The constitutive secretion of latent TGF-β by many cell types in culture suggests that extracellular mechanisms to control the activity of this potent cytokine are important in the pathogenesis of the diseases in which this cytokine may be involved, including fibrotic disorders. In this study, we focused on the αvβ3 integrin, which is recently demonstrated to function as an active receptor for latent TGF-β1 through its interaction with latency-associated peptide-β1, and investigated the involvement of this integrin in the pathogenesis of scleroderma. Scleroderma fibroblasts exhibited increased αvβ3 expression compared with normal fibroblasts in vivo and in vitro. In scleroderma fibroblasts, ERK pathway was constitutively activated and such abnormality induced the up-regulation of αvβ3. Transient overexpression of αvβ3 in normal fibroblasts induced the increase in the promoter activity of human α2(I) collagen gene and the decrease in that of human MMP-1 gene. These effects of αvβ3 were almost completely abolished by the treatment with anti-TGF-β Ab or TGF-β1 antisense oligonucleotide. Furthermore, the addition of anti-αvβ3 Ab reversed the expression of type I procollagen protein and MMP-1 protein, the promoter activity of human α2(I) collagen gene, and the myofibroblastic phenotype in scleroderma fibroblasts. These results suggest that the up-regulated expression of αvβ3 contributes to the establishment of autocrine TGF-β loop in scleroderma fibroblasts, and this integrin is a potent target for the treatment of scleroderma.

Systemic sclerosis (SSc)3 or scleroderma is an acquired disorder that typically results in the fibrosis of the skin and internal organs (1). Previous findings indicate that the pathogenesis of this disorder includes inflammation, autoimmune attack, and vascular damage, leading to fibroblast activation (2). However, cultured SSc fibroblasts, which are free from such environmental factors, continue to produce the excessive amount of extracellular matrix (ECM) proteins (3, 4), suggesting that SSc fibroblasts establish a constitutive self-activation system once activated. One of major cytokines that may be involved in this process is TGF-β1 (5), and the principal effect of this cytokine on mesenchymal cells is the stimulation of ECM deposition. This notion is supported by the following previous findings: 1) SSc fibroblasts express the elevated levels of TGF-β receptors, and this correlates with the increased expression of α2(I) collagen mRNA (6, 7, 8, 9); and 2) the blockade of TGF-β signaling with anti-TGF-β Ab or anti-TGF-β1 antisense oligonucleotide abolishes the increased expression of human α2(I) collagen mRNA in SSc fibroblasts (7).

TGF-β1 is normally secreted as a complex composed of three proteins, including the bioactive peptide of TGF-β1, a latency-associated peptide-β1 (LAP-β1), and a latent TGF-β binding protein-1. TGF-β1 forms a complex with LAP-β1 noncovalently, which is called the small latent complex (SLC), and in this configuration TGF-β1 is unable to bind to its receptors. SLC is joined by a latent TGF-β binding protein-1, the N-terminal region of which is covalently cross-linked to ECM proteins by transglutaminase, and the complex of all three proteins is called the large latent complex (10). The constitutive secretion of latent TGF-β1 by many cell types in culture suggests that there are extracellular mechanisms to control the activity of this potent cytokine. Although these processes are not fully understood, recent reports demonstrated that cell surface molecules or secreted extracellular molecules can activate latent TGF-β1. Specifically, the αvβ6 integrin and thrombospondin (TSP)-1 have been implicated in activation of latent TGF-β1 through nonproteolytic mechanisms (11, 12). In addition, plasmin has been proposed to lead to the activation of latent TGF-β1 through proteolytic degradation of LAP-β1 (13). The αvβ8 integrin has also been demonstrated to be able to activate latent TGF-β1 by membrane-type 1-matrix metalloproteinase (MMP)-dependent degradation of LAP-β1 (14). Thus, normal TGF-β function is thought to be largely controlled by its activation from the latent state.

LAP-β1 contains an RGD motif that is recognized by αv-containing integrins, including αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8 (11, 14, 15, 16). Although all of these αv-containing integrins bind to LAP-β1 and have the potential to modulate the localization and possibly activation of SLC, only αvβ6 and αvβ8, both of which are not expressed in dermal fibroblasts, have been demonstrated to be able to activate SLC (11, 14). Especially, αvβ6-mediated activation of SLC was demonstrated to play an important role in response to tissue injury because the epithelium-restricted β6−/− mice showed only a minor fibrotic response of lung to bleomycin administration compared with wild-type mice (11). Although there have been no reports that indicate the activation of SLC by other αv-containing integrins, such as αvβ1, αvβ3, and αvβ5, a disease process associated with both αvβ3 and TGF-β1 has been implicated in animal models of neointima formation in mechanically injured vessels and in restenosis after angioplasty (17, 18, 19, 20, 21, 22). In the early phase of neointima formation, mRNA levels of TGF-β1, TGF-β receptor type I, TGF-β receptor type II, αv subunit, and β3 subunit are elevated in injured vessels (21). However, the pretreatment of anti-αvβ3-blocking Ab or a small peptide antagonist inhibits neointima formation by promoting apoptosis of smooth muscle cells and preventing migration of these cells, angiogenesis, and excessive ECM deposition (17, 18, 20, 22). Interestingly, the pretreatment of anti-αvβ3 Ab dramatically reduces the accumulation of TGF-β1 protein in injured vessels (22), suggesting that the role of αvβ3 as an SLC receptor contributes to this process. These previous findings stimulate our interest to investigate whether αvβ3 is involved in the pathogenesis of fibrotic disorders.

This study is undertaken to clarify the involvement of αvβ3 in the pathogenesis of SSc. First, we compared the expression levels of αvβ3 between normal and SSc fibroblasts in vivo and in vitro. We then investigated the effect of transiently overexpressed αvβ3 on the promoter activity of the human α2(I) collagen gene or human MMP-1 gene. Furthermore, we determined the effect of anti-αvβ3 Ab on the phenotype of SSc fibroblasts. The results suggest that the up-regulated expression of αvβ3 contributes to the establishment of autocrine TGF-β loop in SSc fibroblasts, and anti-αvβ3 Ab reverses the myofibroblastic phenotype of those cells. To our knowledge, this is the first report that indicates the possibility of regulating fibrotic disorders, especially SSc, by targeting this integrin.

Recombinant human TGF-β1, recombinant human epidermal growth factor (EGF), and recombinant human platelet-derived growth factor (PDGF)-AA were obtained from R&D Systems. Actinomycin D and Ab for β-actin were purchased from Sigma-Aldrich. Abs for αv, β3, phospho-ERK1/2, and ERK2 were obtained from Santa Cruz Biotechnology. Abs for αvβ3 and MMP-1 were obtained from Chemicon International. Ab for type I collagen was purchased from Southern Biotechnology Associates. Anti-Smad2/3 Ab (S66220) was purchased from BD Transduction Laboratories. αv cDNA was a gift from Dr. J. C. Loftus (Mayo Clinic, Scottsdale, AZ). β3 cDNA was a gift from Dr. T. E. O’Toole (Research Institute of Scripps Clinic, La Jolla, CA).

Human dermal fibroblasts were obtained by skin biopsy from the affected areas (dorsal forearm) of 10 patients with diffuse cutaneous SSc and <2 years of skin thickening. Control fibroblasts were obtained by skin biopsy from 10 healthy donors. Institutional approval and informed consent were obtained from all subjects. Control donors were matched with each SSc patient for age, sex, and biopsy site, and control and patient samples were processed in parallel. Primary explant cultures were established in 25-cm2 culture flasks in MEM with 10% FCS, 2 mM l-glutamine, and 50 μg/ml amphotericin as described previously (9). Fibroblast cultures independently isolated from different individuals were maintained as monolayers at 37°C in 95% air, 5% CO2, and studied between the third and sixth subpassages.

Cells were cultured to confluence in MEM supplemented with 10% FCS. After incubation for 24 h in serum-free medium (MEM plus 0.1% BSA), cells were washed with PBS at 4°C and solubilized in lysis buffer (1% Triton X-100 in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 3 mM MgCl2, 1 mM CaCl2 containing 10 μg/ml leupeptin, pepstatin, and aprotinin, and 1 mM PMSF). The lysates were incubated 30 min at 4°C and then centrifuged for 15 min at 4°C. Protein concentrations of lysates were determined using Bio-Rad protein assay reagent. Proteins were subjected to SDS-PAGE and transferred to nitrocellulose membranes as described previously (9). Membranes were incubated overnight with the indicated Abs, washed, and incubated for 1 h with secondary Abs. After washing, visualization was performed by ECL (Amersham Life Science) according to the manufacturer’s recommendations. The densities of bands were measured with a densitometer.

Cells were cultured to confluence in MEM supplemented with 10% FCS. After incubation for 24 h in serum-free medium, cells were washed with PBS. Then, cells were incubated with membrane-impermeant NHS-LC-biotin (Pierce) dissolved at 0.5 mg/ml in PBS at 37°C for 30 min. The cells were washed with cold PBS and harvested into lysis buffer as described above. αvβ3 was immunoprecipitated using anti-β3 Ab. Immune complexes were collected using protein A-agarose and subjected to immunoblotting using streptavidin coupled to HRP (Amersham Biosciences).

Cells were grown to confluence in MEM supplemented with 10% FCS and then incubated for 24 h in serum-free medium before the addition of the indicated reagent. Two micrograms of extracted total RNA was subjected to electrophoresis on 1% agarose/formaldehyde gels and blotted onto nylon filters (Roche). The filters were UV cross-linked, prehybridized, and sequentially hybridized with DNA probe for GAPDH and RNA probes for integrin αv and β3 subunits as described previously (9). The membrane was then washed and exposed to x-ray film.

Immunohistochemical staining on paraffin-embedded sections was performed using a Vectastain ABC kit (Vector Laboratories) according to the manufacturer’s instructions as described previously (23). Two micrometer-thick sections were mounted on silane-coated slides, then deparaffinized by xylene, and rehydrated through a graded series of ethyl alcohol and PBS. The sections were then incubated with Abs against αv or β3 diluted 100 times in PBS overnight at 4°C. The immunoreactivity was visualized by diaminobenzidine. The sections were then counterstained with hematoxylin. We used the following grading system: + for slight staining, 3+ for strong staining, and 2+ for staining between + and 3+.

Kinase assays were performed as described previously (24). Briefly, cells were lysed in buffer containing 20 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, and 1 mM PMSF. Total protein (200 μg) samples were subjected to immunoprecipitation using anti-phospho-ERK (Thr202/Tyr204) Ab. The immunoprecipitate pellets were incubated with 1 μg of Elk-1 fusion protein in the presence of 100 μM ATP and a kinase buffer containing 25 mM Tris-HCl (pH 7.5), 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2. The reaction was terminated with SDS loading buffer. The levels of phosphorylated Elk-1 were analyzed by immunoblotting using anti-phospho-Elk-1 Abs.

A −772 COL1A2/chloramphenicol acetyltransferase (CAT) construct consisting of the human α2(I) collagen gene fragment (+58 to −772 bp relative to the transcription start site) linked to the CAT reporter was generated as described previously (25). Expression vector of dominant-negative mutant of ERK2 (DN ERK2) is a gift from Dr. D. Templeton (Case Western Reserve University, Cleveland, Ohio) (26, 27). Expression vector of constitutive active mutant of MEK1 (CA MEK1) is a gift from Dr. R. J. Davis (University of Massachusetts Medical School, Worcester, Massachusetts) (28). Plasmid used in transient transfection assays were purified twice on CsCl gradients. At least two different plasmid preparations were used for each experiment.

Cells were grown to 50% confluence in 100-mm dishes in MEM with 10% FCS. The medium was replaced with serum-free medium, and after 4-h incubation cultures were transfected with 2 μg of −772 COL1A2/CAT constructs, along with 2 or 4 μg of β3 expression vector or corresponding empty construct (pCDM8), using FuGENE6 (Roche) as described previously (9). To control for minor variations in transfection efficiency, 1 μg of pSV-β-galactosidase vector (Promega) was included in all transfections. After 72-h incubation, cell extracts were prepared by the Reporter Lysis Buffer (Promega). Extracts, normalized for protein content, were incubated with butyl-CoA and 14C-chloramphenicol for 90 min at 37°C. Butylated chloramphenicol was extracted using an organic solvent (a 2:1 mixture of tetramethylpentadecane and xylene) and quantitated by scintillation counting. Each experiment was performed in duplicate.

Quiescent cells cultured in 4-well LAB TEK chambers (Nunc) were treated with 10 μg/ml anti-αvβ3 Ab or preimmune mouse IgG for 48 h. Then, cells were fixed with 3.7% formaldehyde, permeabilized with 0.5% Triton X-100 in PBS, and blocked with 10% FCS in PBS containing 0.5% Triton X-100 as described previously (9). Cells were stained with anti-α-smooth muscle actin Ab, washed, and incubated with FITC-conjugated rabbit anti-mouse IgG (Sigma-Aldrich). The nuclei were counterstained for 5 min with 4′,6-diamidino-2-phenylindole (DAPI; 0.2 μg/ml in PBS) (SigmaAldrich). To visualize the fluorescence, a Zeiss microscope was used.

We used a pan-specific neutralizing TGF-β Ab (R&D Systems), which has been shown to specifically inhibit the activity of TGF-β1, β2, and β3. We also used a TGF-β1 19-mer antisense oligonucleotide (GAGGGCGGCATGGGGAGG), which overlaps the promoter and transcriptional start site of the TGF-β1 gene. This same sequence, which is specific for the TGF-β1 isoform, has been found to be sufficient to block TGF-β1 transcription in vitro (29) and in vivo (30). A sense oligonucleotide served as a control.

Two oligonucleotides containing biotin on the 5′-nucleotide of the sense strand were used (31). The sequences of these oligonucleotides are as follows: 1) 3xCAGA oligo, 5′-TCGAGAGCCAGACAAGGAGCCAGACAAGGAGCCAGACACTCGAG, which is trimer of CAGA motif; and 2) 3xCAGA-M oligo, 5′-TCGAGAGCTACATAAAAAGCTACATATTTAGCTACATACTCGA, which is trimer of CAGA motif mutated. These oligonucleotides were annealed to their respective complementary oligonucleotides, and double-stranded oligonucleotides were gel-purified and used. Cell lysate was prepared using lysis buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 50 mM NaF, 1 mM PMSF, 1 mM Na3VO4, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 μg/ml pepstatin. Five micrograms of poly(dI-dC) competitor was incubated with 500 μg of cell lysate for 30 min at 4°C, followed by 1-h incubation with 500 pmol of each double-stranded oligonucleotide. After the incubation, 65 μl of streptavidin-agarose (Sigma-Aldrich) was added to the reaction and incubated at 4°C for overnight. The protein-DNA-streptavidin-agarose complex was washed three times with lysis buffer, resuspended in the sample buffer for electrophoresis, boiled for 3 min, spun briefly, and the supernatants were subjected to Western blotting with anti-Smad2/3 Ab (S66220). The specific binding of Smad3 with 3xCAGA oligo was confirmed by the experiments using 3xCAGA-M oligo. The binding of Smad3 with 3xCAGA-M oligo was not observed in the presence or absence of TGF-β1 (data not shown).

Statistical analysis was conducted with the Mann-Whitney U test for comparison of means. p values <0.05 were considered significant.

As an initial experiment, we compared the expression levels of αv and β3 subunit proteins between normal and SSc fibroblasts using whole cell lysates by immunoblotting. As shown in Fig. 1, A and B, the expression levels of αv subunit protein were ∼2.7 times higher in SSc fibroblasts than normal fibroblasts. The expression levels of β3 subunit protein were also ∼5.2 times higher in SSc fibroblasts than normal fibroblasts. To function as active receptors, integrins have to be present on the cell surface as dimers. Therefore, we next determined the cell surface levels of αvβ3 in normal and SSc fibroblasts. To this end, cell surface proteins were labeled with biotins, and immunoprecipitation was performed using anti-β3 Ab. As shown in Fig. 1,C, cell surface levels of αvβ3 were markedly elevated in SSc fibroblasts compared with normal fibroblasts. These bands were confirmed to be αv or β3 by a reprobing analysis using anti-αv or β3 Abs (data not shown). Although β3 subunit can interact with two kinds of α subunits, such as αv and αIIb, αIIb subunit is not precipitated by anti-β3 Ab in dermal fibroblasts. This is consistent with previous reports that αIIb subunit is mainly expressed in platelets, but not in dermal fibroblasts (32). The expression levels of αv and β3 subunit mRNAs in normal and SSc fibroblasts were also determined by Northern blotting. As shown in Fig. 2, the levels of αv mRNA were ∼2.3 times higher in SSc fibroblasts than normal fibroblasts. Similarly, the expression levels of β3 mRNA were ∼4.3 times higher in SSc fibroblasts than normal fibroblasts.

FIGURE 1.

Expression levels of αvβ3 in normal and SSc fibroblasts. A, Whole cell lysates were electrophoresed through a 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with anti-αv, β3, or β-actin Abs. B, The protein levels quantitated by scanning densitometry are shown relative to those in normal fibroblasts (100 arbitrary units (AU)). Data are expressed as the mean ± SD of five independent experiments. ∗, p < 0.05 vs normal fibroblasts. C, Cell surface proteins were labeled with biotin, and whole cell lysates were immunoprecipitated using anti-β3 Ab. All blots show one representative of five independent experiments.

FIGURE 1.

Expression levels of αvβ3 in normal and SSc fibroblasts. A, Whole cell lysates were electrophoresed through a 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with anti-αv, β3, or β-actin Abs. B, The protein levels quantitated by scanning densitometry are shown relative to those in normal fibroblasts (100 arbitrary units (AU)). Data are expressed as the mean ± SD of five independent experiments. ∗, p < 0.05 vs normal fibroblasts. C, Cell surface proteins were labeled with biotin, and whole cell lysates were immunoprecipitated using anti-β3 Ab. All blots show one representative of five independent experiments.

Close modal
FIGURE 2.

Expression levels of αv and β3 mRNAs in normal and SSc fibroblasts. A and B, Two micrograms of extracted total RNA was subjected to electrophoresis on 1% agarose/formaldehyde gels, blotted onto nylon filters, and hybridized with DNA probe for GAPDH and RNA probes for integrin αv (A) and β3 (B) subunits. One representative of five independent experiments is shown. C, The mRNA levels quantitated by scanning densitometry and corrected for the level of GAPDH in the same samples are shown relative to those in normal fibroblasts (100 AU). Data are expressed as the mean ± SD of five independent experiments. ∗, p < 0.05 vs normal fibroblasts.

FIGURE 2.

Expression levels of αv and β3 mRNAs in normal and SSc fibroblasts. A and B, Two micrograms of extracted total RNA was subjected to electrophoresis on 1% agarose/formaldehyde gels, blotted onto nylon filters, and hybridized with DNA probe for GAPDH and RNA probes for integrin αv (A) and β3 (B) subunits. One representative of five independent experiments is shown. C, The mRNA levels quantitated by scanning densitometry and corrected for the level of GAPDH in the same samples are shown relative to those in normal fibroblasts (100 AU). Data are expressed as the mean ± SD of five independent experiments. ∗, p < 0.05 vs normal fibroblasts.

Close modal

The steady-state level of mRNA can be affected by the level of gene transcription and/or the stability of mRNA. To determine whether the up-regulated expression of αv and β3 mRNAs takes place at the transcriptional level or posttranscriptional level, cells were treated with actinomycin D for 4 or 8 h before RNA extraction. As shown in Fig. 3, there was no difference in the stability of αv and β3 mRNAs between normal and SSc fibroblasts. These results indicate that the expression of αv and β3 is up-regulated at the transcriptional level in SSc fibroblasts.

FIGURE 3.

Time course of integrin αv and β3 subunit mRNA degradation in normal and SSc fibroblasts. Actinomycin D (400 ng/ml) was added to stop all de novo RNA transcription. Cells were harvested at the indicated times after actinomycin D treatment and analyzed for integrin αv, β3, and GAPDH mRNA by Northern blot analysis. Plots show the linear regression of the percentage of remaining αv mRNA (A) and β3 mRNA (B) relative to time 0 and after normalization to the GAPDH mRNA.

FIGURE 3.

Time course of integrin αv and β3 subunit mRNA degradation in normal and SSc fibroblasts. Actinomycin D (400 ng/ml) was added to stop all de novo RNA transcription. Cells were harvested at the indicated times after actinomycin D treatment and analyzed for integrin αv, β3, and GAPDH mRNA by Northern blot analysis. Plots show the linear regression of the percentage of remaining αv mRNA (A) and β3 mRNA (B) relative to time 0 and after normalization to the GAPDH mRNA.

Close modal

Previous reports demonstrated that the sustained activation of ERK can specifically control the expression of β3 subunit in a variety of human and mouse cell lines, including mouse fibroblasts, mouse macrophages, mouse and human endothelial cells, and human K-562 erythroleukemia cells (33, 34). Based on this concept, we next investigated the effect of UO126, a specific inhibitor of MEK, on the expression levels of β3 subunit in SSc fibroblasts. As shown in Fig. 4,A, UO126 reduced the expression levels of β3 subunit protein in a dose- and time-dependent manner in SSc fibroblasts, whereas the same treatment did not affect the expression levels of β3 subunit protein in normal fibroblasts. This effect of UO126 on β3 subunit protein was paralleled with that on β3 subunit mRNA in those cells (Fig. 4,B). To further confirm the involvement of the MEK-ERK pathway in the up-regulated expression of β3 in SSc fibroblasts, the effect of transiently overexpressed DN ERK2 was determined. As shown in Fig. 4,C, DN ERK2 significantly reduced the levels of β3 subunit protein in SSc fibroblasts, whereas it showed no effect on the levels of β3 subunit protein in normal fibroblasts. We also demonstrated that the transient overexpression of CA MEK1 induced the up-regulation of β3 subunit protein in normal fibroblasts (Fig. 4 D). In contrast, the treatment of UO126 and transient overexpression of DN ERK2 or CA MEK1 did not affect the levels of αv subunit in normal and SSc fibroblasts (data not shown). Taken together, these results suggest that β3 subunit is up-regulated by sustained activation of the MEK-ERK pathway in SSc fibroblasts.

FIGURE 4.

The effect of the inhibition of the MEK-ERK pathway on the expression levels of β3 subunit in normal and SSc fibroblasts. A, Confluent quiescent cells were treated with the indicated concentration of UO126 or the equal amount of vehicle (DMSO) for 24 or 48 h. Whole cell lysates were subjected to immunoblotting using anti-β3 Ab or anti-β-actin Ab. B, Under the same condition described above, the levels of β3 mRNA were determined by Northern blotting. C, Cells were transfected with the indicated amount of DN ERK2 or empty vector, and incubated for 72 h. Whole cell lysates were subjected to immunoblotting using anti-β3 Ab or anti-β-actin Ab. D, Cells were transfected with the indicated amount of expression vector of CA MEK1 or empty vector, and incubated for 72 h. Whole cell lysates were subjected to immunoblotting using anti-β3 Ab or anti-β-actin Ab. All blots show one representative of five independent experiments.

FIGURE 4.

The effect of the inhibition of the MEK-ERK pathway on the expression levels of β3 subunit in normal and SSc fibroblasts. A, Confluent quiescent cells were treated with the indicated concentration of UO126 or the equal amount of vehicle (DMSO) for 24 or 48 h. Whole cell lysates were subjected to immunoblotting using anti-β3 Ab or anti-β-actin Ab. B, Under the same condition described above, the levels of β3 mRNA were determined by Northern blotting. C, Cells were transfected with the indicated amount of DN ERK2 or empty vector, and incubated for 72 h. Whole cell lysates were subjected to immunoblotting using anti-β3 Ab or anti-β-actin Ab. D, Cells were transfected with the indicated amount of expression vector of CA MEK1 or empty vector, and incubated for 72 h. Whole cell lysates were subjected to immunoblotting using anti-β3 Ab or anti-β-actin Ab. All blots show one representative of five independent experiments.

Close modal

To confirm the hypothesis described above, we compared the phosphorylation levels of ERK1/2 between normal and SSc fibroblasts. As shown in Fig. 5,A, the levels were marginal in normal fibroblasts, whereas the constitutive phosphorylation of ERK, ranging from moderate to strong, was observed in SSc fibroblasts. In SSc fibroblasts, the phosphorylation of ERK1/2 was not affected by the addition of EGF or PDGF-AA, which can induce the rapid and strong phosphorylation of ERK 1/2 in normal fibroblasts (Fig. 5, B and C). Furthermore, the kinase activity of ERK1/2 was markedly elevated in SSc fibroblasts compared with normal fibroblasts (Fig. 5 D). These results indicate that the ERK pathway is constitutively activated in SSc fibroblasts.

FIGURE 5.

Constitutive activation of ERK1/2 in SSc fibroblasts. A, Whole cell lysates prepared from confluent quiescent cells were subjected to immunoblotting using anti-phospho-ERK1/2 Ab. The same membrane was stripped and reprobed with anti-ERK2 Ab. B and C, Confluent quiescent cells were treated with 20 μg/ml EGF (B) or 10 μg/ml PDGF-AA (C) for the indicated period of time, and the levels of phospho-ERK1/2 and total ERK2 were analyzed by immunoblotting. D, Whole cell lysates prepared from confluent quiescent cells were immunoprecipitated using anti-phospho-ERK Ab. The immunoprecipitate pellets were incubated with 1 μg of Elk-1 fusion protein and 100 μM ATP. The levels of phosphorylated Elk-1 were analyzed by immunoblotting using anti-phospho-Elk-1 Ab. All blots show one representative of five independent experiments.

FIGURE 5.

Constitutive activation of ERK1/2 in SSc fibroblasts. A, Whole cell lysates prepared from confluent quiescent cells were subjected to immunoblotting using anti-phospho-ERK1/2 Ab. The same membrane was stripped and reprobed with anti-ERK2 Ab. B and C, Confluent quiescent cells were treated with 20 μg/ml EGF (B) or 10 μg/ml PDGF-AA (C) for the indicated period of time, and the levels of phospho-ERK1/2 and total ERK2 were analyzed by immunoblotting. D, Whole cell lysates prepared from confluent quiescent cells were immunoprecipitated using anti-phospho-ERK Ab. The immunoprecipitate pellets were incubated with 1 μg of Elk-1 fusion protein and 100 μM ATP. The levels of phosphorylated Elk-1 were analyzed by immunoblotting using anti-phospho-Elk-1 Ab. All blots show one representative of five independent experiments.

Close modal

To investigate the distribution of αv and β3 subunit proteins in vivo, immunohistochemical staining was performed against five skin sections from each of normal and SSc groups. Representative results are shown in Fig. 6, and the results are summarized in Table I. Regarding the epidermis, blood vessels, and smooth muscles, there were no differences in immunoreactivity for the anti-αv Ab and anti-β3 Ab between normal and SSc skin sections. The expression of the αv subunit protein was moderate in the blood vessels, and weak in the epidermis and smooth muscles. The expression of the β3 subunit protein was moderate in these tissues. However, the spindle-shaped cells, especially those between thickened collagen bundles in the middle and deep dermis, demonstrated strong immunoreactivity for the αv and β3 subunits in SSc dermal sections, whereas those in normal dermal sections were weakly stained. These results were consistent with the results for cultured fibroblasts described above.

FIGURE 6.

Integrin αv and β3 subunit protein expression in normal and SSc dermal sections. Paraffin sections of normal and SSc dermal tissue were subjected to immunohistochemical analysis with anti-αv Ab and anti-β3 Ab. The immunoreactivity was visualized by diaminobenzidine. The sections were counterstained with hematoxylin. Original magnification, ×200.

FIGURE 6.

Integrin αv and β3 subunit protein expression in normal and SSc dermal sections. Paraffin sections of normal and SSc dermal tissue were subjected to immunohistochemical analysis with anti-αv Ab and anti-β3 Ab. The immunoreactivity was visualized by diaminobenzidine. The sections were counterstained with hematoxylin. Original magnification, ×200.

Close modal
Table I.

Results of immunohistochemical staining for integrin αv and β3 subunits

SubjectsAge/SexDisease Duration (years)EpidermisBlood VesselSmooth MuscleFibroblasts
αvβ3αvβ3αvβ3αvβ3
Patients           
 1 57/Fa ++ ++ ++ ++ +++ +++ 
 2 61/F 0.5 ++ ++ ++ +++ +++ 
 3 31/F ++ ++ ++ ++ +++ +++ 
 4 32/F ++ ++ ++ +++ +++ 
 5 27/F ++ ++ ++ +++ +++ 
Controls           
 1 57/F  ++ ++ ++ ++ 
 2 61/F  ++ ++ ++ 
 3 31/F  ++ ++ ++ 
 4 32/F  ++ ++ ++ 
 5 27/F  ++ ++ ++ ++ 
SubjectsAge/SexDisease Duration (years)EpidermisBlood VesselSmooth MuscleFibroblasts
αvβ3αvβ3αvβ3αvβ3
Patients           
 1 57/Fa ++ ++ ++ ++ +++ +++ 
 2 61/F 0.5 ++ ++ ++ +++ +++ 
 3 31/F ++ ++ ++ ++ +++ +++ 
 4 32/F ++ ++ ++ +++ +++ 
 5 27/F ++ ++ ++ +++ +++ 
Controls           
 1 57/F  ++ ++ ++ ++ 
 2 61/F  ++ ++ ++ 
 3 31/F  ++ ++ ++ 
 4 32/F  ++ ++ ++ 
 5 27/F  ++ ++ ++ ++ 
a

F, Female.

Because αvβ3 functions as an active receptor for SLC, we next investigated whether the up-regulated expression of αvβ3 is involved in the phenotypical alteration of SSc fibroblasts. To this end, we transiently overexpressed αvβ3 in normal fibroblasts and investigated the promoter activity of human α2(I) collagen gene and human MMP-1 gene. Because αv subunit is excessively expressed in the cytoplasm as monomer, and the cell surface expression levels of αvβ3 is controlled by the levels of β3 subunit (34, 35), we first confirmed that transient overexpression of β3 subunit is sufficient to induce the up-regulation of cell surface αvβ3 (Fig. 7,A). Under the same condition, the promoter activities were determined. As shown in Fig. 7,B, left panel, the promoter activity of human α2(I) collagen gene was significantly increased in proportion to the cell surface levels of αvβ3. In contrast, the promoter activity of human MMP-1 gene was significantly decreased in inverse relation to the cell surface levels of αvβ3 (Fig. 7 B, right panel). These results indicate that the up-regulated expression of αvβ3 induces the deposition of type I collagen by coordinating the expression of type I collagen and MMP-1, suggesting that the up-regulated expression of αvβ3 contributes to the phenotypical alteration of SSc fibroblasts.

FIGURE 7.

The effect of transiently overexpressed αvβ3 on the promoter activity of human α2(I) collagen gene and human MMP-1 gene in normal fibroblasts. Normal fibroblasts were transfected with the indicated amount of β3 expression vector or empty vector, along with 2 μg of −772 COL1A2/CAT promoter or human MMP-1 promoter and 1 μg of pSV-β-galactosidase vector, and incubated for 72 h. A, The cell surface levels of αvβ3 were determined by biotinylation and immunoprecipitation. One representative of five independent experiments is shown. B, Values represent the promoter activities of human α2(I) collagen gene (left panel) or human MMP-1 gene (right panel) relative to those of mock transfectants (100 AU). Data are expressed as the mean ± SD of five independent experiments. ∗, p < 0.05 vs mock transfectants under the same conditions.

FIGURE 7.

The effect of transiently overexpressed αvβ3 on the promoter activity of human α2(I) collagen gene and human MMP-1 gene in normal fibroblasts. Normal fibroblasts were transfected with the indicated amount of β3 expression vector or empty vector, along with 2 μg of −772 COL1A2/CAT promoter or human MMP-1 promoter and 1 μg of pSV-β-galactosidase vector, and incubated for 72 h. A, The cell surface levels of αvβ3 were determined by biotinylation and immunoprecipitation. One representative of five independent experiments is shown. B, Values represent the promoter activities of human α2(I) collagen gene (left panel) or human MMP-1 gene (right panel) relative to those of mock transfectants (100 AU). Data are expressed as the mean ± SD of five independent experiments. ∗, p < 0.05 vs mock transfectants under the same conditions.

Close modal

We have proposed that the activation of SSc fibroblasts may be a result of the stimulation by autocrine TGF-β1 (6, 7). To verify the involvement of αvβ3 in this model, we investigated the effect of anti-TGF-β Ab or TGF-β1 antisense oligonucleotide on the promoter activity of human α2(I) collagen gene or human MMP-1 gene in β3 transfectants. As shown in Fig. 8, the increased promoter activity of human α2(I) collagen gene or the decreased promoter activity of human MMP-1 gene in β3 transfectants were almost completely reversed by the treatment of anti-TGF-β Ab or TGF-β1 antisense oligonucleotide. These results suggest that the up-regulated expression of αvβ3 contribute to the establishment of autocrine TGF-β loop in normal fibroblasts. The TGF-β isoform that was primarily responsible for the activation of β3 transfectants may be TGF-β1, because TGF-β1 antisense oligonucleotide reversed the promoter activities to a similar extent as achieved with anti-TGF-β Ab, which neutralizes TGF-β1, β2, β3, and β5.

FIGURE 8.

The effect of anti-TGF-β Ab or TGF-β1 antisense oligonucleotide on the promoter activity of human α2(I) collagen gene or human MMP-1 gene in β3 transfectants. Transfection was performed as described in Fig. 7 in the presence of anti-TGF-β Ab, preimmune IgG, TGF-β1 antisense oligonucleotide, or TGF-β1 sense oligonucleotide. Values represent the promoter activity relative to that of mock transfectants in the presence of preimmune IgG or TGF-β1 sense oligonucleotide (100 AU). Data are expressed as the mean ± SD of five independent experiments. ∗, p < 0.05 vs mock transfectants under the same conditions.

FIGURE 8.

The effect of anti-TGF-β Ab or TGF-β1 antisense oligonucleotide on the promoter activity of human α2(I) collagen gene or human MMP-1 gene in β3 transfectants. Transfection was performed as described in Fig. 7 in the presence of anti-TGF-β Ab, preimmune IgG, TGF-β1 antisense oligonucleotide, or TGF-β1 sense oligonucleotide. Values represent the promoter activity relative to that of mock transfectants in the presence of preimmune IgG or TGF-β1 sense oligonucleotide (100 AU). Data are expressed as the mean ± SD of five independent experiments. ∗, p < 0.05 vs mock transfectants under the same conditions.

Close modal

Because the activation of latent TGF-β is an indispensable process for the establishment of autocrine TGF-β loop, the results described above imply that αvβ3 is involved in the activation process of latent TGF-β1. Based on the evidence that αv-containing integrins, such as αvβ6 and αvβ8, activate latent TGF-β1 on the cell surface, we made a hypothesis that αvβ3 also activates latent TGF-β1 on the cell surface. To clarify this point, we cocultured normal or SSc fibroblasts with TMLC cells, mink lung epithelial reporter cells stably expressing a portion of the plasminogen activator inhibitor-1 promoter (36) (Fig. 9 A). The luciferase activity was significantly elevated in TMLC cells cocultured with SSc fibroblasts compared with those with normal fibroblasts (∼4-fold increase; p < 0.05). This increase was significantly reduced by anti-αvβ3 Ab (∼50% reduction) and completely abolished by anti-TGF-β Ab. We also did coculture assays with inserts to separate TMLC cells and normal or SSc fibroblasts while allowing soluble molecules to pass. In the absence of contact, SSc fibroblasts caused a slight induction of luciferase activity, which was similar to the induction level observed in normal fibroblasts. These results indicate that latent TGF-β is activated on the cell surface of SSc fibroblasts, but that at least a small amount of the active TGF-β formed is freely diffusible, and suggest that this activation process was partially attributed to αvβ3.

FIGURE 9.

The involvement of αvβ3 in the self-activation system in SSc fibroblasts. A, Normal and SSc fibroblasts were mixed with TMLC cells at a ratio of 1:1 and cocultured in confluence. In experiments without cell-cell contact, normal and SSc fibroblasts were cocultured with TMLC cells separately by using inserts. After 24-h incubation, the luciferase activities were determined. In some experiments, assays were performed in the presence of anti-αvβ3 Ab (anti-αvβ3, 10 μg/ml) or anti-TGF-β Ab (anti-TGF-β, 10 μg/ml). Values represent the luciferase activity relative to that of TMLC cells cocultured with normal fibroblasts in the absence of contact (100 AU). ∗, p < 0.05 vs normal fibroblasts under the same conditions. ∗∗, p < 0.05 vs SSc fibroblasts cocultured with TMLC cells in the absence of anti-αvβ3 Ab or anti-TGF-β Ab. B, The −772 COL1A2/CAT promoter activities were determined in the presence of anti-αvβ3 Ab or preimmune IgG in normal and SSc fibroblasts either stimulated or unstimulated with exogenous TGF-β1. Values represent the CAT activity relative to that of unstimulated normal fibroblasts treated with preimmune IgG (100 AU). ∗, p < 0.05 vs unstimulated SSc fibroblasts treated with preimmune IgG. C, Confluent quiescent fibroblasts were treated with anti-αvβ3 Ab or preimmune IgG for 48 h. Whole cell lysates were analyzed by immunoblotting using anti-type I collagen Ab or anti-MMP-1 Ab. One representative of five independent experiments is shown (left panels). The levels of type I procollagen proteins were defined as the mean band density of α1(I) and α2(I) procollagen proteins, which were quantitated by scanning densitometry. Values represent the band density relative to the mean value of normal fibroblasts treated with preimmune IgG (100 AU) (right panels). Data are expressed as the mean ± SD of five independent experiments. ∗, p < 0.05 vs normal fibroblasts under the same condition. ∗∗, p < 0.05 vs SSc fibroblasts treated with preimmune IgG. D, Confluent quiescent fibroblasts were treated with anti-αvβ3 Ab or preimmune IgG for 48 h. Whole cell lysates were incubated with biotin-labeled oligonucleotides. Proteins bound to these nucleotides were isolated with streptavidin-agarose beads, and Smad3 was detected by immunoblotting. In some experiments, cells were stimulated with TGF-β1 (2 ng/ml) for the last 3 h. One representative of five independent experiments is shown (left panel). Values represent the band density relative to the mean value of normal fibroblasts treated with preimmune IgG and TGF-β1 (100 AU) (right panel). Data are expressed as the mean ± SD of five independent experiments. ∗, p < 0.05 vs SSc fibroblasts treated with preimmune IgG.

FIGURE 9.

The involvement of αvβ3 in the self-activation system in SSc fibroblasts. A, Normal and SSc fibroblasts were mixed with TMLC cells at a ratio of 1:1 and cocultured in confluence. In experiments without cell-cell contact, normal and SSc fibroblasts were cocultured with TMLC cells separately by using inserts. After 24-h incubation, the luciferase activities were determined. In some experiments, assays were performed in the presence of anti-αvβ3 Ab (anti-αvβ3, 10 μg/ml) or anti-TGF-β Ab (anti-TGF-β, 10 μg/ml). Values represent the luciferase activity relative to that of TMLC cells cocultured with normal fibroblasts in the absence of contact (100 AU). ∗, p < 0.05 vs normal fibroblasts under the same conditions. ∗∗, p < 0.05 vs SSc fibroblasts cocultured with TMLC cells in the absence of anti-αvβ3 Ab or anti-TGF-β Ab. B, The −772 COL1A2/CAT promoter activities were determined in the presence of anti-αvβ3 Ab or preimmune IgG in normal and SSc fibroblasts either stimulated or unstimulated with exogenous TGF-β1. Values represent the CAT activity relative to that of unstimulated normal fibroblasts treated with preimmune IgG (100 AU). ∗, p < 0.05 vs unstimulated SSc fibroblasts treated with preimmune IgG. C, Confluent quiescent fibroblasts were treated with anti-αvβ3 Ab or preimmune IgG for 48 h. Whole cell lysates were analyzed by immunoblotting using anti-type I collagen Ab or anti-MMP-1 Ab. One representative of five independent experiments is shown (left panels). The levels of type I procollagen proteins were defined as the mean band density of α1(I) and α2(I) procollagen proteins, which were quantitated by scanning densitometry. Values represent the band density relative to the mean value of normal fibroblasts treated with preimmune IgG (100 AU) (right panels). Data are expressed as the mean ± SD of five independent experiments. ∗, p < 0.05 vs normal fibroblasts under the same condition. ∗∗, p < 0.05 vs SSc fibroblasts treated with preimmune IgG. D, Confluent quiescent fibroblasts were treated with anti-αvβ3 Ab or preimmune IgG for 48 h. Whole cell lysates were incubated with biotin-labeled oligonucleotides. Proteins bound to these nucleotides were isolated with streptavidin-agarose beads, and Smad3 was detected by immunoblotting. In some experiments, cells were stimulated with TGF-β1 (2 ng/ml) for the last 3 h. One representative of five independent experiments is shown (left panel). Values represent the band density relative to the mean value of normal fibroblasts treated with preimmune IgG and TGF-β1 (100 AU) (right panel). Data are expressed as the mean ± SD of five independent experiments. ∗, p < 0.05 vs SSc fibroblasts treated with preimmune IgG.

Close modal

To investigate whether blockade of αvβ3 reverses the SSc phenotype, we first focused on the promoter activity of human α2(I) collagen gene. As shown in Fig. 9,B, anti-αvβ3 Ab significantly reduced the increased basal promoter activity in SSc fibroblasts, whereas it had no significant effects on the basal and the TGF-β1-induced promoter activity in normal fibroblasts and the TGF-β1-induced promoter activity in SSc fibroblasts. These results indicate that blockade of αvβ3 inhibit the autocrine TGF-β signaling in SSc fibroblasts without affecting the effect of exogenous TGF-β1 stimulation. To further confirm these findings, we investigated the effect of anti-αvβ3 Ab on the expression of type I procollagen protein and MMP-1 protein in normal and SSc fibroblasts. As shown in Fig. 9,C, the expression levels of type I procollagen protein were elevated, and those of MMP-1 protein were decreased in SSc fibroblasts compared with normal fibroblasts. The blockage of αvβ3 by anti-αvβ3 Ab reversed the expression levels of these proteins. Moreover, we examined the effect of anti-αvβ3 Ab on the activation state of Smad3/4 pathway. In our previous report, we demonstrated that the phosphorylation level of Smad3 and the DNA binding ability of Smad3 were significantly elevated in SSc fibroblasts, and that the phosphorylation level of Smad3 was completely correlated with the DNA binding ability of Smad3 (37). Based on these data, we performed the DNA affinity precipitation assay. Consistent with our previous report (37), as shown in Fig. 9,D, the constitutive DNA-Smad3 binding was detected in SSc fibroblasts, and the marked DNA-Smad3 binding were detected in normal fibroblasts treated with TGF-β1. The treatment of anti-αvβ3 Ab partially decreased the DNA-Smad3 binding in scleroderma fibroblasts, whereas the same treatment did not affect the DNA-Smad3 binding in normal fibroblasts either treated or untreated with TGF-β1. We finally performed immunofluorescence using anti-α-smooth muscle actin Ab to determine the effect of anti-αvβ3 Ab on the expression levels of α-smooth muscle actin and the morphology of cells (Fig. 10). In SSc fibroblasts, ∼60% cells showed the morphological changes of cellular hypertrophy and well-formed α-smooth muscle actin fibers, which are characteristics of myofibroblasts. However, after the treatment of anti-αvβ3 Ab, the percentage of cells with these features were reduced to ∼30%. In contrast, in normal fibroblasts, cells with these features were <5% in the presence or absence of anti-αvβ3 Ab. These results indicate that anti-αvβ3 Ab can reverse the myofibroblastic phenotype of SSc fibroblasts.

FIGURE 10.

The effect of anti-αvβ3 Ab on the expression levels of α-smooth muscle actin and the morphology of cells in normal and SSc fibroblasts. Confluent quiescent cells were treated with anti-αvβ3 Ab or preimmune mouse IgG for 48 h. Cells were stained with anti-α-smooth muscle actin Ab, washed, and incubated with FITC-conjugated rabbit anti-mouse IgG (green). The nuclei were counterstained with DAPI (blue).

FIGURE 10.

The effect of anti-αvβ3 Ab on the expression levels of α-smooth muscle actin and the morphology of cells in normal and SSc fibroblasts. Confluent quiescent cells were treated with anti-αvβ3 Ab or preimmune mouse IgG for 48 h. Cells were stained with anti-α-smooth muscle actin Ab, washed, and incubated with FITC-conjugated rabbit anti-mouse IgG (green). The nuclei were counterstained with DAPI (blue).

Close modal

Throughout this study, cells were grown in medium with 10% FCS and then cultured with serum-free medium for 24 h before stimulation or harvest. Because 10% FCS contains large amounts of TGF-β and other factors that can modulate type I procollagen and MMP-1 production, it may be required to grow fibroblasts for several days with serum-free medium before experiments. To clarify this point, normal and SSc fibroblasts were grown to confluence in the presence of 10% FCS and then cultured with serum-free medium for 24, 48, 72, 96, or 120 h. As shown in Fig. 11, there was no significant difference in the levels of type I procollagen and MMP-1 between these five groups in both normal and SSc fibroblasts. These results indicate that the 24-h incubation with serum-free medium is enough to completely remove the effect of FCS on the expression levels of type I procollagen and MMP-1.

FIGURE 11.

The 24-h incubation with serum-free medium is enough to exclude the effect of FCS on the expression of type I procollagen protein and MMP-1 protein. Confluent quiescent fibroblasts were incubated with serum-free medium for the indicated period of time. Then, the whole cell lysates were subjected to immunoblotting using anti-type I collagen Ab or anti-MMP-1 Ab.

FIGURE 11.

The 24-h incubation with serum-free medium is enough to exclude the effect of FCS on the expression of type I procollagen protein and MMP-1 protein. Confluent quiescent fibroblasts were incubated with serum-free medium for the indicated period of time. Then, the whole cell lysates were subjected to immunoblotting using anti-type I collagen Ab or anti-MMP-1 Ab.

Close modal

Dermal fibroblast activation is one of the important processes in the pathogenesis of SSc (2, 3, 4, 5). We have reported that the activation of SSc fibroblasts may be a result of the stimulation by autocrine TGF-β1, and the up-regulated expression of TGF-β receptors may contribute to this process (6, 7, 8, 9). However, the biological effect of various cytokines, including TGF-β1, is mainly determined by the incidence of cytokine-receptor interaction, which is modulated by the concentration and activity of cytokines and/or their receptors. Therefore, the concentration and/or activity of TGF-β1 as well as the expression levels of its receptors are important aspects in the pathogenesis of SSc. Although we previously reported that there was no significant difference in the levels of total (latent + active) and active TGF-β1 protein in conditioned media between cultured normal and SSc fibroblasts (7), the recruitment and/or activation of latent TGF-β1 in the pericellular region may enhance the incidence of the interaction between active TGF-β1 and its receptors, leading to the activation of SSc fibroblasts. One of candidates that can mediate this process is the αv-containing integrins. These integrins function as an active receptor for LAP-β1 and have the potential to modulate the localization and possibly activation of SLC (11, 14, 15, 16). Because previous reports demonstrated that the expression of αvβ3 was up-regulated in injured vessels, and the blockade of this integrin reduced the TGF-β1 accumulation and the ECM deposition in those tissues (22), we speculated that the up-regulated expression of αvβ3 may contribute to the establishment of autocrine TGF-β loop in SSc fibroblasts. This notion is verified by the following present findings: 1) the expression of αvβ3 is up-regulated in SSc fibroblasts in vivo and in vitro; 2) transient overexpression of αvβ3 in normal fibroblasts induces the increase in the promoter activity of human α2(I) collagen gene and the decrease in that of human MMP-1 gene, and these effects are almost completely abolished by anti-TGF-β Ab or TGF-β1 antisense oligonucleotide; and 3) anti-αvβ3 Ab reverses the expression of type I procollagen protein and MMP-1 protein, the promoter activity of human α2(I) collagen gene, and the myofibroblastic phenotype in SSc fibroblasts.

So far, two mechanisms have been reported in the activation of SLC. One is the conformational change of LAP leading to activation of SLC. This nonproteolytic process is thought to be dependent on an intrinsic ability of LAP to adopt different conformations (38). TSP-1 and αvβ6 have been demonstrated to be involved in this process (11, 12). SLC binds to TSP-1 through the N terminus of LAP, and such interaction induces a conformational change and a subsequent activation of SLC, although the active TGF-β molecules remains bound to TSP-1 (12). SLC also interact with αvβ6 through the C terminus of LAP, but such interaction is not sufficient for its activation. Following the binding, αvβ6 requires the interaction with actin cytoskeleton to activate bound SLC (11). The other mechanism is the proteolysis of LAP which, results in the release of active TGF-β from SLC. Proteases such as plasmin, metalloproteases, aspartic proteases, and cysteine proteases have been reported to be involved in this process (13, 15, 39). Especially, αvβ8 has recently been shown to generate TGF-β1 activity by localizing the SLC to the cell surface, thereby permitting membrane-type 1-MMP proteolytic cleavage of LAP-β1 to liberate the active TGF-β1 (14). Interestingly, αvβ3 expression is associated with enhanced cell surface proteolytic activity by MMP-2 (40) and MMP-9 (41, 42), for which LAP-β1 has been shown to be a substrate, presenting a potential mechanism to generate TGF-β1 activity from the αvβ3-dependent activation of MMP-2 and/or MMP-9. In addition, the interaction of LAP-β1 with up-regulated αvβ3 may strongly initiate αvβ3-specific intracellular signaling events commonly associated with integrin-ligand ligation, which may subsequently induce a potential mechanism for establishing autocrine TGF-β loop.

There is clear evidence of the importance of αvβ6 to regulate TGF-β1 activity from the observations of β6-knockout mice (11). Thus, to prove the role of αvβ3 in fibrotic disorders, it is very important to determine whether there is a defect in TGF-β1 activation in β3-knockout mice using appropriate models, such as the bleomycin model of dermal fibrosis or pulmonary fibrosis. β3-knockout mice were previously established as an animal model of human Glanzmann’s thrombasthenia (43), which is induced by the loss of the platelet integrin αIIbβ3. Observational studies of these mice have implicated αvβ3 in several physiological roles, including embryo implantation, angiogenesis, and bone resorption (43, 44, 45). To our knowledge, however, a defect in TGF-β1 activation has not been studied in these mice. Alternatively, it is also informative to investigate the activity of TGF-β1 in animal models treated with anti-αvβ3 Ab or to determine whether β3-transgenic mice develop any kind of fibrotic disorders, including SSc.

During the preparation of this manuscript, Kim et al. (34) reported that sustained ERK activity is associated with β3 induction and subsequent cell surface expression of αvβ3 in osteoclasts, which may contribute to the acceleration of bone resorption. Furthermore, it has been reported that the blockade of αvβ3 reduces bone resorption in vitro and prevents osteoporosis in vivo (46). Because bone resorption is a process that is stimulated by TGF-β1 (42, 47), these previous results regarding osteoclasts are paralleled with the present data as follows: 1) the expression of αvβ3 may be up-regulated through sustained ERK activation in SSc fibroblasts; 2) transient overexpression of αvβ3 increases the promoter activity of human α2(I) collagen gene in a TGF-β1-dependent manner in normal fibroblasts; and 3) anti-αvβ3 Ab reduced the promoter activity of human α2(I) collagen gene in SSc fibroblasts. These results imply that dermal fibroblasts and osteoclasts have a similar mechanism of regulating tissue remodeling by αvβ3. Thus, the possible association of αvβ3 with latent TGF-β activation is a novel finding that strongly supports the understanding of αvβ3-associated biological processes.

In summary, this study demonstrated that up-regulated expression of αvβ3 contributed to the establishment of autocrine TGF-β loop in SSc fibroblasts. Although in vivo studies using animal models are needed in the future, the present data suggest that this integrin can be a potent target in developing the treatment of SSc.

We thank Drs. Joseph C. Loftus, Timothy E. O’Toole, Dennis Templeton, and Roger J. Davis for providing us with αv cDNA, β3 cDNA, expression vector of DN ERK2, and expression vector of CA MEK1.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported in part by a grant for scientific research from Japanese Ministry of Education (10770391), and by Project Research for Progressive Systemic Sclerosis from the Japanese Ministry of Health and Welfare.

3

Abbreviations used in this paper: SSc, systemic sclerosis; ECM, extracellular matrix; LAP-β1, latency-associated peptide-β1; SLC, small latent complex; TSP, thrombospondin; MMP, matrix metalloproteinase; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; CAT, chloramphenicol acetyltransferase; DN ERK2, dominant-negative mutant of ERK2; CA MEK1, constitutive active mutant of MEK1; DAPI, 4′,6-diamidino-2-phenylindole; AU, arbitrary unit.

1
LeRoy, E. C..
1992
. Systemic sclerosis (scleroderma). J. B. Wyngaarden, and L. H. Smith, and J. C. Bennett, eds.
Cecil Text Book of Medicine
19th Ed.
1530
-1535. WB Saunders, Philadelphia.
2
Korn, J. H..
1989
. Immunologic aspects of scleroderma.
Curr. Opin. Rheumatol.
1
:
479
-484.
3
LeRoy, E. C..
1974
. Increased collagen synthesis by scleroderma skin fibroblasts in vitro: a possible defect in the regulation or activation of the scleroderma fibroblast.
J. Clin. Invest.
54
:
880
-889.
4
Jelaska, A., M. Arakawa, G. Broketa, J. H. Korn.
1996
. Heterogeneity of collagen synthesis in normal and systemic sclerosis skin fibroblasts: increased proportion of high collagen-producing cells in systemic sclerosis fibroblasts.
Arthritis Rheum.
39
:
1338
-1346.
5
LeRoy, E. C., E. A. Smith, M. B. Kahaleh, M. Trojanowska, R. M. Silver.
1989
. A strategy for determining the pathogenesis of systemic sclerosis: is transforming growth factor-β the answer?.
Arthritis Rheum.
32
:
817
-825.
6
Kawakami, T., H. Ihn, W. Xu, E. Smith, C. LeRoy, M. Trojanowska.
1998
. Increased expression of TGF-β receptors by scleroderma fibroblasts: evidence for contribution of autocrine TGF-β signaling to scleroderma phenotype.
J. Invest. Dermatol.
110
:
47
-51.
7
Ihn, H., K. Yamane, M. Kubo, K. Tamaki.
2001
. Blockade of endogenous transforming growth factor-β signaling prevents up-regulated collagen synthesis in scleroderma fibroblasts: association with increased expression of transforming growth factor-β receptors.
Arthritis Rheum.
44
:
474
-480.
8
Yamane, K., H. Ihn, M. Kubo, K. Tamaki.
2002
. Increased transcriptional activities of transforming growth factor-β receptors in scleroderma fibroblasts.
Arthritis Rheum.
46
:
2421
-2428.
9
Asano, Y., H. Ihn, K. Yamane, M. Kubo, K. Tamaki.
2004
. Impaired Smad7-Smurf-mediated negative regulation of transforming growth factor (TGF)-β signaling in scleroderma fibroblasts.
J. Clin. Invest.
113
:
253
-264.
10
Annes, J. P., J. S. Munger, D. B. Rifkin.
2003
. Making sense of latent TGF-β activation.
J. Cell Sci.
116
:
217
-224.
11
Munger, J. S., X. Huang, H. Kawakatsu, M. J. Griffiths, S. L. Dalton, J. Wu, J. F. Pittet, N. Kaminski, C. Garat, M. A. Matthay, et al
1999
. The integrin αvβ6 binds and activates latent TGF-β1: a mechanism for regulating pulmonary inflammation and fibrosis.
Cell
96
:
319
-328.
12
Crawford, S. E., V. Stellmach, J. E. Murphy-Ullrich, S. M. Ribeiro, J. Lawler, R. O. Hynes, G. P. Boivin, N. Bouck.
1998
. Thrombospondin-1 is a major activator of TGF-β1 in vivo.
Cell
93
:
1159
-1170.
13
Lyons, R. M., L. E. Gentry, A. F. Purchio, H. L. Moses.
1990
. Mechanism of activation of latent recombinant transforming growth factor-β1 by plasmin.
J. Cell Biol.
110
:
1361
-1367.
14
Mu, D., S. Cambier, L. Fjellbirkeland, J. L. Baron, J. S. Munger, H. Kawakatsu, D. Sheppard, V. C. Broaddus, S. L. Nishimura.
2002
. The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1.
J. Cell Biol.
157
:
493
-507.
15
Munger, J. S., J. G. Harpel, F. G. Giancotti, D. B. Rifkin.
1998
. Interactions between growth factors and integrins: latent forms of transforming growth factor-β are ligands for the integrin αvβ1.
Mol. Biol. Cell
9
:
2627
-2638.
16
Ludbrook, S. B., S. T. Barry, C. J. Delves, C. M. T. Horgan.
2003
. The integrin αvβ3 is a receptor for the latency-associated peptides of transforming growth factors β1 and β3.
Biochem. J.
369
:
311
-318.
17
Choi, E. T., L. Engel, A. D. Callow, S. Sun, J. Trachtenberg, S. Santoro, E. S. Ryan.
1994
. Inhibition of neointimal hyperplasia by blocking αvβ3 integrin with a small peptide antagonist GpenGRGDSPCA.
J. Vasc. Surg.
19
:
125
-134.
18
Matsuno, H., J. M. Stassen, J. Vermylen, H. Deckmyn.
1994
. Inhibition of integrin function by a cyclic RGD-containing peptide prevents neointima formation.
Circulation
90
:
2203
-2206.
19
Hoshiga, M., C. E. Alpers, L. L. Smith, C. M. Giachelli, S. M. Schwartz.
1995
. αvβ3 integrin expression in normal and atherosclerotic artery.
Circ. Res.
77
:
1129
-1135.
20
Srivatsa, S. S., L. A. Fitzpatrick, P. W. Tsoa, T. M. Reilly, D. R. Holmes, Jr, R. S. Schwartz, S. A. Mousa.
1997
. Selective αvβ3 integrin blockade potently limits neointimal hyperplasia and lumen stenosis following deep coronary arterial stent injury: evidence for the functional importance of integrin αvβ3 and osteopontin expression during neointima formation.
Cardiovasc. Res.
36
:
408
-428.
21
Ward, M. R., A. Agrotis, P. Kanellakis, R. Dilley, G. Jennings, A. Bobik.
1997
. Inhibition of protein tyrosine kinases attenuates increases in expression of transforming growth factor-β isoforms and their receptors following arterial injury.
Arterioscler. Thromb. Vasc. Biol.
17
:
2461
-2470.
22
Coleman, K. R., G. A. Braden, M. C. Willingham, D. C. Sane.
1999
. Vitaxin, a humanized monoclonal antibody to the vitronectin receptor (αvβ3), reduces neointimal hyperplasia and total vessel area after balloon injury in hypercholesterolemic rabbits.
Circ. Res.
84
:
1268
-1276.
23
Kubo, M., H. Ihn, K. Yamane, K. Tamaki.
2001
. Up-regulated expression of transforming growth factor-β receptors in dermal fibroblasts in skin sections from patients with localized scleroderma.
Arthritis Rheum.
44
:
731
-734.
24
Ihn, H., K. Tamaki.
2002
. Mitogenic activity of dermatofibrosarcoma protuberans is mediated via an extracellular signal related kinase dependent pathway.
J. Invest. Dermatol.
119
:
954
-960.
25
Ihn, H., K. Ohnishi, T. Tamaki, E. C. LeRoy, M. Trojanowska.
1996
. Transcriptional regulation of the human α2(I) collagen gene: combined action of upstream stimulatory and inhibitory cis-acting elements.
J. Biol. Chem.
271
:
26717
-26723.
26
David, M., E. Petricoin, 3rd, C. Benjamin, R. Pine, M. J. Weber, A. C. Larner.
1995
. Requirement for MAP kinase (ERK2) activity in interferon α- and interferon β-stimulated gene expression through STAT proteins.
Science
269
:
1721
-1723.
27
Zanke, B. W., E. A. Rubie, E. Winnett, J. Chan, S. Randall, M. Parsons, K. Boudreau, M. McInnis, M. Yan, D. J. Templeton, J. R. Woodgett.
1996
. Mammalian mitogen-activated protein kinase pathways are regulated through formation of specific kinase-activator complexes.
J. Biol. Chem.
271
:
29876
-29881.
28
Wartmann, M., R. J. Davis.
1994
. The native structure of the activated Raf protein kinase is a membrane- bound multi-subunit complex.
J. Biol. Chem.
269
:
6695
-6701.
29
Falanga, V., A. S. Greenberg, S. M. Ochoa, A. B. Roberts, A. Falabella, Y. Yamaguchi.
1998
. Stimulation of collagen synthesis by anabolic steroid stanozol.
J. Invest. Dermatol.
111
:
1193
-1197.
30
Brunet, C. L., P. M. Sharpe, M. W. J. Ferguson.
1995
. Inhibition of TGF-β3 (but not TGF-β1 or TGF-β2) activity prevents normal and mouse embryonic palate fusion.
Int. J. Dev. Biol.
39
:
345
-355.
31
Yagi, K., M. Furuhashi, H. Aoki, D. Goto, H. Kuwano, K. Sugamura, K. Miyazono, M. Kato.
2002
. c-myc is a downstream target of the Smad pathway.
J. Biol. Chem.
277
:
6266
-6272.
32
Du, X., M. H. Ginsberg.
1997
. Integrin αIIbβ3 and platelet function.
Thromb. Haemost.
78
:
96
-100.
33
Woods, D., H. Cherwinski, E. Venetsanakos, A. Bhat, S. Gysin, M. Humbert, P. F. Bray, V. L. Saylor, M. McMahon.
2001
. Induction of β3-integrin gene expression by sustained activation of the Ras-regulated Raf-MEK-extracellular signal-regulated kinase signaling pathway.
Mol. Cell Biol.
21
:
3192
-3205.
34
Kim, H. H., W. J. Chung, S. W. Lee, P. J. Chung, J. W. You, H. J. Kwon, S. Tanaka, Z. H. Lee.
2003
. Association of sustained ERK activity with integrin β3 induction during receptor activator of nuclear factor κB ligand (RANKL)-directed osteoclast differentiation.
Exp. Cell Res.
289
:
368
-377.
35
Sheppard, D., D. S. Cohen, A. Wang, M. Busk.
1992
. Transforming growth factor-β differentially regulates expression of integrin subunits in guinea pig airway epithelial cells.
J. Biol. Chem.
267
:
17409
-17414.
36
Abe, M., J. G. Harpel, C. N. Metz, I. Nunes, D. J. Loskutoff, D. B. Rifkin.
1994
. An assay for transforming growth factor-β using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct.
Anal. Biochem.
216
:
276
-284.
37
Asano, Y., H. Ihn, K. Yamane, M. Jinnin, Y. Mimura, K. Tamaki.
2004
. Phosphatidylinositol 3-kinase is involved in α2(I) collagen gene expression in normal and scleroderma fibroblasts.
J. Immunol.
172
:
7123
-7135.
38
McMahon, G. A., J. D. Dignam, L. E. Gentry.
1996
. Structural characterization of the latent complex between transforming growth factor-β1 and β1-latency-associated peptide.
Biochem. J.
313
:
343
-351.
39
Schultz-Cherry, S., J. Lawler, J. E. Murphy-Ullrich.
1994
. The type 1 repeats of thrombospondin 1 activate latent transforming growth factor-β.
J. Biol. Chem.
269
:
26783
-26788.
40
Brooks, P. C., S. Stromblad, L. C. Sanders, T. L. von Schalscha, R. T. Aimes, W. G. Stetler-Stevenson, J. P. Quigley, D. A. Cheresh.
1996
. Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin αvβ3.
Cell
85
:
683
-693.
41
Rolli, M., E. Fransvea, J. Pilch, A. Saven, B. Felding-Habermann.
2003
. Activated integrin αvβ3 cooperates with metalloproteinase MMP-9 in regulating migration of metastatic breast cancer cells.
Proc. Natl. Acad. Sci. USA
100
:
9482
-9487.
42
Karsdal, M. A., P. Hjorth, K. Henriksen, T. Kirkegaard, K. L. Nielsen, H. Lou, J. M. Delaisse, N. T. Foged.
2003
. Transforming growth factor-β controls human osteoclastogenesis through the p38 MAPK and regulation of RANK expression.
J. Biol. Chem.
278
:
44975
-44987.
43
Hodivala-Dilke, K. M., K. P. McHugh, D. A. Tsakiris, H. Rayburn, D. Crowley, M. Ullman-Cullere, F. P. Ross, B. S. Coller, S. Teitelbaum, R. O. Hynes.
1999
. β3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival.
J. Clin. Invest.
103
:
229
-238.
44
McHugh, K. P., K. Hodivala-Dilke, M. H. Zheng, N. Namba, J. Lam, D. Novack, X. Feng, F. P. Ross, R. O. Hynes, S. L. Teitelbaum.
2000
. Mice lacking β3 integrins are osteosclerotic because of dysfunctional osteoclasts.
J. Clin. Invest.
105
:
433
-440.
45
Feng, X., D. V. Novack, R. Faccio, D. S. Ory, K. Aya, M. I. Boyer, K. P. McHugh, F. P. Ross, S. L. Teitelbaum.
2001
. A Glanzmann’s mutation in β3 integrin specifically impairs osteoclast function.
J. Clin. Invest.
107
:
1137
-1144.
46
Engleman, V. W., G. A. Nickols, F. P. Ross, M. A. Horton, D. W. Griggs, S. L. Settle, P. G. Ruminski, S. L. Teitelbaum.
1997
. A petidomimetic antagonist of the αvβ3 integrin inhibits bone resorption in vitro and prevents osteoporosis in vivo.
J. Clin. Invest.
99
:
2284
-2292.
47
Chin, S. L., S. A. Johnson, J. Quinn, D. Mirosavljevic, J. T. Price, A. C. Dudley, D. M. Thomas.
2003
. A role for αv integrin subunit in TGF-β-stimulated osteoclastogenesis.
Biochem. Biophys. Res. Commun.
307
:
1051
-1058.