Key points are not available for this paper at this time.
All-trans-retinoic acid (RA) plays a crucial role in survival and differentiation of neurons. For elucidating signaling mechanisms involved in RA-induced neuronal differentiation, we have selected SH-SY5Y cells, which are an established in vitro cell model for studying RA signaling. Here we report that RA-induced neuronal differentiation of SH-SY5Y cells is coupled with increased expression/activation of TGase andin vivo transamidation and activation of RhoA. In addition, RA promotes formation of stress fibers and focal adhesion complexes, and activation of ERK1/2, JNK1, and p38α/β/γ MAP kinases. Using C-3 exoenzyme (RhoA inhibitor) or monodansylcadaverine (TGase inhibitor), we show that transamidated RhoA regulates cytoskeletal rearrangement and activation of ERK1/2 and p38γ MAP kinases. Further, by using stable SH-SY5Y cell lines (overexpressing wild-type, C277S mutant, and antisense TGase), we demonstrate that transglutaminase activity is required for activation of RhoA, ERK1/2, JNK1, and p38γ MAP kinases. Activated MAP kinases differentially regulate RA-induced neurite outgrowth and neuronal marker expression. The results of our studies suggest a novel mechanism of RA signaling, which involves activation of TGase and transamidation of RhoA. RA-induced activation of TGase is proposed to induce multiple signaling pathways that regulate neuronal differentiation. All-trans-retinoic acid (RA) plays a crucial role in survival and differentiation of neurons. For elucidating signaling mechanisms involved in RA-induced neuronal differentiation, we have selected SH-SY5Y cells, which are an established in vitro cell model for studying RA signaling. Here we report that RA-induced neuronal differentiation of SH-SY5Y cells is coupled with increased expression/activation of TGase andin vivo transamidation and activation of RhoA. In addition, RA promotes formation of stress fibers and focal adhesion complexes, and activation of ERK1/2, JNK1, and p38α/β/γ MAP kinases. Using C-3 exoenzyme (RhoA inhibitor) or monodansylcadaverine (TGase inhibitor), we show that transamidated RhoA regulates cytoskeletal rearrangement and activation of ERK1/2 and p38γ MAP kinases. Further, by using stable SH-SY5Y cell lines (overexpressing wild-type, C277S mutant, and antisense TGase), we demonstrate that transglutaminase activity is required for activation of RhoA, ERK1/2, JNK1, and p38γ MAP kinases. Activated MAP kinases differentially regulate RA-induced neurite outgrowth and neuronal marker expression. The results of our studies suggest a novel mechanism of RA signaling, which involves activation of TGase and transamidation of RhoA. RA-induced activation of TGase is proposed to induce multiple signaling pathways that regulate neuronal differentiation. retinoic acid mitogen-activated protein MAP kinase extracellular-regulated kinase fetal bovine serum c-Jun amino-terminal kinase glutathione S-transferase myelin basic protein phosphate-buffered saline transglutaminase monodansylcadaverine Retinoic acid (RA),1 a metabolic derivative of vitamin A, plays a crucial role in the development and differentiation of the nervous system (1White J.C. Highland M. Kaiser M. Clagett-Dame M. Dev. Biol. 2000; 220: 263-284Crossref PubMed Scopus (158) Google Scholar). During normal embryogenesis, there is a requirement for precise timing of the exposure of embryonal structures to RA and a coordinated pattern of expression of the retinoic acid receptor (RAR) and retinoic acid X receptor (RXR) isoforms (2Smith S.M. Dickman E.D. Power S.C. Lancman J. J. Nutr. 1998; 128: 467S-470SCrossref PubMed Google Scholar). 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Neuroscience. 2001; 102: 481-491Crossref PubMed Scopus (110) Google Scholar). the signaling mechanisms involved differentiation are we that RA-induced neuronal differentiation in SH-SY5Y cells is associated with increased expression/activation of TGase, to transamidation of RhoA. RhoA is and promotes formation of stress fibers and focal adhesion The transamidated RhoA promotes activation of ERK1/2 and p38γ MAP which regulate for expression neuronal differentiation. Cell from and from RhoA, JNK1, and and protein from MAP kinase myelin basic protein from TGase and from and from and from and the signal used for of proteins from C-3 and from SH-SY5Y cells in with and and in a cell lines of SH-SY5Y cells, with TGase, an inactive TGase activity to a the active or an antisense TGase and RA-induced expression of from of in the of and to or treated with RA or in for as in the with or different agents and RA treated SH-SY5Y cells in and and The for and used for of MAP JNK1, or with the and with kinase and and in of kinase of or of and of The reaction for by the of to on and proteins to and to by the with JNK1, or The expression of TGase, neuronal marker protein and by treated with or RA in for the with the cells in and by for The protein to the of on to with and The with to the used for of the RhoA activation using the as Schwartz M.A. EMBO J. 1999; PubMed Scopus Google Scholar). In cells with of and and The for of cell for with of protein the RhoA binding of on The in and proteins on Activated RhoA by with RhoA. The of Rho to the of RhoA in cell for the of Rho activity in different studies on stress and focal adhesion formation using SH-SY5Y cells on in with with cells with for to for with cells with for and with for with and for an and with with and with an of the with a the of RhoA and pathways on RA-induced neurite SH-SY5Y cells with C-3 monodansylcadaverine inhibitor), of and or of RA in with with or different the cell using a The cell used for neuronal marker expression and transglutaminase and SH-SY5Y cells and of protein from the used for the of transglutaminase as (13Singh U.S. Erickson J.W. Cerione R.A. Biochemistry. 1995; 34: 15863-15871Crossref PubMed Scopus (85) Google Scholar, Cerione R. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). SH-SY5Y cells, to in treated (or with RA for of in the of as (15Singh U.S. Kunar M.T. Kao Y.L. Baker K.M. EMBO J. 2001; 20: 2413-2423Crossref PubMed Scopus (80) Google Scholar). and with RhoA (or to and the with a signal proteins by are by a for RA-induced cellular differentiation is coupled with increased expression/activation of TGase in cell lines J. Lesort M. Johnson G.V. Neuroscience. 2001; 102: 481-491Crossref PubMed Scopus (110) Google Scholar, M.A. Singh U.S. Lee D.A. Boehm J.E. Combs C. Zgola M.M. Page R.L. Cerione R.A. J. Biol. Chem. 2001; 276: 33582-33587Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). the role of TGase in RA-induced neuronal differentiation in SH-SY5Y cells, we the expression of TGase and neuronal in to RA shown in and of TGase expression of RA which associated with increased transglutaminase activity of of of protein, with of of of protein, RA the expression of and from to of and of neuronal marker that RA-induced expression of neuronal associated with increased expression/activation of TGase, which involved in neuronal differentiation. we that RhoA is an in vivo substrate of TGase and RA promotes transamidation of RhoA in cells (15Singh U.S. Kunar M.T. Kao Y.L. Baker K.M. EMBO J. 2001; 20: 2413-2423Crossref PubMed Scopus (80) Google Scholar). the of RA-induced expression of TGase in SH-SY5Y cells promotes transamidation of RhoA, we in vivo of SH-SY5Y cells using of RhoA by and of RhoA. shown in A, there increased of RhoA of RA which with the increased transglutaminase activity of TGase transamidated RhoA is the binding a protein of by from RA cells as RhoA that transamidated RhoA as an a cell vivo transamidated cells with and and activation of RhoA shown in we of or RhoA the of the role of TGase in activation of RhoA, we have used monodansylcadaverine a known TGase D. A. L. Biochem. J. Scopus Google Scholar), and as shown in A, RA-induced transglutaminase SH-SY5Y cells with RA in the or of for and to the binding RA the of RhoA (or activation of of and in The RA-induced activation of RhoA by used stable SH-SY5Y cell lines wild-type, C277S mutant, and antisense TGase J. Lesort M. Johnson G.V. Neuroscience. 2001; 102: 481-491Crossref PubMed Scopus (110) Google Scholar) for the role of TGase in RhoA activation and of TGase in a significant activation of RhoA with the C277S or cell change in RhoA activation RA activation of RhoA in a cell line which is to that of normal SH-SY5Y a of RhoA activation in and significant in RhoA activation in C277S and antisense cell lines RA Further, we the expression of TGase in different cell lines RA of TGase expression in cells, and in in TGase expression in and C277S cell TGase expression in the or of RA in antisense results that TGase required for RA-induced activation of RhoA in SH-SY5Y of the of RhoA activation is increased rearrangement A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3843) Google Scholar). the of RhoA on cytoskeletal SH-SY5Y cells treated with RA in the or of C-3 exoenzyme (RhoA inhibitor) or for and with stress and focal adhesion increased stress formation and focal adhesion formation in to RA of of RA required to induce the in stress and focal adhesion of cells with C-3 or RA-induced formation of stress fibers and focal adhesion results that transamidation of RhoA required for activation and cytoskeletal rearrangement in to RA in SH-SY5Y studies suggest that pathways a role in neuronal differentiation M. M. N. J. 1999; PubMed Scopus Google Scholar). the is involved in RA-induced neuronal differentiation of SH-SY5Y cells, MAP kinase and activity by in vitro kinase shown in RA significant activation of ERK1/2, JNK1, and p38γ MAP ERK1/2 and p38γ MAP kinase activation and activation RA activation of on of ERK1/2, JNK1, and and an of protein in different the RA-induced activation of MAP kinases are by TGase and of RhoA, we the of and C-3 exoenzyme on activation of MAP kinases. shown in RA-induced activation of ERK1/2, and p38γ by a role of transglutaminase activity in the other of cells with C-3 exoenzyme RA-induced activation of and the activation of ERK1/2, on that transamidated RhoA involved in RA-induced we that or C-3 exoenzyme on activation of or RA-induced of TGase expression A, Using stable SH-SY5Y cell lines (overexpressing wild-type, C277S mutant, and antisense TGase), we the role of TGase in activation of MAP kinases. of TGase the activation of ERK1/2, JNK1, and p38γ MAP kinase and with as shown in and activation in the C277S and cells, transglutaminase activity of TGase plays an important role in with RA significant activation of ERK1/2, JNK1, and p38γ MAP kinase in and with cell and TGase and with TGase cell cell of C277S or antisense TGase RA-induced activation of ERK1/2, JNK1, and of TGase on the activation of and MAP kinases. with RA a activation of in and cell and on activation of MAP kinase in cell that RA-induced activation of ERK1/2, JNK1, and p38γ MAP kinases by the the role of TGase and transamidated RhoA in RA-induced neurite SH-SY5Y cells treated with RA in the or of or C-3 exoenzyme and in phenotype RA neuronal differentiation as shown in by the of the cell and the of which of differentiation of RA RA-induced neurite outgrowth by and phenotype change in cells transamidated RhoA neuronal marker we treated cells with or C-3 exoenzyme for the expression of and by RA-induced expression of and by and change in cells that TGase is required in RA-induced neuronal differentiation, RhoA involved in RA-induced neurite outgrowth and neuronal marker and MAP kinases RA-induced expression of and SH-SY5Y cells or treated with different as in the to and for and TGase as in the to The for as a The cell used for the of transglutaminase are for the role of MAP kinases in RA-induced neuronal differentiation, SH-SY5Y cells treated with RA or RA inhibitor), or inhibitor), and the and neuronal marker expression RA-induced neurite outgrowth and of expression by and or on RA-induced neurite RA-induced expression of the of the is involved in RA-induced expression/activation of TGase, we the of on TGase expression and RA-induced of TGase expression by of the and RA-induced TGase activity by the other is that RA of and expression of neuronal is regulated by expression of TGase and the activation of MAP kinases. have that RA TGase GTP binding and transglutaminase activities the protein expression of TGase in cells, of other proteins U.S. Cerione R.A. J. Biol. Chem. 1996; 271: 27292-27298Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). cells, there change in the expression of TGase, and the inactive of TGase to the active RA expression and activation of TGase in SH-SY5Y cells important which RA signaling in SH-SY5Y cells, is the phenotype change and expression of the neuronal SH-SY5Y cells neuronal differentiation and of and expression in to RA and is the protein in the and proteins of the neurite required for of and and have been shown to for neuronal differentiation S. J. Mol. Cell Neurosci. 2001; 17: PubMed Scopus Google Scholar, J.W. J. Cell Biol. PubMed Scopus Google Scholar). that RA-induced neuronal differentiation associated with increased expression/activation of The state of differentiation of in is to to neuronal T. 2000; PubMed Scopus Google Scholar). that TGase is involved in apoptosis M.A. Singh U.S. Lee D.A. Boehm J.E. Combs C. Zgola M.M. Page R.L. Cerione R.A. J. Biol. Chem. 2001; 276: 33582-33587Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and neurite outgrowth J. Lesort M. Johnson G.V. Neuroscience. 2001; 102: 481-491Crossref PubMed Scopus (110) Google Scholar) are with our that RA-induced expression/activation of TGase a crucial role in of neuronal differentiation. For studying the signaling that are by TGase in RA-induced neuronal differentiation, we have used which as a of TGase and is used for of in vivo transamidation (15Singh U.S. Kunar M.T. Kao Y.L. Baker K.M. EMBO J. 2001; 20: 2413-2423Crossref PubMed Scopus (80) Google Scholar, A. G. M. R.A. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). with polyamines for and is and apoptosis in cells for a and the function of TGase and of M.A. Singh U.S. Lee D.A. Boehm J.E. Combs C. Zgola M.M. Page R.L. Cerione R.A. J. Biol. Chem. 2001; 276: 33582-33587Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). In SH-SY5Y cells, RA-induced transglutaminase activity and apoptosis when used in serum RhoA is vivo substrate of TGase, and has been shown that RA of cells to transamidation of RhoA and promotes formation of stress fibers and focal adhesion complexes, which are of RhoA activation (15Singh U.S. Kunar M.T. Kao Y.L. Baker K.M. EMBO J. 2001; 20: 2413-2423Crossref PubMed Scopus (80) Google Scholar). In SH-SY5Y cells, RA transamidation of RhoA to as shown by binding and RA-induced activation of RhoA by for a role of transglutaminase activity in activation of RhoA. The of transglutaminase activity and of RhoA activation a to the in vitro and in vivo of transamidation the role of TGase in RhoA we used SH-SY5Y stable cell wild-type, C277S mutant, and antisense The of TGase activation of RhoA and significant change RA of RA activation of RhoA in the cells C277S or antisense results suggest that transglutaminase activity is required for RA-induced of RhoA in SH-SY5Y The outgrowth of the in to is by R. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). of and and the are by the of the E. J. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). The Rho family of of the are important of the signaling pathways that the of to the regulation of the in neuronal 2001; 11: PubMed Scopus Google Scholar). of and the formation of and in neuroblastoma structures by of C-3 which such as neurite R. S. S. L. Mol. Cell. Biol. 1997; 17: PubMed Scopus Google Scholar). The increased formation of stress fibers and focal adhesion in RA treated SH-SY5Y cells by and C-3 exoenzyme that RhoA transamidation is required for cytoskeletal RA of cells shown to stress formation and increased cell adhesion G. R. E. G. E. Int. J. Cancer. 1998; PubMed Scopus Google Scholar). RhoA is a in the of cytoskeletal and in the transamidated state to the induce formation of stress fibers and focal adhesion in SH-SY5Y cells A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3843) Google Scholar). focal adhesion RA-induced stress formation in is that following activation by RhoA exhibits differentiation of focal adhesion and formation of stress fibers in RA activation of ERK1/2, JNK1, and p38γ in SH-SY5Y cells, which by that transglutaminase activity is involved in the activation and using the C-3 which the of RhoA by K. T. N. S. J. Cell Biol. PubMed Scopus Google Scholar), we the role of RhoA in RA-induced activation of MAP kinases. The of the C-3 exoenzyme on ERK1/2 and p38γ a of RhoA in activation ERK1/2 is shown to regulate neuronal differentiation by expression M. M. N. J. 1999; PubMed Scopus Google Scholar), and p38γ the factors and which on the and C. M.A. A. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, M. Dev. 2001; PubMed Scopus Google Scholar). ERK1/2, and RA expression of required for neuronal differentiation. of the C277S or antisense promotes activation of ERK1/2, JNK1, and p38γ MAP that transglutaminase activity is required The activation of ERK1/2, JNK1, and p38α/β/γ in TGase cells by RA to the of which to TGase and in to RA RA-induced neurite outgrowth and neuronal marker expression in SH-SY5Y cells is by TGase and RhoA we treated SH-SY5Y cells with RA C-3 and RA shown in and RA neurite outgrowth and expression of neuronal and which by that TGase is involved in RA-induced neuronal differentiation, which is with studies J. Lesort M. Johnson G.V. Neuroscience. 2001; 102: 481-491Crossref PubMed Scopus (110) Google Scholar). the other C-3 exoenzyme neurite or expression of and that activation of RhoA in RA-induced neurite or expression of neuronal expression and of proteins is to to the and of neuronal processes J.W. J. Cell Biol. PubMed Scopus Google Scholar, E.A. Lee J. 1991; PubMed Scopus Google Scholar). The neurite as in our to the of other signaling such as and Rac1, which by RA of SH-SY5Y cells is a that RhoA promotes axonal as by studies Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of cells with and RA-induced neurite outgrowth and expression of the expression of the other of neurite outgrowth as well as the expression of to cells shown by the that apoptosis a The regulation of neurite outgrowth and expression of neuronal by MAP kinase the of multiple signaling in RA-induced neuronal differentiation. report S. Lee J.C. Biochem. 1997; PubMed Scopus Google Scholar) that the that p38γ neurite outgrowth and expression of and in RA-induced neuronal differentiation. RA-induced activation of the that the involved in of expression/activation of we used of and and the expression/activation of TGase in to shown in of SH-SY5Y cells with and on RA-induced expression or activation of TGase, that are In our studies suggest that RA on TGase the expression/activation of TGase in SH-SY5Y cells, in cells, RA promotes activation of TGase the expression U.S. Cerione R.A. J. Biol. Chem. 1996; 271: 27292-27298Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). using a stable cell line of SH-SY5Y cells (overexpressing wild-type, C277S mutant, and antisense and TGase we have that TGase is required for neurite outgrowth and expression of neuronal a novel mechanism of RA signaling, which involves activation of TGase, to transamidation of RhoA. RhoA is and promotes cytoskeletal rearrangement and activation of MAP kinases. RA-induced activation of TGase and transamidation of RhoA as a for signaling which are proposed to an important role in neuronal differentiation of SH-SY5Y cells for of the Johnson of for stable cell lines of SH-SY5Y that C277S mutant, and antisense TGase, and of for
Singh et al. (Sat,) studied this question.