RESUMO
In canonical pathway, Wnt3A has been known to stabilize ß-catenin through the dissociation between ß-catenin and glycogen synthase kinase-3ß (GSK-3ß) that suppresses the phosphorylation and degradation of ß-catenin. In non-canonical signaling pathway, Wnt was known to activate Rho GTPases and to induce cell migration. The cross-talk between canonical and non-canonical pathways by Wnt signaling; however, has not been fully elucidated. Here, we revealed that Wnt3A induces not only the phosphorylation of GSK-3ß and accumulation of ß-catenin but also RhoA activation in RAW264.7 and HEK293 cells. Notably, sh-RhoA and Tat-C3 abolished both the phosphorylation of GSK-3ß and accumulation of ß-catenin. Y27632, an inhibitor of Rho-associated coiled coil kinase (ROCK) and si-ROCK inhibited both GSK-3ß phosphorylation and ß-catenin accumulation. Furthermore, active domain of ROCK directly phosphorylated the purified recombinant GSK-3ß in vitro. In addition, Wnt3A-induced cell proliferation and migration, which were inhibited by Tat-C3 and Y27632. Taken together, we propose the cross-talk between canonical and non-canonical signaling pathways of Wnt3A, which induces GSK-3ß phosphorylation and ß-catenin accumulation through RhoA and ROCK activation. J. Cell. Physiol. 232: 1104-1113, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Proteína Wnt3A/farmacologia , beta Catenina/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Amidas/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Quimiocinas/metabolismo , Células HEK293 , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Piridinas/farmacologia , Células RAW 264.7 , Proteínas Recombinantes de Fusão/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
Transforming growth factor (TGF)-ß1 plays several roles in a variety of cellular functions. TGF-ß1 transmits its signal through Smad transcription factor-dependent and -independent pathways. It was reported that TGF-ß1 activates NF-κB and RhoA, and RhoA activates NF-κB in several kinds of cells in a Smad-independent pathway. However, the activation molecular mechanism of NF-κB by RhoA upon TGF-ß1 has not been clearly elucidated. We observed that RhoA-GTP level was increased by TGF-ß1 in RAW264.7 cells. RhoA-GDP and RhoGDI were bound to N- and C-terminal domains of IKKγ, respectively. Purified IKKγ facilitated GTP binding to RhoA complexed with RhoGDI. Furthermore, Dbs, a guanine nucletotide exchange factor of RhoA much more enhanced GTP binding to RhoA complexed with RhoGDI in the presence of IKKγ. Indeed, si-IKKγ abolished RhoA activation in response to TGF-ß1 in cells. However, TGF-ß1 stimulated the release of RhoA-GTP from IKKγ and Rho-associated kinase (ROCK), an active RhoA effector protein, directly phosphorylated IKKß in vitro, whereas TGF-ß1-activated kinase 1 activated RhoA upon TGF-ß1 stimulation. Taken together, our data indicate that IKKγ facilitates RhoA activation via a guanine nucletotide exchange factor, which in turn activates ROCK to phosphorylate IKKß, leading to NF-κB activation that induced the chemokine expression and cell migration upon TGF-ß1.
Assuntos
Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Movimento Celular/fisiologia , Quimiocinas/biossíntese , Quimiocinas/genética , Ativação Enzimática/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Quinase I-kappa B/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação/fisiologia , Estrutura Terciária de Proteína , Fator de Crescimento Transformador beta1/genética , Proteínas rho de Ligação ao GTP/genética , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/fisiologia , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Phagocytosis occurs primarily through two main processes in macrophages: the Fcγ receptor- and the integrin αMß2-mediated processes. Complement C3bi-opsonized particles are known to be engulfed through integrin αMß2-mediated process, which is regulated by RhoA GTPase. C3 toxin fused with Tat-peptide (Tat-C3 toxin), an inhibitor of the Rho GTPases, was shown to markedly inhibit the phagocytosis of serum (C3bi)-opsonized zymosans (SOZs). However, 8CPT-2Me-cAMP, an activator of exchange protein directly activated by cAMP (Epac, Rap1 guanine nucleotide exchange factor), restored the phagocytosis of the SOZs that was previously inhibited by the Tat-C3 toxin. In addition, a constitutively active form of Rap1 GTPase (CA-Rap1) also restored the phagocytosis that was previously reduced by a dominant negative form of RhoA GTPase (DN-RhoA). This suggests that Rap1 can replace the function of RhoA in the phagocytosis. Inversely, CA-RhoA rescued the phagocytosis that was suppressed by DN-Rap1. These findings suggest that both RhoA and Rap1 GTPases collectively regulate the phagocytosis of SOZs. In addition, filamentous actin was reduced by the Tat-C3 toxin, which was again restored by 8CPT-2Me-cAMP. Small interfering profilin suppressed the phagocytosis, suggesting that profilin is essential for the phagocytosis of SOZs. Furthermore, 8CPT-2Me-cAMP increased the co-immunoprecipitation of profilin with Rap1, whereas Tat-C3 toxin decreased that of profilin with RhoA. Co-immunoprecipitations of profilin with actin, Rap1, and RhoA GTPases were augmented in the presence of GTPγS rather than GDP. Therefore, we propose that both Rap1 and RhoA GTPases regulate the formation of filamentous actin through the interaction between actin and profilin, thereby collectively inducing the phagocytosis of SOZs in macrophages.
Assuntos
Complemento C3b/farmacologia , Macrófagos/metabolismo , Fagocitose/fisiologia , Zimosan/farmacologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Difosfato/farmacologia , Macrófagos/citologia , Camundongos , Mutação , Fagocitose/efeitos dos fármacos , Profilinas/genética , Profilinas/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTPRESUMO
Transforming growth factor (TGF)-ß1 regulates diverse cellular functions. Particularly, TGF-ß1 induces monocyte migration to sites of injury or inflammation in early period, whereas TGF-ß1 inhibits cell migration in late phase. In this study, we attempted to understand how TGF-ß1 suppresses cell migration in late phase. We found that TGF-ß1 of short exposure induces the production of chemokines, such as macrophage inflammatory protein (MIP)-1α, by Raw 264.7 cells. However, knock-down of small GTPase RhoA by sh-RhoA inhibited the production of MIP-1α and macrophage migration, suggesting that RhoA is essential for expression of this chemokine. An activator of Epac (exchange proteins directly activated by cAMP; a guanine nucleotide exchange factor of Rap1), 8CPT-2Me-cAMP which leads to Rap1 activation abrogated MIP-1α expression and macrophage migration. Indeed, GTP-RhoA and GTP-Rap1 levels were reciprocally regulated in a time-dependent manner following TGF-ß1 stimulation. 8CPT-2Me-cAMP suppressed GTP-RhoA levels, whereas si-Rap1 augmented GTP-RhoA levels and cell migration. TGF-ß1 produced cAMP in late period and si-RNAs of Epac1 and Epac2 reduced GTP-Rap1 levels leading to promotion of GTP-RhoA levels. Furthermore, si-RNA of ARAP3 (Rap-dependent RhoGAP) increased GTP-RhoA level and cell migration. Therefore, we propose the mechanism that prolonged TGF-ß1 treatment produce cAMP, which activates sequentially Epac, Rap1 and ARAP3, resulting in suppression of RhoA, chemokine expression, and macrophage migration. Contrary to the general concept that Rap1 stimulates cell migration, we demonstrated in this study that Rap1 inhibits cell migration by suppression of RhoA activity in response to TGF-ß1.
Assuntos
Movimento Celular , Fator de Crescimento Transformador beta1/farmacologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Quimiocina CCL3/metabolismo , Quimiocinas/metabolismo , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Camundongos , Modelos Biológicos , Tionucleotídeos/farmacologiaRESUMO
Severe fever with thrombocytopenia syndrome (SFTS) is an arthropod-borne viral disease with a high mortality rate with high fever and thrombocytopenia. We investigated the clinical and epidemiological characteristics and viral genotypes from 2019 to 2021 in Gangwon Province, Korea. Of the 776 suspected cases, 62 were SFTS. The fatality rate was 11.5-28.6% (average rate, 19.4%), and the frequent clinical symptoms were high fever (95.2%), thrombocytopenia (95.2%), and leukopenia (90.3%). Hwacheon had the highest incidence rate per 100,000 persons at 8.03, followed by Inje and Yanggu (7.37 and 5.85, respectively). Goseong, Yangyang, and Hoengseong had rates of 2 or higher; Samcheok, Hongcheon, Jeongsen, and Yeonwol were 1.70-1.98, and Wonju, Gangneung, and Donghae were slightly lower, ranging from 0.31 to 0.74. Of the 57 cases with identified genotypes, eight genotypes (A, B1, B2, B3, C, D, E, and F) were detected, and the B2 genotype accounted for 54.4% (31 cases), followed by the A genotype at 22.8% (13 cases). The B2 and A genotypes were detected throughout Gangwon Province, and other genotypes, B1, B3, C, D, and F, were discovered in a few regions. In particular, genotype A could be further classified into subtypes. In conclusion, SFTS occurred throughout Gangwon Province, and Hwacheon had the highest incidence density. Multiple genotypes of SFTS were identified, with B2 and A being the most common. These findings provide important insights for the understanding and management of SFTS in this region.
RESUMO
cAMP induces neurite outgrowth in the rat pheochromocytoma cell line 12 (PC12). In particular, di-butyric cAMP (db-cAMP) induces a greater number of primary processes with shorter length than the number induced by nerve growth factor (NGF). db-cAMP up- and down-regulates GTP-RhoA levels in PC12 cells in a time-dependent manner. Tat-C3 toxin stimulates neurite outgrowth, whereas lysophosphatidic acid (LPA) and constitutively active (CA)-RhoA reduce neurite outgrowth, suggesting that RhoA inactivation is essential for the neurite outgrowth from PC12 cells stimulated by cAMP. In this study, the mechanism by which RhoA is inactivated in response to cAMP was examined. db-cAMP induces phosphorylation of RhoA and augments the binding of RhoA with Rho guanine nucleotide dissociation inhibitor (GDI). Moreover, RhoA (S188D) mimicking phosphorylated RhoA induces greater neurite outgrowth than RhoA (S188A) mimicking dephosphorylated form does. Additionally, db-cAMP increases GTP-Rap1 levels, and dominant negative (DN)-Rap1 and DN-Rap-dependent RhoGAP (ARAP3) block neurite outgrowth induced by db-cAMP. DN-p190RhoGAP and the Src inhibitor PP2 suppress neurite outgrowth, whereas transfection of c-Src and p190RhoGAP cDNAs synergistically stimulate neurite outgrowth. Taken together, RhoA is inactivated by phosphorylation of itself, by p190RhoGAP which is activated by Src, and by ARAP3 which is activated by Rap1 during neurite outgrowth from PC12 cells in response to db-cAMP.
Assuntos
Neuritos/fisiologia , Neurônios/citologia , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Bucladesina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Complemento C3/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Imunoprecipitação , Mutagênese , Fator de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Células PC12 , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Ratos , Transfecção , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Although the migration of hepatic stellate cells (HSCs) is important for hepatic fibrosis, the regulation of this migration is poorly understood. Notably, transforming growth factor (TGF)ß1 induces monocyte migration to sites of injury or inflammation during the early phase, but inhibits cell migration during the late phase. In the present study, the role of transforming protein RhoA signaling in TGFß1induced HSC migration was investigated. TGFß1 was found to increase the protein and mRNA levels of smooth muscle actin and collagen type I in HSCT6 cells. The level of RhoAGTP in TGFß1stimulated cells was significantly higher than that in control cells. Furthermore, the phosphorylation of cofilin and formation of filamentous actin (Factin) were more marked in TGFß1stimulated cells than in control cells. Additionally, TGFß1 induced the activation of nuclear factorκB, and the expression of extracellular matrix proteins and several cytokines in HSCT6 cells. The active form of Rap1 (Rap1 V12) suppressed RhoAGTP levels, whereas the dominantnegative form of Rap1 (Rap1 N17) augmented RhoAGTP levels. Therefore, the data confirmed that Rap1 regulated the activation of RhoA in TGFß1stimulated HSCT6 cells. These findings suggest that TGFß1 regulates Rap1, resulting in the suppression of RhoA, activation of and formation of Factin during the migration of HSCs.
Assuntos
Células Estreladas do Fígado/citologia , Fator de Crescimento Transformador beta1/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Movimento Celular , Células Estreladas do Fígado/metabolismo , Fosforilação , RatosRESUMO
Zinc is an essential element required for cell division, migration, and proliferation. Under zinc-deficient conditions, proliferation and differentiation of neural progenitors are significantly impaired. Adipose-derived mesenchymal stem cells (AD-MSCs) are multipotent stem cells that can differentiate into neurons. The aim of this study was to evaluate the effect of zinc on AD-MSC proliferation and differentiation. We initially examined the effect of zinc on stem cell proliferation at the undifferentiated stage. AD-MSCs showed high proliferation rates on day 6 in 30 µM and 100 µM of ZnCl2. Zinc chelation inhibited AD-MSC proliferation via downregulation of ERK1/2 activity. We then assessed whether zinc was involved in cell migration and neurite outgrowth during differentiation. After three days of neuronal differentiation, TUJ-1-positive cells were observed, implying that AD-MSCs had differentiated into early neuron or neuron-like cells. Neurite outgrowth was increased in the zinc-treated group, while the CaEDTA-treated group showed diminished, shrunken neurites. Furthermore, we showed that zinc promoted neurite outgrowth via the inactivation of RhoA and led to the induction of neuronal gene expression (MAP2 and nestin) in differentiated stem cells. Taken together, zinc promoted AD-MSC proliferation and affected neuronal differentiation, mainly by increasing neurite outgrowth.
RESUMO
Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47(PHOX). Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47(PHOX) may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.
Assuntos
Macrófagos/metabolismo , Proteínas Opsonizantes/metabolismo , Fagocitose , Transdução de Sinais , Superóxidos/metabolismo , Zimosan/sangue , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Membrana Celular , Citosol , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Antígeno de Macrófago 1/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/ultraestrutura , Camundongos , Quinase de Cadeia Leve de Miosina/metabolismo , Proteínas Opsonizantes/sangue , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Acetato de Tetradecanoilforbol/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
Fibrillar amyloid-beta (fAß) peptide causes neuronal cell death, which is known as Alzheimer's disease. One of the mechanisms for neuronal cell death is the activation of microglia which releases toxic compounds like reactive oxygen species (ROS) in response to fAß. We observed that fAß rather than soluble form blocked BV2 cell proliferation of microglial cell line BV2, while N-acetyl-l-cysteine (NAC), a scavenger of superoxide, prevented the cells from death, suggesting that cell death is induced by ROS. Indeed, both fAß1-42 and fAß25-35 induced superoxide production in BV2 cells. fAß25-35 produced superoxide, although fAß25-35 is not phagocytosed into BV2 cells. Thus, superoxide production by fAß does not seem to be dependent on phagocytosis of fAß. Herein we studied how fAß produces superoxide in BV2. Transfection of dominant negative (DN) RhoA (N19) cDNA plasmid, small hairpin (sh)-RhoA forming plasmid, and Y27632, an inhibitor of Rho-kinase, abrogated the superoxide formation in BV2 cells stimulated by fAß. Furthermore, fAß elevated GTP-RhoA level as well as Rac1 and Cdc42. Tat-C3 toxin, sh-RhoA, and Y27632 inhibited the phosphorylation of p47(PHOX). Moreover, peritoneal macrophages from p47(PHOX) (-/-) knockout mouse could not produce superoxide in response to fAß. These results suggest that RhoA closely engages in the regulation of superoxide production induced by fAß through phosphorylation of p47(PHOX) in microglial BV2 cells.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Microglia/citologia , Superóxidos/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Animais , Linhagem Celular , Camundongos , Microglia/metabolismo , Dados de Sequência Molecular , NADPH Oxidases/metabolismo , FosforilaçãoRESUMO
Phagocytic NADPH oxidase plays a critical role in superoxide generation in macrophage cells. Small GTPases, including Rac1 and Rac2, have been implicated in the regulation of NADPH oxidase activity. Rap1, which has no effect in a cell-free system of oxidase activation, recently has been proven to colocalize with cytochrome b(558). In addition, neutrophils from rap1A(-/-) mice reduce fMLP-stimulated superoxide production. Here, we tried to determine whether Rap1 also plays a role in the production of superoxide. IgG-opsonized zymosan (IOZ) particles treatment induced Rap1 activation and superoxide generation. Knock-down of Rap1 by si-Rap1 suppressed IOZ-induced superoxide formation. Sh-RhoA also reduced superoxide levels, but 8CPT-2Me-cAMP, an activator of Epac1 (a guanine nucleotide exchange factor (GEF) of Rap1), could recover the levels to the control value. When cells were stimulated by IOZ, Rap1 and Rac1 were translocated to the membrane, and then interacted with p22(phox). 8CPT-2Me-cAMP rescued sh-RhoA-induced reduction of the interaction between Rac1 and p22(phox), and enhanced lysophosphatidic acid (LPA)-induced increase of their interaction. Moreover, Rac1 activity was increased by both LPA and 8CPT-2Me-cAMP when treated with IOZ particles. Si-Vav2 impaired GTP-Rac1 levels in response to 8CPT-2Me-cAMP/IOZ. Phosphorylation of RhoA activates Rac1 in response to IOZ by the enhanced binding of phospho-RhoA to RhoGDI, leading to the release of Rac1 from the Rac1-RhoGDI complex. In conclusion, IOZ treatment induces Rap1 activation and phosphorylation of RhoA, which in turn cause Rac1 activation and promote Rac1 translocation to the membrane leading to binding with p22(phox) that activates NADPH oxidase and produces superoxide.
Assuntos
Imunoglobulina G/metabolismo , Macrófagos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Superóxidos/metabolismo , Zimosan/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Linhagem Celular , Ativação Enzimática , Imunoprecipitação , Camundongos , FagocitoseRESUMO
Amyloid-beta (Abeta) is one of the main factors to cause Alzheimer's disease. Although fibrillar Abeta (fAbeta) activates microglial cells that release toxic compounds to induce partial neuronal death, the mechanism of interaction between Abeta and microglia remains unclear. Therefore, we examined the interaction of microglial cells (BV2) and fAbeta on a gelatin-precoated plate. The binding was markedly enhanced by RhoA inactivation using Tat-C3, dominant negative RhoA, and si-RhoA. To identify the receptor for fAbeta, we tested various antibodies to mask receptors. Among them, anti-beta2-integrin antibody mostly suppressed cell binding to fAbeta. The incremental binding of cells induced by RhoA inhibition was also blocked by addition of anti-beta2-integrin antibody. These results suggest that RhoA inhibition stimulates beta2-integrin-mediated cell interaction to fAbeta.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Antígenos CD18/metabolismo , Microglia/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Antígenos CD18/imunologia , Linhagem Celular , Humanos , Camundongos , Microglia/citologia , Microglia/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/genética , Transfecção/métodos , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Brief treatment with transforming growth factor (TGF)-beta1 stimulated the migration of macrophages, whereas long-term exposure decreased their migration. Cell migration stimulated by TGF-beta1 was markedly inhibited by 10 mug/mL Tat-C3 exoenzyme. TGF-beta1 increased mRNA and protein levels of macrophage inflammatory protein (MIP)-1alpha in the initial period, and these effects also were inhibited by 10 mug/mL Tat-C3 and a dominant-negative (DN)-RhoA (N19RhoA). Cycloheximide, actinomycin D, and antibodies against MIP-1alpha and monocyte chemoattractant protein-1 (MCP-1) abolished the stimulation of cell migration by TGF-beta1. These findings suggest that migration of these cells is regulated directly and indirectly via the expression of chemokines such as MIP-1alpha and MCP-1 mediated by RhoA in response to TGF-beta1. TGF-beta1 activated RhoA in the initial period, and thereafter inactivated them, suggesting that the inactivation of RhoA may be the cause of the reduced cell migration in response to TGF-beta1 at later times. We therefore attempted to elucidate the molecular mechanism of the inactivation of RhoA by TGF-beta1. First, TGF-beta1 phosphorylated RhoA via protein kinase A, leading to inactivation of RhoA. Second, wild-type p190 Rho GTPase activating protein (p190RhoGAP) reduced and DN-p190RhoGAP reversed the reduction of cell migration induced by TGF-beta, suggesting that it inactivated RhoA via p190 Rho GAP.