Genetic and Pharmacological Inhibition of Rho-associated Kinase II Enhances Adipogenesis

Abstract

Rho-associated kinase (ROCK) regulates reorganization of actin cytoskeleton. During adipogenesis, the structure of filamentous actin is converted from long stress fibers to cortical actin, suggesting that the ROCK is involved in adipogenesis. Two ROCK isoforms have been identified: ROCK-I and ROCK-II. However, pharmacological inhibitors of ROCK cannot distinguish two ROCK isoforms. In the present study, we examined the role of ROCK in adipogenesis and actin cytoskeleton using genetic and pharmacological approaches. Y-27632, which inhibits the activity of both ROCK isoforms, enhanced adipogenesis through the up-regulation of adipogenic transcription factors in 3T3-L1 cells. Furthermore, Y-27632 restored inhibition of adipogenesis by lysophosphatidic acid, which activates Rho. Regarding actin cytoskeleton, Y-27632 disrupted stress fibers in 3T3-L1 preadipocytes. Next, we analyzed adipogenesis of mouse embryonic fibroblasts (MEFs) derived from ROCK-I and ROCK-II knock-out mice, respectively. Adipogenesis of ROCK-II (-/-) MEFs was markedly enhanced compared with wild-type MEFs while that of ROCK-I (-/-) MEFs was not. In contrast to pharmacological approaches, no obvious alteration was found in actin cytoskeleton of ROCK-II (-/-) MEFs compared with wild-type MEFs. In 3T3-L1 cells, knockdown of ROCK-II by RNA interference enhanced the expression of adipogenic transcription factors while that of ROCK-I did not. Moreover, Y-27632 inhibited IRS-1 serine phosphorylation and enhanced Akt phosphorylation in 3T3-L1 preadipocytes. Similarly, Akt phosphorylation in ROCK-II (-/-) MEFs was augmented compared with wild-type MEFs. In conclusion, inhibition of ROCK-II, not ROCK-I, enhances adipogenesis accompanied by the up-regulation of adipogenic transcription factors. Augmentation of insulin signaling may contribute to the enhancement of adipogenesis. Rho-associated kinase (ROCK) regulates reorganization of actin cytoskeleton. During adipogenesis, the structure of filamentous actin is converted from long stress fibers to cortical actin, suggesting that the ROCK is involved in adipogenesis. Two ROCK isoforms have been identified: ROCK-I and ROCK-II. However, pharmacological inhibitors of ROCK cannot distinguish two ROCK isoforms. In the present study, we examined the role of ROCK in adipogenesis and actin cytoskeleton using genetic and pharmacological approaches. Y-27632, which inhibits the activity of both ROCK isoforms, enhanced adipogenesis through the up-regulation of adipogenic transcription factors in 3T3-L1 cells. Furthermore, Y-27632 restored inhibition of adipogenesis by lysophosphatidic acid, which activates Rho. Regarding actin cytoskeleton, Y-27632 disrupted stress fibers in 3T3-L1 preadipocytes. Next, we analyzed adipogenesis of mouse embryonic fibroblasts (MEFs) derived from ROCK-I and ROCK-II knock-out mice, respectively. Adipogenesis of ROCK-II (-/-) MEFs was markedly enhanced compared with wild-type MEFs while that of ROCK-I (-/-) MEFs was not. In contrast to pharmacological approaches, no obvious alteration was found in actin cytoskeleton of ROCK-II (-/-) MEFs compared with wild-type MEFs. In 3T3-L1 cells, knockdown of ROCK-II by RNA interference enhanced the expression of adipogenic transcription factors while that of ROCK-I did not. Moreover, Y-27632 inhibited IRS-1 serine phosphorylation and enhanced Akt phosphorylation in 3T3-L1 preadipocytes. Similarly, Akt phosphorylation in ROCK-II (-/-) MEFs was augmented compared with wild-type MEFs. In conclusion, inhibition of ROCK-II, not ROCK-I, enhances adipogenesis accompanied by the up-regulation of adipogenic transcription factors. Augmentation of insulin signaling may contribute to the enhancement of adipogenesis. Adipogenesis is an area of intense interest because of the large and growing prevalence of obesity, insulin resistance, and type 2 diabetes (1Spiegelman B.M. Flier J.S. Cell. 2001; 104: 531-543Abstract Full Text Full Text PDF PubMed Scopus (1988) Google Scholar, 2Flier J.S. Cell. 2004; 116: 337-350Abstract Full Text Full Text PDF PubMed Scopus (973) Google Scholar). Studies with in vitro models, such as 3T3-L1 and 3T3-F442A cell lines, have clarified the mechanisms of adipogenesis (3Hwang C.S. Loftus T.M. Mandrup S. Lane M.D. Annu. Rev. Cell Dev. Biol. 1997; 13: 231-259Crossref PubMed Scopus (213) Google Scholar, 4Rosen E.D. Spiegelman B.M. Annu. Rev. Cell Dev. Biol. 2000; 16: 145-171Crossref PubMed Scopus (1071) Google Scholar). In 3T3-L1 cells, adipocyte differentiation is a process regulated by multiple transcription factors, principally the CCAAT/enhancer-binding protein (C/EBP) 2The abbreviations used are:C/EBPCCAAT/enhancer-binding proteinIRSinsulin receptor substrateERKextracellular signal-related kinaseMAPKmitogen-activated protein kinaseCScalf serumFBSfetal bovine serumDMEMDulbecco's modified Eagle mediumBSAbovine serum albuminPI3Kphosphatidylinositol 3-kinaseIGF-1insulin-like growth factor-1ROCKRho-associated kinasePPARγproliferator-activated receptor γLPAlysophosphatidic acid. family and peroxisome proliferator-activated receptor γ (PPARγ). In the presence of hormonal inducers, the expression of C/EBPβ and C/EBPδ temporarily increases (5Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1420) Google Scholar), followed by the expression of PPARγ and C/EBPα (6Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (2024) Google Scholar, 7Lane M.D. Tang Q.Q. Jiang M.S. Biochem. Biophys. Res. Commun. 1999; 266: 677-683Crossref PubMed Scopus (243) Google Scholar). A cooperative interaction between PPARγ and C/EBPα drives the expression of genes that are necessary for the generation and maintenance of the adipogenic phenotype such as lipid accumulation and insulin sensitivity (8Wu Z. Rosen E.D. Brun R. Hauser S. Adelmant G. Troy A.E. McKeon C. Darlington G.J. Spiegelman B.M. Mol. Cell. 1999; 3: 151-158Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar). CCAAT/enhancer-binding protein insulin receptor substrate extracellular signal-related kinase mitogen-activated protein kinase calf serum fetal bovine serum Dulbecco's modified Eagle medium bovine serum albumin phosphatidylinositol 3-kinase insulin-like growth factor-1 Rho-associated kinase proliferator-activated receptor γ lysophosphatidic acid. During adipogenesis, adipocytes morphologically change from fibroblastic cells to round and lipid-laden cells. It is well known that actin cytoskeleton regulates the morphology of the cells (9Jaffe A.B. Hall A. Annu. Rev. Cell Dev. Biol. 2005; 21: 247-269Crossref PubMed Scopus (2414) Google Scholar). Concerning adipogenesis and actin cytoskeleton, it has been reported that adipocyte differentiation results in the conversion of filamentous actin (F-actin) from stress fibers and lamellipodia to cortical actin structures (10Kanzaki M. Pessin J.E. J. Biol. Chem. 2001; 276: 42436-42444Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). During the past several decades, many studies have shed light on the molecular mechanisms of actin cytoskeletal regulation. Members of the Rho family are essential regulatory components of the signaling pathway that directs reorganization of actin cytoskeleton. Small GTPase Rho contributes to many cellular functions such as cell motility, adhesion, and cytokinesis through reorganization of actin cytoskeleton. Rho is activated by extracellular signals such as lysophosphatidic acid (LPA). The actions of Rho are mediated by downstream Rho effectors. One of these effectors is Rho-associated kinase (ROCK) (11Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3927) Google Scholar, 12Riento K. Ridley A.J. Nat. Rev. Mol. Cell. Biol. 2003; 4: 446-456Crossref PubMed Scopus (1601) Google Scholar). Two ROCK isoforms have been identified: ROCK-I (also known as ROKβ) and ROCK-II (also known as Rho kinase and ROKα) (13Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar, 14Leung T. Manser E. Tan L. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 15Ishizaki T. Maekawa M. Fujisawa K. Okawa K. Iwamatsu A. Fujita A. Watanabe N. Saito Y. Kakizuka A. Morii N. Narumiya S. EMBO J. 1996; 15: 1885-1893Crossref PubMed Scopus (798) Google Scholar, 16Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (949) Google Scholar). ROCK mediates Rho signaling and reorganizes actin cytoskeleton through phosphorylation of several substrates that contribute to the assembly of actin filaments and contractility. Elucidation of the role of ROCK has been facilitated by the introduction of ROCK inhibitors, Y-27632 and fasudil. Studies with these inhibitors have revealed that ROCK regulates various physiological and pathological processes (17Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. Maekawa M. Narumiya S. Nature. 1997; 389: 990-994Crossref PubMed Scopus (2573) Google Scholar, 18Itoh K. Yoshioka K. Akedo H. Uehata M. Ishizaki T. Narumiya S. Nat. Med. 1999; 5: 221-225Crossref PubMed Scopus (561) Google Scholar, 19Shimokawa H. Seto M. Katsumata N. Amano M. Kozai T. Yamawaki T. Kuwata K. Kandabashi T. Egashira K. Ikegaki I. Asano T. Kaibuchi K. Takeshita A. Cardiovasc. Res. 1999; 43: 1029-1039Crossref PubMed Scopus (285) Google Scholar, 20Sawada N. Itoh H. Ueyama K. Yamashita J. Doi K. Chun T.H. Inoue M. Masatsugu K. Saito T. Fukunaga Y. Sakaguchi S. Arai H. Ohno N. Komeda M. Nakao K. Circulation. 2000; 101: 2030-2033Crossref PubMed Scopus (186) Google Scholar), while they cannot refer to the isoform-specific role of ROCK because ROCK inhibitors inhibit the activity of both ROCK-I and ROCK-II. Furthermore, ROCK inhibitors may potentially inhibit other protein kinases such as protein kinase A (12Riento K. Ridley A.J. Nat. Rev. Mol. Cell. Biol. 2003; 4: 446-456Crossref PubMed Scopus (1601) Google Scholar). Several studies have reported that Rho-ROCK pathway is involved in adipogenesis (21Sordella R. Jiang W. Chen G.C. Curto M. Settleman J. Cell. 2003; 113: 147-158Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 22McBeath R. Pirone D.M. Nelson C.M. Bhadriraju K. Chen C.S. Dev. Cell. 2004; 6: 483-495Abstract Full Text Full Text PDF PubMed Scopus (3509) Google Scholar). However, the role of ROCK in adipogenesis still remains to be clarified. We show here the effects of inhibition of ROCK on adipogenesis and examine the molecular mechanisms using pharmacological and genetic approaches. Especially, the adipogenic analysis using MEFs derived from ROCK-I or ROCK-II knock-out mice and ROCK-I or ROCK-II siRNA can provide the evidence about the physiological role of ROCK and the isoform-specific role of ROCK in adipogenesis. Materials—Y-27632 and pioglitazone (AD-4833) were provided by Mitsubishi Pharma (Osaka, Japan) and Takeda Pharmaceutical Company (Osaka, Japan), respectively. Insulin was purchased from Roche Applied Science (Mannheim, Germany). 3-Isobutyl-1-methylxanthine (IBMX) and dexamethasone were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). LPA and wortmannin were obtained from Sigma-Aldrich (Tokyo, Japan). Fasudil was obtained from Calbiochem. Polyclonal antibodies against ROCK-I, ROCK-II, C/EBPα, C/EBPδ, PPARγ, and IRS-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies against C/EBPβ and adiponectin were obtained from Affinity BioReagents (Golden, CO). The against was purchased from Polyclonal antibodies against and were purchased from Cell Inc. and antibodies and were purchased from Cell and cells provided by H. and M. were and adipocytes as Lane M.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). cells were for 2 as in was with and insulin for 2 The cells were in with insulin for 2 and with to was in and to of The medium was other the cells were with in for and with for 2 were with to was with and by the W. PubMed Scopus Google Scholar). of and of generation of ROCK-I and ROCK-II knock-out mice was reported Y. J. Ishizaki T. H. M. Y. Narumiya S. J. Cell Biol. 2005; PubMed Scopus Google Scholar, J. Ishizaki T. M. K. H. M. Narumiya S. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar). ROCK-II knock-out mice of a and genetic were to the for MEFs were from of as H. T. R. Komeda K. A. N. Y. J. Y. J. Y. R. S. K. T. Mol. Cell. Biol. 2001; 21: PubMed Scopus Google Scholar). of MEFs was with dexamethasone and insulin for and with to Small RNA were purchased from CA). were 3T3-L1 cells by a of with the cells were with the medium for and on for The cells were the siRNA was used as the of The of the were and of Cell and were with and in a acid, and the of transcription factors, cells were in a and were for were for protein the and to were to and to Science were with Japan) and with the with were with antibodies and with as by the The on the was and with Japan). cells were with and as 3T3-L1 cells were for with and in and for with in The cells were by in for and with bovine serum albumin for was with CA). were obtained with a RNA was using was by ROCK-I, ROCK-II, PPARγ, and C/EBPα these and were and and and and was using as by CA). of were to of are as was used to with the were as ROCK and Adipogenesis and of ROCK on protein expression of ROCK in 3T3-L1 cells by analysis In 3T3-L1 and adipocytes both ROCK-I and ROCK-II were The expression of two ROCK isoforms did not change adipogenesis. we actin cytoskeleton by and During adipogenesis, the structure of filamentous actin was converted from long stress to cortical actin suggesting that Rho-ROCK pathway is involved in adipogenesis examine the effects of ROCK inhibitors on adipogenesis, 3T3-L1 cells were to Y-27632, and in the presence of dexamethasone and Y-27632, or pioglitazone was to from to Insulin was to from the of differentiation to we the lipid accumulation by and Y-27632 enhanced lipid accumulation in a Y-27632 or lipid markedly compared with that ROCK inhibitors adipogenesis. with T. C. J. PubMed Google Scholar, N. H. M. Inoue G. K. T. Watanabe M. S. Nakao K. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Mol. Google Scholar), insulin and pioglitazone enhanced lipid respectively. to the ROCK principally adipogenesis, Y-27632 was in various of 3T3-L1 adipogenesis by dexamethasone and A and The of Y-27632 enhanced lipid while Y-27632 did not it In of and Y-27632 enhanced lipid with or Y-27632 did not cell adipogenesis compared with of Y-27632 3T3-L1 adipogenesis. in the presence of dexamethasone and was from to 2 to to to or to respectively. adipocytes were and with and of cells are lipid accumulation was by the of in with are as from compared with dexamethasone and compared with dexamethasone and cell adipogenesis. or Y-27632 was from to in the presence of dexamethasone and of MEFs was by cells the are as from of Y-27632 on the of ROCK inhibitors enhanced lipid we examined the expression of adipogenic transcription factors C/EBPβ and C/EBPδ and C/EBPα and PPARγ by analysis A and In the presence of dexamethasone and Y-27632 with a of augmented the expression of C/EBPβ C/EBPα and PPARγ while it that of C/EBPδ compared with the insulin did not the expression of C/EBPβ and C/EBPδ 2 while it enhanced that of C/EBPα and PPARγ pioglitazone augmented the expression of C/EBPβ and C/EBPα while it of C/EBPδ and The effects of insulin and pioglitazone on the expression of adipogenic factors were with N. H. M. Inoue G. K. T. Watanabe M. S. Nakao K. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, E.D. P. Spiegelman B.M. Genes Dev. 2000; PubMed Google Scholar). Furthermore, we examined protein expression of adipogenic genes the of and with the Y-27632 enhanced the expression of adipogenic transcription C/EBPβ PPARγ and C/EBPα and adipogenic and adiponectin in Y-27632 the expression of C/EBPδ of LPA and Y-27632 on and activates GTPase Rho S. C. M. Chun J. P. J.S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). It has been reported that LPA the stress in which is inhibited by Y-27632 T. Ishizaki T. M. C. K. T. Y. T. H. Narumiya S. J. Cell Biol. 2002; PubMed Scopus Google Scholar, A.J. Hall A. Cell. Full Text PDF PubMed Scopus (3927) Google Scholar). we examined the of actin cytoskeleton by LPA and Y-27632 in 3T3-L1 We 3T3-L1 with or LPA the of Y-27632 or and the effects on actin cytoskeleton by cells with LPA to 3T3-L1 cells the cells and stress fibers while change was inhibited by In cells LPA we not stress fibers or actin in the cell We examined the effects of Y-27632 on 3T3-L1 cells by LPA and Y-27632 were to from to In the presence of and Y-27632 enhanced adipogenesis while LPA inhibited it with a in 3T3-F442A cells S. C. M. Chun J. P. J.S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). In 3T3-L1 cells, LPA inhibited adipogenesis in a with of and Y-27632 restored inhibition with of and and Adipogenesis of ROCK-II (-/-) and ROCK-I (-/-) between ROCK and adipogenesis, mouse embryonic fibroblasts (MEFs) from ROCK-II ROCK-II ROCK-II and ROCK-I ROCK-I ROCK-I (-/-) mice were to the adipogenic of ROCK-I and ROCK-II protein was by analysis and ROCK-II protein in ROCK-I ROCK-I and ROCK-I (-/-) mice did not between Similarly, ROCK-I protein in ROCK-II and (-/-) mice did not between results show that is no in expression of the other ROCK adipogenic we examined lipid accumulation by and the expression of adipogenic transcription factors by ROCK-II (-/-) MEFs the enhancement of lipid accumulation compared with wild-type MEFs and of PPARγ and C/EBPα were markedly enhanced compared with wild-type MEFs and ROCK-II MEFs the enhancement of lipid accumulation compared with wild-type MEFs and of PPARγ and C/EBPα were enhanced compared with wild-type MEFs and Y-27632 did not lipid accumulation in ROCK-II (-/-) and adipogenic the of actin cytoskeleton in ROCK-II (-/-) MEFs did not change compared with wild-type MEFs The of stress cells was not In ROCK-I (-/-) adipogenesis was not enhanced compared with wild-type MEFs. accumulation and of PPARγ and C/EBPα did not between Furthermore, Y-27632 enhanced lipid accumulation in ROCK-I (-/-) MEFs and of ROCK-I (-/-) MEFs. MEFs were and with and of cells are Y-27632 was in the differentiation medium of ROCK-I (-/-) MEFs. of were to analysis using antibodies against ROCK-I, ROCK-II, and lipid accumulation was by the of in with are as from compared with ROCK-I MEFs. expression of for PPARγ and C/EBPα from ROCK-I and (-/-) MEFs. are as not in of of ROCK on of inhibition of ROCK-II enhances adipogenesis in 3T3-L1 cells, we examined the of ROCK-I or ROCK-II knockdown on 3T3-L1 adipogenesis using siRNA We siRNA for ROCK-I or ROCK-II 3T3-L1 preadipocytes. 3T3-L1 cells were introduction of We the knockdown of ROCK-I or ROCK-II expression by analysis with the introduction of ROCK-I and ROCK-II siRNA in and A and was no obvious of the expression of the other ROCK we examined the expression of adipogenic transcription factors by ROCK-II siRNA enhanced the expression of PPARγ and C/EBPα compared with the and ROCK-I siRNA did not the expression of PPARγ and C/EBPα of of ROCK on the mechanisms of downstream signaling of It is known that ROCK with IRS-1 and serine S. Y. N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, N. P. K. C.S. Kaibuchi K. H. Cell 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, N. Ito M. A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We 3T3-L1 with insulin the of Y-27632 inhibited IRS-1 and IRS-1 phosphorylation and markedly enhanced Akt phosphorylation compared with A and Regarding Y-27632 enhanced phosphorylation and inhibited phosphorylation the of insulin We examined an of the enhancement of 3T3-L1 adipogenesis by and Y-27632 were to from to with of and inhibited the enhancement of differentiation by Y-27632 in the presence of dexamethasone and We examined Akt phosphorylation in ROCK-II knock-out MEFs. In ROCK-II (-/-) and Akt phosphorylation was enhanced compared with wild-type MEFs and In ROCK-I is no in Akt phosphorylation between of knock-out of ROCK on Akt phosphorylation in MEFs. Akt phosphorylation in ROCK-II (-/-) MEFs. ROCK-II and (-/-) MEFs were with for serum analysis was using antibodies against and are of of Akt was to Akt are as from compared with ROCK-II MEFs. Akt phosphorylation in ROCK-I (-/-) MEFs. ROCK-I and (-/-) MEFs were with insulin for serum analysis was using antibodies against and are of of Akt was to Akt are as from not In the present study, we show that inhibition of ROCK enhances adipogenesis. It is to the isoform-specific role of ROCK by of ROCK In to pharmacological approaches, we examined the adipogenesis of both ROCK-I (-/-) MEFs and ROCK-II (-/-) MEFs. Furthermore, we examined the effects of of ROCK-I or ROCK-II on 3T3-L1 adipogenesis by RNA the of is the that inhibition of ROCK-II enhances adipogenesis and Akt results that ROCK-II an role in adipogenesis and insulin We examined the ROCK expression in 3T3-L1 cells by ROCK isoforms are the of adipogenesis. A that ROCK-II was in 3T3-L1 adipocytes N. P. K. C.S. Kaibuchi K. H. Cell 2005; Full Text Full Text PDF PubMed Scopus Google while the expression of ROCK-I has not been Next, we examined the effects of ROCK inhibitors on lipid accumulation in 3T3-L1 cells. We found that two ROCK inhibitors, Y-27632 and lipid respectively. that inhibition of ROCK enhances adipogenesis. We Y-27632 adipogenesis. with Y-27632 enhances adipogenesis. results that inhibition of ROCK enhances the expression of adipogenic transcription factors. Y-27632 enhances the expression of adipogenic transcription factors, C/EBPα, and PPARγ in the presence of dexamethasone and which that inhibition of ROCK enhances adipogenesis through the up-regulation of adipogenic transcription factors. Y-27632 enhances the expression of adipogenic such as and We the effects of LPA and Y-27632 on adipogenesis and actin cytoskeleton. the Rho-ROCK pathway regulates actin cytoskeleton, we examined the effects of LPA and Y-27632 on actin cytoskeleton with We that stress is augmented by LPA and that it is inhibited by Y-27632 in 3T3-L1 preadipocytes. results provide the evidence that LPA activates the Rho-ROCK pathway and Y-27632 inhibits the we that LPA inhibits adipogenesis in 3T3-L1 cells with a in 3T3-F442A cells. Furthermore, we that Y-27632 inhibition in a results that the Rho-ROCK pathway an role in adipogenesis. the isoform-specific role of ROCK and to the effects of ROCK inhibitors, we examined adipogenesis of ROCK-I (-/-) and ROCK-II (-/-) MEFs. We that of ROCK-II enhances adipogenesis and in that of ROCK-I not adipogenesis. Furthermore, Y-27632 enhances lipid accumulation in ROCK-I (-/-) MEFs and not lipid accumulation in ROCK-II (-/-) MEFs. It that inhibition of ROCK-II enhances adipogenesis. In 3T3-L1 cells, we examined the of knockdown of ROCK-I or ROCK-II on adipogenesis using We that ROCK-II siRNA enhances the expression of adipogenic transcription factors while ROCK-I siRNA not results from the pharmacological and genetic that inhibition of ROCK-II, enhances adipogenesis. We the mechanisms of the enhancement of adipogenesis by inhibition of We that Y-27632 inhibits IRS-1 serine phosphorylation and markedly enhances Akt phosphorylation the of insulin in 3T3-L1 preadipocytes. it has been reported that signaling is for adipogenesis. MEFs derived from IRS-1 (-/-) mice not have differentiation H. T. R. Komeda K. A. N. Y. J. Y. J. Y. R. S. K. T. Mol. Cell. Biol. 2001; 21: PubMed Scopus Google Scholar). IRS-1 and knock-out mice M.D. K. Genes Dev. 2002; 16: PubMed Scopus Google Scholar). Akt differentiation in 3T3-L1 cells R.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, we that an of the enhancement of adipogenesis by of insulin signaling may contribute to the enhancement of Concerning we that Y-27632 enhances phosphorylation and inhibits phosphorylation in cell Regarding and a of evidence about the molecular between and adipogenesis has been N. P. P. J. J. K. A. Narumiya S. G. C. Mol. 2001; 15: Google Scholar, M. A. E. 1991; PubMed Scopus Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. P. Spiegelman B.M. 1996; PubMed Scopus (949) Google Scholar, J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). However, in adipogenesis are still In to pharmacological approaches, we that in ROCK-II (-/-) MEFs and in ROCK-II Akt phosphorylation is enhanced compared with that in wild-type MEFs while in ROCK-I is no in Akt phosphorylation between It the evidence that of ROCK-II enhances Akt Regarding the ROCK and insulin we that a ROCK IRS-1 serine phosphorylation and increases Akt phosphorylation in 3T3-L1 preadipocytes. It is known that ROCK with IRS-1 and serine S. Y. N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, N. P. K. C.S. Kaibuchi K. H. Cell 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, N. Ito M. A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In studies have reported that IRS-1 serine phosphorylation regulates insulin signaling in cells N. Ito M. A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google and fibroblasts H. T. J.E. S. A. 2001; PubMed Scopus Google Scholar). However, it has been reported that ROCK IRS-1 serine and regulates insulin signaling in 3T3-L1 adipocytes N. P. K. C.S. Kaibuchi K. H. Cell 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The between these that IRS-1 serine phosphorylation regulates insulin signaling on cell be a in the of insulin signaling by IRS-1 serine phosphorylation between and analysis of both ROCK-I (-/-) and ROCK-II (-/-) MEFs revealed that of ROCK-II, not ROCK-I, enhances adipogenesis. The isoform-specific role of ROCK has been still both in vitro and in of ROCK-I results in the and while of ROCK-II results in growth and fetal However, it has been reported that ROCK-II knock-out mice in the not the phenotype and Y. S. S. Narumiya S. Genes 2005; PubMed Scopus Google Scholar). Concerning actin cytoskeletal it that ROCK-I and ROCK-II actin assembly for and In that inhibition of ROCK-II, not ROCK-I, enhances adipogenesis is a isoform-specific role of In the present the and evidence that inhibition of ROCK-II enhances adipogenesis accompanied by the up-regulation of adipogenic transcription factors and that inhibition of ROCK-II enhances Akt results that inhibition of ROCK-II enhances adipogenesis, in of insulin these the that ROCK-II is a for the of obesity, insulin resistance, and type 2