Increased expression of integrin alphavbeta5 induces the myofibroblastic differentiation of dermal fibroblasts

Abstract

The biological effect of cytokines is mainly determined by the cytokine-receptor interaction, which is modulated by the concentration and the activity of cytokines and/or their receptors. Because alphav-containing integrins can bind to and/or activate latent TGF-beta, these integrins have been thought to be involved in the pathogenesis of fibrotic disorders. Our recent observations that alphavbeta5 is up-regulated in scleroderma fibroblasts and that the transient overexpression of alphavbeta5 increases the human alpha2(I) collagen gene expression in normal fibroblasts suggest the involvement of alphavbeta5 in the self-activation system in scleroderma fibroblasts. In this study, we established stable transfectants with alphavbeta5 using normal dermal fibroblasts and demonstrated that such cells differentiated into myofibroblasts by the stimulation of autocrine TGF-beta. This observation is explained by 1) alphavbeta5 recruiting latent TGF-beta1 on the cell surface, 2) endogenous active TGF-beta localizing on the cell surface, and 3) alphavbeta5 interacting with TGF-beta receptors. Furthermore, blockade of alphavbeta5 reversed the myofibroblastic phenotype in scleroderma fibroblasts. These data identify a novel mechanism for the establishment of autocrine TGF-beta signaling in dermal fibroblasts by the up-regulation of alphavbeta5 and suggest the possibility of regulating fibrotic disorders, especially scleroderma, by targeting this integrin.

Figure 1

Confirmation of the establishment of β5-transfectants. Total RNAs were extracted from confluent quiescent cells, and the levels of β5 mRNA (A) or αv mRNA (C) were determined by Northern blotting. Cell surface proteins were labeled with biotin, and whole-cell lysates were subjected to immunoprecipitation using goat anti-β5 antibody (B, left panel), goat anti-β1 antibody (D), or goat anti-β3 antibody (E). Precipitated proteins were subjected to SDS-PAGE, and Western blots were prepared. The blots were probed with streptavidin coupled to horseradish peroxidase. A parallel immunoprecipitation performed with preimmune goat IgG failed to show integrin immunoreactivities (B, right panel). The bands in immunoprecipitation were confirmed to be the indicated subunits by a reprobing analysis using specific antibodies to αv-, β1-, β3-, or β5-subunits (data not shown), whereas the identity of α1, α2, α3, and α5 was determined by the size on a gel. Experiments were repeated five times with similar results.

Figure 2

Assay of mock and β5-transfectant attachment to vitronectin or type I collagen. A: Cells were incubated at 37°C for 1 hour in the wells coated with vitronectin or type I collagen. After centrifugation, attached cells were stained, and the absorbance of 595 nm was measured. B: Cells were preincubated at 37°C for 1 hour with anti-αvβ5 antibody or preimmune IgG, and cell attachment was determined. The mean from five separate experiments is shown.

Figure 3

Myofibroblastic differentiation of β5-transfectants. A: mRNA levels of α2(I) collagen and GAPDH genes were analyzed by Northern blotting. DNA probes against α2(I) collagen hybridized to cellular transcripts of 5.2 and 5.7 kb. One representative of five independent experiments is shown. B: The −353COL1A2 promoter activities were determined by CAT assays. Values represent the CAT activity relative to that of mock transfectants (100 arbitrary units [AU]). The means ± SD of five independent experiments are shown. *P < 0.05 versus mock transfectants. The expression of α-SMA protein was determined by immunoblotting (C) and by immunofluorescence (D). Experiments were repeated five times with similar results.

Figure 4

Constitutive activation of TGF-β/Smad signaling by endogenous TGF-β stimulation in β5-transfectants. Cells were transfected with 2 μg of −353COL1A2 promoter in the presence of anti-TGF-β antibody (anti-TGF-β) or preimmune IgG (A) or in the presence of TGF-β1 antisense oligonucleotide (AS-TGF-β1) or TGF-β1 sense oligonucleotide (S-TGF-β1) (B). Values represent the CAT activity relative to that of mock transfectants treated with preimmune IgG (A) or treated with S-TGF-β1 (B) (100AU). The means ± SD of five independent experiments are shown. *P < 0.05 versus β5-transfectants treated with preimmune IgG (A) or S-TGF-β1 (B). Whole-cell lysates derived from multiple clones of each transfectant were subjected to immunoprecipitation using anti-Smad3 antibody, and phospho-Smad3 was detected by immunoblotting using anti-phospho-serine antibody (C). The same membrane was stripped and reprobed with anti-Smad2/3 antibody (left panel). A parallel immunoprecipitation performed with preimmune mouse IgG failed to show Smad3 immunoreactivity (right panel). D: Nuclear extracts derived from multiple clones of each transfectant were incubated with biotin-labeled oligonucleotides. Proteins bound to these nucleotides were isolated with streptavidin-agarose beads, and Smad3 was detected by immunoblotting. All blots show one representative of five independent experiments.

Figure 5

Myofibroblastic differentiation of β5-transfectants is dependent on both TGF-β signaling and integrin/FAK signaling. A: Whole-cell lysates derived from multiple clones of each transfectant were subjected to immunoblotting using anti-397Y-phospho-FAK antibody. The same membrane was stripped and reprobed with anti-FAK antibody. B: Quiescent fibroblasts were treated with the indicated reagent for 48 hours. Whole-cell lysates were subjected to immunoblotting using anti-α-SMA antibody and anti-β-actin antibody. C: Cells were transfected with 2 μg of expression vector of Y397F-FAK or corresponding empty vector. After a 48-hour incubation, whole-cell lysates were subjected to immunoblotting using anti-α-SMA antibody and anti-β-actin antibody. D: Quiescent cells were treated with 10 μmol/L TGF-β1 antisense oligonucleotide (AS-TGF-B1) or TGF-β1 sense oligonucleotide (S-TGF-β1) for 48 hours. Whole-cell lysates were subjected to immunoblotting using anti-α-SMA antibody and anti-β-actin antibody. E: Quiescent cells were treated with 10 μmol/L TGF-β1 antisense oligonucleotide (AS-TGF-B1) or TGF-β1 sense oligonucleotide (S-TGF-β1) for 48 hours. Whole-cell lysates were subjected to immunoblotting using anti-397Y-phospho-FAK antibody. The same membrane was stripped and reprobed with anti-FAK antibody. All experiments were performed using multiple clones of each transfectants, and one representative is shown. In right panels of B through D, the protein levels quantitated by scanning densitometry are shown relative to those in mock transfectants treated with dimethylsulfoxide (DMSO), empty vector, or S-TGF-β1, respectively (1 arbitrary unit [AU]). Data are expressed as the means ± SD of five independent experiments. *P < 0.05 versus mock transfectants under the same condition. **P < 0.05 versus β5-transfectants treated with DMSO, empty vector, or S-TGF-β1.

Figure 6

SLC is recruited and activated by its interaction with αvβ5 in β5-transfectants. A: Northern blotting was performed with DNA probes for TGF-β1 and GAPDH genes. Experiments were repeated five times with similar results. B: Confluent quiescent cells were incubated at 4°C for 1 hour with ³⁵S-labeled SLC or mutated SLC (mSLC) in the presence or absence of cold SLC. Cell surface-bound SLC was removed by extraction buffer and quantitated by scintillation counting. In some experiments, assays were performed in the presence of RGD peptide (10 μg/ml), RGE peptide (10 μg/ml), anti-αvβ5 antibody (anti-αvβ5, 10 μg/ml), or preimmune mouse IgG (IgG, 10 μg/ml). Values represent the scintillation count relative to that of mock transfectants treated with SLC (100AU). *P < 0.05 versus mock transfectants under the same condition. C: Cells were cultured in MEM containing 10 μmol/L AS-TGF-β1 for 48 hours. Then, the medium was changed to serum free-medium containing 10 μmol/L AS-TGF-β1, and cells were transfected with 2 μg of -353COL1A2 promoter. Cells were stimulated with the indicated concentration of TGF-β1 or SLC for the last 24 hours, and the CAT activities were determined. In some experiments, SLC stimulation was performed in the presence of RGD peptide (10 μg/ml), RGE peptide (10 μg/ml), anti-αvβ5 antibody (anti-αvβ5, 10 μg/ml), or preimmune mouse IgG (IgG, 10 μg/ml). Values represent the CAT activity relative to that of mock transfectants treated with S-TGF-β1 (100AU). The means ± SD of five independent experiments are shown. *P < 0.05 versus β5-transfectants treated with AS-TGF-β1. ^#P < 0.05 versus β5-transfectants treated with AS-TGF-β1, SLC, and RGE peptide. ^$P < 0.05 versus β5-transfectants treated with AS-TGF-β1, SLC, and preimmune mouse IgG.

Figure 7

SLC is activated on the cell surface of β5-transfectants. A: Transfectant and TMLC cells were mixed at a ratio of 1:1 and co-cultured in confluence. In experiments without cell-cell contact, either transfectant or TMLC cells were co-cultured separately by using inserts. After a 24-hour incubation, the luciferase activities were determined. In some experiments, assays were performed in the presence of anti-αvβ5 antibody (anti-αvβ5, 10 μg/ml), preimmune mouse IgG (M-IgG, 10 μg/ml), anti-TGF-β antibody (anti-TGF-β, 10 μg/ml), or preimmune rabbit IgG (R-IgG, 10 μg/ml). Values represent the luciferase activity relative to that of TMLC cells co-cultured with mock transfectants in the absence of contact (100AU). *P < 0.05 versus mock transfectants under the same condition. B: Mock or β5-transfectants were transfected with 2 μg of -353COL1A2 promoter in the presence of appropriate reagents. Values represent the CAT activity relative to that of β5-transfectants treated with DMSO (100AU). The means ± SD of five independent experiments are shown.

Figure 8

Interaction between αvβ5 and TGF-β receptors. A: Whole-cell lysates were subjected to immunoprecipitation using anti-clathrin antibody, and αv, β5, TβRI, TβRII, or clathrin were detected by immunoblotting. B: Whole-cell lysates were subjected to immunoprecipitation using anti-clathrin antibody (CL) or anti-caveolin antibody (CV), and β5, clathrin, or caveolin levels in precipitated proteins were determined by immunoblotting (left panels). Residual (unprecipitated) proteins were also subjected to immunoblotting using anti-clathrin antibody or anti-caveolin antibody to confirm the efficiency of immunoprecipitation (right panels). C: Immunoprecipitation was performed using anti-β5 antibody, and β5, TβRI, or TβRII was detected by immunoblotting. All blots show one representative of five independent experiments.

Figure 9

The treatment of anti-αvβ5 antibody reverses the myofibroblastic phenotype in scleroderma fibroblasts. A: Whole-cell lysates prepared from quiescent normal and scleroderma fibroblasts were subjected to immunoblotting using anti-α-SMA antibody and anti-β-actin antibody. B: Quiescent normal and scleroderma fibroblasts were treated with anti-αvβ5 antibody (10 μg/ml) or preimmune mouse IgG (10 μg/ml) for 48 hours. Whole-cell lysates were subjected to immunoblotting using anti-α-SMA antibody and anti-β-actin antibody. Experiments were repeated five times with similar results.