected in this study, including PI3K, Akt, mTORC1, mTORC2, and ERK. FAK and Src are nonreceptor protein tyrosine kinases that function cooperatively. Indeed, many stimuli induce FAK autophosphorylation on Tyr397, creating a high-affinity binding site for the Src homology 2 domain of Src. Upon binding to FAK, Src is autophosphorylated at Tyr418 in its catalytic domain, thereby stabilizing its active conformation. Then, Src phosphorylates Tyr576 and Tyr577 within the activation loop of the kinase domain of FAK, promoting maximal activation of FAK catalytic activity. In line with this model, knockdown of FAK inhibited HLA-II Ab-induced phosphorylation of Src at Tyr418, and chemical inhibitors of Src family kinases abrogated the increase in the phosphorylation of FAK on Tyr576 and Tyr577. We therefore propose that FAK/Src activation is one of the earliest events leading to downstream signaling, migration, and proliferation triggered by Ab-mediated ligation of HLA-II molecules in primary human aortic EC.

PI3K catalyzes the formation of PIP3, a second messenger that coordinates the localization and activation of several downstream signaling molecules, including Akt (28, 52). Pretreatment of starved Ad-CIITA–infected or TNF-α/IFN-γ–pretreated EC with either the dual PI3K/mTOR inhibitor LY294002 or A66, a selective antagonist of the p110α subunit of the PI3K, suppressed HLA-II Ab-stimulated phosphorylation of p85 PI3K Tyr458, Akt Thr308, and Akt Ser473 and abolished phosphorylation of mTOR Ser2448, S6K Thr389, and S6RP Ser240/244. Because in some cell types PI3K activation leads to Raf-mediated MEK/ERK (63), it is of interest that A66 did not prevented HLA-II Ab-induced phosphorylation of ERK Thr202/Tyr204, indicating that PI3K does not contribute to ERK activation in EC in the context of HLA-II signaling. Interestingly, dual PI3K/mTOR inhibitor LY294002 caused ERK hyperphosphorylation, which suggests that there is negative feedback via mTORC2. Importantly, pretreatment with A66 or LY294002 did not affect HLA-II Ab-induced FAK/Src activation, reinforcing our conclusion that Src/FAK is upstream of PI3K/Akt in EC activated by ligation of HLA-II (Fig. 9E). It has been reported that overactivation of ERK mediated by inhibition of mTORC2 was shown to involve Akt-mediated inhibitory phosphorylation of c-Raf (64). To test this hypothesis, we determined the effect of Akt inhibitors on HLA-II Ab-stimulated phosphorylation of c-Raf-1 at Ser259. Inhibition of mTORC2 (by 24 h exposure to rapamycin) inhibited AKT phosphorylation at Ser473 completely but did not prevent HLA-II–induced AKT phosphorylation at Thr308, which is the key site in the T loop for AKT activation. Furthermore, treatment of EC with allosteric (MK-2206) or active-site (GDC-0068) inhibitors of Akt did not affect HLA-II Ab-stimulated phosphorylation of c-Raf-1 Ser259 or ERK Thr202/Tyr204 (Fig. 5E, 5F). Our data suggest the mechanism by which mTORC2 inhibition results in ERK overactivation is predominantly Akt-independent.

The PI3K/Akt/mTOR network is a key signaling pathway in the regulation of cell metabolism, migration, survival, and proliferation (49) that plays a critical role in HLA class I Ab-induced EC activation in chronic rejection (27, 31, 33, 34) but its function and regulation in the context of HLA-II signaling were virtually unknown. mTOR functions as a catalytic subunit in two structurally distinct multi protein complexes, mTORC1 and mTORC2 (65, 66). mTORC1, a complex of mTOR characterized by the substrate-binding subunit, phosphorylates and controls at least two regulators of protein synthesis, the 40S ribosomal protein subunit S6K and the inhibitor of protein synthesis 4E-binding protein 1 (6770). mTORC1 is acutely inhibited by rapamycin and everolimus whereas mTORC2, characterized by Rictor, is not inhibited by short exposure to this agent (71, 72). In this study, we demonstrate that Ab-mediated ligation of HLA-II molecules expressed in Ad-CIITA–infected or TNF-α/IFN-γ–pretreated EC induces a marked increase in mTORC1 and mTORC2, as revealed by the increase in the phosphorylation of S6K Thr389 and Akt Ser473, respectively. Using pharmacological and genetic approaches, we demonstrate that mTOR plays a key role in proliferation and migration in EC stimulated by Ab-mediated ligation of HLA-II molecules. Subsequently, we explored in depth the relationship between mTOR complexes (mTORC1 and mTORC2) and their upstream elements (FAK/Src, PI3K, and ERK) and downstream targets, including S6K, S6RP, and Akt, following ligation of HLA-II molecules. The upstream and downstream elements in mTOR signaling in EC stimulated by Ab-mediated ligation of HLA-II molecules identified in this study are summarized in Fig. 9E.

Mounting evidence indicates that the mTOR complexes not only stimulate growth-promoting signaling but also mediate negative feedback loops that restrain signaling through upstream or parallel pathways in a cell context–specific manner (63). Suppression of these feedback loops by inhibitors of mTOR causes compensatory overactivation of signaling nodes, including ERK, that potentially oppose the antiproliferative effects of the mTOR inhibitors, thus leading to drug resistance. The elucidation of feedback loops that regulate the outputs of signaling networks has emerged as an area of fundamental importance for the rationale design of effective inhibitors or combination of inhibitors to prevent transplant rejection. In the present study, we produced several lines of evidence demonstrating that suppression of mTORC2 leads to overactivation of ERK in response to Ab-mediated ligation of class II molecules in EC. Specifically, long-term term exposure to either rapamycin or everolimus and treatment with the mTOR active-site inhibitor PP242 or the dual PI3K/mTOR inhibitor LY294002, all of which suppress both mTORC1- and mTORC2-enhanced ERK activation. In agreement with this conclusion, the siRNA-mediated knockdown of Rictor, an essential component of the mTORC2 complex, also promoted ERK overactivation. We verified that all of these interventions blocked the phosphorylation of Akt at Ser473, a site targeted by mTORC2. In contrast, short-term treatment with either rapamycin or everolimus or knockdown of Raptor, which inhibits mTORC1 but not mTORC2, did not have any detectable effect on ERK activation in response to Ab-mediated HLA-II engagement in EC. It is noteworthy that long-term treatment with rapamycin, everolimus, or knockdown Rictor with siRNA inhibited HLA class I Ab-induced phosphorylation of ERK (33, 35), thus implying an important difference in the feedback loops that fine-tune EC signaling through HLA class I and HLA-II molecules. Collectively, these findings identify a novel feedback loop in EC cells challenged with Ab directed against HLA-II by which ERK activation is attenuated through a mTORC2-mediated pathway but is unleashed by multiple treatments that interfere with mTORC2 activity (Fig. 9E, feedback loop 1).

In APCs, engagement of MHC class II molecules by the TCR (or mimicked by Abs) induces bidirectional intracellular signaling. For example, in addition to activation of ERK, Abs to HLA-II increase p38 MAPK phosphorylation in monocytes leading to cytokine production (73). ERK and JNK are also strongly phosphorylated in B cells after treatment with anti–HLA-II Abs (74), as are the NFAT and c-Fos pathways (75). Dendritic cell apoptosis after ligation of HLA-DR is regulated by protein kinase C (76), and superantigen stimulation of MHC class II in this cell type also activates NF-κB/MyD88 (77). Whether these pathways are also activated in allograft vascular EC in the presence of anti-donor HLA-II Abs remains to be determined.

Our studies also demonstrate that exposure of Ad-CIITA–infected or TNF-α/IFN-γ–pretreated EC to MEK inhibitors opposed the overactivation of ERK induced by suppression of mTORC2 inhibition. These results show that the enhanced ERK activation induced by long-term treatment with rapamycin or everolimus can be prevented by cotreatment with MEK inhibitors. Further studies are required to determine the effect of class II signaling in an in vivo allograft model. However, given the increasing importance of everolimus in combating transplant rejection, the findings presented in this study suggest a novel therapeutic strategy, that is, cotreatment with PI3K/mTORC2 and MEK inhibitors in the prevention or treatment of HLA-II Ab-mediated TV.

We express thanks for the cooperation of One Legacy and all of the organ and tissue donors and their families for giving the gift of life and the gift of knowledge by generous donations.

This work was supported by National Institute of Allergy and Infectious Diseases Grant R01 AI 042819, National Institutes of Health Grant U01 AI-124319-01, and by a Novartis investigator-initiated research grant (to E.F.R.). E.R. was supported by National Institutes of Health Grants R01 DK100405, P30 DK41301, and P01 CA163200 and by Department of Veterans Affair Merit Award 1I01BX001473.

The online version of this article contains supplemental material.

Abbreviations used in this article:

Ad-CIITA

adenovirus-based vector encoding CIITA

AMR

Ab-mediated rejection

DSA

donor-specific HLA Ab

EC

endothelial cell

FAK

focal adhesion kinase

HLA-II

HLA class II

LSD

least significant difference

mIgG

mouse IgG

MOI

multiplicity of infection

mTOR

mammalian target of rapamycin

mTORC

mTOR complex

PFA

paraformaldehyde

siRNA

small interfering RNA

S6K

p70 ribosomal S6 kinase

S6RP

S6 ribosomal protein

TV

transplant vasculopathy.

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The authors have no financial conflicts of interest.

Supplementary data