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Dihydrosphingosine 1-phosphate stimulates MMP1 gene expression via activation of ERK1/2-Ets1 pathway in human fibroblasts

Shizhong Bu^*, Masayoshi Yamanaka^*,1, Huiping Pei^*, Alicja Bielawska, Jacek Bielawski, Yusuf A. Hannun, Lina Obeid and Maria Trojanowska^*,2

^* Division of Rheumatology and Immunology and the
Department of Biochemistry and Molecular Biology, Medical University of South Carolina and the
Division of General Internal Medicine, Ralph H. Johnson Veterans Affairs Hospital, Charleston, South Carolina

²Correspondence: Division of Rheumatology & Immunology, Medical University of South Carolina, 96 Jonathan Lucas St., Suite 912, P.O. Box 250637, Charleston, SC 29425, USA. E-mail: trojanme{at}musc.edu

SPECIFIC AIMS

Studies have shown that sphingosine kinase 1 (SphK1) controls expression of TIMP1 (tissue inhibitor of metalloproteinase 1) gene in human fibroblast, suggesting that SphK1 may regulate cellular pathways involved in extracellular matrix (ECM) remodeling. To further investigate the involvement of SphK1 in this process, we focused on regulation of MMP1 (matrix metalloproteinase 1), a principal enzyme responsible for degradation of interstitial collagens.

PRINCIPAL FINDINGS

1. SphK1 is required for the TNF stimulation of MMP1 in dermal fibroblasts through activation of the ERK1/2-Ets1 and NF-B pathways
To determine the effect of SphK1 overexpression on MMP1 production, human dermal fibroblasts were transduced with adenoviral vector carrying SphK1 gene (SphK1Ad). SphK1 overexpression resulted in up-regulation of MMP1 protein and mRNA, as well as promoter activity in a dose-dependent and time-dependent manner, with a potency comparable to an MMP1 inducer, TNF. To investigate the nature of the signaling pathways involved in the SphK1-dependent up-regulation of MMP1, specific pharmacologic inhibitors were used. The inhibitor of ERK1/2, UO 126, and the inhibitor of NFB, SN50, abrogated SphK1-dependent stimulation of MMP1, whereas inhibitors of JNK and p38 pathways had no effect. Furthermore, SphK1 activated ERK1/2 and induced threonine phosphorylation of ERK1/2 effector, Ets1, consistent with the previously established role of this pathway in MMP1 gene activation (Fig. 1 ). Reduction of endogenous SphK1 expression by RNA interference prevented TNF stimulation of MMP1, indicating that SphK1 is required for the TNF induction of MMP1.

View larger version (24K): [in this window] [in a new window]   Figure 1. MMP-1 stimulation by SphK1 is mediated through activation of ERK1/2-Ets1 signaling pathway. A) Cells were transduced for 24 h with 50 or 100 m.o.i. of control or SphK1Ad in the presence or absence of 10 µM of ERK1/2 inhibitor UO 126 (UO), JNK inhibitor SP600125 (SP), or p38 inhibitor SB203580 (SB). MMP1 protein levels were analyzed by Western blot. B) SphK1 induces phospho-ERK1/2 in a time-dependent manner. Cells were transduced with 50 m.o.i. of control or Sphk1Ad for the indicated time periods. Phospho- and total Erk1/2, phospho- and total p38, and phospho- and total JNK were analyzed by Western blot. Treatment for 20 min with 0.7M of NaCl (O-S) was used as positive control. C) SphK1 and TNF stimulate Ets1 phosphorylation. Cells were either transduced with 50 m.o.i. of SphK1 or stimulated with 10 ng/mL of TNF for 24 h. Ets1 threonine phosphorylation was analyzed by IP/Western. D) Ets1 phosphorylation depends on activation of ERK 1/2. Cells were either transduced with 50 m.o.i. of SphK1 or stimulated with 10ng/mL of TNF for 24 h in the presence or absence of 10 µM of ERK1/2 inhibitor UO 126 (UO).

2. Dihydrosphingosine-1-phosphate (dhS1P) activates ERK1/2 and stimulates MMP1 production in dermal fibroblasts
SphK is a conserved lipid kinase that catalyzes formation of sphingosine-1 phosphate (S1P) and dihydrosphingosine 1-phosphate (dhS1P). A role for S1P as a regulator of inter- and intracellular signaling is well established, but little is known about the biologic function of dhS1P. Sphingolipid analyses revealed that overexpressed SphK1 potently increased intracellular dhS1P levels (>100-fold) as well as the levels of S1P (10-fold). To determine which of these sphingolipids is a mediator of SphK1-dependent activation of ERK1/2 and up-regulation of MMP1, fibroblasts were stimulated with increasing doses (25–500 nM) of exogenously added dhS1P, dhSph, S1P, C₆-ceramide, and Sph. Only dhS1P (25–500 nM), and dhSph (75–500 nM) stimulated MMP1 production in a dose-dependent manner. Furthermore, SphK1-specific siRNA abolished dhSph-mediated induction of MMP1 and phosphoERK1/2, while induction by dhS1P was not affected, suggesting that the effects of exogenous dhSph required conversion to dhS1P (Fig. 2 ). The stimulatory effect of dhS1P was sensitive to pertussis toxin, suggesting a receptor-mediated mechanism. In contrast, S1P, but not dhS1P stimulated the induction of COX-2, demonstrating selective actions of these two closely related bioactive lipids.

View larger version (27K): [in this window] [in a new window]   Figure 2. Effect of dhS1P and other related sphingolipids on MMP1 protein levels. A) Fibroblasts were treated with increasing concentrations of indicated sphingolipids for 24 h. MMP1 protein levels were determined by Western blot. B) The output signal was quantified using NIH Image and the data are summarized graphically (n=2). C) Cells were pretreated with 200 nM of nonsilencing or SphK1-specific siRNAs for 24 h and then stimulated with 500 nM of dhS1P or dhSph for additional 24 h. MMP1, p-ERK/2, and total ERK1/2 were analyzed by Western blot. D) Cells were treated with increasing doses (25–500 nM) of S1P or dhS1P for 24 h. COX-2 protein levels were analyzed by Western blot.

CONCLUSIONS AND SIGNIFICANCE

The findings of this study demonstrate for the first time that distinct biologic responses induced by a pleiotropic cytokine TNF are mediated via two different lipid mediators generated by the action of SphK1, dhS1P and S1P. S1P, but not dhS1P, regulates induction of a proinflammatory mediator COX-2. In contrast, dhS1P, but not S1P induces MMP1, a key enzyme in matrix degradation. This is the first report of selective function of dhS1P as compared with the better studied S1P. The nature of the putative dhS1P receptor and the mode of action of this novel lipid mediator remain to be established.

View larger version (30K): [in this window] [in a new window]   Figure 3. S1P and dhS1P mediate distinct biologic response.

FOOTNOTES

¹ Current address: Department of Dermatology, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma, 371-8511, Japan.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4646fje;

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