Phosphatidylinositol 3-kinase is involved in alpha2(I) collagen gene expression in normal and scleroderma fibroblasts

TGF-beta is implicated in the pathogenesis of fibrotic disorders. It has been shown that Smad3 promotes the human alpha2(I) collagen (COL1A2) gene expression by TGF-beta1 in human dermal fibroblasts. Here, we investigated the role of phosphatidylinositol 3-kinase (PI3K) in the COL1A2 gene expression in normal and scleroderma fibroblasts. In normal fibroblasts, the PI3K inhibitor, LY294002, significantly decreased the basal and the TGF-beta1-induced increased stability of COL1A2 mRNA. The TGF-beta1-induced COL1A2 promoter activity, but not the basal activity, was significantly attenuated by LY294002 or the dominant negative mutant of p85 subunit of PI3K, while the constitutive active mutant of p110 subunit of PI3K did not affect the basal or the TGF-beta1-induced COL1A2 promoter activity. LY294002 significantly decreased the phosphorylation of Smad3 induced by TGF-beta1. Furthermore, the transient overexpression of 2xFYVE, which induces the mislocalization of FYVE domain proteins, decreased the TGF-beta1-induced Smad3 phosphorylation to a similar extent to LY294002. In scleroderma fibroblasts, the blockade of PI3K significantly decreased the mRNA stability and the promoter activity of the COL1A2 gene. Furthermore, LY294002 and the transient overexpression of 2xFYVE completely diminished the constitutive phosphorylation of Smad3. These results indicate that 1) the basal activity of PI3K is necessary for the COL1A2 mRNA stabilization in normal and scleroderma fibroblasts, 2) there is an unidentified FYVE domain protein specifically interacting with Smad3, and 3) the basal activity of PI3K and the FYVE domain protein are indispensable for the efficient TGF-beta/Smad3 signaling in normal fibroblasts and for the establishment of the constitutive activation of TGF-beta/Smad3 signaling in scleroderma fibroblasts.     

Modulation of transforming growth factor-beta (TGF-beta) signaling by endogenous sphingolipid mediators

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that plays a critical role in tissue repair and fibrosis. Sphingolipid signaling has been shown to regulate a variety of cellular processes and has been implicated in collagen gene regulation. The present study was undertaken to determine whether endogenous sphingolipids are involved in the TGF-beta signaling pathway. TGF-beta treatment induced endogenous ceramide levels in a time-dependent manner within 5-15 min of cell stimulation. Using human fibroblasts transfected with a alpha2(I) collagen promoter/reporter gene construct (COL1A2), C(6)-ceramide (10 microm) exerted a stimulatory effect on basal and TGF-beta-induced activity of this promoter. Next, to define the effects of endogenous sphingolipids on TGF-beta signaling we employed ectopic expression of enzymes involved in sphingolipid metabolism. Sphingosine 1-phosphate phosphatase (YSR2) stimulated basal COL1A2 promoter activity and cooperated with TGF-beta in activation of this promoter. Furthermore, overexpression of YSR2 resulted in the pronounced increase of COL1A1 and COL1A2 mRNA levels. Conversely, overexpression of sphingosine kinase (SPHK1) inhibited basal and TGF-beta-stimulated COL1A2 promoter activity. These results suggest that endogenous ceramide, but not sphingosine or sphingosine 1-phosphate, is a positive regulator of collagen gene expression. Mechanistically, we demonstrate that Smad3 is a target of YSR2. TGF-beta-induced Smad3 phosphorylation was elevated in the presence of YSR2. Cotransfection of YSR2 with wild-type Smad3, but not with the phosphorylation-deficient mutant of Smad3 (Smad3A), resulted in a dramatic increase of COL1A2 promoter activity. In conclusion, this study demonstrates a direct role for the endogenous sphingolipid mediators in regulating the TGF-beta signaling pathway.     

Identification of novel TGF-beta /Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach

Despite major advances in the understanding of the intimate mechanisms of transforming growth factor-beta (TGF-beta) signaling through the Smad pathway, little progress has been made in the identification of direct target genes. In this report, using cDNA microarrays, we have focussed our attention on the characterization of extracellular matrix-related genes rapidly induced by TGF-beta in human dermal fibroblasts and attempted to identify the ones whose up-regulation by TGF-beta is Smad-mediated. For a gene to qualify as a direct Smad target, we postulated that it had to meet the following criteria: (1) rapid (30 min) and significant (at least 2-fold) elevation of steady-state mRNA levels upon TGF-beta stimulation, (2) activation of the promoter by both exogenous TGF-beta and co-transfected Smad3 expression vector, (3) up-regulation of promoter activity by TGF-beta blocked by both dominant-negative Smad3 and inhibitory Smad7 expression vectors, and (4) promoter transactivation by TGF-beta not possible in Smad3(-/-) mouse embryo fibroblasts. Using this stringent approach, we have identified COL1A2, COL3A1, COL6A1, COL6A3, and tissue inhibitor of metalloproteases-1 as definite TGF-beta/Smad3 targets. Extrapolation of this approach to other extracellular matrix-related gene promoters also identified COL1A1 and COL5A2, but not COL6A2, as novel Smad targets. Together, these results represent a significant step toward the identification of novel, early-induced Smad-dependent TGF-beta target genes in fibroblasts.     

Abrogation of transforming growth factor-beta signaling by SMAD7 inhibits collagen gel contraction of human dermal fibroblasts

Human fibroproliferative disorders like hypertrophic scarring of the skin are characterized by increased contractility and excess extracellular matrix synthesis. A beneficial role of transforming growth factor (TGF)-beta in wound healing was proposed; however, chronic stimulation by this cytokine leads to fibrosis. In the present report, the intracellular TGF-beta signaling in fibroblasts derived from hypertrophic scars and normal skin was examined. In an attempt to intervene in profibrogenic TGF-beta functions, ectopic expression of Smad7 or dominant negative Smads3/4 completely inhibited contractility of scar-derived and normal fibroblasts after suspension in collagen gels. Both cell types displayed constitutive Smad2/3 phosphorylation and (CAGA)9-MLP-Luc activity with expression and phosphorylation of Smad3 being predominant in hypertrophic scar-derived fibroblasts. Down-regulation of intrinsic signaling with various TGF-beta antagonists, e.g. soluble TGF-beta receptor, latency-associated peptide, and anti-TGF-beta1 antibodies, confirms autocrine TGF-beta stimulation of both cell populations. Further, Smad7 expression inhibited alpha1 (I) collagen and alpha-smooth muscle actin expression. In summary, our data indicate that autocrine TGF-beta/Smad signaling is involved in contractility and matrix gene expression of fibroblasts from normal and hypertrophic scars. Smad7 inhibits these processes and may exert beneficial effects on excessive scar formation.