Upon stimulation, cutaneous sensory nerves release neuropeptides such as substance P (SP), which modulate responses in the skin by activating a number of target cells via neurokinin receptors. We have demonstrated that SP preferentially binds to the NK-1R on human dermal microvascular cells, resulting in increased intracellular Ca2+ and induction of ICAM-1 and VCAM-1 expression. In the current studies, we identify specific elements in the regulatory regions of ICAM-1 and VCAM-1 genes as necessary and sufficient for SP-dependent transcriptional activation. SP treatment of human dermal microvascular endothelial cells leads to coincident activation and binding of the transcription factor NF-AT to the −191/−170 region of the ICAM-1 gene (a region bound by activated p65/p65 homodimers in response to TNF-α), and NF-κB (p65/p50) to tandem NF-κB binding sites at −76/−52 of the VCAM-1 gene. The SP-elicited intracellular Ca2+ signal was required for activation and subsequent binding of both NF-AT and NF-κB. The transacting factor induction by SP was specific, since a selective NK-1R antagonist blocked SP activation and subsequent NF-AT and NF-κB activation and binding. These data demonstrate coincident activation of NF-AT and NF-κB via SP-induced intracellular Ca2+ mobilization and indicate a crucial role for neuropeptides in modulating localized cutaneous inflammatory responses.
Neuropeptides such as substance P (SP)3 are released by sensory C fibers in the skin, where they exert a variety of modulatory actions, including vasodilatation, activation, and modulation of various immune cells (1, 2). SP mediates its effects on target cells by binding to cell surface G protein-coupled neurokinin receptors (3, 4, 5). SP has a high affinity receptor, NK-1R, which we have previously detected in HDMEC (6). Engagement of the NK-1R with SP results in an increase in intracellular Ca2+ levels, followed by increased expression of ICAM-1 and VCAM-1 in the dermal microvasculature (6, 7). However, little is known of the regulatory events by which SP modulates adhesion molecule gene expression.
The final targets of cell surface signals are often the activated proteins that associate with DNA regulatory elements and regulate transcription. Adhesion molecules ICAM-1 and VCAM-1 are highly regulated at the transcriptional level by a number of mediators (8, 9, 10, 11, 12, 13, 14, 15, 16, 17). TNF-α induces ICAM-1 and VCAM-1 gene expression via activation of distinct members of the Rel family (14, 18, 19, 20, 21). In most cells, NF-κB is retained in the cytoplasm in an inactive form by the inhibitory protein IκB, which, in turn, is regulated by the IκB kinase or IKK (22). Following a variety of extracellular stimuli, NF-κB dissociates from IκB, translocates to the nucleus, and activates target genes. Activation of NF-κB is thus IKK activation dependent. The minimal DNA binding domain of NF-κB (p65/p65) on the ICAM-1 promoter corresponds exactly to the NF-AT1 high affinity consensus (TGGAAA) site (23, 24, 25). Within particular NF-κB DNA binding domains, an NF-AT monomer can bind to the 5′ half-site of the DNA binding domain, and a second NF-AT monomer can bind to the symmetrical 3′ half-site, which is why NF-AT transcription factors are often termed monomeric Rel proteins (reviewed in Refs. 26). NF-AT was originally described as an activator of IL-2 transcription (27) and is now known to be expressed in most immune cells, playing a pivotal role, like NF-κB, in the transcription of genes critical for inflammatory responses (reviewed in Ref. 26).
NF-AT nuclear translocation is initiated by stimulation of receptors coupled to calcium mobilization (28, 29). The phosphatase calcineurin, which controls the translocation of NF-AT proteins from the cytoplasm to the nucleus of activated cells (28, 29), is the major target of the immunosuppressive drug cyclosporin A (CsA). Thus, the inhibition of calcineurin activity by CsA prevents activation and subsequent DNA binding by NF-AT. Recently, CsA has been shown to be an NK-1 and NK-2 receptor antagonist and was capable of blocking SP-elicited phosphoinositide turnover in human lymphoblastoid and astrocytoma cells (30). In addition, SP-mediated induction of NF-κB-dependent IL-8 gene expression in astrocytoma cells was blocked by CsA (31). Thus, CsA may inhibit Ca2+-dependent SP-mediated gene expression at the level of NF-AT-transacting factor activation and/or at the level of extracellular NK receptor blockade.
Elucidation of the mechanisms by which SP modulates adhesion molecule gene transcription in microvascular endothelial cells is crucial to understanding the interplay between the nervous and immune systems, processes that probably play a significant role during the evolution of inflammation. In this study we demonstrate the coincident activation by SP of p65/p50 heterodimers binding to VCAM-1 tandem NF-κB sites and NF-AT binding to the ICAM-1 p65 homodimer site, both occurring as a consequence of Ca2+ mobilization after NK-1R engagement by SP.
Materials and Methods
Cells and reagents
HDMEC isolated from foreskins were obtained from the cell culture facility of the Emory Skin Disease Research Core Center (Atlanta, GA) (32). Experiments were conducted with cells in passages 3–5. HDMEC were cultured on a gelatinized (0.1%) surface in MCDB 131 (Life Technologies, Gaithersburg, MD) supplemented with 10% normal human serum (Irvine Scientific, Santa Ana, CA), 5 ng/ml epidermal growth factor (Clonetics, San Diego, CA), 1 mg/ml hydrocortisone acetate (Sigma, St. Louis, MO), 100 U/ml penicillin, 250 μg/ml amphotericin B, and 100 μg/ml streptomycin (Life Technologies). Lyophilized SP (Peninsula Laboratories, Belmont, CA) was diluted in the appropriate volume of HDMEC assay medium immediately before use. In selected studies, 1 μM NK-1 receptor antagonist GR82334 (Peninsula Laboratories, Belmont, CA) was added to cultured EC 20 min before the addition of SP or TNF-α. CsA (Sandimmune injection, Sandoz/Novartis, East Hanover, NJ) in vehicle Cremophor EL (polyoxyethylated castor oil) was purchased from Sigma and was diluted in HDMEC medium to 0.1, 1, or 10 μM for dose dependence studies. For all subsequent studies, CsA was diluted to 1 μM in HDMEC medium. As a control, vehicle alone (Cremophor EL) from Sandoz/Novartis was diluted identically to CsA in HDMEC medium. Lyophilized human recombinant TNF-α was obtained from R & D Systems (Minneapolis, MN). TNF-α (300 U/ml) served as a positive control for HDMEC ICAM-1 and VCAM-1 induction. mAb 84H10 recognizing human ICAM-1 was provided by Dr. Stephen Shaw (National Institutes of Health, Bethesda, MD), and mAb P3C4 directed against human VCAM-1 was a gift from Dr. Elizabeth Wayner (University of Minnesota, Minneapolis, MN). The anti-TNF-α blocking study used a combination of 10 μg/ml each of anti-TNF receptor 1 and anti-TNF receptor 2 Abs added simultaneously. Both Abs, purchased from Santa Cruz Biotechnology (Santa Cruz, CA), were diluted in HDMEC medium and incubated with HDMEC for 20 min at 37°C in 5% CO2 before the addition of TNF-α or SP. Abs against AP-1 (c-Fos and c-Jun) as well as Rel and NF-AT family members were purchased from Santa Cruz Biotechnology and added to nuclear lysates at a concentration of 100 ng/μl. The selective intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′-tetraacetic acid-acetoxymethyl (BAPTA-AM) was purchased from Sigma and diluted to a concentration of 50 μM in HDMEC medium. HDMEC were incubated in the presence of BAPTA-AM for 20 min at 37°C in 5% CO2 before SP or TNF-α addition.
Measurement of SP-induced ICAM-1 and VCAM-1 on CsA-treated HDMEC by ELISA
HDMEC were plated onto 96-well plates and upon reaching 80% confluence were left untreated or were treated with 100 nM SP for 18 h for ICAM-1 induction or 10 nM SP for 16 h for VCAM-1 induction, optimal concentrations and durations of SP for induction of ICAM-1 and VCAM-1 in HDMECs as previously determined in our laboratories (6, 7). HDMEC treated with 300 U/ml TNF-α for 16 h were used as a positive control for ICAM-1 and VCAM-1 cell surface induction. HDMEC cell adhesion molecule expression was assessed using mAbs 84H10 for ICAM-1 or P3C4 for VCAM-1 induction by ELISA as described previously (10). Results represent the mean of three values ± SD for each variable tested, and results are representative of three independent assays.
Reporter constructs generated for CAM deletional analyses
The (−1162/+1) bp ICAM-1 chloramphenicol acetyltransferase (CAT) construct (pBS CATP) has been described previously (9) and was used as a template for the generation of ICAM-1-based constructs. The (−288/+20) bp VCAM-1 CAT construct was provided by Dr. Douglas Dean (Washington University, St. Louis, MO) and has been characterized previously (12). VCAM-1- and ICAM-1-based derivative 5-deletional and heterologous promoter/reporter plasmids as well as site-directed mutagenesis of specific transcription factor DNA binding sites were designed and constructed using the techniques and strategies described previously (9, 11, 33, 34, 35, 36). Fragments of the ICAM-1 or VCAM-1 5′-regulatory regions were isolated by appropriate restriction enzyme digestion or generated by PCR and cloned into pBRAMScat2 (37), which contains the CAT reporter gene under control of the herpes simplex virus minimal thymidine kinase promoter and multiple cloning sites designed for analysis of eukaryotic enhancers. Inserts and ligation sites of all constructs generated were confirmed by sequence analysis via the dideoxynucleotide chain termination method using the Sequenase Quick-Denature Kit (Amersham, Arlington Heights, IL).
Transient transfection of reporter gene constructs and analysis of CAT expression
HDMEC were transiently transfected with ICAM-1, VCAM-1, and control CAT reporter gene constructs as described previously (36). Briefly, HDMEC were transfected with a concentration of 20 μg of plasmid DNA in the presence of 500 ng/ml DEAE-dextran (Sigma) at 85% confluence for 30 min at 37°C in 5% CO2. After 30 min, HDMEC medium containing 8 μM chloroquine was added, and the transfection was continued for an additional 2 h. After exposure to plasmid DNA for 2.5 h, the medium was replenished. At 24 h post-transfection, cells were left untreated or were treated for 18 h with 100 nM SP for ICAM-1, for 16 h with 10 nM SP for VCAM-1 induction, or for 16 h with 300 U/ml TNF-α as a positive control for both ICAM-1 and VCAM-1 induction. In selected experiments, HDMEC were pretreated for 20 min with the NK-1R antagonist GR82334 (Peninsula Laboratories), CsA (Sandoz/Novartis), or the CsA vehicle Cremophor before SP or TNF-α addition. Assays and normalization protocols for transfection efficiency (using cotransfection of a β-galactosidase control vector), transfection lysate protein concentrations, and CAT expression were performed as described previously (11, 33).
Preparation of nuclear extracts and gel mobility shift analysis
Confluent HDMEC were either left untreated or were treated for 4 h with TNF-α or SP for CAM induction. In selected experiments, HDMEC were incubated in the presence of the NK-1R antagonist GR82334 or CsA for 20 min before the addition of SP or TNF-α. For TNF-α blocking studies, anti-TNF-α receptor 1 and 2 Abs (see Cells and reagent) were added to HDMEC 20 min before the addition of SP or TNF-α. Nuclear extracts were prepared as described previously (33). Oligonucleotides encompassing the single NF-κB region of ICAM-1 (wild-type NF-κB/ICAM) and the tandem NF-κB regions of VCAM-1 (wild-type NF-κB/VCAM) were designed, annealed, and extended as previously described (33). All oligonucleotides were synthesized by the Emory University Microchemical Facility. The DNA-protein binding reaction was performed as previously described (11). Briefly, 3–4 μg of HDMEC nuclear extracts, binding buffer, and 1 × 105 cpm of radiolabeled oligonucleotide probe were incubated together for 30 min at room temperature. For the competition assays and supershift experiments, an excess of the appropriate unlabeled oligonucleotide (50 ng) or Ab (1 μg) was added to the binding reaction prior to the addition of radiolabeled probe. Unlabeled irrelevant competitor oligonucleotides incubated in excess (50 ng) with lysate were (−88/−68) of the ICAM-1 gene or (−42/−22) of the VCAM-1 gene. For some NF-AT supershift experiments, SP-treated HDMEC nuclear lysates were incubated with probe DNA for 30 min, followed by the addition of the anti- NF-AT1 Ab (1 μg). Samples were subjected to electrophoresis on a native 4% polyacrylamide gel for 4 h at 120 V and 10–12 A. Gels were dried, and autoradiography was performed at −70°C for 1–3 days. Autoradiographs were scanned on a La Cie scanner (La Cie, Beaverton, OR) using Adobe Photoshop software (Adobe Systems, Mountain View, CA). The digitized image was subsequently labeled in Microsoft Power Point (Microsoft, Redmond, WA) and printed on a high resolution laser printer. Each figure represents a computer-generated image of the autoradiograph, and each is typical of the autoradiograph in the context of relative band and background densities.
SP induction of HDMEC ICAM-1 and VCAM-1 cell surface expression is CsA sensitive
The effects of pretreatment with CsA and NK-1R antagonists on the SP-mediated induction of cell surface ICAM-1 and VCAM-1 expression on HDMEC was examined by ELISA (Fig. 1). Optimal concentrations and durations of SP treatment for induction of ICAM-1 and VCAM-1 on HDMEC were previously established in our laboratories (6, 7) and were used in these and subsequent studies. Untreated HDMEC have a low basal expression of cell surface ICAM-1 (Fig. 1,A), which is up-regulated 5-fold when HDMEC are treated with 100 nM SP for 18 h. This specific SP-mediated ICAM-1 induction is abrogated when HDMEC are pretreated with an NK-1R antagonist (Fig. 1,A). CsA was found to dose-dependently block SP induction of ICAM-1 on HDMEC, whereas pretreatment with the CsA vehicle had no blocking effect on SP-mediated ICAM-1 induction (Fig. 1,A). TNF-α strongly induced HDMEC cell surface ICAM-1 and served as a positive control (Fig. 1,A). TNF-α-mediated ICAM-1 induction was unaffected when HDMEC were pretreated with either the NK-1R antagonist or 1 μM CsA, demonstrating specificity and differential receptor signaling pathways for the two stimuli as well as a nontoxic effect of reagents (Fig. 1,A). Untreated HDMEC show little or no constitutive VCAM-1 expression (Fig. 1,B). HDMEC treated with 10 nM SP for 16 h resulted in a 7-fold induction of VCAM-1 cell surface expression, which was abrogated when HDMEC were pretreated with the NK-1R antagonist (Fig. 1,B). CsA in a dose-dependent fashion was capable of blocking SP-induced VCAM-1 on HDMEC (Fig. 1,B). Again, the CsA vehicle had no blocking effect on SP/VCAM-1 induction on HDMEC (Fig. 1,B). As with ICAM-1, TNF-α-treated HDMEC displayed high levels of cell surface VCAM-1 expression, which were unaffected by pretreatment with either the NK-1R antagonist or 1 μM CsA (Fig. 1 B). Pretreatment of HDMEC with CsA vehicle had no effect on the ability of TNF-α to induce ICAM-1 or VCAM-1 expression (data not shown). These data demonstrate that SP/NK-1R-mediated up-regulation of ICAM-1 and VCAM-1 cell surface expression is CsA sensitive and occurs via receptors and signaling pathways distinct from those used by TNF-α.
Identification of a SP-responsive region between bp −191 and −170 of the ICAM-1 gene promoter by 5′-deletional analysis in HDMEC
Our previous studies in HDMEC revealed that upon a 3-h incubation with SP (100 nM), mRNA basal steady state levels increased by 3-fold (6). To determine molecular mechanisms responsible for the SP-mediated ICAM-1 gene transcription, a sequential series of 5′-deletional ICAM-1 promoter-based reporter gene constructs were transiently transfected into HDMEC (Fig. 2). Analysis of respective CAT expression in untreated, SP-treated, and TNF-α-treated cells revealed a basal activity of the −384/+1 ICAM-1 construct that was increased after treatment with SP and TNF-α by a factor of 3 (Fig. 2). While the −277/+1 construct demonstrated comparable constitutive, SP-induced, and TNF-α-induced expression, the −182/+1 construct showed a loss of induced transcriptional activity with both SP and TNF-α stimulation (Fig. 2). The −182/+1 construct interrupts a p65/p65 homodimer-binding modified NF-κB site on the ICAM-1 promoter that is critical for TNF-α induction of ICAM-1, as previously described by our laboratory (36). The promoterless construct, pCAT BASIC, served as a negative control for transfection and CAT reporter gene expression. Both SP- and TNF-α-induced reporter gene expression were restored when the −191/−170 TK-CAT construct was transfected into HDMEC (Fig. 2, C and D), yet the TK-CAT minimal promoter alone displayed no inducibility upon SP or TNF-α treatment. These findings indicate that a critical gene regulatory region necessary and sufficient for SP-inducible expression is located between bp −191 and −170 of the ICAM-1 promoter.
NF-AT1, but not AP-1, is a component of the SP-inducible ICAM-1 DNA binding complex
To demonstrate the formation of a specific DNA-protein complex, the newly identified SP-responsive element of the ICAM-1 promoter was used (−191/−170) in an EMSA. This site had previously been identified by our laboratory as containing a modified NF-κB binding site that bound a p65 homodimeric complex upon stimulation of cells with TNF-α and PMA (34, 36). Comparison of this minimal sequence to that of known transcription factor binding sites revealed a strong homology to the NF-AT family DNA binding domain overlapping the 5′ portion of the p65 homodimeric modified NF-κB binding site (26). We therefore assessed whether NF-κB or NF-AT was part of the SP-induced DNA binding complex (Fig. 3 A), and whether complex formation, if any, displayed CsA sensitivity. Untreated HDMEC displayed little or no complex formation (lane 1). TNF-α-treated HDMEC nuclear lysates served as a positive control for complex binding to this ICAM-1 promoter region. TNF-α-treated complex formation (lane 2) was supershifted with the anti-RelA Ab (lane 3), but not the anti-p50 Ab (lane 4), as previously described in HDMEC (36). The TNF-α-treated HDMEC complexes were unaffected upon addition of anti-NF-AT1 (lane 5) and anti-NF-AT family (lane 6) Abs.
Pretreatment of HDMEC with CsA (1 or 10 μM) had no effect on TNF-α-mediated complex formation (lanes 7 and 8, respectively). The specificity of TNF-α-induced complexes was demonstrated by competition with excess cold identical oligonucleotide, but not with excess cold irrelevant oligonucleotide (data not shown). Nuclear lysates from SP-treated HDMEC incubated with the −191/−170 ICAM-1 labeled probe (lane 9) displayed specific complex formation distinct from that with TNF-α-treated HDMEC (lane 2). The specificity of the SP-induced complex formation was demonstrated by competition with excess cold identical oligonucleotide (lane 10). SP complex formation disappeared upon incubation with anti-NF-AT1 Ab (lane 11) and with the broad spectrum NF-AT Ab (lane 12), which cross-reacts with all NF-AT family members.
To determine specificity of complex formation, HDMEC were preincubated with a selective NK-1R antagonist that completely prevented complex formation (Fig. 3 A, lane 13). HDMEC pretreated with CsA (1 or 10 μM) displayed no SP-dependent complex formation as well (lanes 14 and 15). The 1 μM dose of CsA was chosen as optimal for subsequent experiments. These data demonstrate that the SP-induced ICAM-1-binding complex contains a protein(s) recognized by anti-NF-AT1 and anti-NF-AT family member-specific Abs. This specific SP-induced NF-AT-containing complex, which binds the region −191/−170 of the ICAM-1 promoter, can be blocked by both CsA and a selective NK-1R antagonist. These data also demonstrate that two independent transcription factor complexes, p65 homodimers induced by TNF-α and an NF-AT-containing complex induced by SP, can bind to the same critical regulatory region (−191/−170) of the ICAM-1 gene, and that the activation of these two transcription factors occurs in a mutually independent fashion.
Typically, NF-AT associates cooperatively with heterologous DNA binding proteins, particularly AP-1. The SP-inducible NF-AT-containing complex was unaffected when incubated in the presence of Abs to AP-1 members, anti-c-Fos, or anti-c-Jun (Fig. 3,B). Disappearance of the SP-induced complex when using anti-NF-AT Abs seen in Fig. 3,A presumptively occurred through physical interference of the bound Ab with the DNA recognition motif. The NF-AT-containing complex activated by SP was visibly supershifted upon incubation with an anti-NF-AT1 Ab (Fig. 3 B, lane 5) when the order of addition of reagents was altered, such that the protein/DNA complex was allowed to form before addition of the anti-NF-AT1 Ab.
Although enhancer trap reporter expression studies had shown that the −191/−170 region was sufficient to confer both SP and TNF-α inducibility (Fig. 2, C and D), we tested whether other regions of the wild-type ICAM-1 promoter might be contributing to native SP transcription induction of ICAM-1. To do so, we compared SP and TNF-α inducibility of CAT reporter expression in HDMEC after transfection with either wild type −384/+1 ICAM CAT or a derivative plasmid, mutκB−384/+1 ICAM CAT, in which the overlapping regions of the NF-AT and NF-κB consensus binding sites (Fig. 2,A) had been mutated by site-directed mutagenesis. Mutagenesis limited to nucleotides critical for both NF-AT and p65 binding completely abrogated both SP and TNF-α inducibility (data not shown). Since EMSA data demonstrate that SP induces only NF-AT and not p65 binding to the critical −191/−170 region (Fig. 3, A and B), and since mutation of the NF-AT binding site abrogates all SP reporter gene inducibility, we conclude that not only are NF-AT activation and binding to this site necessary for SP induction of ICAM-1 expression, but also that no other regions of the native ICAM-1 promoter within the −384/+1 fragment are capable of independently mediating gene responsiveness to SP.
Rel family members do not comprise the SP-inducible ICAM-1 DNA binding complex
To further elucidate which proteins comprise the SP-induced DNA binding complex, the SP-responsive region (−191/−170) of the ICAM-1 promoter was used in an EMSA. Since TNF-α is known to induce NF-κB (Rel family) member binding to (−191/−170) of the same region of the ICAM-1 promoter, we therefore investigated whether Rel family members were part of the SP-induced complex formation in HDMEC (Fig. 4). Nuclear lysates from untreated HDMEC show no complex formation (lane 1). SP-treated HDMEC complexes (lane 2) are unaffected by the addition of Abs against Rel family members anti-p65/RelA (lane 3), anti-p50 (lane 4), anti-c-Rel (lane 5), and anti-RelB (lane 6). HDMEC pretreated with CsA, followed by SP addition, display no complex formation (lane 7). Thus, SP-mediated complex formation, which binds to the ICAM-1 promoter region (−191/−170), does not contain Rel family member proteins, as defined by anti-Rel Abs.
Deletional analysis of the VCAM-1 gene promoter reveals a SP-responsive region between bp −88 and −32 in HDMEC
Our previous studies demonstrated a significant induction of VCAM-1 mRNA expression by HDMEC after SP treatment (7). We thus investigated SP-mediated VCAM-1 transcription induction via 5′-deletional VCAM-1 promoter-based reporter gene constructs after transient transfection into HDMEC. Analysis of the respective CAT expression in untreated cells revealed an expected lack of basal activity of the −288/+20 VCAM-1 construct, and induction of CAT expression was seen after treatment with SP and TNF-α (Fig. 5). The −88/+20 5′ deletion construct demonstrated comparable SP- and TNF-α-induced expression equivalent to the −288/+20 construct, whereas the −32/+20 construct showed a loss of SP and TNF-α inducibility (Fig. 5). We conclude that the required transcriptional regulatory region necessary for SP-mediated VCAM-1 induction is located between bp −88 and −32 of the VCAM-1 promoter.
The VCAM-1 SP-responsive element binds p65 and p50, but not NF-AT
The identified SP-responsive region of the VCAM-1 promoter between regions bp −88 and −32 contains the previously reported tandem NF-κB regions (14, 38) at position −76/−52. Since our analysis of the SP-induced ICAM-1 expression had implicated a similar region, this VCAM-1 region was used as radiolabeled probe along with HDMEC nuclear lysates in EMSAs (Fig. 6). Nuclear lysates from untreated HDMEC demonstrated no complex formation (lane 1). SP-induced complex formation (lane 2) was abrogated upon preincubation of HDMEC with an NK-1R antagonist (lane 3) as well as by competition with excess cold oligonucleotide (lane 8). In contrast to SP-induced ICAM-1 binding complexes, anti-NF-AT1 (lane 4) and anti-NF-AT (lane 5) family member-specific Abs did not affect SP-induced VCAM-1 complex formation. Moreover, both anti-p65 (lane 6) and anti-p50 (lane 7) supershifted the SP-induced complexes. TNF-α-treated nuclear extracts from HDMEC (lane 9) were unaffected by the addition of anti-NF-AT family Ab (lane 10), yet TNF-α-mediated complex formation was supershifted upon incubation with anti-p65-specific (lane 11) and anti-p50-specific (lane 12) Abs, as previously reported (20). TNF-α-treated complex formation specificity was demonstrated by preincubation with excess cold oligonucleotide (lane 13). Therefore, the SP-mediated VCAM-1 binding complex contains p65 and p50, but not NF-AT family members, as shown by anti-p65, anti-p50, and anti-NF-AT family member Abs. In separate experiments, we demonstrated that Abs against other Rel family members, including c-Rel, RelB, and p52, did not shift or affect SP-induced complex formation bound to the −76/−52 region of the VCAM-1 promoter (data not shown).
SP-induction of p65 and p50 binding to the VCAM-1 SP-responsive element is TNF-α independent
SP is known to induce cytokine production in various immune cells, including TNF-α production in mast cells (39). TNF-α is the only defined cytokine capable of transcriptionally inducing VCAM-1 in HDMEC (16), although VCAM-1 can be induced by other cytokines (e.g., IL-1) in different types of endothelial cells, such as human umbilical vein endothelial cells (17). Since SP and TNF-α-induced identical NF-κB complexes binding to the −76/−52 region of the VCAM-1 promoter in HDMEC, it was possible that SP was inducing autocrine TNF-α expression and stimulation of these cells. To test this possibility, we used Abs against human TNF-receptors 1 and -2 added just before SP treatment to block any subsequent TNF-α stimulation of HDMEC. We then assessed the induction of specific nuclear protein-DNA complex formation using the −76/−52 region of the VCAM-1 promoter (Fig. 7). Untreated HDMEC nuclear lysates lacked specific complex formation (lane 1). TNF-α-treated complex formation (lane 2) was abrogated when HDMEC were pretreated with anti-TNF-α blocking Abs (lane 3). The TNF-α-specific complex (lane 2) was supershifted by anti-p65 (lane 4) and anti-p50 (lane 5), as shown above. TNF-α-treated complex formation supershifts (lanes 4 and 5) by Abs to p65 and p50 were abrogated when HDMEC were preincubated with anti-TNF-α blocking Abs (lanes 6 and 7). Therefore, TNF-α blocking Abs are capable of abrogating TNF-α-induced p65 and p50 binding to −76/−52 of the VCAM-1 promoter. SP-treated complex formation (lane 8) was unaffected by HDMEC pretreatment with anti-TNF-α blocking Abs (lane 9). The SP-induced complex was supershifted by anti-p65 (lane 10) and anti-p50 (lane 11) Abs, and these supershifts were unaffected by HDMEC pretreated with anti-TNF-α blocking Abs (lanes 12 and 13). However, SP-treated complex formation was blocked by HDMEC pretreatment with a selective NK-1R antagonist (lane 14). These data indicate that SP-mediated p65 and p50 complex binding to the SP-responsive VCAM-1 element is a direct effect of SP, and that SP-dependent activation of NF-κB does not involve autocrine stimulation by HDMEC-derived TNF-α.
SP-induced NF-AT activation and binding to the −191/−170 region of the ICAM-1 promoter is intracellular Ca2+ dependent
We next determined whether an SP- or TNF-α-elicited intracellular Ca2+ mobilization was required for transcription factor activation and binding to the ICAM-1 −191/−170 region by pretreating HDMEC with the membrane-permeable selective intracellular calcium chelator BAPTA-AM (Fig. 8). Untreated HDMEC (lane 1) displayed no specific complex formation. SP-induced complex formation (lane 3) was abrogated when HDMEC were pretreated with BAPTA-AM (lane 2). Specificity of the SP-induced complex was demonstrated by incubation with cold excess identical oligonucleotide (lane 4), while excess cold irrelevant oligonucleotide (ICAM-1 −88/−68) had no effect on this complex (data not shown). TNF-α-stimulated p65 homodimer binding to the identical region on the ICAM-1 gene (lane 6) was unaffected when HDMEC were pretreated with BAPTA-AM (lane 5). Thus, SP-induced NF-AT activation and binding to −191/−170 of the ICAM-1 promotor, but not TNF-α-induced p65 homodimer activation and binding to the same region, is intracellular Ca2+ mobilization dependent.
SP-induced, but not TNF-α-induced, p65/p50 heterodimer activation and binding to the VCAM-1 −76/−52 region is intracellular Ca2+ mobilization dependent
We have demonstrated that SP and TNF-α stimulation of HDMEC induces identical NF-κB family member binding to the −76/−52 region of the VCAM-1 promoter and that SP induction of such binding was TNF-α independent. We next assessed whether the SP- or TNF-α-stimulated p65/p50 heterodimer activation and binding to the VCAM-1 −76/−52 region was dependent upon the mobilization of intracellular calcium (Fig. 9). Untreated HDMEC nuclear lysates displayed no specific complex formation (lane 1). SP-induced complex formation (lanes 2 and 4) was inhibited when HDMEC were pretreated with BAPTA-AM before the addition of SP (lane 3). The specificity of SP-induced complex formation is demonstrated when lysates are incubated in the presence of cold excess −76/−52 oligonucleotide (lane 5), but not irrelevant −42/−22 oligonucleotide (data not shown). In contrast, TNF-α-elicited complex formation (lane 6) was unaffected when HDMEC were pretreated with BAPTA-AM (lane 7). These data indicate that although both SP and TNF-α stimulation of HDMEC are capable of inducing the activation of identical transcription factors that bind to the same region (−76/−52) of the VCAM-1 gene, the signaling mechanisms are distinct. SP induction of p65/p50 heterodimers requires intracellular calcium mobilization, whereas TNF-α induction of the identical NF-κB complex is not calcium mobilization dependent.
SP has been well defined as a key player in the induction of peripheral inflammation (40). SP can modulate leukocyte effector activities such as lymphocyte proliferation, cytotoxicity, and Ig production (1, 41); mast cell degranulation (42); macrophage and polymorphonuclear leukocyte activation (43, 44); and cytokine production by monocytes (45, 46, 47). We have previously demonstrated that SP can directly activate mast cells and keratinocytes to secrete TNF-α and IL-1, respectively (39, 48). SP activation in the CNS of the transcription factor NF-κB and IL-8 (κB-dependent) gene expression in human astrocytoma cells has been recently reported (31). SP-mediated transcription factor induction and subsequent inflammatory gene expression involving neuropeptides secreted by the peripheral nervous system have not, as of yet, been described.
We have previously demonstrated a difference in concentration dependence induction of mRNA and cell surface HDMEC ICAM-1 and VCAM-1 by SP (6, 7). Optimal induction of VCAM-1 occurred at a 10-fold lower concentration of SP (10 nM) compared with ICAM-1 induction by SP (100 nM). We also demonstrated that SP-mediated induction of VCAM-1 occurred at earlier time points, 12–16 vs 16–18 h for ICAM-1 induction (6, 7). In vivo studies have indicated a preferential infiltration of eosinophils upon injection of SP into human skin (49). Neutrophils represent >70% of the immune cells in blood, yet these cells lack the integrin VLA-4, which is present on eosinophils. One can speculate that SP-mediated eosinophil influx is regulated at the level of VCAM-1/VLA-4 interactions, because SP induces VCAM-1 at lower concentrations and earlier time points, and preferentially activates the integrin VLA-4. It would thus appear from our data that in HDMEC the p65/p50 heterodimers required for VCAM-1 induction display a threshold for activation by SP that is not be seen for the p65 homodimers required for ICAM-1 induction. Indeed, our data demonstrate that at 10-fold higher SP concentrations, NF-AT1, rather than p65 homodimers, is activated and mediates the induction of ICAM-1 expression. Why SP selectively activates p65/p50 heterodimer, but not p65 homodimer, formation and binding in HDMEC is currently under further investigation.
Our previous transcriptional work focused on the identification and characterization of the ICAM-1 5′ regulatory region (9, 10, 35, 36) as well as additional characterization of the VCAM-1 5′-regulatory region (11, 17, 33, 50). In this study we have identified the minimal DNA binding domains within the regulatory regions of ICAM-1 and VCAM-1 that confer SP-mediated transcriptional activity in HDMEC. In addition, we have identified which SP-activated transcription factors associate with the DNA binding domains of the ICAM-1 and VCAM-1 regulatory regions in HDMEC. We demonstrate that 5′-regulatory regions between −191/−170 of the ICAM-1 gene and −76/−52 of the VCAM-1 gene are necessary for activation-dependent transcription by SP in HDMEC. Both regulatory regions, −191/−170 of ICAM-1 and −76/−52 of VCAM-1, contain previously defined NF-κB binding domains (14, 18, 21, 37). Multiple differences exist between the κB regions of the ICAM-1 and VCAM-1 promoters. The VCAM-1 promoter contains two tandem consensus κB regions at positions −73 and −58, and TNF-α induces preferential p65/p50 heterodimer binding to these sites (38). The ICAM-1 promoter contains a single nonconsensus κB region to which TNF-α induces preferential binding of a p65 homodimer (19, 36). The ICAM-1 κB nonconsensus region is flanked by sequences that are required for TNF-α-induced p65 homodimer binding (36), whereas flanking sequences have not been shown to be necessary for TNF-α-mediated p65/p50 heterodimer binding to the tandem consensus region on the VCAM-1 promoter. We demonstrate SP-mediated activation and binding of NF-AT to the −191/−170 of ICAM-1 and NF-κB (p65/p50) to the −76/−52 of VCAM-1 genes. These events can be specifically blocked by both the NK-1R antagonist and CsA. Typically, NF-AT is associated with heterologous DNA binding proteins, especially AP-1 (reviewed in Refs. 48). SP-driven NF-AT/ICAM-1 appears in vitro to lack cooperative binding with transcription factors of the AP-1 family. It is possible that in vivo, cooperation with other transcription factors may occur. However, site-directed mutagenesis of the NF-AT site, which overlaps with the 5′ portion of the p65 homodimer NF-κB binding site, abrogates both SP- and TNF-α-induced transcription, even within the context of the remaining native ICAM-1 promoter. Our data also indicate that the SP-elicited intracellular Ca2+ mobilization is required for NF-AT and NF-κB activation and binding to their respective consensus sites on the ICAM-1 and VCAM-1 genes. In contrast, the TNF-α-activated NF-κB pathway was Ca2+ independent, again demonstrating that SP and TNF-α transduce via distinct signaling pathways. Recent reports have demonstrated an intracellular Ca2+ mobilization requirement for NF-κB activation and κB-dependent gene expression in cell types other than EC and by signals other than neuropeptides (51, 52).
Our model of activation and differential regulation of microvascular endothelial cell adhesion molecule expression by the neuropeptide SP differs from the traditional TNF-α-induced NF-κB-mediated transcriptional induction of adhesion molecules ICAM-1 and VCAM-1 in microvascular endothelial cells (Fig. 10). The neuropeptide SP upon binding to its G protein-coupled NK-1R elicits an intracellular calcium signal. We have demonstrated that this Ca2+ signal results in differential transcription factor activation and binding to previously defined regions of ICAM-1 and VCAM-1. As a consequence of the SP-induced calcium signal, the phosphatase calcineurin becomes activated and dephosphorylates NF-AT (reviewed in Ref. 26). We propose that NF-AT then translocates to the nucleus and binds its high affinity TGGAAA consensus site on the ICAM-1 promoter, a site that is colocalized with the 5′ portion of the modified NF-κB site that is used with TNF-α activation of p65 homodimers. By an unknown mechanism, the SP-elicited calcium signal also leads to the activation of the NF-κB pathway, resulting in nuclear translocation of the p65/p50 heterodimer, which binds to its high affinity tandem κB sites on the VCAM-1 promoter. This SP-induced activation of NF-κB heterodimers does not extend to the activation of sufficient quantities of p65 homodimers for the binding and activation of transcription through the ICAM-1-modified κB site. Instead, SP treatment induces NF-AT activation and ICAM-1 DNA binding. These transcriptional activation events lead to ICAM-1 and VCAM-1 HDMEC cell surface expression, and these pathways of gene expression induction can be specifically blocked by both NK-1R and CsA.
In summary, our studies demonstrate that the neuropeptide SP is capable of directly activating intracellular Ca2+-responsive transcription factors NF-AT and NF-κB, resulting in induction of ICAM-1 and VCAM-1 gene expression in microvascular endothelial cells. These findings indicate that neuropeptides released in skin can activate transcription factors and modulate adhesion molecule expression, contributing to inflammation and wound healing.
We acknowledge generation of ICAM-1 promoter 5′-deletional constructs by Naotaka Shibagaki and Lian-Jie Li, generation of ICAM-1-based site-directed mutagenesis constructs by Lani Paxton, and generation of VCAM-1 promoter 5′-deletional constructs by Jens Gille. We thank Neera Bahl for the isolation of primary microvascular endothelial cells and Andrew Neish and Melissa Brown for review and discussion of the manuscript.
This work was supported by National Institutes of Health Grants AR40678 (to J.A.), AR41206 and AR42687 (to S.W.C.), A141493 (to J.A. and S.W.C.), and RO3AR44969 (to C.A.A.) and a Veterans Administration Merit Review Award (to J.C.A.).
Abbreviations used in this paper: SP, substance P; NK-1R, neurokinin receptor-1; HDMEC, human dermal microvascular endothelial cells; IκB, inhibitor of NF-κB; IKK, IκB kinase; CsA, cyclosporin A; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′-tetraacetic acid-acetoxymethyl; CAT, chloramphenicol acetyltransferase; TK, thymidine kinase; VLA-4, very late Ag-4.