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Increased Expression of Integrin _v3 Contributes to the Establishment of Autocrine TGF- Signaling in Scleroderma Fibroblasts¹

Yoshihide Asano^*, Hironobu Ihn^2,, Kenichi Yamane^*, Masatoshi Jinnin^*, Yoshihiro Mimura^* and Kunihiko Tamaki^*

^* Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan; and Department of Dermatology and Plastic and Reconstructive Surgery, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan

	Abstract

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The constitutive secretion of latent TGF- 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 _v3 integrin, which is recently demonstrated to function as an active receptor for latent TGF-1 through its interaction with latency-associated peptide-1, and investigated the involvement of this integrin in the pathogenesis of scleroderma. Scleroderma fibroblasts exhibited increased _v3 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 _v3. Transient overexpression of _v3 in normal fibroblasts induced the increase in the promoter activity of human 2(I) collagen gene and the decrease in that of human MMP-1 gene. These effects of _v3 were almost completely abolished by the treatment with anti-TGF- Ab or TGF-1 antisense oligonucleotide. Furthermore, the addition of anti-_v3 Ab reversed the expression of type I procollagen protein and MMP-1 protein, the promoter activity of human 2(I) collagen gene, and the myofibroblastic phenotype in scleroderma fibroblasts. These results suggest that the up-regulated expression of _v3 contributes to the establishment of autocrine TGF- loop in scleroderma fibroblasts, and this integrin is a potent target for the treatment of scleroderma.

	Introduction

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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-1 (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- receptors, and this correlates with the increased expression of 2(I) collagen mRNA (6, 7, 8, 9); and 2) the blockade of TGF- signaling with anti-TGF- Ab or anti-TGF-1 antisense oligonucleotide abolishes the increased expression of human 2(I) collagen mRNA in SSc fibroblasts (7).

TGF-1 is normally secreted as a complex composed of three proteins, including the bioactive peptide of TGF-1, a latency-associated peptide-1 (LAP-1), and a latent TGF- binding protein-1. TGF-1 forms a complex with LAP-1 noncovalently, which is called the small latent complex (SLC), and in this configuration TGF-1 is unable to bind to its receptors. SLC is joined by a latent TGF- 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-1 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-1. Specifically, the _v6 integrin and thrombospondin (TSP)-1 have been implicated in activation of latent TGF-1 through nonproteolytic mechanisms (11, 12). In addition, plasmin has been proposed to lead to the activation of latent TGF-1 through proteolytic degradation of LAP-1 (13). The _v8 integrin has also been demonstrated to be able to activate latent TGF-1 by membrane-type 1-matrix metalloproteinase (MMP)-dependent degradation of LAP-1 (14). Thus, normal TGF- function is thought to be largely controlled by its activation from the latent state.

LAP-1 contains an RGD motif that is recognized by _v-containing integrins, including _v1, _v3, _v5, _v6, and _v8 (11, 14, 15, 16). Although all of these _v-containing integrins bind to LAP-1 and have the potential to modulate the localization and possibly activation of SLC, only _v6 and _v8, both of which are not expressed in dermal fibroblasts, have been demonstrated to be able to activate SLC (11, 14). Especially, _v6-mediated activation of SLC was demonstrated to play an important role in response to tissue injury because the epithelium-restricted 6^–/– 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 _v-containing integrins, such as _v1, _v3, and _v5, a disease process associated with both _v3 and TGF-1 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-1, TGF- receptor type I, TGF- receptor type II, _v subunit, and ₃ subunit are elevated in injured vessels (21). However, the pretreatment of anti-_v3-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-_v3 Ab dramatically reduces the accumulation of TGF-1 protein in injured vessels (22), suggesting that the role of _v3 as an SLC receptor contributes to this process. These previous findings stimulate our interest to investigate whether _v3 is involved in the pathogenesis of fibrotic disorders.

This study is undertaken to clarify the involvement of _v3 in the pathogenesis of SSc. First, we compared the expression levels of _v3 between normal and SSc fibroblasts in vivo and in vitro. We then investigated the effect of transiently overexpressed _v3 on the promoter activity of the human 2(I) collagen gene or human MMP-1 gene. Furthermore, we determined the effect of anti-_v3 Ab on the phenotype of SSc fibroblasts. The results suggest that the up-regulated expression of _v3 contributes to the establishment of autocrine TGF- loop in SSc fibroblasts, and anti-_v3 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.

	Materials and Methods

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Reagents

Recombinant human TGF-1, 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 -actin were purchased from Sigma-Aldrich. Abs for _v, ₃, phospho-ERK1/2, and ERK2 were obtained from Santa Cruz Biotechnology. Abs for _v3 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. _v cDNA was a gift from Dr. J. C. Loftus (Mayo Clinic, Scottsdale, AZ). 3 cDNA was a gift from Dr. T. E. O’Toole (Research Institute of Scripps Clinic, La Jolla, CA).

Cell cultures

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.

Immunoblotting using whole cell lysates

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.

Biotinylation and immunoprecipitation

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. _v3 was immunoprecipitated using anti-3 Ab. Immune complexes were collected using protein A-agarose and subjected to immunoblotting using streptavidin coupled to HRP (Amersham Biosciences).

RNA preparation and Northern blot analysis

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 _v and ₃ subunits as described previously (9). The membrane was then washed and exposed to x-ray film.

Immunohistochemical stainings

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 _v or ₃ 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⁺.

ERK activity assays

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 -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 -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.

Plasmid construction

A –772 COL1A2/chloramphenicol acetyltransferase (CAT) construct consisting of the human 2(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.

Transient transfection

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 3 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--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.

Immunofluorescence

Quiescent cells cultured in 4-well LAB TEK chambers (Nunc) were treated with 10 µg/ml anti-_v3 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--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.

Anti-TGF- Abs or antisense TGF-1 oligonucleotide

We used a pan-specific neutralizing TGF- Ab (R&D Systems), which has been shown to specifically inhibit the activity of TGF-1, 2, and 3. We also used a TGF-1 19-mer antisense oligonucleotide (GAGGGCGGCATGGGGAGG), which overlaps the promoter and transcriptional start site of the TGF-1 gene. This same sequence, which is specific for the TGF-1 isoform, has been found to be sufficient to block TGF-1 transcription in vitro (29) and in vivo (30). A sense oligonucleotide served as a control.

DNA affinity precipitation

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-1 (data not shown).

Statistical analysis

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

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Expression levels of _v3 in cultured normal and SSc fibroblasts

As an initial experiment, we compared the expression levels of _v and ₃ 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 _v subunit protein were 2.7 times higher in SSc fibroblasts than normal fibroblasts. The expression levels of ₃ 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 _v3 in normal and SSc fibroblasts. To this end, cell surface proteins were labeled with biotins, and immunoprecipitation was performed using anti-3 Ab. As shown in Fig. 1C, cell surface levels of _v3 were markedly elevated in SSc fibroblasts compared with normal fibroblasts. These bands were confirmed to be _v or ₃ by a reprobing analysis using anti-_v or ₃ Abs (data not shown). Although ₃ subunit can interact with two kinds of subunits, such as _v and _IIb, _IIb subunit is not precipitated by anti-₃ Ab in dermal fibroblasts. This is consistent with previous reports that _IIb subunit is mainly expressed in platelets, but not in dermal fibroblasts (32). The expression levels of _v and ₃ subunit mRNAs in normal and SSc fibroblasts were also determined by Northern blotting. As shown in Fig. 2, the levels of _v mRNA were 2.3 times higher in SSc fibroblasts than normal fibroblasts. Similarly, the expression levels of ₃ mRNA were 4.3 times higher in SSc fibroblasts than normal fibroblasts.

View larger version (42K): [in this window] [in a new window]   FIGURE 1. Expression levels of v3 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-v, 3, or -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-3 Ab. All blots show one representative of five independent experiments.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Expression levels of v and 3 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 v (A) and 3 (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.

Comparison of the stability of _v and ₃ mRNAs between normal and SSc fibroblasts

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 _v and ₃ 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 _v and ₃ mRNAs between normal and SSc fibroblasts. These results indicate that the expression of _v and ₃ is up-regulated at the transcriptional level in SSc fibroblasts.

View larger version (13K): [in this window] [in a new window]   FIGURE 3. Time course of integrin v and 3 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 v, 3, and GAPDH mRNA by Northern blot analysis. Plots show the linear regression of the percentage of remaining v mRNA (A) and 3 mRNA (B) relative to time 0 and after normalization to the GAPDH mRNA.

The treatment with UO126 decreases the expression levels of ₃ subunit in SSc fibroblasts

Previous reports demonstrated that the sustained activation of ERK can specifically control the expression of ₃ 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 ₃ subunit in SSc fibroblasts. As shown in Fig. 4A, UO126 reduced the expression levels of ₃ subunit protein in a dose- and time-dependent manner in SSc fibroblasts, whereas the same treatment did not affect the expression levels of ₃ subunit protein in normal fibroblasts. This effect of UO126 on ₃ subunit protein was paralleled with that on ₃ subunit mRNA in those cells (Fig. 4B). To further confirm the involvement of the MEK-ERK pathway in the up-regulated expression of ₃ in SSc fibroblasts, the effect of transiently overexpressed DN ERK2 was determined. As shown in Fig. 4C, DN ERK2 significantly reduced the levels of ₃ subunit protein in SSc fibroblasts, whereas it showed no effect on the levels of ₃ subunit protein in normal fibroblasts. We also demonstrated that the transient overexpression of CA MEK1 induced the up-regulation of ₃ subunit protein in normal fibroblasts (Fig. 4D). In contrast, the treatment of UO126 and transient overexpression of DN ERK2 or CA MEK1 did not affect the levels of _v subunit in normal and SSc fibroblasts (data not shown). Taken together, these results suggest that ₃ subunit is up-regulated by sustained activation of the MEK-ERK pathway in SSc fibroblasts.

View larger version (58K): [in this window] [in a new window]   FIGURE 4. The effect of the inhibition of the MEK-ERK pathway on the expression levels of 3 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-3 Ab or anti--actin Ab. B, Under the same condition described above, the levels of 3 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-3 Ab or anti--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-3 Ab or anti--actin Ab. All blots show one representative of five independent experiments.

To confirm the hypothesis described above, we compared the phosphorylation levels of ERK1/2 between normal and SSc fibroblasts. As shown in Fig. 5A, 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. 5D). These results indicate that the ERK pathway is constitutively activated in SSc fibroblasts.

View larger version (71K): [in this window] [in a new window]   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.

Distribution of _v and ₃ subunit proteins in normal and SSc skin sections

To investigate the distribution of _v and ₃ 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-_v Ab and anti-₃ Ab between normal and SSc skin sections. The expression of the _v subunit protein was moderate in the blood vessels, and weak in the epidermis and smooth muscles. The expression of the ₃ 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 _v and ₃ 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.

View larger version (113K): [in this window] [in a new window]   FIGURE 6. Integrin v and 3 subunit protein expression in normal and SSc dermal sections. Paraffin sections of normal and SSc dermal tissue were subjected to immunohistochemical analysis with anti-v Ab and anti-3 Ab. The immunoreactivity was visualized by diaminobenzidine. The sections were counterstained with hematoxylin. Original magnification, x200.

View this table: [in this window] [in a new window]   Table I. Results of immunohistochemical staining for integrin v and 3 subunits

Transient overexpression of _v3 increased the promoter activity of human ₂(I) collagen gene and reduced the promoter activity of human MMP-1 gene in normal fibroblasts

Because _v3 functions as an active receptor for SLC, we next investigated whether the up-regulated expression of _v3 is involved in the phenotypical alteration of SSc fibroblasts. To this end, we transiently overexpressed _v3 in normal fibroblasts and investigated the promoter activity of human 2(I) collagen gene and human MMP-1 gene. Because _v subunit is excessively expressed in the cytoplasm as monomer, and the cell surface expression levels of _v3 is controlled by the levels of ₃ subunit (34, 35), we first confirmed that transient overexpression of ₃ subunit is sufficient to induce the up-regulation of cell surface _v3 (Fig. 7A). Under the same condition, the promoter activities were determined. As shown in Fig. 7B, left panel, the promoter activity of human 2(I) collagen gene was significantly increased in proportion to the cell surface levels of _v3. In contrast, the promoter activity of human MMP-1 gene was significantly decreased in inverse relation to the cell surface levels of _v3 (Fig. 7B, right panel). These results indicate that the up-regulated expression of _v3 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 _v3 contributes to the phenotypical alteration of SSc fibroblasts.

View larger version (28K): [in this window] [in a new window]   FIGURE 7. The effect of transiently overexpressed v3 on the promoter activity of human 2(I) collagen gene and human MMP-1 gene in normal fibroblasts. Normal fibroblasts were transfected with the indicated amount of 3 expression vector or empty vector, along with 2 µg of –772 COL1A2/CAT promoter or human MMP-1 promoter and 1 µg of pSV--galactosidase vector, and incubated for 72 h. A, The cell surface levels of v3 were determined by biotinylation and immunoprecipitation. One representative of five independent experiments is shown. B, Values represent the promoter activities of human 2(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.

The up-regulated expression of _v3 may contribute to the establishment of autocrine TGF- loop in dermal fibroblasts

We have proposed that the activation of SSc fibroblasts may be a result of the stimulation by autocrine TGF-1 (6, 7). To verify the involvement of _v3 in this model, we investigated the effect of anti-TGF- Ab or TGF-1 antisense oligonucleotide on the promoter activity of human 2(I) collagen gene or human MMP-1 gene in ₃ transfectants. As shown in Fig. 8, the increased promoter activity of human 2(I) collagen gene or the decreased promoter activity of human MMP-1 gene in ₃ transfectants were almost completely reversed by the treatment of anti-TGF- Ab or TGF-1 antisense oligonucleotide. These results suggest that the up-regulated expression of _v3 contribute to the establishment of autocrine TGF- loop in normal fibroblasts. The TGF- isoform that was primarily responsible for the activation of 3 transfectants may be TGF-1, because TGF-1 antisense oligonucleotide reversed the promoter activities to a similar extent as achieved with anti-TGF- Ab, which neutralizes TGF-1, 2, 3, and 5.

View larger version (27K): [in this window] [in a new window]   FIGURE 8. The effect of anti-TGF- Ab or TGF-1 antisense oligonucleotide on the promoter activity of human 2(I) collagen gene or human MMP-1 gene in 3 transfectants. Transfection was performed as described in Fig. 7 in the presence of anti-TGF- Ab, preimmune IgG, TGF-1 antisense oligonucleotide, or TGF-1 sense oligonucleotide. Values represent the promoter activity relative to that of mock transfectants in the presence of preimmune IgG or TGF-1 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.

_v3 activates latent TGF- on the cell surface of SSc fibroblasts

Because the activation of latent TGF- is an indispensable process for the establishment of autocrine TGF- loop, the results described above imply that _v3 is involved in the activation process of latent TGF-1. Based on the evidence that _v-containing integrins, such as _v6 and _v8, activate latent TGF-1 on the cell surface, we made a hypothesis that _v3 also activates latent TGF-1 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. 9A). 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-_v3 Ab (50% reduction) and completely abolished by anti-TGF- 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- is activated on the cell surface of SSc fibroblasts, but that at least a small amount of the active TGF- formed is freely diffusible, and suggest that this activation process was partially attributed to _v3.

View larger version (34K): [in this window] [in a new window]   FIGURE 9. The involvement of v3 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-v3 Ab (anti-v3, 10 µg/ml) or anti-TGF- Ab (anti-TGF-, 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-v3 Ab or anti-TGF- Ab. B, The –772 COL1A2/CAT promoter activities were determined in the presence of anti-v3 Ab or preimmune IgG in normal and SSc fibroblasts either stimulated or unstimulated with exogenous TGF-1. 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-v3 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 1(I) and 2(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-v3 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-1 (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-1 (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.

Blockade of _v3 reverses the SSc phenotype

To investigate whether blockade of _v3 reverses the SSc phenotype, we first focused on the promoter activity of human 2(I) collagen gene. As shown in Fig. 9B, anti-_v3 Ab significantly reduced the increased basal promoter activity in SSc fibroblasts, whereas it had no significant effects on the basal and the TGF-1-induced promoter activity in normal fibroblasts and the TGF-1-induced promoter activity in SSc fibroblasts. These results indicate that blockade of _v3 inhibit the autocrine TGF- signaling in SSc fibroblasts without affecting the effect of exogenous TGF-1 stimulation. To further confirm these findings, we investigated the effect of anti-_v3 Ab on the expression of type I procollagen protein and MMP-1 protein in normal and SSc fibroblasts. As shown in Fig. 9C, 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 _v3 by anti-_v3 Ab reversed the expression levels of these proteins. Moreover, we examined the effect of anti-_v3 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. 9D, the constitutive DNA-Smad3 binding was detected in SSc fibroblasts, and the marked DNA-Smad3 binding were detected in normal fibroblasts treated with TGF-1. The treatment of anti-_v3 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-1. We finally performed immunofluorescence using anti--smooth muscle actin Ab to determine the effect of anti-_v3 Ab on the expression levels of -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 -smooth muscle actin fibers, which are characteristics of myofibroblasts. However, after the treatment of anti-_v3 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-_v3 Ab. These results indicate that anti-_v3 Ab can reverse the myofibroblastic phenotype of SSc fibroblasts.

View larger version (35K): [in this window] [in a new window]   FIGURE 10. The effect of anti-v3 Ab on the expression levels of -smooth muscle actin and the morphology of cells in normal and SSc fibroblasts. Confluent quiescent cells were treated with anti-v3 Ab or preimmune mouse IgG for 48 h. Cells were stained with anti--smooth muscle actin Ab, washed, and incubated with FITC-conjugated rabbit anti-mouse IgG (green). The nuclei were counterstained with DAPI (blue).

View larger version (59K): [in this window] [in a new window]   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.

	Discussion

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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 _v6 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- molecules remains bound to TSP-1 (12). SLC also interact with _v6 through the C terminus of LAP, but such interaction is not sufficient for its activation. Following the binding, _v6 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- 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, _v8 has recently been shown to generate TGF-1 activity by localizing the SLC to the cell surface, thereby permitting membrane-type 1-MMP proteolytic cleavage of LAP-1 to liberate the active TGF-1 (14). Interestingly, _v3 expression is associated with enhanced cell surface proteolytic activity by MMP-2 (40) and MMP-9 (41, 42), for which LAP-1 has been shown to be a substrate, presenting a potential mechanism to generate TGF-1 activity from the _v3-dependent activation of MMP-2 and/or MMP-9. In addition, the interaction of LAP-1 with up-regulated _v3 may strongly initiate _v3-specific intracellular signaling events commonly associated with integrin-ligand ligation, which may subsequently induce a potential mechanism for establishing autocrine TGF- loop.

There is clear evidence of the importance of _v6 to regulate TGF-1 activity from the observations of 6-knockout mice (11). Thus, to prove the role of _v3 in fibrotic disorders, it is very important to determine whether there is a defect in TGF-1 activation in 3-knockout mice using appropriate models, such as the bleomycin model of dermal fibrosis or pulmonary fibrosis. 3-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 _IIb3. Observational studies of these mice have implicated _v3 in several physiological roles, including embryo implantation, angiogenesis, and bone resorption (43, 44, 45). To our knowledge, however, a defect in TGF-1 activation has not been studied in these mice. Alternatively, it is also informative to investigate the activity of TGF-1 in animal models treated with anti-_v3 Ab or to determine whether 3-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 3 induction and subsequent cell surface expression of _v3 in osteoclasts, which may contribute to the acceleration of bone resorption. Furthermore, it has been reported that the blockade of _v3 reduces bone resorption in vitro and prevents osteoporosis in vivo (46). Because bone resorption is a process that is stimulated by TGF-1 (42, 47), these previous results regarding osteoclasts are paralleled with the present data as follows: 1) the expression of _v3 may be up-regulated through sustained ERK activation in SSc fibroblasts; 2) transient overexpression of _v3 increases the promoter activity of human 2(I) collagen gene in a TGF-1-dependent manner in normal fibroblasts; and 3) anti-_v3 Ab reduced the promoter activity of human 2(I) collagen gene in SSc fibroblasts. These results imply that dermal fibroblasts and osteoclasts have a similar mechanism of regulating tissue remodeling by _v3. Thus, the possible association of _v3 with latent TGF- activation is a novel finding that strongly supports the understanding of _v3-associated biological processes.

In summary, this study demonstrated that up-regulated expression of _v3 contributed to the establishment of autocrine TGF- 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.

	Acknowledgments

 
We thank Drs. Joseph C. Loftus, Timothy E. O’Toole, Dennis Templeton, and Roger J. Davis for providing us with _v cDNA, ₃ cDNA, expression vector of DN ERK2, and expression vector of CA MEK1.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
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.

² Address correspondence and reprint requests to Dr. Hironobu Ihn, Department of Dermatology and Plastic and Reconstructive Surgery, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto City, Kumamoto 860-8556, Japan. E-mail address: ihn-der{at}kaiju.medic.kumamoto-u.ac.jp

³ Abbreviations used in this paper: SSc, systemic sclerosis; ECM, extracellular matrix; LAP-1, latency-associated peptide-1; 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.

Received for publication January 6, 2005. Accepted for publication September 23, 2005.

	References

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