Sphingosine 1-phosphate stimulates smooth muscle cell differentiation and proliferation by activating separate serum response factor co-factors.
Sphingosine 1-phosphate (S1P) is a lipid agonist that regulates smooth muscle cell (SMC) and endothelial cell functions by activating several members of the S1P subfamily of G-protein-coupled Edg receptors. We have shown previously that SMC differentiation is regulated by RhoA-dependent activation of serum response factor (SRF). Because S1P is a strong activator of RhoA, we hypothesized that S1P would stimulate SMC differentiation. Treatment of primary rat aortic SMC cells with S1P activated RhoA as measured by precipitation with a glutathione S-transferase-rhotekin fusion protein. In SMC and 10T1/2 cells, S1P treatment up-regulated the activities of several transiently transfected SMC-specific promoters, and these effects were inhibited by the Rho-kinase inhibitor, Y-27632. S1P also increased smooth muscle alpha-actin protein levels in SMC but had no effect on SRF binding to the smooth muscle alpha-actin CArG B element. Quantitative reverse transcriptase-PCR showed that S1P treatment of SMC or 10T1/2 cells did not increase the mRNA level of either of the recently identified SRF co-factors, myocardin or myocardin-related transcription factor-A (MRTF-A). MRTF-A protein was expressed highly in SMC and 10T1/2 cultures, and importantly the effects of S1P were inhibited by a dominant negative form of MRTF-A indicating that S1P may regulate the transcriptional activity of MRTF-A. Indeed, S1P treatment increased the nuclear localization of FLAG-MRTF-A, and the effect of MRTF-A overexpression on smooth muscle alpha-actin promoter activity was inhibited by dominant negative RhoA. S1P also stimulated SMC growth by activating the early growth response gene, c-fos. This effect was not attenuated by Y-27632 but could be inhibited by the MEK inhibitor, UO126. S1P enhanced SMC growth through ERK-mediated phosphorylation of the SRF co-factor, Elk-1, as measured by gel shift and Elk-1 activation assays. Taken together these results demonstrate that S1P activates multiple signaling pathways in SMC and regulates proliferation by ERK-dependent activation of Elk-1 and differentiation by RhoA-dependent activation of MRTF-A.
Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Kashelle
K
Lockman
Jeremiah S
JS
Hinson
Matt D
MD
Medlin
Dionne
D
Morris
Joan M
JM
Taylor
Christopher P
CP
Mack
D000577:Amides / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D019204:GTP-Binding Proteins; D005799:Genes, Dominant; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008246:Lysophospholipids / Q000378:metabolism*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D041681:NIH 3T3 Cells; D009570:Nitriles / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D026362:Serum Response Factor / Q000378:metabolism*; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D015854:Up-Regulation; D050807:ets-Domain Protein Elk-1; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D000099251:Myocardin
D000577:Amides; D051017:Apoptosis Regulatory Proteins; D002070:Butadienes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D019204:GTP-Binding Proteins; D005982:Glutathione Transferase; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids; D020928:Mitogen-Activated Protein Kinases; D009570:Nitriles; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011725:Pyridines; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D026362:Serum Response Factor; D013110:Sphingosine; D015534:Trans-Activators; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D020742:rhoA GTP-Binding Protein; D000099251:Myocardin; C494113:Elk1 protein, mouse; C494114:Elk1 protein, rat; C498395:Rtkn protein, mouse; C498396:Rtkn protein, rat; C113580:U 0126; C108830:Y 27632; C060506:sphingosine 1-phosphate