Glucocorticoid Receptor-mediated Expression of Caldesmon Regulates Cell Migration via the Reorganization of the Actin Cytoskeleton

Glucocorticoids (GCs) play important roles in numerous cellular processes, including growth, development, homeostasis, inhibition of inflammation, and immunosuppression. Here we found that GC-treated human lung carcinoma A549 cells exhibited the enhanced formation of the thick stress fibers and focal adhesions, resulting in suppression of cell migration. In a screen for GC-responsive genes encoding actin-interacting proteins, we identified caldesmon (CaD), which is specifically up-regulated in response to GCs. CaD is a regulatory protein involved in actomyosin-based contraction and the stability of actin filaments. We further demonstrated that the up-regulation of CaD expression was controlled by glucocorticoid receptor (GR). An activated form of GR directly bound to the two glucocorticoid-response element-like sequences in the human CALD1 promoter and transactivated the CALD1 gene, thereby up-regulating the CaD protein. Forced expression of CaD, without GC treatment, also enhanced the formation of thick stress fibers and focal adhesions and suppressed cell migration. Conversely, depletion of CaD abrogated the GC-induced phenotypes. The results of this study suggest that the GR-dependent up-regulation of CaD plays a pivotal role in regulating cell migration via the reorganization of the actin cytoskeleton. Glucocorticoids (GCs) play important roles in numerous cellular processes, including growth, development, homeostasis, inhibition of inflammation, and immunosuppression. Here we found that GC-treated human lung carcinoma A549 cells exhibited the enhanced formation of the thick stress fibers and focal adhesions, resulting in suppression of cell migration. In a screen for GC-responsive genes encoding actin-interacting proteins, we identified caldesmon (CaD), which is specifically up-regulated in response to GCs. CaD is a regulatory protein involved in actomyosin-based contraction and the stability of actin filaments. We further demonstrated that the up-regulation of CaD expression was controlled by glucocorticoid receptor (GR). An activated form of GR directly bound to the two glucocorticoid-response element-like sequences in the human CALD1 promoter and transactivated the CALD1 gene, thereby up-regulating the CaD protein. Forced expression of CaD, without GC treatment, also enhanced the formation of thick stress fibers and focal adhesions and suppressed cell migration. Conversely, depletion of CaD abrogated the GC-induced phenotypes. The results of this study suggest that the GR-dependent up-regulation of CaD plays a pivotal role in regulating cell migration via the reorganization of the actin cytoskeleton. Glucocorticoids (GCs) 2The abbreviations used are: GC, glucocorticoid; CaD, caldesmon; GR, glucocorticoid receptor; GRE, GC-response element; DEX, dexamethasone; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SRF, serum-response factor; RBD, Rho binding domain; siRNA, small interference RNA; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; GFP, green fluorescent protein; MR, mineralocorticoid receptor; ChIP, chromatin immunoprecipitation; qPCR, quantitative PCR; SMC, smooth muscle cell; cytD, cytochalasin D; CHX, cycloheximide; MRTF, myocardin-related transcription factor; MLC, myosin light chain. strongly affect numerous biological processes, including cell growth, development, homeostasis, inhibition of inflammation, and immunosuppression (1Munck A. Guyre P.M. Holbrook N.J. Endocr. Rev. 1984; 5: 25-44Crossref PubMed Scopus (2470) Google Scholar, 2Barnes P.J. Clin. 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Upon ligand binding, GR translocates into the nucleus, binds to the cognate DNA element (GC response element, GRE) within the promoter regions of target genes, and then activates or represses their transcription (10Schoneveld O.J. Gaemers I.C. Lamers W.H. Biochim. Biophys. Acta. 2004; 1680: 114-128Crossref PubMed Scopus (254) Google Scholar). Despite the many reports on the biological effects of GCs, their effect on cell motility has been little studied. Cell migration is an essential cellular process for embryogenesis, organogenesis, immune response, and wound healing (12Ridley A.J. Schwartz M.A. Burridge K. Firtel R.A. Ginsberg M.H. Borisy G. Parsons J.T. Horwitz A.R. Science. 2003; 302: 1704-1709Crossref PubMed Scopus (3870) Google Scholar). It is also involved in various pathological events, such as cancer progression (13Condeelis J. Segall J.E. Nat. Rev. Cancer. 2003; 3: 921-930Crossref PubMed Scopus (756) Google Scholar, 14Wang W. Goswami S. Sahai E. Byskoff J.B. Segall J.E. 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We found that the CALD1 is expression of CaD was the suppression of cell motility. These results that CaD plays a pivotal role in cell migration in response to GCs, and light on the mechanisms the effects of GCs. Dulbecco's modified Eagle's and from bovine and from from FBS, was J.A. Res. 1984; Google Scholar). cytochalasin and from and from and was and as J. N. K. J. Cell Sci. 1993; PubMed Google Scholar). The primary and and from and from and from light light and from Cell and from and myosin from The and from Cell human lung carcinoma cell A549 of was in and in a and of human glucocorticoid receptor was by from A549 cells as a a form of the of the was C. Biol. 2007; PubMed Scopus Google Scholar). The was to the of the sequences of GR and by The into the expression A549 cells expression the or cell GFP, or A549 cells by a and expression The and by the of enhanced GFP, K. J. Biol. 2007; Full Text Full Text PDF PubMed Scopus Google or into the the by A549 cells the in the of for The cells and the cells interference for human CaD was not and CaD target human GR human and a The cells the for and then used in and grown on and in phosphate-buffered for and then in bovine in The cells primary in by the as actin or was to the or was cytochalasin treatment, cells for cells in for the the A549 cells an cell motility the of cells a of cell contraction the a of The cells and the the and migration was in a The cells or other for was used as the from cell the cells for The cells in and in a The was The to the and to migration to the cells on the of the The cells that to the and a light Cell for and then an of treatment, was for to Cell the and in cell was used as the for cell contraction Proc. Sci. S. A. 70: PubMed Scopus Google Scholar, P. S. C. P. S. P. Biol. Scholar). was from A549 cells and The was The sequences used in this study are in The by to the expression in cells by in The by in and the a The was primary in in by the as was used to the and the was to the expression in Rho Rho binding was a Rho to the A549 cells or for PBS, and then in of cell of for The bound Rho protein was by The in the from the and the cell by and a The of protein was by promoter of the human CALD1 to was by and into The for the promoter of the human CALD1 was K. Cell Res. 2007; PubMed Scopus Google Scholar). the into the two sequences in the promoter as for for A549 cells and for of the In some cells the the cells and the and the and was a chromatin to the A549 cells or for The cells and the DNA and bound by the or The promoter to of the human CALD1 gene, which the two was from the DNA the and The promoter to of the GC-responsive and was the and as a J. Physiol. 2002; PubMed Scopus Google Scholar). The promoter to of the gene, which not was also the and The on an and the DNA was the in and The for and and and and and and used as The of DNA in for The then to the which for was to the for Glucocorticoids Cell and the in a the effects of on cell migration and the effects of in FBS, we by J.A. Res. 1984; Google Scholar) and used The cells for and to for the to the effect of on cell In our we that cell not cell this the and glucocorticoid suppressed the motility of human cell lung carcinoma A549 and including and not changes in cell migration the formation of thick stress fibers in and A549 such changes in cells the other and also enhanced the formation of focal adhesions, which located of the stress as by Because of the cell A549 cells the changes in the actin in response to GC not we used A549 cells in the and as a as a molecular by the and the and they cell motility and the of the actin C. A. Full Text PDF PubMed Scopus Google Scholar, L. C. Genes Dev. PubMed Scopus Google Scholar). In the Rho stress formation and focal C. A. Full Text PDF PubMed Scopus Google Scholar, L. C. Genes Dev. PubMed Scopus Google Scholar). We the of Rho and in response to not affect the Rho as by a binding by the expression of the Rho and or by the of and myosin light that the of thick stress formation and focal is of the Rho Glucocorticoids the of then the expression of actin-interacting in response to and found that the CaD and the expression of CaD protein Because has a for GR A. R. J. Biochem. PubMed Scopus Google CaD expression the cells the stress fibers located the cell and the of actin within the GC formation of the thick stress fibers the cell CaD was in the and to the in the the thick stress fibers in the cells The stress fibers are of and which are for cell contraction and for on the stress fibers W. Full Text PDF PubMed Scopus Google Scholar, P. P. J. Cell Biol. PubMed Scopus Google Scholar, S. H. J. 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The myosin was the actin fibers in the thick stress fibers in a CaD, which was the thick stress the myosin in cells was located the of the fibers and was the the of CaD and myosin within the of the results suggest that the suppression of cell migration by is a of thick stress formation and focal via the up-regulation of CaD Glucocorticoid Caldesmon the molecular the CaD qPCR, we a of CaD expression and found that CaD a and then of CaD protein treatment, and the up-regulated expression was for the up-regulation of CaD protein the cells a potent The of CaD was in the of that protein was not We then the of GR in the up-regulation of potent GR not a mineralocorticoid receptor the up-regulation of CaD and also the thick stress formation and suppression of cell migration and These results suggest that GR is a critical of CaD We this by GR in A549 cells GR In cells of GR, the up-regulation of CaD was and We that the CALD1 is transactivated by the or myocardin-related transcription in and H. K. H. M. K. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Cell Res. 2007; PubMed Scopus Google Scholar). depletion of not the up-regulation of CaD in A549 cells and that this is not involved in the CaD response to the other expression of a GR, C. Biol. 2007; PubMed Scopus Google up-regulated the CaD A549 which of the expression of the CaD protein cells Because of up-regulation of the promoter in the by GC the expression of and in response to Because is a form of GR that we that into the without ligand The cells exhibited thick stress formation without as cells results suggest that the up-regulation of CaD expression is the CALD1 via the within the of the CALD1 by GR, we the human CALD1 promoter in a We that the human CALD1 has two promoter to as the and K. H. R. K. E. K. Proc. Sci. S. A. PubMed Scopus Google Scholar). We the of the two promoter the promoter to the of the promoter in A549 cells was that of the We also the expression of the two of CaD by as for the and CaD and to the CaD We found the to the was also a the of and CaD to GC and the CaD expression These results suggest that the the of the two CALD1 and that the CALD1 promoter is for the GC-induced up-regulation of We used the to the promoter The promoter was activated by treatment, and this GR depletion also the promoter of strongly enhanced the promoter by These results the that the of the promoter to the up-regulation of CaD expression by the GC-responsive within the CALD1 promoter we a of promoter or the promoter to a to promoter the was not of the element to which is a pivotal for transcription of the CALD1 gene, not affect the promoter of the two sequences the to to which are the in or to in of the the was in response to not These results that GR the CALD1 promoter via and the GR binding to and in we a chromatin of the CALD1 promoter or from not that GR binds to the two sequences in response to in We also that GR into the of the A549 cells not We further GR binding to the promoter by an in or in GR protein and in GR bound to and The of GR for was and was for results that GR binds directly to the two sequences within the CALD1 promoter and the CALD1 Caldesmon Cell via the of and the expression of CaD the effects of on the reorganization of the actin and cell migration. We A549 cells The of CaD CaD in cells in the The expression of also in response to as that of of up-regulation of the promoter in the by GC In the we the thick stress fibers that the cell as GC the thick stress fibers of CaD actin by from N. E. S. H. Biochem. J. PubMed Scopus Google Scholar, D. L. J.A. E. 2001; Full Text Full Text PDF PubMed Scopus (756) Google Scholar) and agents D. C. J. L. J. Cell Biol. PubMed Scopus Google Scholar). The thick stress fibers in and cells the cytochalasin adhesions also enhanced in cells the enhanced stress formation and focal a effect on cell in the migration was that of the in the of We further the effect of up-regulated CaD on actomyosin-based cell The was in and to that in the cells myosin These results suggest that up-regulated CaD also actomyosin-based in the expression of CaD the effect of on the reorganization of the actin and on cell of expression of on cell migration and effect of expression of on cell migration. The migration of or A549 cells was in a migration or without effect of expression of CaD on cell or A549 cells or for The cells for and then for Cell contraction was from in cell DEX, We the effect of CaD depletion on cell migration and the actin cytoskeleton. for human CaD the CaD protein in A549 cells The depletion of CaD the thick stress formation and The depletion of CaD also suppression of cell migration the cells changes in cell migration in the of These the of a pivotal role for CaD in thick stress formation and suppression of cell migration. Conversely, expression of the effects of the Because the target of CaD is located in the of the human CaD the CaD, not which the The cells an in the of CaD the stress fibers and exhibited the We further the cell motility in or cells on and or without GC The of cells by the of cells in and the migration of cells or and The cells migration without the migration of the cells by We the migration of the various by and we found that the of cells the cells and The expression of the in cells CaD depletion the migration by These results suggest that the reorganization of the actin and the of A549 cells depend on the expression of of the CALD1 are to the transcription of numerous GC-responsive genes, such as and receptor and (10Schoneveld O.J. Gaemers I.C. Lamers W.H. Biochim. Biophys. Acta. 2004; 1680: 114-128Crossref PubMed Scopus (254) Google Scholar). GC-responsive genes are CaD expression was by GC and involved in the of the actin F. J. S. B. A. Proc. Sci. S. A. PubMed Scopus Google Scholar, F. S. F. A. J. Cell Biol. PubMed Scopus Google molecular and biological In this we a for the CALD1 The of the CALD1 has been mainly in of the of CaD in The transcription of the CALD1 is regulated by the of a of the and and transcription W. M. S. M. Y. S. K. K. K. K. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). is a identified for transcription of the and smooth genes D. L. J.A. E. 2001; Full Text Full Text PDF PubMed Scopus (756) Google and the in the actin genes, including the CALD1 K. Cell Res. 2007; PubMed Scopus Google Scholar, K. J. Cell Biol. 2007; PubMed Scopus Google Scholar). and that the GC-induced CaD expression on GR not on These results that the is not for GC-induced of the CALD1 in A549 and that GR directly binds to the two sequences in the human CALD1 promoter Because the and sequences are the transcription of the CALD1 the sequences a GC various (1Munck A. Guyre P.M. Holbrook N.J. Endocr. Rev. 1984; 5: 25-44Crossref PubMed Scopus (2470) Google Scholar, 3Turnbull A.V. Rivier C.L. Physiol. Rev. 1999; 79: 1-71Crossref PubMed Scopus (1037) Google Scholar, E. M. Endocr. Rev. 1998; PubMed Scopus Google Scholar). are also to essential for the of GC-responsive such as and the to tissues J.A. K. Schmid W. A. G. E. K. G. Genes Dev. 9: PubMed Scopus Google Scholar, H.M. Endocr. Rev. PubMed Scopus Google Scholar). A549 which from lung the potent to the up-regulation of CaD of cell The expression of CaD in A549 cells the up-regulation of CaD is in cells an of CaD, such as not inhibition of cell migration by in cells that CaD the that the suppression of cell migration and the reorganization of the actin involved in the of GC-responsive tissues and processes within GC-induced of Caldesmon Cell via the of the in a the role of CaD in smooth muscle contraction is K. J. Biol. Full Text PDF PubMed Google our to role in cells has been results that the suppression of cell migration is directly to the of thick stress formation and focal The stress fibers the and for cell morphology and motility W. Full Text PDF PubMed Scopus Google Scholar, P. P. J. Cell Biol. PubMed Scopus Google Scholar, S. H. J. Cell Sci. 2007; PubMed Scopus Google Scholar). The focal adhesions for the cell to the and to the stress fibers B. B. Cell 2004; PubMed Scopus Google Scholar). the of stress formation their by of actin or myosin the focal adhesions P. P. J. Cell Biol. PubMed Scopus Google Scholar). In A549 the actin are in the cell and the stress fibers are the cell In the stress fibers in GC-treated cells are and and the to by enhanced focal adhesions and CaD has a in regulating the actin cytoskeleton, via of K. K. M. K. Kakiuchi S. Res. 3: Scopus Google Scholar, J. Biol. 1984; Full Text PDF PubMed Google Scholar) and the of actin R. S. F. J. Biol. Full Text PDF PubMed Google Scholar, D. C. J. L. J. Cell Biol. PubMed Scopus Google Scholar). CaD is also to have an actin in (17Sobue K. Muramoto Y. Fujita M. Kakiuchi S. Proc. Sci. S. A. PubMed Scopus Google Scholar, A. J. Biol. Full Text PDF PubMed Google Scholar). The thick stress formation by up-regulated CaD mainly to actin and thick stress fibers in CaD up-regulated cells are cytochalasin and The focal by the thick stress of the stress fibers and focal by and the of the and their and the of the actin (12Ridley A.J. Schwartz M.A. Burridge K. Firtel R.A. Ginsberg M.H. Borisy G. Parsons J.T. Horwitz A.R. Science. 2003; 302: 1704-1709Crossref PubMed Scopus (3870) Google Scholar, 15Pantaloni D. Le Clainche C. Carlier M.F. Science. 2001; 292: 1502-1506Crossref PubMed Scopus (565) Google Scholar). The stress fibers and focal adhesions are to the for the of cell adhesions the D. Le Clainche C. Carlier M.F. 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