Involvement of the Serum Response Factor Coactivator Megakaryoblastic Leukemia (MKL) in the Activin-regulated Dendritic Complexity of Rat Cortical Neurons*
2010; Elsevier BV; Volume: 285; Issue: 43 Linguagem: Inglês
10.1074/jbc.m110.118745
ISSN1083-351X
AutoresMitsuru Ishikawa, Naoki Nishijima, Jun Shiota, Hiroyuki Sakagami, Kunihiro Tsuchida, Miho Mizukoshi, Mamoru Fukuchi, Masaaki Tsuda, Akiko Tabuchi,
Tópico(s)NF-κB Signaling Pathways
ResumoDynamic changes in neuronal morphology and transcriptional regulation play crucial roles in the neuronal network and function. Accumulating evidence suggests that the megakaryoblastic leukemia (MKL) family members, which function not only as actin-binding proteins but also as serum response factor (SRF) transcriptional coactivators, regulate neuronal morphology. However, the extracellular ligands and signaling pathways, which activate MKL-mediated morphological changes in neurons, remain unresolved. Here, we demonstrate that in addition to MKL1, MKL2, highly enriched in the forebrain, strongly contributes to the dendritic complexity, and this process is triggered by stimulation with activin, a member of the transforming growth factor β (TGF-β) superfamily. Activin promoted dendritic complexity in a SRF- and MKL-dependent manner without drastically affecting MKL localization and protein levels. In contrast, activin promoted the nuclear export of suppressor of cancer cell invasion (SCAI), which is a corepressor for SRF and MKL. Furthermore, overexpression of SCAI blocked activin-induced SRF transcriptional responses and dendritic complexity. Collectively, these results strongly suggest that activin-SCAI-MKL signaling is a novel pathway that regulates the dendritic morphology of rat cortical neurons by excluding SCAI from the nucleus and activating MKL/SRF-mediated gene expression. Dynamic changes in neuronal morphology and transcriptional regulation play crucial roles in the neuronal network and function. Accumulating evidence suggests that the megakaryoblastic leukemia (MKL) family members, which function not only as actin-binding proteins but also as serum response factor (SRF) transcriptional coactivators, regulate neuronal morphology. However, the extracellular ligands and signaling pathways, which activate MKL-mediated morphological changes in neurons, remain unresolved. Here, we demonstrate that in addition to MKL1, MKL2, highly enriched in the forebrain, strongly contributes to the dendritic complexity, and this process is triggered by stimulation with activin, a member of the transforming growth factor β (TGF-β) superfamily. Activin promoted dendritic complexity in a SRF- and MKL-dependent manner without drastically affecting MKL localization and protein levels. In contrast, activin promoted the nuclear export of suppressor of cancer cell invasion (SCAI), which is a corepressor for SRF and MKL. Furthermore, overexpression of SCAI blocked activin-induced SRF transcriptional responses and dendritic complexity. Collectively, these results strongly suggest that activin-SCAI-MKL signaling is a novel pathway that regulates the dendritic morphology of rat cortical neurons by excluding SCAI from the nucleus and activating MKL/SRF-mediated gene expression. IntroductionSerum response factor (SRF) 4The abbreviations used are: SRFserum response factorCREcAMP response elementCREBcAMP response element-binding proteinLucluciferaseMKLmegakaryoblastic leukemiaSCAIsuppressor of cancer cell invasionSRFΔdominant negative SRF mutant. is a transcription factor that binds to a consensus sequence CC(A/T)6GG (called the CArG box), which is present in the promoters of several immediate-early or cytoskeletal genes (1Treisman R. EMBO J. 1995; 14: 4905-4913Crossref PubMed Scopus (346) Google Scholar). Several studies have demonstrated that deletion mutants of SRF in the mouse brain result in the attenuation of activity-dependent expression of immediate-early genes, impair synaptic plasticity and learning (2Ramanan N. Shen Y. Sarsfield S. Lemberger T. Schütz G. Linden D.J. Ginty D.D. Nat. Neurosci. 2005; 8: 759-767Crossref PubMed Scopus (173) Google Scholar, 3Etkin A. Alarcón J.M. Weisberg S.P. Touzani K. Huang Y.Y. Nordheim A. Kandel E.R. Neuron. 2006; 50: 127-143Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), reduce neurite outgrowth, and show abnormality of pathfindings and neuronal migration (4Alberti S. Krause S.M. Kretz O. Philippar U. Lemberger T. Casanova E. Wiebel F.F. Schwarz H. Frotscher M. Schütz G. Nordheim A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 6148-6153Crossref PubMed Scopus (119) Google Scholar, 5Knöll B. Kretz O. Fiedler C. Alberti S. Schütz G. Frotscher M. Nordheim A. Nat. Neurosci. 2006; 9: 195-204Crossref PubMed Scopus (134) Google Scholar). These findings strongly suggest that SRF plays an important role in neuronal development and plasticity (6Knöll B. Nordheim A. Trends Neurosci. 2009; 32: 432-442Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). SRF-dependent transcription is controlled by at least two different types of coactivators. One comprises the ternary complex factors, which mainly regulate the immediate-early genes such as c-fos (7Buchwalter G. Gross C. Wasylyk B. Gene. 2004; 324: 1-14Crossref PubMed Scopus (291) Google Scholar). The other type of coactivator comprises megakaryoblastic leukemia (MKL) family members. The MKL family consists of megakaryocytic acute leukemia/megakaryoblastic leukemia 1/myocardin-related transcription factor-A/basic SAP, and coiled-coil domain (MAL/MKL1/MRTF-A/BSAC) and MKL2/MRTF-B (8Wang D.Z. Li S. Hockemeyer D. Sutherland L. Wang Z. Schratt G. Richardson J.A. Nordheim A. Olson E.N. Proc. Natl. Acad. U.S.A. 2002; 99: 14855-14860Crossref PubMed Scopus (395) Google Scholar, 9Sasazuki T. Sawada T. Sakon S. Kitamura T. Kishi T. Okazaki T. Katano M. Tanaka M. Watanabe M. Yagita H. Okumura K. Nakano H. J. Biol. Chem. 2002; 277: 28853-28860Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 10Cen B. Selvaraj A. Burgess R.C. Hitzler J.K. Ma Z. Morris S.W. Prywes R. Mol. Cell. Biol. 2003; 23: 6597-6608Crossref PubMed Scopus (243) Google Scholar, 11Selvaraj A. Prywes R. J. Biol. Chem. 2003; 278: 41977-41987Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 12Cen B. Selvaraj A. Prywes R. J. Cell. Biochem. 2004; 93: 74-82Crossref PubMed Scopus (132) Google Scholar, 13Pipes G.C. Creemers E.E. Olson E.N. Genes Dev. 2006; 20: 1545-1556Crossref PubMed Scopus (385) Google Scholar). In nonneuronal cells, MKL1 is primarily activated by actin rearrangement stimulated with the activation of Rho GTPases; that is, release of MKL1 from G-actin enables MKL1 to translocate to the nucleus where it binds to and activates SRF (14Miralles F. Posern G. Zaromytidou A.I. Treisman R. Cell. 2003; 113: 329-342Abstract Full Text Full Text PDF PubMed Scopus (1030) Google Scholar). MKL2, in addition to MKL1, regulates a set of cytoskeletal genes (11Selvaraj A. Prywes R. J. Biol. Chem. 2003; 278: 41977-41987Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 12Cen B. Selvaraj A. Prywes R. J. Cell. Biochem. 2004; 93: 74-82Crossref PubMed Scopus (132) Google Scholar). Furthermore, suppressor of cancer cell invasion (SCAI) has recently been identified as an MKL-interacting cofactor that inhibits cancer cell invasion (15Brandt D.T. Baarlink C. Kitzing T.M. Kremmer E. Ivaska J. Nollau P. Grosse R. Nat. Cell Biol. 2009; 11: 557-568Crossref PubMed Scopus (98) Google Scholar). Therefore, SRF-driven transcriptional regulation appears to be more complicated because of the presence of novel MKL-interacting cofactors. However, it remains unclear as to whether MKL1, MKL2, and SCAI play different roles in several biological processes through the regulation of their own target genes in neurons.The transforming growth factor β (TGF-β) superfamily plays important roles in various cellular processes associated with cell growth, differentiation, and development (16Massagué J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2053) Google Scholar, 17Derynck R. Akhurst R.J. Nat. Cell Biol. 2007; 9: 1000-1004Crossref PubMed Scopus (300) Google Scholar, 18Tsuchida K. Nakatani M. Hitachi K. Uezumi A. Sunada Y. Ageta H. Inokuchi K. Cell. Commun. Signal. 2009; 7: 15Crossref PubMed Scopus (131) Google Scholar). To regulate such a wide variety of cellular functions, TGF-β binds to specific receptors and activates a multitude of signals, including the canonical pathway that promotes phosphorylation and nuclear translocation of Smad2 and Smad3 (19Xu L. Biochim. Biophys. Acta. 2006; 1759: 503-513Crossref PubMed Scopus (77) Google Scholar) and the noncanonical pathway that activates ERK, p38 MAP kinases, or Rho GTPases (20Zhang Y.E. Cell Res. 2009; 19: 128-139Crossref PubMed Scopus (1284) Google Scholar). Several lines of evidence suggest that TGF-β signaling also regulates SRF (21Qiu P. Feng X.H. Li L. J. Mol. Cell. Cardiol. 2003; 35: 1407-1420Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 22Morita T. Mayanagi T. Sobue K. J. Cell Biol. 2007; 179: 1027-1042Crossref PubMed Scopus (215) Google Scholar). Recently, a study showed that TGF-β1, a member of the TGF-β superfamily, activates SRF-dependent cytoskeletal genes or Smad3-dependent genes via nuclear translocation of MKL. This process may be involved in epithelial-mesenchymal transition, which occurs during embryonic development and tumor progression (22Morita T. Mayanagi T. Sobue K. J. Cell Biol. 2007; 179: 1027-1042Crossref PubMed Scopus (215) Google Scholar). However, it is unknown whether MKL family members mediate TGF-β signaling in the brain.In neurons, MKL1 regulates SRF-mediated transcription through the RhoA or brain-derived neurotrophic factor (BDNF)-induced ERK1/2 signaling pathway (23Tabuchi A. Estevez M. Henderson J.A. Marx R. Shiota J. Nakano H. Baraban J.M. J. Neurochem. 2005; 94: 169-180Crossref PubMed Scopus (34) Google Scholar, 24Kalita K. Kharebava G. Zheng J.J. Hetman M. J. Neurosci. 2006; 26: 10020-10032Crossref PubMed Scopus (72) Google Scholar). In addition, MKL1 is highly expressed in the forebrain and regulates the neuronal morphology of cortical or hippocampal neurons (5Knöll B. Kretz O. Fiedler C. Alberti S. Schütz G. Frotscher M. Nordheim A. Nat. Neurosci. 2006; 9: 195-204Crossref PubMed Scopus (134) Google Scholar, 25Shiota J. Ishikawa M. Sakagami H. Tsuda M. Baraban J.M. Tabuchi A. J. Neurochem. 2006; 98: 1778-1788Crossref PubMed Scopus (31) Google Scholar). MKL1 may also be involved in nerve growth factor (NGF)-induced axonal growth of sensory neurons (26Wickramasinghe S.R. Alvania R.S. Ramanan N. Wood J.N. Mandai K. Ginty D.D. Neuron. 2008; 58: 532-545Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). These observations imply that MKL family members participate in the regulation of neuronal morphology through regulating actin-based morphological changes or by controlling the expression of SRF-targeted cytoskeletal genes. In contrast to several reports on the function of MKL1, the expression and function of MKL2 in the brain remain unresolved. Furthermore, there is little information describing the kinds of extracellular ligands that potentially act upstream of the MKL family to regulate dendritic morphology. In the present study, we first investigated the pattern of MKL2 expression in the developing and adult brain and assessed whether MKL2 regulates SRF transcription and the morphology of rat cortical neurons. Furthermore, we found that activin, a member of the TGF-β superfamily, promoted dendritic complexity through nuclear export of SCAI and removing SCAI repressor function for MKL/SRF gene expression.DISCUSSIONThis study examining MKL expression and function in neurons has yielded several important findings. Quantitative PCR analysis with cDNA derived from several tissues revealed that, unlike the broad distribution of MKL1 mRNA, MKL2 mRNA is enriched in the brain (Fig. 1, A and B). In addition, the in situ hybridization study showed that MKL2 is selectively expressed in several forebrain areas (Fig. 1, C and D). Characterization of the developmental profile of MKL2 mRNA showed that its expression rises during the postnatal week and is sustained into adulthood (Fig. 1, F–H). Taking our previous study into consideration, the up-regulation of MKL2 expression is similar to that of MKL1 expression during brain development (25Shiota J. Ishikawa M. Sakagami H. Tsuda M. Baraban J.M. Tabuchi A. J. Neurochem. 2006; 98: 1778-1788Crossref PubMed Scopus (31) Google Scholar). Although several studies including ours indicate that MKL1 and MKL2 function as the SRF coactivators and modulators of neuronal morphology (5Knöll B. Kretz O. Fiedler C. Alberti S. Schütz G. Frotscher M. Nordheim A. Nat. Neurosci. 2006; 9: 195-204Crossref PubMed Scopus (134) Google Scholar, 23Tabuchi A. Estevez M. Henderson J.A. Marx R. Shiota J. Nakano H. Baraban J.M. J. Neurochem. 2005; 94: 169-180Crossref PubMed Scopus (34) Google Scholar, 25Shiota J. Ishikawa M. Sakagami H. Tsuda M. Baraban J.M. Tabuchi A. J. Neurochem. 2006; 98: 1778-1788Crossref PubMed Scopus (31) Google Scholar, 41O'Sullivan N.C. Pickering M. Di Giacomo D. Loshcer J.S. Murphy K.J. Cereb. Cortex. 2010; 20: 1915-1925Crossref PubMed Scopus (26) Google Scholar, and this study), MKL2 appears to be expressed in different areas of the brain and is more confined to the forebrain compared with the levels of MKL1 in the brain. As such, it is plausible that MKL1 and MKL2 play different roles in neuronal development and function. Moreover, MKL1 mutant mice display a different phenotype from MKL2, i.e. the failure to maintain a differentiated state of mammary myoepithelial cells during lactation (42Li S. Chang S. Qi X. Richardson J.A. Olson E.N. Mol. Cell. Biol. 2006; 26: 5797-5808Crossref PubMed Scopus (148) Google Scholar, 43Sun Y. Boyd K. Xu W. Ma J. Jackson C.W. Fu A. Shillingford J.M. Robinson G.W. Hennighausen L. Hitzler J.K. Ma Z. Morris S.W. Mol. Cell. Biol. 2006; 26: 5809-5826Crossref PubMed Scopus (130) Google Scholar). A null mutation in the MKL2 gene displays an embryonic lethality phenotype, due to a spectrum of cardiovascular defects (44Li J. Zhu X. Chen M. Cheng L. Zhou D. Lu M.M. Du K. Epstein J.A. Parmacek M.S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 8916-8921Crossref PubMed Scopus (120) Google Scholar) or because of the cardiac outflow tract (45Oh J. Richardson J.A. Olson E.N. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15122-15127Crossref PubMed Scopus (124) Google Scholar). To understand precisely the functional differences between MKL1 and MKL2 in the brain, further studies aimed at finding molecules that interact with the MKL family members in the brain and the assessment of the role of the MKL1/2-SRF signaling pathway on neuronal development and function in vivo, especially in conditional MKL knock-out mice, are warranted.We have demonstrated that activin is an extracellular ligand upstream of MKL/SRF that promotes the dendritic complexity of cortical neurons (FIGURE 5, FIGURE 6 and supplemental Fig. S5). Activin-induced dendritic complexity was observed to change slowly (supplemental Fig. S3) and was dependent on de novo protein and RNA synthesis (Fig. 4). In addition, MKL up-regulated the promoter of the β-actin gene, which is a cytoskeletal SRF target gene in cortical neurons (supplemental Fig. S9). In relation to dendritic spine morphology, activin increases the spine length of hippocampal neurons in a protein and RNA synthesis-independent manner (36Shoji-Kasai Y. Ageta H. Hasegawa Y. Tsuchida K. Sugino H. Inokuchi K. J. Cell Sci. 2007; 120: 3830-3837Crossref PubMed Scopus (51) Google Scholar). Therefore, our observation that activin promotes dendritic complexity may represent a late phase phenomenon that occurs after an increase in the number or strength of synaptic contacts that are made during the early phase of response to activin.What is the mechanism by which activin can transduce the signal to MKL in regulating dendritic morphology? We found that both MKL1 and MKL2 are localized to both the cytoplasm and the nucleus in cortical neurons, and a drastic change in the nuclear/cytoplasmic ratio of the total amounts of MKL was not observed even when cells were stimulated with activin (supplemental Fig. S7). The expression levels of MKL1 and MKL2 were also not altered (data not shown). On the other hand, the cytoplasmic localization of SCAI, a repressor for MKL/SRF transcription, was observed to increase by activin-stimulated neurons (Fig. 7). Furthermore, overexpression of SCAI blocked activin-induced SRF transcriptional activation and dendritic complexity (Fig. 8). Collectively, these findings suggest that the exclusion of SCAI from the nucleus, rather than direct modulation of MKL, plays a major role in activin-induced dendritic complexity. These observations represent evidence that SCAI influences neuronal morphology and that its nucleocytoplasmic shuttling is regulated by extracellular ligands.We found that C3 transferase, which is a Rho inhibitor, dominant negative SRF, and MKL1 or MKL2 siRNA reduced the dendritic complexity of neurons in the presence or absence of activin (FIGURE 5, FIGURE 6 and supplemental Fig. S8). Conversely, follistatin, an inhibitor of activin, did not affect dendritic complexity of neurons in the absence of activin (Fig. 3). MKL is regulated by Rho and actin remodeling (14Miralles F. Posern G. Zaromytidou A.I. Treisman R. Cell. 2003; 113: 329-342Abstract Full Text Full Text PDF PubMed Scopus (1030) Google Scholar, 46Stern S. Debre E. Stritt C. Berger J. Posern G. Knöll B. J. Neurosci. 2009; 29: 4512-4518Crossref PubMed Scopus (54) Google Scholar). ERK1/2-mediated phosphorylation, which partially depends upon Rho, can modulate the transcriptional function of the MKL family (14Miralles F. Posern G. Zaromytidou A.I. Treisman R. Cell. 2003; 113: 329-342Abstract Full Text Full Text PDF PubMed Scopus (1030) Google Scholar, 47Muehlich S. Wang R. Lee S.M. Lewis T.C. Dai C. Prywes R. Mol. Cell. Biol. 2008; 28: 6302-6313Crossref PubMed Scopus (84) Google Scholar). Thus, these findings indicate that constitutive MKL/SRF transcription in unstimulated cortical neurons, at least in part, contributes to dendritic complexity in a Rho-dependent manner.BDNF and NGF are the upstream signals that regulate neuronal morphology. Recent evidence suggests that BDNF and NGF also activate MKL/SRF transcriptional responses (24Kalita K. Kharebava G. Zheng J.J. Hetman M. J. Neurosci. 2006; 26: 10020-10032Crossref PubMed Scopus (72) Google Scholar, 26Wickramasinghe S.R. Alvania R.S. Ramanan N. Wood J.N. Mandai K. Ginty D.D. Neuron. 2008; 58: 532-545Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Although it is unknown whether BDNF and NGF affect dendritic morphology in an MKL-dependent manner, it would be interesting to examine whether such neurotrophin signals and activin signals converge on dendritic morphology via a common pathway, such as the cytoplasmic retention of SCAI, or convey distinct information spatiotemporally.Activin is known to regulate a variety of biological processes, especially brain functions such as adult neurogenesis and anxiety-related behavior (38Ageta H. Murayama A. Migishima R. Kida S. Tsuchida K. Yokoyama M. Inokuchi K. PLoS One. 2008; 3: e1869Crossref PubMed Scopus (85) Google Scholar, 48Zheng F. Adelsberger H. Müller M.R. Fritschy J.M. Werner S. Alzheimer C. Mol. Psychiatry. 2009; 14: 332-346Crossref PubMed Scopus (42) Google Scholar, 49Kitamura T. Saitoh Y. Takashima N. Murayama A. Niibori Y. Ageta H. Sekiguchi M. Sugiyama H. Inokuchi K. Cell. 2009; 139: 814-827Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). Elucidating whether such activin-regulated neuronal function is also mediated by the MKL family is important and may shed light on elucidating the novel mechanism by which structural alteration links to gene expression for neuronal plasticity. IntroductionSerum response factor (SRF) 4The abbreviations used are: SRFserum response factorCREcAMP response elementCREBcAMP response element-binding proteinLucluciferaseMKLmegakaryoblastic leukemiaSCAIsuppressor of cancer cell invasionSRFΔdominant negative SRF mutant. is a transcription factor that binds to a consensus sequence CC(A/T)6GG (called the CArG box), which is present in the promoters of several immediate-early or cytoskeletal genes (1Treisman R. EMBO J. 1995; 14: 4905-4913Crossref PubMed Scopus (346) Google Scholar). Several studies have demonstrated that deletion mutants of SRF in the mouse brain result in the attenuation of activity-dependent expression of immediate-early genes, impair synaptic plasticity and learning (2Ramanan N. Shen Y. Sarsfield S. Lemberger T. Schütz G. Linden D.J. Ginty D.D. Nat. Neurosci. 2005; 8: 759-767Crossref PubMed Scopus (173) Google Scholar, 3Etkin A. Alarcón J.M. Weisberg S.P. Touzani K. Huang Y.Y. Nordheim A. Kandel E.R. Neuron. 2006; 50: 127-143Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), reduce neurite outgrowth, and show abnormality of pathfindings and neuronal migration (4Alberti S. Krause S.M. Kretz O. Philippar U. Lemberger T. Casanova E. Wiebel F.F. Schwarz H. Frotscher M. Schütz G. Nordheim A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 6148-6153Crossref PubMed Scopus (119) Google Scholar, 5Knöll B. Kretz O. Fiedler C. Alberti S. Schütz G. Frotscher M. Nordheim A. Nat. Neurosci. 2006; 9: 195-204Crossref PubMed Scopus (134) Google Scholar). These findings strongly suggest that SRF plays an important role in neuronal development and plasticity (6Knöll B. Nordheim A. Trends Neurosci. 2009; 32: 432-442Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). SRF-dependent transcription is controlled by at least two different types of coactivators. One comprises the ternary complex factors, which mainly regulate the immediate-early genes such as c-fos (7Buchwalter G. Gross C. Wasylyk B. Gene. 2004; 324: 1-14Crossref PubMed Scopus (291) Google Scholar). The other type of coactivator comprises megakaryoblastic leukemia (MKL) family members. The MKL family consists of megakaryocytic acute leukemia/megakaryoblastic leukemia 1/myocardin-related transcription factor-A/basic SAP, and coiled-coil domain (MAL/MKL1/MRTF-A/BSAC) and MKL2/MRTF-B (8Wang D.Z. Li S. Hockemeyer D. Sutherland L. Wang Z. Schratt G. Richardson J.A. Nordheim A. Olson E.N. Proc. Natl. Acad. U.S.A. 2002; 99: 14855-14860Crossref PubMed Scopus (395) Google Scholar, 9Sasazuki T. Sawada T. Sakon S. Kitamura T. Kishi T. Okazaki T. Katano M. Tanaka M. Watanabe M. Yagita H. Okumura K. Nakano H. J. Biol. Chem. 2002; 277: 28853-28860Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 10Cen B. Selvaraj A. Burgess R.C. Hitzler J.K. Ma Z. Morris S.W. Prywes R. Mol. Cell. Biol. 2003; 23: 6597-6608Crossref PubMed Scopus (243) Google Scholar, 11Selvaraj A. Prywes R. J. Biol. Chem. 2003; 278: 41977-41987Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 12Cen B. Selvaraj A. Prywes R. J. Cell. Biochem. 2004; 93: 74-82Crossref PubMed Scopus (132) Google Scholar, 13Pipes G.C. Creemers E.E. Olson E.N. Genes Dev. 2006; 20: 1545-1556Crossref PubMed Scopus (385) Google Scholar). In nonneuronal cells, MKL1 is primarily activated by actin rearrangement stimulated with the activation of Rho GTPases; that is, release of MKL1 from G-actin enables MKL1 to translocate to the nucleus where it binds to and activates SRF (14Miralles F. Posern G. Zaromytidou A.I. Treisman R. Cell. 2003; 113: 329-342Abstract Full Text Full Text PDF PubMed Scopus (1030) Google Scholar). MKL2, in addition to MKL1, regulates a set of cytoskeletal genes (11Selvaraj A. Prywes R. J. Biol. Chem. 2003; 278: 41977-41987Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 12Cen B. Selvaraj A. Prywes R. J. Cell. Biochem. 2004; 93: 74-82Crossref PubMed Scopus (132) Google Scholar). Furthermore, suppressor of cancer cell invasion (SCAI) has recently been identified as an MKL-interacting cofactor that inhibits cancer cell invasion (15Brandt D.T. Baarlink C. Kitzing T.M. Kremmer E. Ivaska J. Nollau P. Grosse R. Nat. Cell Biol. 2009; 11: 557-568Crossref PubMed Scopus (98) Google Scholar). Therefore, SRF-driven transcriptional regulation appears to be more complicated because of the presence of novel MKL-interacting cofactors. However, it remains unclear as to whether MKL1, MKL2, and SCAI play different roles in several biological processes through the regulation of their own target genes in neurons.The transforming growth factor β (TGF-β) superfamily plays important roles in various cellular processes associated with cell growth, differentiation, and development (16Massagué J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2053) Google Scholar, 17Derynck R. Akhurst R.J. Nat. Cell Biol. 2007; 9: 1000-1004Crossref PubMed Scopus (300) Google Scholar, 18Tsuchida K. Nakatani M. Hitachi K. Uezumi A. Sunada Y. Ageta H. Inokuchi K. Cell. Commun. Signal. 2009; 7: 15Crossref PubMed Scopus (131) Google Scholar). To regulate such a wide variety of cellular functions, TGF-β binds to specific receptors and activates a multitude of signals, including the canonical pathway that promotes phosphorylation and nuclear translocation of Smad2 and Smad3 (19Xu L. Biochim. Biophys. Acta. 2006; 1759: 503-513Crossref PubMed Scopus (77) Google Scholar) and the noncanonical pathway that activates ERK, p38 MAP kinases, or Rho GTPases (20Zhang Y.E. Cell Res. 2009; 19: 128-139Crossref PubMed Scopus (1284) Google Scholar). Several lines of evidence suggest that TGF-β signaling also regulates SRF (21Qiu P. Feng X.H. Li L. J. Mol. Cell. Cardiol. 2003; 35: 1407-1420Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 22Morita T. Mayanagi T. Sobue K. J. Cell Biol. 2007; 179: 1027-1042Crossref PubMed Scopus (215) Google Scholar). Recently, a study showed that TGF-β1, a member of the TGF-β superfamily, activates SRF-dependent cytoskeletal genes or Smad3-dependent genes via nuclear translocation of MKL. This process may be involved in epithelial-mesenchymal transition, which occurs during embryonic development and tumor progression (22Morita T. Mayanagi T. Sobue K. J. Cell Biol. 2007; 179: 1027-1042Crossref PubMed Scopus (215) Google Scholar). However, it is unknown whether MKL family members mediate TGF-β signaling in the brain.In neurons, MKL1 regulates SRF-mediated transcription through the RhoA or brain-derived neurotrophic factor (BDNF)-induced ERK1/2 signaling pathway (23Tabuchi A. Estevez M. Henderson J.A. Marx R. Shiota J. Nakano H. Baraban J.M. J. Neurochem. 2005; 94: 169-180Crossref PubMed Scopus (34) Google Scholar, 24Kalita K. Kharebava G. Zheng J.J. Hetman M. J. Neurosci. 2006; 26: 10020-10032Crossref PubMed Scopus (72) Google Scholar). In addition, MKL1 is highly expressed in the forebrain and regulates the neuronal morphology of cortical or hippocampal neurons (5Knöll B. Kretz O. Fiedler C. Alberti S. Schütz G. Frotscher M. Nordheim A. Nat. Neurosci. 2006; 9: 195-204Crossref PubMed Scopus (134) Google Scholar, 25Shiota J. Ishikawa M. Sakagami H. Tsuda M. Baraban J.M. Tabuchi A. J. Neurochem. 2006; 98: 1778-1788Crossref PubMed Scopus (31) Google Scholar). MKL1 may also be involved in nerve growth factor (NGF)-induced axonal growth of sensory neurons (26Wickramasinghe S.R. Alvania R.S. Ramanan N. Wood J.N. Mandai K. Ginty D.D. Neuron. 2008; 58: 532-545Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). These observations imply that MKL family members participate in the regulation of neuronal morphology through regulating actin-based morphological changes or by controlling the expression of SRF-targeted cytoskeletal genes. In contrast to several reports on the function of MKL1, the expression and function of MKL2 in the brain remain unresolved. Furthermore, there is little information describing the kinds of extracellular ligands that potentially act upstream of the MKL family to regulate dendritic morphology. In the present study, we first investigated the pattern of MKL2 expression in the developing and adult brain and assessed whether MKL2 regulates SRF transcription and the morphology of rat cortical neurons. Furthermore, we found that activin, a member of the TGF-β superfamily, promoted dendritic complexity through nuclear export of SCAI and removing SCAI repressor function for MKL/SRF gene expression.
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