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• W2022702828 abstract "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). In addition, RA plays an important role in the function of the adult brain, which has been shown to synthesize RA and express retinoid receptors, as well as cellular-binding proteins (3Zetterstrom R.H. Lindqvist E. Mata de Urquiza A. Tomac A. Eriksson U. Perlmann T. Olson L. Eur. J. Neurosci. 1999; 11: 407-416Crossref PubMed Scopus (216) Google Scholar). Many studies on a variety of embryogenic neuronal cell types have shown that RA can stimulate both neurite number and length (4Maden M. Methods Mol. Biol. 1999; 97: 491-509PubMed Google Scholar) and neurite outgrowth in amphibian spinal cord (5Hunter K. Maden M. Summerbell D. Eriksson U. Holder N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3666-3670Crossref PubMed Scopus (85) Google Scholar). SH-SY5Y cells, a neuroblastic subclone of the neuroblastoma cell line SK-N-SH, withdraw from the cell cycle and exhibit a distinct neuronal phenotype when treated with different agents such as neurotrophic factors (6Kaplan D.R. Matsumoto K. Lucarelli E. Thiele C.J. Neuron. 1993; 11: 321-331Abstract Full Text PDF PubMed Scopus (314) Google Scholar), retinoic acid (6Kaplan D.R. Matsumoto K. Lucarelli E. Thiele C.J. Neuron. 1993; 11: 321-331Abstract Full Text PDF PubMed Scopus (314) Google Scholar), phorbol ester (7Pahlman S. Odelstad L. Larsson E. Grotte G. Nilsson K. Int. J. Cancer. 1981; 28: 583-589Crossref PubMed Scopus (226) Google Scholar), or staurosporine (8Jalava A. Heikkila J. Lintunen M. Akerman K. Pahlman S. FEBS Lett. 1992; 300: 114-118Crossref PubMed Scopus (58) Google Scholar, 9Jalava A. Akerman K. Heikkila J. J. Cell. Physiol. 1993; 155: 301-312Crossref PubMed Scopus (57) Google Scholar). The RA-induced change to a neuronal phenotype in SH-SY5Y cells is associated with increased expression of tissue transglutaminase (TGase) (10Piacentini M. Annicchiarico-Petruzzelli M. Oliverio S. Piredda L. Biedler J.L. Melino E. Int. J. Cancer. 1992; 52: 271-278Crossref PubMed Scopus (110) Google Scholar, 11Zhang J. Lesort M. Guttmann R.P. Johnson G.V. J. Biol. Chem. 1998; 273: 2288-2295Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 12Tucholski J. Lesort M. Johnson G.V. Neuroscience. 2001; 102: 481-491Crossref PubMed Scopus (110) Google Scholar). TGase, a GTP-binding protein, participates in signal transduction pathways as a nonconventional G-protein, and exhibits distinct GTP-binding/hydrolyzing and transglutaminase activities (13Singh U.S. Erickson J.W. Cerione R.A. Biochemistry. 1995; 34: 15863-15871Crossref PubMed Scopus (85) Google Scholar, 14Singh U.S. Cerione R.A. J. Biol. Chem. 1996; 271: 27292-27298Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 15Singh U.S. Kunar M.T. Kao Y.L. Baker K.M. EMBO J. 2001; 20: 2413-2423Crossref PubMed Scopus (80) Google Scholar). Transglutaminase activity (or transamidation function), used to cross-link polyamines such as putrescine, spermine, or spermidine to target proteins, is regulated by the GTP binding activity of TGase (13Singh U.S. Erickson J.W. Cerione R.A. Biochemistry. 1995; 34: 15863-15871Crossref PubMed Scopus (85) Google Scholar,16Antonyak 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 transamidation reaction of TGase has been implicated in a number of biological processes such as axonal regeneration, cellular differentiation, and apoptosis (17Eitan S. Schwartz M. Science. 1993; 261: 106-108Crossref PubMed Scopus (128) Google Scholar, 18Chiocca E.A. Davies P.J. Stein J.P. J. Cell. Biochem. 1989; 39: 293-304Crossref PubMed Scopus (56) Google Scholar, 19Oliverio S. Amendola A., Di Sano F. Farrace M.G. Fesus L. Nemes Z. Piredda L. Spinedi A. Piacentini M. Mol. Cell. Biol. 1997; 17: 6040-6048Crossref PubMed Scopus (125) Google Scholar). The Rho family of small G-proteins, primarily RhoA, Rac1, and Cdc42, are known to have a significant role in neuronal differentiation (20Luo L. Nat. Rev. Neurosci. 2000; 1: 173-180Crossref PubMed Scopus (838) Google Scholar,21Threadgill R. Bobb K. Ghosh A. Neuron. 1997; 19: 625-634Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). RhoA is an in vivo substrate of TGase and is known to have an important role in cytoskeletal rearrangement and regulation of cell morphology and differentiation (15Singh U.S. Kunar M.T. Kao Y.L. Baker K.M. EMBO J. 2001; 20: 2413-2423Crossref PubMed Scopus (80) Google Scholar, 22Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1682) Google Scholar, 23Mack C.P. Somlyo A.V. Hautmann M. Somlyo A.P. Owens G.K. J. Biol. Chem. 2001; 276: 341-347Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 24Paterson H.F. Self A.J. Garrett M.D. Just I. Aktories K. Hall A. J. Cell Biol. 1990; 111: 1001-1007Crossref PubMed Scopus (571) Google Scholar, 25Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3843) Google Scholar). Like other G-proteins, RhoA binds GTP in the active state, and following hydrolysis, returns to an inactive GDP-bound state (26Bourne H.R. Wrischnik L. Kenyon C. Nature. 1990; 348: 678-679Crossref PubMed Scopus (20) Google Scholar). The glutamine residue at position 63 of RhoA (switch II domain) is required for GTP hydrolysis (27Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Crossref PubMed Scopus (355) Google Scholar). After deamidation/transamidation, glutamine 63 is not available for GTP hydrolysis, and RhoA is constitutively activated (28Flatau G. Lemichez E. Gauthier M. Chardin P. Paris S. Fiorentini C. Boquet P. Nature. 1997; 387: 729-733Crossref PubMed Scopus (427) Google Scholar,29Masuda M. Betancourt L. Matsuzawa T. Kashimoto T. Takao T. Shimonishi Y. Horiguchi Y. EMBO J. 2000; 19: 521-530Crossref PubMed Scopus (97) Google Scholar). RA promotes activation of TGase and in vivotransamidation of RhoA (at glutamine 63) in HeLa cells. After transamidation, RhoA binds/activates RhoA-associated kinase-2 (ROCK-2), a downstream target and an effector of GTP-bound RhoA, leading to increased stress fiber and focal adhesion complex formation (15Singh U.S. Kunar M.T. Kao Y.L. Baker K.M. EMBO J. 2001; 20: 2413-2423Crossref PubMed Scopus (80) Google Scholar). Mitogens, tumor promoters, and cell differentiation inducing agents trigger an intracellular signaling cascade, which involves Ras and Rho GTPases and leads to activation of mitogen-activated protein (MAP) kinases (30Aznar S. Lacal J.C. Cancer Lett. 2001; 165: 1-10Crossref PubMed Scopus (289) Google Scholar). Previous studies have suggested that ERK/MAPK pathway is crucial for NGF-induced neuronal differentiation of the cells, since blockade of the ERK/MAPK activation inhibits neurite induction (31Szeberenyi J. Cai H. Cooper G.M. Mol. Cell. Biol. 1990; 10: 5324-5332Crossref PubMed Scopus (275) Google Scholar,32Pang L. Sawada T. Decker S.J. Saltiel A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar), and constitutive activation of the ERK/MAPK pathway results in neurite outgrowth (33Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1854) Google Scholar, 34Encinas M. Iglesias M. Llecha N. Comella J.X. J. Neurochem. 1999; 73: 1409-1421Crossref PubMed Scopus (237) Google Scholar). However, some other findings demonstrate that sustained activation of ERK/MAPK does not induce neurite outgrowth in dorsal root ganglionic (DRG) sensory and sympathetic neurons and SH-SY5Y cells (35Klinz F.J. Wolff P. Heumann R. J. Neurosci. Res. 1996; 46: 720-726Crossref PubMed Scopus (30) Google Scholar, 36Olsson A.K. Nanberg E. Exp. Cell Res. 2001; 265: 21-30Crossref PubMed Scopus (22) Google Scholar). These observations suggest the existence of an additional signaling pathway(s) important for neuronal differentiation. Activation of c-Jun amino-terminal kinase is required for RA-induced neuronal differentiation of P19 embryonal carcinoma cells (37Wang H. Ikeda S. Kanno S. Guang L.M. Ohnishi M. Sasaki M. Kobayashi T. Tamura S. FEBS Lett. 2001; 503: 91-96Crossref PubMed Scopus (34) Google Scholar). The subfamily of p38 MAP kinases includes p38α (38Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2420) Google Scholar), p38β (39Jiang Y. Chen C., Li, Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. Chem. 1996; 271: 17920-17926Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar), p38γ (40Lechner C. Zahalka M.A. Giot J.F. Moller N.P. Ullrich A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4355-4359Crossref PubMed Scopus (278) Google Scholar), and p38δ (41Jiang Y. Gram H. Zhao M. New L., Gu, J. Feng L., Di Padova F. Ulevitch R.J. Han J. J. Biol. Chem. 1997; 272: 30122-30128Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). Sustained activation of p38α promotes neuronal differentiation, and inhibition of p38 α/β by a specific inhibitor SB203580 or by expression of dominant-negative constructs of the p38 pathway blocks neurite outgrowth in PC12 cells (42Morooka T. Nishida E. J. Biol. Chem. 1998; 273: 24285-24288Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Studies have demonstrated that p38γ is highly expressed in human skeletal muscle and appears to function as a signal transducer during differentiation of myoblasts to myotubes (40Lechner C. Zahalka M.A. Giot J.F. Moller N.P. Ullrich A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4355-4359Crossref PubMed Scopus (278) Google Scholar). However, its role in neuronal differentiation is not demonstrated. There is growing evidence that RA-induced neuronal differentiation in SH-SY5Y cells is mediated by TGase. Using stable cell lines of SH-SY5Y, overexpressing wild type, C277S mutant (transglutaminase-defective), and antisense TGase, it has been demonstrated that TGase is necessary and sufficient in promoting neurite outgrowth (12Tucholski J. Lesort M. Johnson G.V. Neuroscience. 2001; 102: 481-491Crossref PubMed Scopus (110) Google Scholar). However, the signaling mechanisms involved during differentiation are not known. Here, we present evidence that RA-induced neuronal differentiation in SH-SY5Y cells is associated with increased expression/activation of TGase, leading to transamidation of RhoA. After transamidation, RhoA is activated and promotes formation of stress fibers and focal adhesion complexes. The transamidated RhoA also promotes activation of ERK1/2 and p38γ MAP kinase, which may regulate nuclear events for promoting gene expression during neuronal differentiation. Cell culture reagents were purchased from Invitrogen. RA, common use reagents, and vinculin antibody were from Sigma; RhoA, GAP-43, actin, ERK 1/2, JNK1, and p38 α/β MAPK antibodies, ATF-2, c-Jun, and protein A/G-agarose were from Santa Cruz Biotechnology; p38γ, δ MAP kinase antibody, myelin basic protein (MBP) were from Upstate Biotechnology; TGase and neurofilament B (NF-B) antibodies were purchased from Neomarkers. Texas Red-X phalloidin and fluorescein-labeled anti-mouse secondary antibody were from Molecular Probes; [32P]ATP and [14C]putrescine were from PerkinElmer Life Sciences and the signal enhancer kit used for autoradiography of 14C-labeled proteins was from AmershamBiosciences. C-3 exoenzyme, PD98059, SB202190, and JNK inhibitor were from CalBiochem. SH-SY5Y cells were grown in Dulbecco's Modified Eagle's Medium supplemented with 10% FBS and antibiotics (penicillin and streptomycin; 1% each) in a 5% CO2humidified incubator at 37 °C. Stable cell lines of SH-SY5Y cells, stably transfected with vector, wild-type TGase, an inactive TGase mutant (C277S, without transamidating activity due to a point mutation within the active site), or an antisense TGase construct (which blocks endogenous and RA-induced expression of TGase) were obtained from Dr. Gail V. Johnson, University of Alabama at Birmingham, Alabama. Cells were grown in the presence of 150 μg/ml G418 and 10% FBS. Cells grown to subconfluence (20%) were untreated or treated with RA at 5 μm or vehicle (Me2SO), in medium containing 3% FBS for 1–4 days, as indicated in the figures. Medium with or without different agents was replaced every day. Untreated and RA treated SH-SY5Y cells were lysed in buffer containing 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mmβ-glycerophosphate, 1 mm sodium vanadate, 10 μg/ml leupeptin and aprotinin, and 1 mm phenylmethylsulfonyl fluoride. The lysate was sedimented at 15,000 ×g for 10 min at 4 °C, and used for immunoprecipitation of p38α/β, -γ, -δ MAP kinase, JNK1, or ERK 1/2. Immunoprecipitates were washed three times with the lysis buffer and two times with kinase buffer (25 mm HEPES, pH 7.4, 5 mmβ-glycerophosphate, 2 mm dithiothreitol, and 10 mm MgCl2) and resuspended in 40 μl of kinase buffer containing 4 μg of GST-ATF-2, GST-c-jun, or 20 μg of MBP, 50 μm ATP, and 10 μCi of [γ-32P]ATP. The reaction mixture was incubated at 30 °C for 20 min, terminated by the addition of 5× Laemmli's sample buffer. Samples were subjected to electrophoresis on SDS-PAGE and proteins were transferred to nitrocellulose membrane and exposed to Kodak x-ray film. Loading differences (between samples) were determined by blotting the membrane with anti-ERK, JNK1, or p38 α/β, -γ, -δ antibodies. The expression level of TGase, neuronal marker growth-associated protein 43 (GAP-43), and neurofilament B (NF-B) were determined by immunoblotting. Cells were treated with or without RA in medium containing 10% FBS for the indicated times. After washing with cold PBS, the cells were harvested in lysis buffer containing 25 mm Tris-HCl (pH 7.4), 100 mmNaCl, 1 mm EDTA, 1mm dithiothreitol, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin and leupeptin. Insoluble material was removed by sedimentation at 10,000 × g for 15 min at 4 °C. The protein concentration was determined according to the Bio-Rad method. Lysates (50 μg of protein) were electrophoresed on SDS-PAGE, transferred to nitrocellulose membrane, blotted with anti-TGase, GAP-43, and NF-B antibody. The blots were reprobed with anti-actin antibody to determine the loading difference between samples. Scanning densitometry was used for semiquantitative analysis of the data. RhoA activation was determined using the Rhotekin-binding assay, as previously described (43Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1369) Google Scholar). In brief, cells were lysed with lysis buffer containing 50 mm Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mm NaCl, 10 mm MgCl2, 10 μg/ml each of leupeptin and aprotinin, and 1 mmphenylmethylsulfonyl fluoride. The lysate was sedimented at 14,000 × g for 5 min. Equal volume of cell lysates was incubated at 4 °C, for 45 min with 30 μg of GST-RBD (GST fusion protein containing the RhoA binding domain of Rhotekin) immobilized on glutathione-Sepharose beads (Sigma). The beads were suspended in Laemmli sample buffer, and eluted proteins were separated on 15% SDS-PAGE. Activated RhoA was detected by Western blotting with monoclonal antibody against RhoA. The amount of RBD-bound Rho was normalized to the total amount of RhoA in cell lysates for the comparison of Rho activity (activated Rho) in different samples. Immunoflourescence studies on stress fiber and focal adhesion complex formation were performed using SH-SY5Y cells grown on chamber slides (Lab-Tek II, Nalgen Nunc Int.) in medium with 3% FBS. After washing with cold PBS, cells were fixed with 3% paraformaldehyde at 21 °C for 20 min. Cells were exposed to 0.2% Triton X-100 for 3 min after washing with PBS. To block nonspecific binding, cells were first incubated with 100 mm glycine for 20 min at room temperature, and then incubated with primary antibody at 21 °C for 30 min (dilutions: vinculin monoclonal antibody, 1:400; Texas Red-X phalloidin, 1:40). After washing three times with PBS, fluorescein-labeled secondary antibody (1:400) was added and incubated for an additional 30 min and washed two times with PBS. Coverslips were mounted with anti-fade medium (ProLong®, Molecular Probes) and slides viewed with an Olympus Provis fluorescence microscope. Pictures of the slides were taken with a digital color camera (Olympus UltraPix 2000 RGB). To study the effect of RhoA and MAPK pathways on RA-induced neurite outgrowth, SH-SY5Y cells were pretreated with C-3 exoenzyme, monodansylcadaverine (MDC), PD98059 (specific MEK inhibitor), SB202190 (specific inhibitor of p38 MAPK), and JNK inhibitor or Me2SO before addition of 5 μm RA in medium with 10% FBS. Medium with or without different inhibitors was replaced every day. After 4 days, the cell photographs were taken using a Nikon digital camera. The cell lysates were used for determining neuronal marker expression and transglutaminase activity. Untreated and RA-treated SH-SY5Y cells were lysed, and 100 μg of protein from the lysate was used for the assay of transglutaminase activity, as previously described (13Singh U.S. Erickson J.W. Cerione R.A. Biochemistry. 1995; 34: 15863-15871Crossref PubMed Scopus (85) Google Scholar, 44Singh U.S., Li, Q. Cerione R. J. Biol. Chem. 1998; 273: 1946-1950Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). SH-SY5Y cells, grown to 40% subconfluence in Dulbecco's modified Eagle's medium containing 3% FBS, were treated (or untreated) with 5 μm RA for 2 days. Labeling (in vivo) of [14C]putrescine (0.5 μCi/ml) was performed overnight in the presence of aminoguanidine (100 mm), as described previously (15Singh U.S. Kunar M.T. Kao Y.L. Baker K.M. EMBO J. 2001; 20: 2413-2423Crossref PubMed Scopus (80) Google Scholar). Cells were lysed and precipitated with RhoA antibody (or GST-RBD beads). Samples were subjected to SDS-PAGE (15%), and the gel was dried after incubating with a signal enhancer kit. Transamidated proteins were detected by autoradiography. Data presented are mean ± S.E. Statistical differences were determined by a Student's ttest for statistical significance (p < 0.05). RA-induced cellular differentiation is coupled with increased expression/activation of TGase in many cell lines (12Tucholski J. Lesort M. Johnson G.V. Neuroscience. 2001; 102: 481-491Crossref PubMed Scopus (110) Google Scholar, 16Antonyak 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). To further study the role of TGase in RA-induced neuronal differentiation in SH-SY5Y cells, we determined the expression level of TGase and neuronal markers in response to RA treatment. As shown in Fig. 1, A and B, up-regulation of TGase expression was observed after 1 day of RA treatment, which was associated with increased transglutaminase activity (∼10-fold, specific activity: 110 nmol of putrescine incorporated/mg of casein/mg of protein, compared with 10.5 nmol of putrescine incorporated/mg of casein/mg of protein, Fig. 1 C). Under similar conditions, RA time-dependently induced the expression of NF-B and GAP-43 from 1 to 4 days of treatment (Fig. 1, A and B), unlike actin (negative control of neuronal marker expression). These data demonstrated that RA-induced expression of neuronal markers was also associated with increased expression/activation of TGase, which might be involved in promoting neuronal differentiation. Previously we demonstrated that RhoA is an in vivo substrate of TGase and RA promotes transamidation of RhoA in HeLa cells (15Singh U.S. Kunar M.T. Kao Y.L. Baker K.M. EMBO J. 2001; 20: 2413-2423Crossref PubMed Scopus (80) Google Scholar). To address the question of whether RA-induced expression of TGase in SH-SY5Y cells promotes transamidation of RhoA, we performed in vivo labeling of SH-SY5Y cells using [14C]putrescine. Incorporation of radiolabeled putrescine into RhoA was detected by immunoprecipitation and subsequent autoradiography of RhoA. As shown in Fig. 2 A, there was increased labeling of [14C]putrescine into RhoA (in vivotransamidation) after 24 h of RA treatment, which was consistent with the increased transglutaminase activity of TGase (Fig. 1 C). To examine whether transamidated RhoA is activated, the Rhotekin binding assay was performed. There was a putrescine-labeled protein of 24 kDa, precipitated by GST-RBD beads from RA (24 h)-treated cells (Fig. 2 B, upper panel) identified as RhoA (lower panel), indicating that transamidated RhoA functions as an activated form. As a negative control, cell lysates prepared fromin vivo transamidated cells were incubated with GST beads, and putrescine incorporation and activation of RhoA were examined. As shown in Fig. 2 C, we could not detect pull-down of [14C]RhoA or activated RhoA (using GST beads), demonstrating the specificity of GST-RBD assays. To determine the role of TGase in activation of RhoA, we have used monodansylcadaverine (MDC), a known TGase inhibitor (45Stenberg P. Curtis C.G. Wing D. Tong Y.S. Credo R.B. Gray A. Lorand L. Biochem. J. 1975; 147: 153-163Crossref Scopus (41) Google Scholar), and as shown in Fig. 3 A, MDC dose-dependently inhibited RA-induced transglutaminase activity. SH-SY5Y cells were pretreated with RA in the absence or presence of MDC (50 μm for 2 h) and were subjected to the Rhotekin binding assay. RA promoted the pull-down of RhoA (or activation of RhoA) after 1 day of treatment and peaked in 2 days. The RA-induced activation of RhoA was blocked by MDC treatment (Fig. 3 B, upper panel). We further used stable SH-SY5Y cell lines overexpressing vector, wild-type, C277S mutant, and antisense TGase (12Tucholski J. Lesort M. Johnson G.V. Neuroscience. 2001; 102: 481-491Crossref PubMed Scopus (110) Google Scholar) for determining the role of TGase in RhoA activation (Fig. 3, C and D). Overexpression of wild-type TGase resulted in a significant activation of RhoA (25-fold increase compared with vector), unlike the C277S mutant- or antisense-overexpressing cell lines, where no change in RhoA activation was observed. RA treatment markedly induced activation of RhoA in a vector cell line (20-fold), which is similar to that of normal SH-SY5Y cells. Only a 1.5-fold increase of RhoA activation was observed in wild type, and no significant changes in RhoA activation were observed in C277S mutant and antisense cell lines after RA treatment. Further, we determined the expression level of TGase in different cell lines (lowermost panel). RA induced up-regulation of TGase expression in vector-transfected cells, and did not result in any detectable changes in TGase expression in wild-type and C277S mutant cell lines. We could not detect any TGase expression in the absence or presence of RA in antisense TGase-overexpressing cells. These results indicated that TGase (transglutaminase activity) was required for RA-induced activation of RhoA in SH-SY5Y cells. One of the hallmarks of RhoA activation is increased cyotskeletal rearrangement (25Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3843) Google Scholar). To determine the effects of RhoA transamidation/activation on cytoskeletal rearrangement, SH-SY5Y cells were treated with RA in the presence or absence of C-3 exoenzyme (RhoA inhibitor) or MDC for 24 h, and immunofluoresence staining was performed with Texas Red-X phalloidin (for stress fiber formation) and vinculin (for focal adhesion complex formation). There was increased stress fiber formation (Fig. 4 A) and focal adhesion complex formation (Fig. 4 B) in response to RA treatment. A minimum of 24 h of RA treatment was required to induce the changes in stress fiber and focal adhesion complex formation. Treatment of cells with C-3 or MDC blocked RA-induced formation of stress fibers (Fig. 4 A) and focal adhesion complexes (Fig. 4 B). These results indicated that transamidation of RhoA was required for activation and inducing cytoskeletal rearrangement in response to RA in SH-SY5Y cells. Recent studies suggest that MAPK pathways play a role in neuronal differentiation (34Encinas M. Iglesias M. Llecha N. Comella J.X. J. Neurochem. 1999; 73: 1409-1421Crossref PubMed Scopus (237) Google Scholar). To determine whether the MAPK pathway is involved in RA-induced neuronal differentiation of SH-SY5Y cells, MAP kinase (ERK1/2, JNK, and p38) activity was determined by in vitro kinase assay. As shown in Fig. 5, RA induced significant activation of ERK1/2, JNK1, and p38γ MAP kinase. ERK1/2 and p38γ MAP kinase activation peaked at 24 h, and JNK activation peaked at 48 h. Under similar conditions, RA induced mild activation of p38α/β, but had no effect on p38δ activation. Corresponding Western blots of ERK1/2, JNK1, and p38α/β, -γ, and -δ showed an equal amount of protein in different samples. To address the question, whether RA-induced activation of MAP kinases are mediated by TGase and resulting transamidation/activation of RhoA, we determined the effect of MDC and C-3 exoenzyme on activation of MAP kinases. As shown in Fig. 6, Aand B, RA-induced activation of ERK1/2, JNK, and p38γ was blocked by MDC treatment, indicating a role of transglutaminase activity in their activation. On the other hand, treatment of cells with C-3 exoenzyme blocked RA-induced activation of p38γ, and partially inhibited the activation of ERK1/2, but had no effect on JNK activation, indicating that transamidated RhoA may not be involved in RA-induced JNK activation. Under similar conditions, we demonstrated that MDC or C-3 exoenzyme had no effect on activation of p38α/β (data not shown) or RA-induced up-regulation of TGase expression (Fig. 6 A, bottom panel). Using stable SH-SY5Y cell lines (overexpressing wild-type, C277S mutant, and antisense TGase), we further examined the role of TGase in activation of MAP kinases. Overexpression of wild-type TGase promoted the activation of ERK1/2, JNK1, and p38γ MAP kinase (2-fold, 2-fold, and 3-fold, respectively, compared with vector) as shown in Fig. 7, A and B. Only basal level activation was observed in the C277S mutant- and antisense-overexpressing cells, indicating transglutaminase activity of TGase plays an important role" @default.
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• W2022702828 title "Tissue Transglutaminase Mediates Activation of RhoA and MAP Kinase Pathways during Retinoic Acid-induced Neuronal Differentiation of SH-SY5Y Cells" @default.
• W2022702828 cites W1495826882 @default.
• W2022702828 cites W1567217875 @default.
• W2022702828 cites W1855950477 @default.
• W2022702828 cites W1949708034 @default.
• W2022702828 cites W1967978186 @default.
• W2022702828 cites W1968164494 @default.
• W2022702828 cites W1969435130 @default.
• W2022702828 cites W1972294274 @default.
• W2022702828 cites W1972404682 @default.
• W2022702828 cites W1975740366 @default.
• W2022702828 cites W1980831535 @default.
• W2022702828 cites W1986178012 @default.
• W2022702828 cites W1989480480 @default.
• W2022702828 cites W1992512418 @default.
• W2022702828 cites W1992604829 @default.
• W2022702828 cites W1994965373 @default.
• W2022702828 cites W1999241144 @default.
• W2022702828 cites W2001399809 @default.
• W2022702828 cites W2003084435 @default.
• W2022702828 cites W2003867300 @default.
• W2022702828 cites W2007660746 @default.
• W2022702828 cites W2011075445 @default.
• W2022702828 cites W2012737818 @default.
• W2022702828 cites W2016291163 @default.
• W2022702828 cites W2018772944 @default.
• W2022702828 cites W2019599792 @default.
• W2022702828 cites W2025444657 @default.
• W2022702828 cites W2029872541 @default.
• W2022702828 cites W2036562186 @default.
• W2022702828 cites W2039339016 @default.
• W2022702828 cites W2043203044 @default.
• W2022702828 cites W2043636830 @default.
• W2022702828 cites W2044964176 @default.
• W2022702828 cites W2056123411 @default.
• W2022702828 cites W2061189162 @default.
• W2022702828 cites W2066780429 @default.
• W2022702828 cites W2072410510 @default.
• W2022702828 cites W2077222940 @default.
• W2022702828 cites W2077623928 @default.
• W2022702828 cites W2082776443 @default.
• W2022702828 cites W2084179274 @default.
• W2022702828 cites W2084634327 @default.
• W2022702828 cites W2084974627 @default.
• W2022702828 cites W2088764696 @default.
• W2022702828 cites W2089748517 @default.
• W2022702828 cites W2091016640 @default.
• W2022702828 cites W2105746559 @default.
• W2022702828 cites W2109110038 @default.
• W2022702828 cites W2118052953 @default.
• W2022702828 cites W2121666423 @default.
• W2022702828 cites W2130172628 @default.
• W2022702828 cites W2130196728 @default.
• W2022702828 cites W2161688885 @default.
• W2022702828 cites W2161954973 @default.
• W2022702828 cites W2164465986 @default.
• W2022702828 cites W2170467038 @default.
• W2022702828 cites W2338091153 @default.
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