Constitutive thrombospondin-1 overexpression contributes to autocrine transforming growth factor-beta signaling in cultured scleroderma fibroblasts

Abstract

The extracellular matrix (ECM) glycoprotein thrombospondin-1 (TSP-1) has been reported to activate the latent complex of transforming growth factor-beta (TGF-beta), the major effects of which in mesenchymal cells is stimulation of the synthesis of ECM. Previous reports suggested the involvement of an autocrine TGF-beta loop in the pathogenesis of scleroderma. In this study, we examined whether TSP-1 plays a role in maintaining the autocrine TGF-beta loop in scleroderma. TSP-1 expression was increased in scleroderma patients compared with in healthy controls in vivo and in vitro. TGF-beta blocking antibody or TGF-beta1 antisense oligonucleotide markedly reduced the up-regulated TSP-1 expression in scleroderma fibroblasts but had little effect on normal fibroblasts. The expression of TSP-1 is up-regulated in scleroderma fibroblasts, possibly at the post-transcriptional level just like in normal fibroblasts stimulated with exogenous TGF-beta1. TSP-1 blocking peptide or antisense oligonucleotide had an inhibitory effect on the up-regulated alpha2I collagen and phosopho-Smad3 levels in scleroderma fibroblasts but had little effects on normal fibroblasts. The transient overexpression of TSP-1 up-regulated alpha2I collagen and phospho-Smad3 levels in normal fibroblasts but had no major effect on scleroderma fibroblasts. Furthermore, these effects of transiently overexpressed TSP-1, which possibly occurred via the activation of latent TGF-beta1, were abolished by the TGF-beta1 antisense oligonucleotide. These results indicate that the constitutive overexpression of TSP-1 may play an important role in autocrine TGF-beta signaling and accumulation of ECM in scleroderma fibroblasts.

Figures

Figure 1

Comparison of TSP-1 expression levels in dermal tissue between scleroderma patients and healthy controls. Paraffin sections of normal and scleroderma dermal tissue were subjected to immunohistochemical analysis as described in Materials and Methods. The immunoreactivity of TSP-1 (arrow) was more strongly detected around the cells between collagen bundles in scleroderma (B) than in normal tissue (A). The cells positive for anti-TSP-1 Ab between collagen bundles were not stained with anti-CD34 Ab (C) or anti-CD68 Ab (D). Original magnification, ×200.

Figure 2

Comparison of levels of type I procollagen expression, p-Smad3 and TSP-1 expression between cultured normal and scleroderma dermal fibroblasts. A: For type I procollagen and TSP-1, culture media were electrophoresed through a 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with anti-type I collagen and anti-TSP-1 Ab. Levels of β-actin are shown as a loading control. For p-Smad3, whole cell lysates were subjected to immunoprecipitation using anti-Smad3 Ab, and p-Smad3 was detected by immunoblotting using anti-phosphoserine Ab. The same membrane was stripped and total levels of smad3 are determined using Smad2/3 Ab. Total Smad3 levels can be appropriately regarded as a loading control. Four separate lanes for normal and scleroderma fibroblast cultures represent 4 separate primary isolates from different individuals. The levels quantitated by scanning densitometry are shown relative to the levels in normal fibroblasts (1.0). Data are expressed as the mean ± SD of five independent experiments (bottom). *P < 0.05, as compared with the value in normal fibroblasts. B: Northern blot analysis was performed to evaluate mRNA expression in normal and scleroderma fibroblasts. Four separate lanes for normal and scleroderma fibroblast cultures represent 4 separate primary isolates from different individuals. One result representative of five independent experiments is shown at the top. α2(I)collagen and TSP-1 mRNA levels quantitated by scanning densitometry and corrected for the levels of GAPDH in the same samples are shown relative to the level in normal fibroblasts (1.0). Data are expressed as the mean ± SD of five independent experiments (bottom). *P < 0.05, as compared with the value in normal fibroblasts.

Figure 3

Effects of anti-TGF-β Ab and TGF-β1 antisense oligonucleotide on TSP-1 protein and mRNA expression in normal and scleroderma fibroblasts. A: The effects of anti-TGF-β Ab or TGF-β1 antisense oligonucleotide on TSP-1 protein expression were investigated by immunoblotting (top). Normal fibroblasts and scleroderma fibroblasts were treated with anti-TGF-β antibody or TGF-β1 antisense oligonucleotide for 48 hours. One result representative of five independent experiments is shown. The levels quantitated by scanning densitometry are shown relative to those in normal fibroblasts treated with preimmune serum or sense oligonucleotide (1.0). Data are expressed as the mean ± SD of five independent experiments (bottom). *P < 0.05 versus control cells treated with TGF-β1 sense oligonucleotide or preimmune IgG. B: The effects of anti-TGF-β blocking Ab and TGF-β1 antisense oligonucleotide on TSP-1 mRNA levels were investigated by Northern blotting (top). Normal and scleroderma fibroblasts were treated with anti-TGF-β blocking Ab or TGF-β1 antisense oligonucleotide for 48 hours. One result representative of five independent experiments is shown. The mRNA levels quantitated by scanning densitometry are shown relative to those in normal fibroblasts treated with preimmune serum or sense oligonucleotide (1.0). Data are expressed as the mean ± SD of five independent experiments (bottom). *P < 0.05 versus control cells treated with TGF-β1 sense oligonucleotide or preimmune IgG.

Figure 4

Exogenous active TGF-β1 increases both TSP-1 protein and mRNA in normal fibroblasts. Human dermal fibroblasts were serum-starved for 24 hours and incubated in the absence or presence of the indicated doses of TGF-β, or for the indicated time in the presence of 2 ng/ml TGF-β. A (I) and B (I): Medium (adjusted based on the protein concentration of the cell lysate) was subjected to immunoblotting with anti-TSP-1 Ab (top). Levels of β-actin are shown as a loading control. TSP-1 protein levels quantitated by scanning densitometry and corrected for the levels of β-actin in the same samples are shown relative to the levels in untreated cells (1.0). Data are expressed as the mean ± SD of five independent experiments (bottom). *P < 0.05, as compared with the value in untreated cells. A (II) and B (II): Northern blot analysis was performed to evaluate TSP-1 mRNA expression (top). Levels of GAPDH mRNA are shown as a loading control. TSP-1 mRNA levels quantitated by scanning densitometry and corrected for the levels of GAPDH in the same samples are shown relative to the levels in untreated cells (1.0). Data are expressed as the mean ± SD of five independent experiments (bottom). * P < 0.05, as compared with the value in untreated cells.

Figure 5

The TSP-1 mRNA stability and promoter activity in cultured normal and scleroderma fibroblasts. A: Normal fibroblasts or scleroderma fibroblasts were serum-starved for 24 hours and incubated in the absence or presence of 2 ng/ml TGF-β for 6 hours, as well as pretreated with 10 μg/ml anti-TGF-β Ab, before the addition of 400 ng/ml actinomycin D. Northern blot analysis was performed to evaluate TSP-1 mRNA expression in untreated normal fibroblasts, normal fibroblasts treated with 2 ng/ml TGF-β1, untreated scleroderma fibroblasts and scleroderma fibroblasts treated with 10 μg/ml anti-TGF-β Ab. Levels of GAPDH mRNA are shown as a loading control. One result representative of five independent experiments is shown. B: Time-dependent degradation of TSP-1 mRNA in untreated normal fibroblasts, normal fibroblasts treated with 2 ng/ml TGF-β1, untreated scleroderma fibroblasts and scleroderma fibroblasts treated with 10 μg/ml anti-TGF-β Ab. The filled circles with a solid line indicate untreated normal fibroblasts. The filled squares with a dashed line indicate normal fibroblasts treated with exogenous 2 ng/ml TGF-β1. The open circles with an irregularly dashed line indicate untreated scleroderma fibroblasts. The open squares with a finely dashed line indicate scleroderma fibroblasts treated with 10 μg/ml anti-TGF-β Ab. *P < 0.05, as compared with the value in untreated normal fibroblasts. Tβ1 antisense, TGF-β1 antisense oligonucleotide. C: Normal and scleroderma fibroblasts were transiently transfected with the TSP-1 promoter-lux (−2200/+754). After incubation for 24 hours, the cells were stimulated with 2 ng/ml TGF-β1 for an additional 18 hours. The luciferase activity was determined as the fold increase relative to that in untreated normal fibroblasts (1.0). Data are expressed as the mean ± SD of five independent experiments.

Figure 6

Effects of TSP-1 blocking peptide and TSP-1 antisense oligonucleotide on type I procollagen protein, α2(I) collagen mRNA expression and Smad3 phosphorylation in normal and scleroderma fibroblasts. A/B: The effects of TSP-1 blocking peptide, TSP-1 antisense oligonucleotide and anti-TSP-1 blocking Ab on type I procollagen protein expression were investigated by immunoblotting (top). Normal fibroblasts and scleroderma fibroblasts were treated with TSP-1 blocking peptide for 96 hours or TSP-1 antisense oligonucleotide/anti-TSP-1 blocking Ab for 48 hours. TSP-1 protein levels in the media are also presented in A to show the efficacy of the TSP-1 antisense oligonucleotide. One result representative of five independent experiments is shown. The levels quantitated by scanning densitometry are shown relative to those in normal fibroblasts treated with a control peptide or sense oligonucleotide (1.0). Data are expressed as the mean ± SD of five independent experiments (bottom). *P < 0.05 versus control cells treated with control peptide or TSP-1 sense oligonucleotide. C: The effect of TSP-1 blocking peptide and TSP-1 antisense oligonucleotide on the α2(I)collagen mRNA levels was investigated by Northern blotting (top). Normal and scleroderma fibroblasts were treated with TSP-1 blocking peptide for 96 hours or TSP-1 antisense oligonucleotide for 48 hours. TSP-1 mRNA levels are also shown. One result representative of five independent experiments is shown. The mRNA levels quantitated by scanning densitometry are shown relative to those in normal fibroblasts treated with a control peptide or sense oligonucleotide (1.0). Data are expressed as the mean ± SD of five independent experiments (bottom). *P < 0.05 versus control cells treated with a control peptide or TSP-1 sense oligonucleotide. D: The effect of TSP-1 blocking peptide or TSP-1 antisense oligonucleotide on Smad3 phosphorylation level (top). Normal and scleroderma fibroblasts were treated with TSP-1 blocking peptide for 96 hours or TSP-1 antisense oligonucleotide for 48 hours. One result representative of five independent experiments is shown. The levels quantitated by scanning densitometry are shown relative to those in normal fibroblasts treated with a control peptide or sense oligonucleotide (1.0). Data are expressed as the mean ± SD of five independent experiments (bottom). *P < 0.05 versus cells treated with a control peptide or TSP-1 sense oligonucleotide.

Figure 7

Effects of transient TSP-1 overexpression on the levels of type I procollagen expression, and Smad3 phosphorylation in normal fibroblasts. A: Type I procollagen protein expression in the culture medium of normal or scleroderma fibroblasts transfected with the indicated doses of empty vector or TSP-1 expression plasmid, as well as pre-treated with TGF-β1 sense or antisense oligonucleotide 24 hours before the transfection. The medium (adjusted based on the protein concentration of cell lysates) was subjected to immunoblotting with anti-type I collagen Ab. TSP-1 protein levels in the medium are also shown. One representative result of five independent experiments is shown at the top. The levels quantitated by scanning densitometry are shown relative to those in untreated normal fibroblasts (1.0). Data are expressed as the mean ± SD of five independent experiments (bottom). *P < 0.05, as compared with the value in empty vector-transfected fibroblasts pre-treated with TGF-β1 sense oligonucleotide. B: The effects of TSP-1 overexpression on the α2(I) collagen promoter activity. Normal or scleroderma fibroblasts were co-transfected with 3 μg of TSP-1 expression plasmid or empty vector, and 2 μg of various deleted or mutated COL1A2/CAT constructs. The mean ± SD from five separate experiments are shown. *P < 0.05, as compared with the fold-increase relative to the control (vector-transfected normal fibroblasts) on transfection with −3500 COL1A2/CAT constructs. C: Smad3 phosphorylation levels in normal fibroblasts transfected with indicated doses of empty vector or TSP-1 expression vector, as well as pretreated with TGF-β1 sense or antisense oligonucleotide (top). The transfected cells were also stimulated with exogenous 2 μg/ml of TGF-β for 1 hour before protein extraction (lanes 4 and 5). One result representative of five independent experiments is shown. The levels quantitated by scanning densitometry are shown relative to those in empty vector-transfected fibroblasts pre-treated with TGF-β1 sense oligonucleotide (1.0). Data are expressed as the mean ± SD of five independent experiments (bottom). *P < 0.05 as compared with the value in empty vector-transfected fibroblasts pre-treated with TGF-β1 sense oligonucleotide.

Figure 8

Exogenous latent TGF-β1 induced the COL1A2 promoter activity more potently in the TSP-1-overexpressing fibroblasts than control fibroblasts. A: Normal fibroblasts were cultured in MEM containing 10 μmol/L TGF-β1 antisense oligonucleotide (antisense oligo) for 48 hours. Then, the cells were co-transfected with 3 μg of empty vector or TSP-1 expression vector and 2 μg of −773 COL1A2-Lux construct. The fibroblasts were stimulated with the indicated concentrations of TGF-β1 or latent TGF-β1 for the last 24 hours, and the luciferase activity was determined. In some experiments, latent TGF-β1 stimulation was performed in the presence of TSP-1 blocking peptide (1 μmol/L). The mean ± SD from five separate experiments are shown. *P < 0.05 versus control cells treated with 10 μmol/L TGF-β1 sense oligonucleotide. L-TGF-β1 represents latent TGF-β1. B: Normal fibroblasts were cultured in MEM containing 10 μmol/L TGF-β1 antisense oligonucleotide (antisense oligo) for 48 hours. Then, the cells were co-transfected with 3 μg of empty vector or TSP-1 expression vector and 2 μg of p3TP-Lux construct. The fibroblasts were stimulated with the indicated concentrations of TGF-β1 or latent TGF-β1 for the last 24 hours, and the luciferase activity was determined. In some experiments, latent TGF-β1 stimulation was performed in the presence of TSP-1 blocking peptide (1 μmol/L). The mean ± SD from 5 separate experiments are shown. *P < 0.05 versus control cells treated with 10 μmol/L TGF-β1 sense oligonucleotide. L-TGF-β1 represents latent TGF-β1.