Blood Lipid Mediator Sphingosine 1-Phosphate Potently Stimulates Platelet-derived Growth Factor-A and -B Chain Expression through S1P1-Gi-Ras-MAPK-dependent Induction of Krüppel-like Factor 5
2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês
10.1074/jbc.m305025200
ISSN1083-351X
AutoresSoichiro Usui, Naotoshi Sugimoto, Noriko Takuwa, Satoru Sakagami, Shigeo Takata, Shuichi Kaneko, Yoh Takuwa,
Tópico(s)Lipid metabolism and biosynthesis
ResumoPlatelet-derived growth factors (PDGFs), potent mitogens and chemoattractants for mesenchymal cell types, play essential roles in development of several organs including blood vessels, kidney, and lung, and are also implicated in the pathogenesis of atherosclerosis and malignancies. Blood lipid mediator sphingosine 1-phosphate (S1P) regulates migration, proliferation, and apoptosis in a variety of cell types through multiple G protein-coupled receptors of the Edg family, and is necessary for vascular formation at the developmental stage. We found in the present study that S1P induced severalfold increases in the mRNA and protein levels of PDGF-A and -B chains in vascular smooth muscle cells and neointimal cells. S1P stimulation of PDGF mRNA and protein expression was abolished by the small interfering RNA duplexes targeting S1P1/Edg1 receptor subtype. S1P stimulated the small GTPase Ras in a Gi-dependent manner, and activated ERK and p38 MAPK in Gi- and Ras-dependent manners. Pertussis toxin pretreatment, adenovirus-mediated Asn17Ras expression, the MEK inhibitor PD98059, or the p38 MAPK inhibitor SB203580 markedly suppressed PDGF mRNA and protein up-regulation, indicating the involvement of Gi-Ras-ERK/p38 MAPK in S1P stimulation of PDGF expression. S1P stimulated expression of the transcription factor KLF5 in manners dependent on Gi, Ras, and ERK/p38 MAPK. Down-regulation of KLF5 by small interfering RNA duplexes abolished S1P-induced PDGF-A and -B chain expression. On the other hand, overexpression of KLF5 stimulated basal and S1P-induced PDGF expression. Either S1P stimulation or KLF5 overexpression increased the PDGF-B promoter activity in a cis-element-dependent manner. These results reveal the S1P1-triggered, Gi-Ras-ERK/p38 MAPK-KLF5-dependent, stimulatory regulation of PDGF gene transcription in vascular smooth muscle cells. Platelet-derived growth factors (PDGFs), potent mitogens and chemoattractants for mesenchymal cell types, play essential roles in development of several organs including blood vessels, kidney, and lung, and are also implicated in the pathogenesis of atherosclerosis and malignancies. Blood lipid mediator sphingosine 1-phosphate (S1P) regulates migration, proliferation, and apoptosis in a variety of cell types through multiple G protein-coupled receptors of the Edg family, and is necessary for vascular formation at the developmental stage. We found in the present study that S1P induced severalfold increases in the mRNA and protein levels of PDGF-A and -B chains in vascular smooth muscle cells and neointimal cells. S1P stimulation of PDGF mRNA and protein expression was abolished by the small interfering RNA duplexes targeting S1P1/Edg1 receptor subtype. S1P stimulated the small GTPase Ras in a Gi-dependent manner, and activated ERK and p38 MAPK in Gi- and Ras-dependent manners. Pertussis toxin pretreatment, adenovirus-mediated Asn17Ras expression, the MEK inhibitor PD98059, or the p38 MAPK inhibitor SB203580 markedly suppressed PDGF mRNA and protein up-regulation, indicating the involvement of Gi-Ras-ERK/p38 MAPK in S1P stimulation of PDGF expression. S1P stimulated expression of the transcription factor KLF5 in manners dependent on Gi, Ras, and ERK/p38 MAPK. Down-regulation of KLF5 by small interfering RNA duplexes abolished S1P-induced PDGF-A and -B chain expression. On the other hand, overexpression of KLF5 stimulated basal and S1P-induced PDGF expression. Either S1P stimulation or KLF5 overexpression increased the PDGF-B promoter activity in a cis-element-dependent manner. These results reveal the S1P1-triggered, Gi-Ras-ERK/p38 MAPK-KLF5-dependent, stimulatory regulation of PDGF gene transcription in vascular smooth muscle cells. Sphingosine 1-phosphate (S1P) 1The abbreviations used are: S1P, sphingosine 1-phosphate; Edg, endothelial differentiation gene; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; KLF, Krüppel-like factor; MAPK, mitogen-activated protein kinase; PDGF, platelet-derived growth factor; RASM, rat aortic smooth muscle cells; siRNA, small interfering RNA; VSMC, vascular smooth muscle cells; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; 8W-RASM, rat aortic smooth muscle cells from an 8-week Wistar male rat; PTX, pertussis toxin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; GST, glutathione S-transferase; RT, reverse transcription; DMEM, Dulbecco's modified Eagle's medium. is a pleiotropic lysophospholipid mediator present in plasma and is released in large amounts from activated platelets (1Spiegel S. Milstien S. Nat. Rev. Mol. Cell. Biol. 2003; 4: 397-407Crossref PubMed Scopus (1758) Google Scholar, 2Hla T. Lee M.-J. Ancellin N. Paik J.H. Kluk M.J. Science. 2001; 294: 1875-1878Crossref PubMed Scopus (479) Google Scholar, 3Fukushima N. Ishii I. Contos J.J.A. Weiner J.A. Chun J. Annu. Rev. Pharmacol. 2001; 131: 767-771Google Scholar, 4Takuwa Y. Biochim. Biophys. Acta. 2002; 1582: 112-120Crossref PubMed Scopus (153) Google Scholar). S1P exerts diverse activities on various cell types, which include stimulation of gene expression and cell proliferation, suppression of apoptosis, and regulation of cell motility and cytoskeletal reorganization. It is now accepted that many of the S1P actions are mediated through the endothelial differentiation gene (Edg) family G protein-coupled S1P receptor isoforms, which comprise S1P1/Edg1, S1P2/Edg5/AGR16, S1P3/Edg3, S1P4/Edg6, and S1P5/Edg8 (1Spiegel S. Milstien S. Nat. Rev. Mol. Cell. Biol. 2003; 4: 397-407Crossref PubMed Scopus (1758) Google Scholar, 2Hla T. Lee M.-J. Ancellin N. Paik J.H. Kluk M.J. 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Yokomizo T. Sugimoto N. Sakurada S. Shigematsu H. Takuwa Y. Mol. Cell. Biol. 2000; 20: 9247-9261Crossref PubMed Scopus (288) Google Scholar, 9Ryu Y. Takuwa N. Sugimoto N. Skurada S. Usui S. Okamoto H. Matsui O. Takuwa Y. Circ. Res. 2002; 90: 325-332Crossref PubMed Scopus (200) Google Scholar). Intriguingly, a recent study (10Liu Y. Wada R. Yamashita T. Mi Y. Deng C.-X. Hobson J.P. Rosenfeldt H.M. Nava V.E. Chae S.-S. Lee M.-J. Liu G.H. Hla T. Spiegel S. Proia R.L. J. Clin. Invest. 2000; 106: 951-961Crossref PubMed Scopus (993) Google Scholar) revealed a developmental role of S1P in the vascular maturation; in mice with disruption of the S1P1 receptor gene, perivascular accumulation of VSMCs and pericytes at the fetal developmental stage was defective. Platelet-derived growth factor (PDGF)-A and -B chains induce a diverse array of cellular responses including cell proliferation, migration, and cell survival in a variety of mesenchymal cell types (11Heldin C.H. EMBO J. 1992; 11: 4251-4259Crossref PubMed Scopus (317) Google Scholar). Especially, PDGF-B chain has been implicated in the formation of various types of vascular proliferative lesions including atherosclerosis, intimal smooth muscle cell accumulation after angioplasty and in grafted vessels, and accelerated arteriosclerosis in transplanted organs (12Ross R. Nature. 1995; 362: 801-809Crossref Scopus (9989) Google Scholar, 13Schwartz S.M. Circ. Res. 1995; 77: 445-465Crossref PubMed Scopus (898) Google Scholar). Besides the pathogenetic roles of PDGFs and PDGF receptors, developmental roles of PDGFs during fetal morphogenesis were demonstrated; it was shown that mice with ablation of either PDGF-B chain gene or PDGF-B chain-specific receptor PDGF-β gene exhibited defects in recruitment and differentiation of vascular mural cells, i.e. vascular remodeling and maturation, after the initial endothelial tube formation (14Hellstrom M. Kalen M. Lindahl P. Abramsson A. Betsholtz C. Development. 1999; 126: 3047-3055PubMed Google Scholar). It was also shown that the homozygous null mutation of PDGF-A gene led to impaired development of alveolar myofibroblasts (15Bostrom H. Willetts K. Pekny M. Leveen P. Lindaht P. Hedstrand H. Pekna M. Hellstrom M. Gebre-Medhin S. Schalling M. Nilsson M. Kurland S. Tornell J. Heath J.K. Betsholtz C. Cell. 1996; 85: 863-873Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar). Despite the potential importance of PDGFs in vascular occlusive diseases and a variety of physiological processes, only a little is known about the regulator of PDGF expression until recently. We and others have previously shown that angiotensin II, which was implicated in neointima formation after vascular injury, induced expression of PDGF-A and -B in VSMCs (16Deguchi J. Makuuchi M. Nakaoka T. Collins T. Takuwa Y. Circ. Res. 1999; 85: 565-574Crossref PubMed Scopus (43) Google Scholar, 17Itoh H. Mukoyama M. Pratt R.E. Gibbons G.M. Dzau V.J. J. Clin. Invest. 1993; 91: 2268-2274Crossref PubMed Scopus (462) Google Scholar). However, it is largely unknown whether at all and how other extracellular mediators are involved in the regulation of PDGF expression under pathological and physiological conditions. In the present study, we demonstrated that S1P potently stimulates PDGF-A and -B chain mRNA and protein expression in VSMCs. Our results suggest that S1P1 receptor is a major isoform to mediate this S1P action. We also show that the stimulatory action of S1P1 is relayed via Gi, Ras, and other small G proteins, and their downstream protein kinases extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and Rho kinase. The transcription factor Krüppel-like factor (KLF)-5/IKLF/BTEB2 belongs to the family of mammalian Krüppel-like transcription factors (18Bieker J.J. J. Biol. Chem. 2001; 276: 34355-34358Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar). KLF5 is implicated in phenotypic changes of activated VSMCs, which include alterations in the gene expression, in the site of vascular injury (19Watanabe N. Kurbayashi M. Shimomura Y. Kawai-Kowase K. Hoshino Y. Manabe I. Watanabe M. Aikawa M. Kuro-o M. Suzuki T. Yazaki Y. Nagai R. Circ. Res. 1999; 85: 182-191Crossref PubMed Scopus (124) Google Scholar). A recent study (20Shindo T. Manabe I. Fukushima Y. Tobe K. Aizawa K. Miyamoto S. Kawai-Kowase K. Moriyama N. Imai Y. Kawakami H. Hishimatsu H. Ishikawa T. Suzuki T. Morita H. Maemura K. Sata M. Hirata Y. Komukai M. Kagechika H. Kadowaki T. Kurabayashi M. Nagai R. Nat. Med. 2002; 8: 856-863Crossref PubMed Scopus (329) Google Scholar) demonstrated that ablation of KLF5 gene suppressed vascular injury-induced vascular proliferative lesion formation. We observed that KLF5 stimulated PDGF-A and -B chain gene expression in a cis-element-dependent manner and that suppression of S1P-induced KLF5 expression by RNA interference abrogated PDGF mRNA and protein expression. Thus, the present study demonstrates the novel role of S1P-Edg receptor system in the stimulatory regulation of PDGFs, which is exerted at the transcriptional level through mechanisms involving the up-regulation of KLF5. Materials—S1P and other related lipids, were purchased, aliquoted and stored as described previously (8Okamoto H. Takuwa N. Yokomizo T. Sugimoto N. Sakurada S. Shigematsu H. Takuwa Y. Mol. Cell. Biol. 2000; 20: 9247-9261Crossref PubMed Scopus (288) Google Scholar). Pertussis toxin (PTX) was purchased from List Biological. The following antibodies were employed in the present study: mouse monoclonal anti-RhoA (clone 26C4, Santa Cruz), mouse monoclonal anti-Rac antibody (Upstate Biotechnology Inc.), mouse monoclonal anti-Ras (clone 18, Transduction Laboratories), mouse monoclonal anti-ERK1 and -2 antibody (clone 13-6200, Zymed Laboratories Inc.), rabbit polyclonal anti-c-Jun N-terminal kinase (JNK)-1 (C-17), anti-p38 MAPK (C-20) antibodies (Santa Cruz), mouse monoclonal anti-α-tubulin antibody (NEO Markers), rabbit polyclonal anti-PDGF-A (N-30) antibody (Santa Cruz), and mouse monoclonal anti-PDGF-B antibody (Mochida). Mouse monoclonal anti-HA antibody and rabbit polyclonal anti-β galactosidase were purchased from Nacalai Tesque (Kyoto, Japan) and Organon Teknika (West Chester, PA), respectively. Rat monoclonal anti-KLF5 antibody (20Shindo T. Manabe I. Fukushima Y. Tobe K. Aizawa K. Miyamoto S. Kawai-Kowase K. Moriyama N. Imai Y. Kawakami H. Hishimatsu H. Ishikawa T. Suzuki T. Morita H. Maemura K. Sata M. Hirata Y. Komukai M. Kagechika H. Kadowaki T. Kurabayashi M. Nagai R. Nat. Med. 2002; 8: 856-863Crossref PubMed Scopus (329) Google Scholar) was donated by Dr. Kurabayashi (Gunma University, Maebashi, Japan). Cell Culture—Rat neointimal VSMCs and newborn male Wistar rat aortic medial VSMCs (RASM) cells, and RASM cells from an 8-week Wistar male rat (8W-RASM cells) were established and maintained as described previously (9Ryu Y. Takuwa N. Sugimoto N. Skurada S. Usui S. Okamoto H. Matsui O. Takuwa Y. Circ. Res. 2002; 90: 325-332Crossref PubMed Scopus (200) Google Scholar, 16Deguchi J. Makuuchi M. Nakaoka T. Collins T. Takuwa Y. Circ. Res. 1999; 85: 565-574Crossref PubMed Scopus (43) Google Scholar). Two days before each experiment, cells were switched to the respective medium supplemented with 0.1% fatty acid-free bovine serum albumin (Sigma). Cells between the 5th and 15th were used in the present study. Northern Blot Analysis and RT-PCR—Twenty micrograms of total RNA, isolated from VSMCs by the acid-guanidinium isothiocyanate/phenol/chloroform method, was separated by formaldehyde, 1.0% agarose gel electrophoresis and transferred onto a nylon membrane (GeneScreen, PerkinElmer Life Sciences) (21Noda M. Katoh T. Takuwa N. Kumada M. Kurokawa K. Takuwa Y. J. Biol. Chem. 1994; 269: 17911-17917Abstract Full Text PDF PubMed Google Scholar), followed by hybridization with rat PDGF-A and -B chain cDNA probes (22Katayose D. Ohe M. Yamauchi K. Ogata M. Shirto K. Fujita H. Shibahara S. Takishima T. Am. J. Physiol. 1993; 204: L100-L106Google Scholar) labeled with [γ-32P]dCTP by the random priming method. A membrane was rehybridized with 32P-labeled GAPDH cDNA probe (21Noda M. Katoh T. Takuwa N. Kumada M. Kurokawa K. Takuwa Y. J. Biol. Chem. 1994; 269: 17911-17917Abstract Full Text PDF PubMed Google Scholar). The radioactivity of corresponding bands was quantified by Fuji BAS 5000 Bio-Image Analyzer (Fuji Film). PDGF mRNA levels normalized for GAPDH mRNA level (PDGF-A or -B mRNA/GAPDH mRNA radioactivity) were expressed as multiples over a value in unstimulated cells, which was expressed as 1.0 (16Deguchi J. Makuuchi M. Nakaoka T. Collins T. Takuwa Y. Circ. Res. 1999; 85: 565-574Crossref PubMed Scopus (43) Google Scholar, 21Noda M. Katoh T. Takuwa N. Kumada M. Kurokawa K. Takuwa Y. J. Biol. Chem. 1994; 269: 17911-17917Abstract Full Text PDF PubMed Google Scholar). RT-PCR was performed as described (9Ryu Y. Takuwa N. Sugimoto N. Skurada S. Usui S. Okamoto H. Matsui O. Takuwa Y. Circ. Res. 2002; 90: 325-332Crossref PubMed Scopus (200) Google Scholar). The 432-bp fragment (nucleotide 607–1038 of S1P1, when “A” of the initiation codon AUG was numbered as 1) and the 214-bp fragment (nucleotide 353–566) of β-actin were amplified by using the primer pairs: 5′-TATATTCTCTTCTGCACCACC-3′ and 5′-GATGATGGGCCTCTTGAATTT-3′ for S1P1, and 5′-ATCATGTTTGAGACCTTCAACAC-3′ and 5′-GGATCTTCATGAGGTAGTCAGT-3′ for β-actin. The PCR comprised 30 cycles of 0.5 min at 94 °C, 0.5 min at 53 °C for S1P1 and 54 °C for β-actin, and 0.5 min at 72 °C. Determination of Activities of ERK, JNK, and p38 MAPK—The activation of p42 ERK2 was determined by detection of band shift with Western blot analysis of total cell lysate, and the results were quantified as described previously (16Deguchi J. Makuuchi M. Nakaoka T. Collins T. Takuwa Y. Circ. Res. 1999; 85: 565-574Crossref PubMed Scopus (43) Google Scholar). The activation of c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) was determined by the solid-phase kinase assay and immunoprecipitation kinase assay using glutathione S-transferase (GST)-c-Jun and GST-ATF2, respectively, and [γ-32P]ATP as substrates, as described previously (16Deguchi J. Makuuchi M. Nakaoka T. Collins T. Takuwa Y. Circ. Res. 1999; 85: 565-574Crossref PubMed Scopus (43) Google Scholar, 23Hamada K. Takuwa N. Yokoyama K. Takuwa Y. J. Biol. Chem. 1998; 273: 6334-6340Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The quantification of activities of MAPK activities was performed by densitometry of the corresponding bands using the Quantity one image analyzing system (PDI, Inc.) for ERK and determination of radioactivity using Fuji BAS 5000 Bio-Image Analyzer for JNK and p38 MAPK, as described previously (16Deguchi J. Makuuchi M. Nakaoka T. Collins T. Takuwa Y. Circ. Res. 1999; 85: 565-574Crossref PubMed Scopus (43) Google Scholar, 23Hamada K. Takuwa N. Yokoyama K. Takuwa Y. J. Biol. Chem. 1998; 273: 6334-6340Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The results were expressed as multiples over a value in unstimulated cells, which was expressed as 1.0. Pull-down Assay of Rho, Rac, and Ras—The amounts of GTP-bound forms of RhoA and Rac were determined as described in detail previously (8Okamoto H. Takuwa N. Yokomizo T. Sugimoto N. Sakurada S. Shigematsu H. Takuwa Y. Mol. Cell. Biol. 2000; 20: 9247-9261Crossref PubMed Scopus (288) Google Scholar, 26Sakurada S. Okamoto H. Takuwa N. Sugimoto N. Takuwa Y. Am. J. Physiol. 2001; 281: C571-C578Crossref PubMed Google Scholar). The amount of a GTP-bound form of Ras was determined as described by others (27Marais R. Light Y. Mason C. Paterson H. Paterson M.F. Marshall C.J. Science. 1998; 280: 109-112Crossref PubMed Scopus (401) Google Scholar). Briefly, cell extracts prepared in the same way as for RhoA and Rac were incubated with GST-c-Raf-(51–101) bound to glutathione-Sepharose 4B beads (Amersham Biosciences) at 4 °C for 45 min. The beads were washed as described for RhoA and Rac assay, and Ras bound to beads was analyzed by Western blotting using monoclonal anti-Ras antibody (Transduction Laboratories). A portion (1/100) of cell extracts was also analyzed for the amount of total Ras by Western blotting. The quantification of small G proteins was performed by densitometry as described for MAPK activities in the present study. Plasmids, Adenoviruses and Gene Transfer—Replication-deficient adenoviruses containing each of the genes of Asn17Ras, Asn17Rac, Myc-tagged Asn19RhoA, and LacZ were described and amplified as described previously (8Okamoto H. Takuwa N. Yokomizo T. Sugimoto N. Sakurada S. Shigematsu H. Takuwa Y. Mol. Cell. Biol. 2000; 20: 9247-9261Crossref PubMed Scopus (288) Google Scholar, 16Deguchi J. Makuuchi M. Nakaoka T. Collins T. Takuwa Y. Circ. Res. 1999; 85: 565-574Crossref PubMed Scopus (43) Google Scholar, 25Sugimoto N. Takuwa N. Okamoto H. Sakurada S. Takuwa Y. Mol. Cell. Biol. 2003; 23: 1534-1545Crossref PubMed Scopus (234) Google Scholar). Adenoviruses containing either Myc-tagged human Ala183,Phe185-JNK1 gene (16Deguchi J. Makuuchi M. Nakaoka T. Collins T. Takuwa Y. Circ. Res. 1999; 85: 565-574Crossref PubMed Scopus (43) Google Scholar, 24Mitui H. Takuwa N. Kurokawa K. Exton J.H. Takuwa Y. J. Biol. Chem. 1997; 272: 4904-4910Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) or rat KLF5 (19Watanabe N. Kurbayashi M. Shimomura Y. Kawai-Kowase K. Hoshino Y. Manabe I. Watanabe M. Aikawa M. Kuro-o M. Suzuki T. Yazaki Y. Nagai R. Circ. Res. 1999; 85: 182-191Crossref PubMed Scopus (124) Google Scholar), driven by the CAG promoter that consists of the cytomegalovirus 1E enhancer and chicken β-actin promoter, were generated by use of homologous recombination, as described in detail previously (28Miyake S. Makimura M. Kanegae Y. Harada S. Saito Y. Takamori K. Tokuda C. Saito I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1320-1324Crossref PubMed Scopus (787) Google Scholar). The cells were infected with adenoviruses at a multiplicity of infection of ∼100, and allowed to recover in Dulbecco's modified Eagle's minimal essential medium (DMEM) with 10% fetal calf serum for 3 h, and then serum-deprived before experiments, as described previously (9Ryu Y. Takuwa N. Sugimoto N. Skurada S. Usui S. Okamoto H. Matsui O. Takuwa Y. Circ. Res. 2002; 90: 325-332Crossref PubMed Scopus (200) Google Scholar, 16Deguchi J. Makuuchi M. Nakaoka T. Collins T. Takuwa Y. Circ. Res. 1999; 85: 565-574Crossref PubMed Scopus (43) Google Scholar). This condition conferred expression of LacZ as a marker gene in nearly 100% of transfected cells. The PDGF-B promoter reporter vector Sis-Luc, which contains a 1.0-kilobase pair fragment (–956 to +45 as indicated as the number of base pairs upstream of the TATA box) of the mouse PDGF-B promoter, was described previously (16Deguchi J. Makuuchi M. Nakaoka T. Collins T. Takuwa Y. Circ. Res. 1999; 85: 565-574Crossref PubMed Scopus (43) Google Scholar). Sis-Luc with mutations in the potential KLF5-binding sites was created by a PCR-based method (24Mitui H. Takuwa N. Kurokawa K. Exton J.H. Takuwa Y. J. Biol. Chem. 1997; 272: 4904-4910Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The expression vectors for non-tagged S1P1 and S1P2, pCAGGS-S1P1 and pCAGGS-S1P2, were described previously (8Okamoto H. Takuwa N. Yokomizo T. Sugimoto N. Sakurada S. Shigematsu H. Takuwa Y. Mol. Cell. Biol. 2000; 20: 9247-9261Crossref PubMed Scopus (288) Google Scholar, 25Sugimoto N. Takuwa N. Okamoto H. Sakurada S. Takuwa Y. Mol. Cell. Biol. 2003; 23: 1534-1545Crossref PubMed Scopus (234) Google Scholar). N-terminally HA-tagged S1P1 cDNA was generated by the PCR-based method and ligated onto the mammalian expression vector pME18S at the EcoRI-SpeI sites. β-Galactosidase expression vector, pCAGGS-LacZ, was kindly donated by Dr. I. Saito (University of Tokyo, Institute of Medical Sciences, Tokyo, Japan). The Ras-binding domain (amino acids 51–101) of human c-Raf was obtained by RT-PCR and ligated onto pGEX-2T at the BamHI-EcoRI sites. Rat KLF5 cDNA (19Watanabe N. Kurbayashi M. Shimomura Y. Kawai-Kowase K. Hoshino Y. Manabe I. Watanabe M. Aikawa M. Kuro-o M. Suzuki T. Yazaki Y. Nagai R. Circ. Res. 1999; 85: 182-191Crossref PubMed Scopus (124) Google Scholar) was obtained by RT-PCR and ligated onto pME18S at the EcoRI site. 8W-RASMs were transfected with pCAGGS-S1P1 or -S1P2 using LipofectAMINE (Invitrogen) and selected with 0.2 mg/ml G418 as described previously. RNA Interference—Small interfering RNA duplexes (siRNA) were synthesized by in vitro transcription of RNA from short DNA templates carrying T7 promoter sequences, using a Silencer siRNA construction kit (Ambion). The targeted sequences of S1P1 and KLF5 were 5′-AACTGACTTCAGTGGTGTTCA-3′ (nucleotides 140–160 of rat S1P1) and 5′-AAATTTACCTGCCACTCTGCC-3′ (nucleotides 990–1010 of rat KLF5), respectively. The information of the targeted S1P1 sequence was kindly provided by Ji-Hye Paik (University of Connecticut, Farmington, CT). The nucleotide sequences in sense strands of scrambled RNA duplex were 5′-AATCGCATAGCGTATGTCGTT-3′ (for S1P1) and 5′-TCACGCAGTCATCAACCCTTT-3′ (for KLF5). The synthesized 21-mer sense and antisense RNA strands were hybridized. The VSMCs, which were in a subconfluent state, were transfected by incubating the cells for 4 h with siRNA duplexes at the final concentrations of 25 nm in serum-free Opti-MEM (Invitrogen) by employing OligofectAMINE (Invitrogen), according to the instructions from the manufacturer. After transfection, fetal calf serum-containing DMEM was added to make a final serum concentration of 10% and incubated for 18 h. The cells were then switched to serum-free DMEM, and 24 h later the cells were used for experiments. PDGF-B Promotor Assay—Newborn RASM cells were co-transfected with Sis-Luc and either of the expression vectors encoding KLF5 or an empty vector (pME18S), using LipofectAMINE as described previously (16Deguchi J. Makuuchi M. Nakaoka T. Collins T. Takuwa Y. Circ. Res. 1999; 85: 565-574Crossref PubMed Scopus (43) Google Scholar). Cells were allowed to recover after transfection for 3 h in DMEM containing 10% fetal calf serum and then serum-deprived for 48 h. Cell lysates were prepared, and luciferase activity was measured with a Lumat LB95001 luminometer (Berthold) using the luciferase assay system (Promega), as described previously (16Deguchi J. Makuuchi M. Nakaoka T. Collins T. Takuwa Y. Circ. Res. 1999; 85: 565-574Crossref PubMed Scopus (43) Google Scholar, 24Mitui H. Takuwa N. Kurokawa K. Exton J.H. Takuwa Y. J. Biol. Chem. 1997; 272: 4904-4910Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Luciferase activity was normalized for β-galactosidase activity measured in parallel cultures co-transfected with the β-galactosidase expression plasmid pSV-βgal and either of the expression vectors or empty vector. PDGF Protein Analysis—Proteins in the conditioned media collected from newborn RASM cells were precipitated by trichloroacetic acid and analyzed by Western analysis using anti-PDGF-A chain and -B chain-specific antibodies, as described previously (16Deguchi J. Makuuchi M. Nakaoka T. Collins T. Takuwa Y. Circ. Res. 1999; 85: 565-574Crossref PubMed Scopus (43) Google Scholar). Statistics—The data are presented as the means ± S.E. of three or more determinations. The statistical significance of differences between the two groups was determined by Student's t test, whereas multiple comparisons were analyzed by Scheffe's test. S1P Up-regulates PDGF mRNA Expression in Newborn RASM and Neointimal VSMCs, but Not in 8W-RASM Cells—Newborn RASM expressed a low level of PDGF-B chain mRNA at quiescent states. Stimulation with S1P (100 nm) induced a marked increase in PDGF-B mRNA level, which peaked after 4 h and gradually declined thereafter, but remained elevated for at least 8 h (Fig. 1A, upper). S1P stimulation did not alter GAPDH gene expression. The PDGF-B gene mRNA level normalized for GAPDH mRNA level showed a maximal 5-fold increase above the unstimulated level (Fig. 1A, bottom). Neointimal VSMCs derived from the neointima of injured artery (16Deguchi J. Makuuchi M. Nakaoka T. Collins T. Takuwa Y. Circ. Res. 1999; 85: 565-574Crossref PubMed Scopus (43) Google Scholar) also responded to S1P with induction of PDGF-B mRNA, with a similar time course and to comparable extents as newborn RASM. In RASM cells from an 8-week-old rat (8W-RASM), by contrast, S1P failed to induce any increase in PDGF-B gene expression over the basal unstimulated level. PDGF-A chain mRNA level was in a detectable level in quiescent newborn RASM cells (Fig. 1A). S1P stimulated PDGF-A chain mRNA with a maximal 5-fold increase in newborn RASM cells. As shown in Fig. 1B, the stimulatory effect of S1P on PDGF-B expression in newborn RASM increased dose-dependently with half-maximal and maximal effects obtained at 100 nm and 1 μm, respectively. Besides S1P, dihydro-S1P and, to a lesser extent, sphingosylphosphorylcholine, both of which are weak agonists for S1P receptors (8Okamoto H. Takuwa N. Yokomizo T. Sugimoto N. Sakurada S. Shigematsu H. Takuwa Y. Mol. Cell. Biol. 2000; 20: 9247-9261Crossref PubMed Scopus (288) Google Scholar, 29Okamoto H. Takuwa N. Gonda K. Okazaki H. Chang K. Yatomi Y. Shigematsu H. Takuwa Y. J. Biol. Chem. 1998; 273: 27104-27110Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 31Tamama K. Kon J. Sto K. Tomura H. Kuwabara A. Kimura T. Kanda T. Ohta H. Ui M. Kobayashi I. Okajima F. Biochem. J. 2001; 352: 809-815Google Scholar), induced dose-dependent increases in PDGF-B chain mRNA level, with expected reduced potencies (Fig. 1C). Other sphingolipids, including sphingosine and sphingomyelin, which are not S1P receptor agonists, or lysophosphatidic acid (LPA), which is an agonist for LPA receptor subfamily of the Edg G protein-coupled receptor family (3Fukushima N. Ishii I. Contos J.J.A. Weiner J.A. Chun J. Annu. Rev. Pharmacol. 2001; 131: 767-771Google Scholar), did not induce PDGF-B mRNA expression under our experimental condition (up to 1 μm). Northern analyses of total cellular RNA revealed that newborn and 8W-RASM as well as neointimal VSMCs expressed comparable levels of S1P2 and S1P3 mRNAs (Fig. 1D). However, the expression levels of S1P1 mRNA were quite different among the three cell types; it was abundant in newborn RASM and neointimal VSMCs, whereas it was barely detectable, if any, in 8W-RASM. The results in neointimal cells are consistent with a previous report (5Kluk M.J. Hla T. Circ. Res. 2001; 89: 496-502Crossref PubMed Scopus (152) Google Scholar). The mRNAs of S1P4 or S1P5 were not detectable in either cell type. Newborn RASM cells were stimulated with S1P or left un-stimulated for 4 h, and then actinomycin D, a transcription inhibitor, was added (0 time points) (Fig. 1E). The PDGF-B mRNA level was then monitored over the next 4 h. The PDGF-B mRNA levels in both cell groups decayed with time at comparable rates, with the half-life being 83 and 76 min in the absence and presence of S1P, respectively. The results suggest that S1P up-regulates PDGF-B mRNA level mainly by stimulating transcription, rather than by inhibiting degradation. S1P1 Mediate
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