Initiation and Transduction of Stretch-induced RhoA and Rac1 Activation through Caveolae

Abstract

The Rho family small GTPases play a crucial role in mediating cellular responses to stretch. However, it remains unclear how force is transduced to Rho signaling pathways. We investigated the effect of stretch on the activation and caveolar localization of RhoA and Rac1 in neonatal rat cardiomyocytes. In unstretched cardiomyocytes, RhoA and Rac1 were detected in both caveolar and non-caveolar fractions as assessed using detergent-free floatation analysis. Stretching myocytes for 4 min activated RhoA and Rac1. By 15 min of stretch, RhoA and Rac1 had dissociated from caveolae, and there was decreased coprecipitation of RhoA and Rac1 with caveolin-3. To determine whether compartmentation of RhoA and Rac1 within caveolae was necessary for stretch signaling, we disrupted caveolae with methyl β-cyclodextrin (MβCD). Treatment with 5 mm MβCD for 1 h dissociated both RhoA and Rac1 from caveolae. Under this condition, stretch failed to activate RhoA or Rac1. Stretch-induced actin cytoskeletal organization was concomitantly impaired. Interestingly the ability of stretch to activate extracellular signal-regulated kinase (ERK) was unaffected by MβCD treatment, but ERK translocation to the nucleus was impaired. Stretch-induced hypertrophy was also inhibited. Actin cytoskeletal disruption with cytochalasin-D also prevented stretch from increasing nuclear ERK, whereas actin polymerization with jasplakinolide restored nuclear translocation of activated ERK in the presence of MβCD. We suggest that activation of RhoA or Rac1, localized in a caveolar compartment, is essential for sensing externally applied force and transducing this signal to the actin cytoskeleton and ERK translocation. The Rho family small GTPases play a crucial role in mediating cellular responses to stretch. However, it remains unclear how force is transduced to Rho signaling pathways. We investigated the effect of stretch on the activation and caveolar localization of RhoA and Rac1 in neonatal rat cardiomyocytes. In unstretched cardiomyocytes, RhoA and Rac1 were detected in both caveolar and non-caveolar fractions as assessed using detergent-free floatation analysis. Stretching myocytes for 4 min activated RhoA and Rac1. By 15 min of stretch, RhoA and Rac1 had dissociated from caveolae, and there was decreased coprecipitation of RhoA and Rac1 with caveolin-3. To determine whether compartmentation of RhoA and Rac1 within caveolae was necessary for stretch signaling, we disrupted caveolae with methyl β-cyclodextrin (MβCD). Treatment with 5 mm MβCD for 1 h dissociated both RhoA and Rac1 from caveolae. Under this condition, stretch failed to activate RhoA or Rac1. Stretch-induced actin cytoskeletal organization was concomitantly impaired. Interestingly the ability of stretch to activate extracellular signal-regulated kinase (ERK) was unaffected by MβCD treatment, but ERK translocation to the nucleus was impaired. Stretch-induced hypertrophy was also inhibited. Actin cytoskeletal disruption with cytochalasin-D also prevented stretch from increasing nuclear ERK, whereas actin polymerization with jasplakinolide restored nuclear translocation of activated ERK in the presence of MβCD. We suggest that activation of RhoA or Rac1, localized in a caveolar compartment, is essential for sensing externally applied force and transducing this signal to the actin cytoskeleton and ERK translocation. Mechanical stress is recognized as an important extracellular stimulus facilitating cell growth, one example of which is cardiomyocyte hypertrophy. There is a growing list of signaling pathways that are activated by mechanical stress in cardiomyocytes including protein kinase C, mitogen-activated protein kinases (MAPKs), 1The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; GDI, GDP dissociation inhibitor; MβCD, methyl β-cyclodextrin; MES, 4-morpholineethanesulfonic acid; RBD, Rho-binding domain of rhotekin; PBD, Rac1-binding domain of p21-activated kinase 1; ANF, atrial natriuretic factor. phosphatidylinositol-3 kinase/Akt, Janus kinase/signal transducer and activator of transcription, calcineurin, and nitric oxide synthase (1Ruwhof C. van der Laarse A. Cardiovasc. Res. 2000; 47: 23-37Crossref PubMed Scopus (409) Google Scholar, 2Kim C.H. Cho Y.S. Chun Y.S. Park J.W. Kim M.S. Circ. Res. 2002; 90: E25-E33Crossref PubMed Google Scholar, 3Petroff M.G. Kim S.H. Pepe S. Dessy C. Marban E. Balligand J.L. Sollott S.J. Nat. Cell Biol. 2001; 3: 867-873Crossref PubMed Scopus (265) Google Scholar). There is also evidence that Rho family small GTPases play a central role in mediating the development of stretch-induced cardiac hypertrophy (4Aikawa R. Komuro I. Yamazaki T. Zou Y. Kudoh S. Zhu W. Kadowaki T. Yazaki Y. Circ. Res. 1999; 84: 458-466Crossref PubMed Scopus (163) Google Scholar, 5Aikawa R. Nagai T. Tanaka M. Zou Y. Ishihara T. Takano H. Hasegawa H. Akazawa H. Mizukami M. Nagai R. Komuro I. Biochem. Biophys. Res. Commun. 2001; 289: 901-907Crossref PubMed Scopus (113) Google Scholar). Rho family small GTPases regulate a variety of cellular functions such as cytoskeletal rearrangement, cell contractility, and cell migration (6Sah V.P. Seasholtz T.M. Sagi S.A. Brown J.H. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 459-489Crossref PubMed Scopus (298) Google Scholar). The cytoskeleton communicates with the extracellular matrix through integrins, which have been considered as potential mechanosensors (1Ruwhof C. van der Laarse A. Cardiovasc. Res. 2000; 47: 23-37Crossref PubMed Scopus (409) Google Scholar, 7Aikawa R. Nagai T. Kudoh S. Zou Y. Tanaka M. Tamura M. Akazawa H. Takano H. Nagai R. Komuro I. Hypertension. 2002; 39: 233-238Crossref PubMed Scopus (171) Google Scholar, 8Geiger B. Bershadsky A. Cell. 2002; 110: 139-142Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). There is also evidence that integrins transduce signals via Rho-dependent pathways (9Juliano R.L. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 283-323Crossref PubMed Scopus (498) Google Scholar). However, the molecular mechanisms by which mechanical force is transduced and the steps leading to recruitment of Rho family small GTPases in mechanotransduction in cardiomyocytes are poorly understood. Caveolae are cholesterol- and sphingolipid-enriched plasma membrane invaginations, which are considered to be important platforms for signal transduction (10Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2551) Google Scholar, 11Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5164) Google Scholar, 12Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (924) Google Scholar, 13Anderson R.G.W. Jacobson K. Science. 2002; 296: 1821-1825Crossref PubMed Scopus (1006) Google Scholar). There is considerable support for the notion that signaling molecules converge in caveolae (14Lohn M. Furstenau M. Sagach V. Elger M. Schulze W. Luft F.C. Haller H. Gollasch M. Circ. Res. 2000; 87: 1034-1039Crossref PubMed Scopus (151) Google Scholar, 15Yarbrough T.L. Lu T. Lee H.C. Shibata E.F. Circ. Res. 2002; 90: 443-449Crossref PubMed Scopus (192) Google Scholar, 16Feron O. Dessy C. Opel D.J. Arstall M.A. Kelly R.A. Michel T. J. Biol. Chem. 1998; 273: 30249-30254Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) and that these play an important role in propagating signals in response to agonist treatment in cardiomyocytes. However, the possibility that mechanotransduction signals through caveolar-localized molecular pathways in cardiomyocytes has not been addressed previously. There are two functionally distinct features of caveolae that are critical to their role in signal transduction (17Feron O. Zhao Y.Y. Kelly R.A. Ann. N. Y. Acad. Sci. 1999; 874: 11-19Crossref PubMed Scopus (20) Google Scholar). First, caveolae are considered to function as reservoirs of signaling proteins, with caveolin serving as a negative regulator of these molecules in quiescent cells. Second, caveolae function as clustering sites for ligand-activated G-protein-coupled receptors such as the muscarinic cholinergic or sphingosine 1 phosphate receptor (18Feron O. Smith T.W. Michel T. Kelly R.A. J. Biol. Chem. 1997; 272: 17744-17748Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 19Igarashi J. Michel T. J. Biol. Chem. 2000; 275: 32363-32370Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Several studies indicate that the caveolar scaffolding protein, caveolin-1, can directly interact with signaling molecules (e.g. G-protein subunits, Src family tyrosine kinases, endothelial nitric oxide synthase, protein kinase C, or Ras) within caveolae to negatively regulate their functions (12Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (924) Google Scholar, 20Song K.S. Li S. Okamoto T. Quilliam L. Sargiacomo M. Lisanti M.P. J. Biol. 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Chem. 1998; 273: 3125-3128Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 26Sowa G. Pypaert M. Sessa W.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14072-14077Crossref PubMed Scopus (191) Google Scholar). In light of the findings cited above, we hypothesized that mechanical force might be transduced into cellular signaling pathways in cardiomyocytes via caveolae. In particular, we focused on the localization and activity of Rho family GTPases, RhoA and Rac1, in caveolar microdomains and examined the effect of stretch on their compartmentation and downstream signaling. The data presented here demonstrate that activation of RhoA and Rac1 by stretch requires their proper localization within caveolae and that stretch leads to their dissociation from this compartment. Disruption of caveolae not only prevents RhoA and Rac1 activation but also impairs stretch-induced actin cytoskeletal rearrangement and nuclear localization of ERK. Materials—Anti-caveolin-3, anti-RhoA, anti-Rac1, anti-Rho-GDP dissociation inhibitor (GDI), and anti-focal adhesion kinase (FAK) monoclonal antibodies were purchased from Transduction Laboratory. Anti-RhoA and anti-Rac1 polyclonal antibodies for immunoprecipitation were purchased from Santa Cruz Biotechnology. Anti-phosphospecific ERK1/2 and anti-ERK1/2 polyclonal antibodies were purchased from Cell Signaling. Protein G-Sepharose was from Amersham Biosciences. Fluorescein isothiocyanate-conjugated goat anti-rabbit antibody was from ICN. Jasplakinolide and rhodamine-phalloidin were purchased from Molecular Probes. All other reagents were purchased from Sigma. Cell Culture—Neonatal rat ventricular myocytes were isolated and cultured as described previously (27Hoshijima M. Sah V.P. Wang Y. Chien K.R. Brown J.H. J. Biol. Chem. 1998; 273: 7725-7730Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 28Shubeita H.E. Martinson E.A. Van Bilsen M. Chien K.R. Brown J.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1305-1309Crossref PubMed Scopus (153) Google Scholar). Briefly, hearts were obtained from 1–2-day-old Sprague-Dawley rat pups and digested with collagenase, and myocytes were purified by passage through a Percoll gradient. Cells were plated onto silicon membrane coated with collagen type I (0.5 μg/cm2), which assembled in a biaxial stretch device. Myocytes were maintained overnight in 4:1 Dulbecco's modified Eagle's medium/medium 199, supplemented with 10% horse serum, 5% fetal calf serum, and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). Cells were then serum-starved for 24 h prior to intervention. Purification of Caveolin-rich Membrane Fractions—Caveolin-rich microdomains were isolated using the methods of Song et al. (20Song K.S. Li S. Okamoto T. Quilliam L. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar) with some modifications. Briefly, myocytes were washed with ice-cold phosphate-buffered saline and lysed directly with 600 μl of 0.5 m Na2CO3 (pH 11). Cells were passed 20 times through a 25-gauge needle and sonicated with three 20-s bursts with minimal output of Micro Ultrasonic cell disrupter (Kontes). A 0.54-ml aliquot of cell lysates was adjusted to 45% sucrose by the addition of 0.54 ml of 90% sucrose prepared in saline mm 4-morpholineethanesulfonic mm and the of an The 45% was with ml of sucrose and ml of 5% sucrose in saline m The were for 20 h in an fractions were from the of the gradient. The in were with The were washed with and then with The were by to and to was then using of The for the Rho-binding domain of or Rac1-binding domain of p21-activated kinase 1 into the were from of The protein was purified from as described previously (27Hoshijima M. Sah V.P. Wang Y. Chien K.R. Brown J.H. J. Biol. Chem. 1998; 273: 7725-7730Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Briefly, of were with and purified by to The were were washed with ice-cold phosphate-buffered saline and lysed in μl of cell mm mm mm 10% μg/ml μg/ml were for min to and cell of were with or for min 4 the for three times with mm mm mm 1 mm μg/ml μg/ml the were and with μl of The were for 5 min and then by to and to was using were washed with ice-cold phosphate-buffered saline and lysed with mm mm μg/ml μg/ml 1 mm 1 mm and 1 mm of RhoA or Rac1 from cell lysates was by μl of cell lysates with 1 of or anti-Rac1 polyclonal antibody in the presence of protein G-Sepharose overnight 4 The were by to and with anti-RhoA, anti-Rac1, or on were for 20 min in in phosphate-buffered for min in and in for 20 The silicon membrane was and then with antibodies overnight 4 by antibody and rhodamine-phalloidin for 1 h The membrane was on a and were with a The a on a In and by were times were such that the response was in the for The data were and using on a of Caveolae from is a and cell of We isolated cardiac caveolae from neonatal rat ventricular myocytes using as a for cardiac caveolae. were by the of and by the presence of and of an inhibitor of RhoA and Rac1 that in the The were in fractions 5 and in by the detergent-free In and were detected in fractions The protein obtained using this was also with that of G. J. A. H.C. E. Circ. Res. 2000; 87: PubMed Scopus Google Scholar). of RhoA and Rac1 in to whether stretch to the activation of RhoA and Rac1 in cardiomyocytes. and in activated RhoA and Rac1 in response to stretch The activation of both RhoA and Rac1 min the of stretch and to the by min of stretch RhoA and Rac1 activation 4 min of stretch is also shown in of stretch with or MβCD on the activation of RhoA and Rac1. were plated on but not or were to stretch for 4 with or prior treatment with 5 mm MβCD for 1 were and then were with antibody RhoA or Rac1 the of RhoA or Rac1 by RhoA or Rac1. The were and are as with of RhoA and Rac1 from Caveolae in to in the localization of RhoA and Rac1 in neonatal rat ventricular myocytes to stretch were assessed using the described in 1 to caveolae. RhoA and Rac1 were in both caveolar and non-caveolar fractions in unstretched cardiomyocytes. stretch for 4 min not the of RhoA or Rac1 in caveolar and non-caveolar fractions not However, stretch was applied to the myocytes for 15 of RhoA and Rac1 from the caveolar to a non-caveolar was of the of and Rac1 in caveolar fractions and non-caveolar fractions (9Juliano R.L. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 283-323Crossref PubMed Scopus (498) Google Scholar, D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2551) Google Scholar, 11Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5164) Google Scholar, 12Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (924) Google Scholar) a in the of RhoA and a in the of Rac1 in caveolae. in non-caveolar fractions were was not by stretch of on Stretch-induced RhoA and Rac1 that activation of RhoA and Rac1 by stretch to their dissociation from caveolae that caveolar compartmentation might be necessary for their To this we used with MβCD to caveolae. were with 5 mm MβCD for 1 h prior to stretch. The of this on the activation and of RhoA and Rac1 was then the of caveolae was not by of some in fractions 5 and this treatment was to both RhoA and Rac1 from caveolae The of RhoA and Rac1 with was also examined in immunoprecipitation with RhoA and with Rac1 in unstretched myocytes 15 min of stretch, the of with RhoA and Rac1 was with their dissociation from the caveolar In the of that with RhoA and Rac1 was decreased by MβCD treatment, dissociation of these small GTPases from caveolar microdomains of MβCD on Stretch-induced RhoA and Rac1 ability of stretch to activate RhoA and Rac1 was then assessed in with MβCD. shown in 4 min of stretch activated both RhoA and Rac1 in cells. with MβCD not the activity of RhoA and stretch failed to activate RhoA or Rac1. suggest that the of stretch-induced RhoA and Rac1 activation requires caveolar of MβCD on ERK findings from studies of and that of caveolae in of ERK in the W. M. J. B. B. Lisanti M.P. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, Park M. J. B. Lisanti M.P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). whether ERK was activated in myocytes caveolar was disrupted with MβCD. shown in MβCD treatment not to activation of ERK, was ERK activation by stretch by MβCD treatment of ERK by MβCD in we investigated the localization of ERK by with a ERK a shown in in unstretched and actin were detected in the of stretch the of and nuclear was in of the cells. concomitantly actin In in with MβCD, stretch but was only in the and actin data suggest that to activate RhoA and Rac1 and to the actin cytoskeleton nuclear translocation of ERK. Jasplakinolide of the of the actin cytoskeleton in nuclear translocation of ERK, we the effect of an actin jasplakinolide on the translocation of ERK by stretch. Actin not be detected with in these studies jasplakinolide with for to actin We for an jasplakinolide treatment, the actin cytoskeleton was and stretch to the nuclear translocation of in a of the we that treatment of myocytes with an actin not ERK activation but prevented stretch-induced organization of the actin cytoskeleton and to of ERK in the of is with a with which that nuclear translocation of ERK is by an actin cytoskeleton S.A. R.L. J. Cell Biol. 2001; PubMed Scopus Google Scholar). The molecular RhoA and Rac1 actin cytoskeletal and ERK nuclear translocation remains to be However, ERK signaling in cardiomyocytes has been with the development of by in cell actin and for 24 h in of these treatment with MβCD prevents these MβCD treatment signaling pathways that regulate the data are with a for caveolae in transduction of stretch to growth, via Rac1, and ERK signaling pathways. The findings presented here demonstrate by that stretch the of activated RhoA and Rac1 in cardiomyocytes. that Rho is activated by stretch in and cardiac (4Aikawa R. Komuro I. Yamazaki T. Zou Y. Kudoh S. Zhu W. Kadowaki T. Yazaki Y. Circ. Res. 1999; 84: 458-466Crossref PubMed Scopus (163) Google Scholar, K. S. T. T. Circ. Res. 1999; PubMed Scopus Google Scholar). The by which mechanical force is transduced into activation of downstream signaling remains The data presented here suggest that the compartmentation of RhoA and Rac1 in caveolae play a critical role in mechanotransduction in cardiomyocytes. a detergent-free of caveolae, we that a of RhoA and Rac1 is localized in caveolar microdomains of cardiomyocytes. we demonstrate that mechanical stretch in the dissociation of RhoA and Rac1 from the caveolar and the of with RhoA or Rac1 in We also evidence that MβCD treatment leads to of compartmentation of RhoA and Rac1 within caveolae and that this prevents stretch-induced RhoA and Rac1 data caveolae as the sites which RhoA and Rac1 are activated by stretch. a cellular studies suggest that caveolar and are of their C. J. Scopus Google Scholar). it might be hypothesized that stretch, by caveolar caveolar-localized RhoA and Rac1 to that the of GDP and by B. Bershadsky A. Cell. 2002; 110: 139-142Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). In addition to RhoA and Rac1 MβCD treatment prevents stretch-induced actin cytoskeletal The role of RhoA and Rac1 in activation of molecules for actin cytoskeletal (e.g. Rho p21-activated kinase 1 is M. T. S. N. A. A. T. K. K. S. Science. 1999; PubMed Scopus Google Scholar, N. O. K. K. A. K. E. K. 1998; PubMed Scopus Google Scholar). has also been that a protein in actin cytoskeletal through is a protein M. van B. Mol. Biol. Cell. 2000; PubMed Scopus Google Scholar, Li F. L. D. Y. R. Nat. Cell Biol. 2002; PubMed Scopus Google Scholar). The ability of caveolar disruption to actin cytoskeletal organization from to activate RhoA and Rac1 or from of cytoskeletal molecules to data also suggest that stretch-induced actin cytoskeletal rearrangement a role in the proper localization of ERK. Disruption of caveolae with MβCD not ERK activation by stretch. the that ERK is activated in the presence of MβCD that some stretch-induced signaling pathways signaling pathways are and to ERK activation in the of caveolae MβCD treatment to of activated ERK in response to stretch. The possibility that the ERK localization is to the failed cytoskeletal organization is with that an actin impairs nuclear translocation of ERK in S.A. R.L. J. Cell Biol. 2001; PubMed Scopus Google Scholar). presented here indicate that this also in cardiomyocytes and in response to stretch. Stretch-induced ERK activation and nuclear translocation in have also been to be disrupted via of the actin cytoskeleton from nitric oxide or treatment with L. L. K. H. J.W. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Jasplakinolide the and proper ERK localization in L. L. K. H. J.W. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). in cardiomyocytes, jasplakinolide nuclear translocation of ERK by stretch, in the presence of MβCD. The by which an actin cytoskeleton in the of ERK into the nucleus remains data suggest that organization of the actin cytoskeleton is for mechanotransduction in cardiomyocytes. and G-protein-coupled are to ERK signaling pathways to cardiomyocyte hypertrophy (1Ruwhof C. van der Laarse A. Cardiovasc. Res. 2000; 47: 23-37Crossref PubMed Scopus (409) Google Scholar, T. S. T. Y. T. M. T. S. H. M. J. Mol. Cell 2000; Full Text PDF PubMed Scopus Google Scholar, Circ. Res. 2002; PubMed Scopus Google Scholar). We demonstrate here that MβCD treatment prevents stretch-induced in and cell there are potential molecular by MβCD, findings suggest that the ability of MβCD treatment to RhoA or Rac1 cytoskeletal and ERK translocation nuclear In findings demonstrate that stretch and RhoA and Rac1 from a caveolar in rat cardiomyocytes. via this to organization of the actin a response that to the nuclear localization of ERK. We suggest that proper cellular compartmentation of RhoA and Rac1 within caveolae is necessary for transducing mechanical force into ERK We D. J. and Y. Zhao for and M. for the of the Rho-binding domain We also S. and J. of the of for with of