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...

Objective. Aberrant transforming growth factor ␤ (TGF␤) signaling has been implicated in the pathogenesis of scleroderma (systemic sclerosis [SSc]), but the contribution of specific components in this pathway to SSc fibroblast phenotype remains unclear. This study was undertaken to delineate the role of TGF␤ receptor type I (TGF␤RI) and TGF␤RII in collagen overexpression by SSc fibroblasts.Methods. Primary dermal fibroblasts from SSc patients and healthy adults were studied (n ‫؍‬ 10 matched pairs). Adenoviral vectors were generated for TGF␤RI (AdTGF␤RI), TGF␤RII (AdTGF␤RII), and kinase-deficient TGF␤RII (Ad⌬kRII). TGF␤RI basal protein levels were analyzed by 35 S-methionine labeling/ immunoprecipitation and immunohistochemistry. Type I collagen and TGF␤RII basal protein levels were analyzed by Western blot and newly secreted collagen by 3 H-proline incorporation assay.Results. Analysis of endogenous TGF␤RI and TGF␤RII protein levels revealed that SSc TGF␤RI levels were increased 1.7-fold (P ‫؍‬ 0.008; n ‫؍‬ 7) compared with levels in healthy controls, while TGF␤RII levels were decreased by 30% (P ‫؍‬ 0.03; n ‫؍‬ 7). This increased TGF␤RI:TGF␤RII ratio correlated with SSc collagen overexpression. To determine the consequences of altered TGF␤RI:TGF␤RII ratio on collagen expression, healthy fibroblasts were transduced with AdTGF␤RI or AdTGF␤RII. Forced expression of TGF␤RI in the range corresponding to elevated SSc TGF␤RI levels increased basal collagen expression in a dose-dependent manner, while similar TGF␤RII overexpression had no effect, although transduction of fibroblasts at higher multiplicities of infection led to a marked reduction of basal collagen levels. Blockade of TGF␤ signaling via Ad⌬kRII resulted in ϳ50% inhibition of basal collagen levels in healthy fibroblasts and in 5 of 9 SSc cell lines. A subset of SSc fibroblasts (4 of 9 cell lines) was resistant to this treatment. SSc fibroblasts with the highest levels of TGF␤RI were the least responsive to collagen inhibition via ⌬kRII.Conclusion. This study indicates that an increased TGF␤RI:TGF␤RII ratio may underlie aberrant TGF␤ signaling in SSc and contribute to elevated basal collagen production, which is insensitive to TGF␤ signaling blockade via ⌬kRII.Scleroderma (systemic sclerosis [SSc]) is characterized by excessive deposition of extracellular matrix

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Sphingosine kinase (SphK) is a conserved lipid kinase that catalyzes formation of important regulators of inter- and intracellular signaling, sphingosine-1 phosphate (S1P), and dihydrosphingosine 1-phosphate (dhS1P). In this study, we investigated the role of SphK1 in the regulation of expression of matrix metalloproteinase 1 (MMP1) in dermal fibroblasts, a key event in regulation of extra cellular matrix. We show that overexpression of SphK1 up-regulated MMP1 protein, MMP1 mRNA, and MMP1 promoter activity, and this action of SphK1 required activation of the ERK1/2-Ets1 and NF-kappaB pathways. Furthermore, experiments using SphK1 specific siRNA demonstrated that SphK1 is required for the TNF-alpha stimulation of MMP1. Additional data revealed a specific role of dhS1P, and not S1P, as a mediator of SphK1-dependent activation of ERK1/2 and up-regulation of MMP1. The stimulatory effect of dhS1P was sensitive to pertussis toxin, suggesting a possible involvement of a G-protein-coupled receptor. In contrast, S1P, but not dhS1P, stimulated the induction of COX-2, which demonstrated selective actions of these two closely related bioactive lipids. In conclusion, this study describes a novel mode of SphK1 signaling through generation of dhS1P with a key role in mediating transcriptional responses to TNF-alpha. This is the first report of selective function of dhS1P as compared with the better studied S1P.