Transforming growth factor- (TGF-) signaling plays a pivotal role in extracellular matrix deposition by stimulating collagen production and other extracellular matrix proteins and by inhibiting matrix degradation. The present study was undertaken to define the role of sphingosine kinase (SphK) in TGF- signaling. TGF- markedly up-regulated SphK1 mRNA and protein amounts and caused a prolonged increase in SphK activity in dermal fibroblasts. Concomitantly, TGF- reduced sphingosine-1-phosphate phosphatase activity. Consistent with the changes in enzyme activity, corresponding changes in sphingolipid levels were observed such that sphingosine 1-phosphate (S1P) was increased (ϳ2-fold), whereas sphingosine and ceramide were reduced after 24 h of TGF- treatment. Given the relatively early induction of SphK gene expression in response to TGF-, we examined whether SphK1 may be involved in the regulation of TGF--inducible genes that exhibit compatible kinetics, e.g. tissue inhibitor of metalloproteinase-1 (TIMP-1). We demonstrate that decreasing SphK1 expression by small interfering RNA (siRNA) blocked TGF--mediated up-regulation of TIMP-1 protein suggesting that up-regulation of SphK1 contributes to the induction of TIMP-1 in response to TGF-. The role of SphK1 as a positive regulator of TIMP-1 gene expression was further corroborated by using ectopically expressed SphK1 in the absence of TGF-. Adenovirally expressed SphK1 led to a 2-fold increase of endogenous S1P and to increased TIMP-1 mRNA and protein production. In addition, ectopic SphK1 and TGF- cooperated in TIMP-1 upregulation. Mechanistically, experiments utilizing TIMP-1 promoter constructs demonstrated that the action of SphK1 on the TIMP-1 promoter is through the AP1-response element, consistent with the SphK1-mediated up-regulation of phospho-c-Jun levels, a key component of AP1. Together, these experiments demonstrate that SphK/S1P are important components of the TGF- signaling pathway involved in up-regulation of the TIMP-1 gene. Transforming growth factor- (TGF-)1 is a member of a large growth factor family with diverse functions in embryonic and adult tissues (1, 2). TGF- signaling plays a pivotal role in extracellular matrix (ECM) deposition in fibroblasts by stimulating the production of collagens and other ECM proteins and inhibiting matrix degradation (3). The negative effects of TGF- on ECM degradation are mediated via direct inhibition of the principal matrix-degrading proteinase, metalloproteinase-1 (MMP-1) (4), and stimulation of endogenous inhibitors of MMP-1, including TIMP-1 (5). In healthy tissues, a balance between matrix synthesis and degradation precisely controls ECM homeostasis, and shifting this balance can lead to pathological ECM turnover (6). Excessive matrix degradation occurs in rheumatoid arthritis, osteoarthritis, periodontitis, and tumor invasion (6). An imbalance between the tissue inhibitors of matrix metalloproteinases and matrix metalloproteinases is believed to be one of the mechanisms contributing to thes...
Transforming growth factor-beta (TGF-beta) and connective tissue growth factor (CTGF) are ubiquitously expressed in various forms of tissue fibrosis, including fibrotic diseases of the kidney. To clarify the common and divergent roles of these growth factors in the cells responsible for pathological extracellular matrix (ECM) deposition in renal fibrosis, the effects of TGF-beta and CTGF on ECM expression in primary human mesangial (HMCs) and human proximal tubule epithelial cells (HTECs) were studied. Both TGF-beta and CTGF significantly induced collagen protein expression with similar potency in HMCs. Additionally, alpha(2)(I)-collagen promoter activity and mRNA levels were similarly induced by TGF-beta and CTGF in HMCs. However, only TGF-beta stimulated collagenous protein synthesis in HTECs. HTEC expression of tenascin-C (TN-C) was increased by TGF-beta and CTGF, although TGF-beta was the more potent inducer. Thus both growth factors elicit similar profibrogenic effects on ECM production in HMCs, while promoting divergent effects in HTECs. CTGF induction of TN-C, a marker of epithelial-mesenchymal transdifferentiation (EMT), with no significant induction of collagenous protein synthesis in HTECs, may suggest a more predominant role for CTGF in EMT rather than induction of excessive collagen deposition by HTECs during renal fibrosis.
The mammalian target of rapamycin (mTOR) is a multifunctional protein involved in the regulation of cell growth, proliferation, and differentiation. The goal of this study was to determine the role of mTOR in type I collagen regulation. The pharmacological inhibitor of phosphatidylinositol (PI) 3-kinase, LY294002, significantly inhibited collagen type I protein and mRNA levels. The effects of LY294002 were more pronounced on the collagen ␣1(I) chain, which was inhibited at the transcriptional and mRNA stability levels versus collagen ␣2(I) chain, which was inhibited through a decrease in mRNA stability. In contrast, addition of the PI 3-kinase inhibitor, wortmannin, did not alter type I collagen steady-state mRNA levels. This observation and further experiments using an inactive LY294002 analogue suggested that collagen mRNA levels are inhibited independent of PI 3-kinase. Additional experiments have established that mTOR positively regulates collagen type I synthesis in human fibroblasts. These conclusions are based on results demonstrating that inhibition of mTOR activity using a specific inhibitor, rapamycin, reduced collagen mRNA levels. Furthermore, decreasing mTOR expression by about 50% by using small interfering RNA resulted in a significant decrease of collagen mRNA (75% COL1A1 decrease and 28% COL1A2 decrease) and protein levels. Thus, mTOR plays an essential role in regulating basal expression of collagen type I gene in dermal fibroblasts. Together, our data suggest that the classical PI 3-kinase pathway, which places mTOR downstream of PI 3-kinase, is not involved in mTOR-dependent regulation of type I collagen synthesis in dermal fibroblasts. Because collagen overproduction is a main feature of fibrosis, identification of mTOR as a critical mediator of its regulation may provide a suitable target for drug or gene therapy.A common characteristic of all fibrotic diseases, including scleroderma, is an abnormal accumulation of extracellular matrix proteins. Fibrotic lesions disrupt normal tissue architecture and contribute to organ failure. Type I collagen, the primary component of fibrotic lesions, is a triple helix composed of two ␣1 chains and one ␣2 chain. These chains, although coordinately expressed, are not regulated via the same mechanisms. It is well established that collagen protein degradation, mRNA stability, and transcription are tightly regulated during collagen biosynthesis. Signals from external stimuli such as cytokines (1-3), nutrients (4), and cell interactions (5, 6) modulate these processes via several pathways, including TGF- 1 / p38 (7, 8), PKC (9), and stress-activated protein kinase/c-Jun N-terminal kinase (10, 11). Despite these advances, the pathways controlling collagen biosynthesis are not fully characterized, prompting us to further examine potential signaling molecules involved in type I collagen regulation.Prior studies suggested the involvement of phosphatidylinositol 3-kinase (PI 3-kinase), a ubiquitous lipid kinase, in collagen regulation. For example, Ivarsson et al....
Transforming growth factor beta has been implicated as a mediator of excessive extracellular matrix deposition in scar tissue and fibrosis, including systemic sclerosis. To further characterize the mechanism of collagen gene expression in systemic sclerosis and healthy skin fibroblasts, we examined the role of p38 MAPK signaling in collagen gene regulation by transforming growth factor beta. Treatment of dermal fibroblasts with transforming growth factor beta resulted in a prolonged activation of p38 MAPK. Furthermore, a specific inhibitor of p38 suppressed transforming growth factor beta stimulation of collagen type I mRNA and the alpha2(I) collagen promoter activity. To further probe the role of p38 in collagen regulation by transforming growth factor beta, we utilized an expression vector containing p38alpha cDNA. Ectopic expression of p38alpha enhanced COL1A2 promoter activity and potentiated transforming growth factor beta stimulation of this promoter. The p38 response element in the COL1A2 promoter overlapped with the previously characterized transforming growth factor beta response element. Consistent with these observations, collagen type I mRNA and protein levels were increased in transforming-growth-factor-beta-stimulated fibroblasts transduced with an adenoviral vector expressing p38alpha. To determine the possible role of p38 in abnormal collagen production by systemic sclerosis fibroblasts, p38 protein levels were compared in systemic sclerosis and healthy skin fibroblasts. Both cell types exhibited similar total levels of p38 MAPK and similar kinetics of p38 activation in response to transforming growth factor beta. In conclusion, this study demonstrates a costimulatory role for p38 MAPK in transforming growth factor beta induction of the collagen type I gene. Expression levels and activation status of p38 are not consistently elevated in systemic sclerosis fibroblasts suggesting that the p38 MAPK pathway is not dysregulated in systemic sclerosis fibroblasts.
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