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1.
Arterioscler Thromb Vasc Biol ; 40(3): 714-732, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31996022

RESUMO

OBJECTIVE: Calcification of atherosclerotic plaque is traditionally associated with increased cardiovascular event risk; however, recent studies have found increased calcium density to be associated with more stable disease. 3-hydroxy-3-methylglutaryl coenzymeA reductase inhibitors or statins reduce cardiovascular events. Invasive clinical studies have found that statins alter both the lipid and calcium composition of plaque but the molecular mechanisms of statin-mediated effects on plaque calcium composition remain unclear. We recently defined a macrophage Rac (Ras-related C3 botulinum toxin substrate)-IL-1ß (interleukin-1 beta) signaling axis to be a key mechanism in promoting atherosclerotic calcification and sought to define the impact of statin therapy on this pathway. Approach and Results: Here, we demonstrate that statin therapy is independently associated with elevated coronary calcification in a high-risk patient population and that statins disrupt the complex between Rac1 and its inhibitor RhoGDI (Rho GDP-dissociation inhibitor), leading to increased active (GTP bound) Rac1 in primary monocytes/macrophages. Rac1 activation is prevented by rescue with the isoprenyl precursor geranylgeranyl diphosphate. Statin-treated macrophages exhibit increased activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), increased IL-1ß mRNA, and increased Rac1-dependent IL-1ß protein secretion in response to inflammasome stimulation. Using an animal model of calcific atherosclerosis, inclusion of statin in the atherogenic diet led to a myeloid Rac1-dependent increase in atherosclerotic calcification, which was associated with increased serum IL-1ß expression, increased plaque Rac1 activation, and increased plaque expression of the osteogenic markers, alkaline phosphatase and RUNX2 (Runt-related transcription factor 2). CONCLUSIONS: Statins are capable of increasing atherosclerotic calcification through disinhibition of a macrophage Rac1-IL-1ß signaling axis.


Assuntos
Aterosclerose/tratamento farmacológico , Atorvastatina/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Macrófagos/efeitos dos fármacos , Neuropeptídeos/metabolismo , Placa Aterosclerótica , Calcificação Vascular/enzimologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Idoso , Animais , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos/enzimologia , Macrófagos/patologia , Masculino , Camundongos Knockout para ApoE , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Prenilação , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Estudos Retrospectivos , Transdução de Sinais , Calcificação Vascular/genética , Calcificação Vascular/patologia , Proteínas rac1 de Ligação ao GTP/deficiência , Proteínas rac1 de Ligação ao GTP/genética , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo
2.
Nat Commun ; 10(1): 112, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30631060

RESUMO

Wilms tumor gene on the X chromosome (WTX) is a putative tumor suppressor gene in Wilms tumor, but its expression and functions in other tumors are unclear. Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in women and the second leading cause in men in the United States. We demonstrated that WTX frequently lost in CRC which was highly correlated with cell proliferation, tumor invasion and metastasis. Mechanistically, WTX loss disrupts the interaction between RhoGDIα and CDC42 by losing of the binding with RhoGDIα and triggers the activation of CDC42 and its downstream cascades, which promotes CRC development and liver metastasis. The aberrant upregulation of miR-20a/miR-106a were identified as the reason of WTX loss in CRC both in vivo and in vitro. These study defined the mechanism how miR-20a/miR-106a-mediated WTX loss regulates CRC progression and metastasis, and provided a potential therapeutic target for preventing CRC progression.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias do Colo/genética , MicroRNAs/genética , Proteínas Supressoras de Tumor/genética , Proteína cdc42 de Ligação ao GTP/genética , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Transdução de Sinais/genética , Transplante Heterólogo , Proteínas Supressoras de Tumor/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo
3.
Endocrinology ; 159(8): 3036-3047, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29873699

RESUMO

Rho GDP-dissociation inhibitor (GDIα) inhibits glucose-stimulated insulin secretion (GSIS) in part by locking Rho GTPases in an inactive GDP-bound form. The onset of GSIS causes phosphorylation of GDIα at Ser174, a critical inhibitory site for GDIα, leading to the release of Rho GTPases and their subsequent activation. However, the kinase regulator(s) that catalyzes the phosphorylation of GDIα in islet ß cells remains elusive. We propose that SAD-A, a member of AMP-activated protein kinase-related kinases that promotes GSIS as an effector kinase for incretin signaling, interacts with and inhibits GDIα through phosphorylation of Ser174 during the onset GSIS from islet ß cells. Coimmunoprecipitation and phosphorylation analyses were carried out to identify the physical interaction and phosphorylation site of GDIα by SAD-A in the context of GSIS from INS-1 ß cells and primary islets. We identified GDIα directly binds to SAD-A kinase domain and phosphorylated by SAD-A on Ser174, leading to dissociation of Rho GTPases from GDIα complexes. Accordingly, overexpression of SAD-A significantly stimulated GDIα phosphorylation at Ser174 in response to GSIS, which is dramatically potentiated by glucagonlike peptide-1, an incretin hormone. Conversely, SAD-A deficiency, which is mediated by short hairpin RNA transfection in INS-1 cells, significantly attenuated endogenous GDIα phosphorylation at Ser174. Consequently, coexpression of SAD-A completely prevented the inhibitory effect of GDIα on insulin secretion in islets. In summary, glucose and incretin stimulate insulin secretion through the phosphorylation of GDIα at Ser174 by SAD-A, which leads to the activation of Rho GTPases, culminating in insulin exocytosis.


Assuntos
Glucose/metabolismo , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Animais , Linhagem Celular , Exenatida/farmacologia , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Glucose/farmacologia , Incretinas/farmacologia , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Ratos
4.
Radiother Oncol ; 129(1): 30-37, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29519627

RESUMO

BACKGROUND AND PURPOSE: SKLB-163 is a novel benzothiazole-2-thiol derivative with antitumor activities. This study investigated the effects of SKLB-163 on nasopharyngeal carcinoma (NPC) and its mechanisms. MATERIALS AND METHODS: Rho GDP-dissociation inhibitor (RhoGDI) expression was examined in NPC cell lines by western blot. Effects of SKLB-163 on proliferation, migration and radiosensitivity were assessed by MTT, wound healing and colony formation assays in vitro. Anti-tumor and anti-metastatic effects, and radiosensitizing effects of SKLB-163 were evaluated in a NPC lung metastatic nude mouse model and a subcutaneous xenograft mouse model. Effects of SKLB-163 on proliferation and apoptosis were assessed by PCNA immunohistochemistry and TUNEL assay in vivo. Key molecules in RhoGDI/c-Jun N-terminal kinases-1 (JNK-1) pathway were examined by western blot. RESULTS: RhoGDI was up-regulated in NPC cell lines. SKLB-163 inhibited proliferation and migration, and increased radiosensitivity of NPC cells. SKLB-163 inhibited tumor growth and metastases, and sensitized tumor to irradiation. The radiosensitizing effects were correlated with induction of apoptosis and suppression of proliferation. The molecular mechanism was the down-regulation of RhoGDI and activation of JNK-1 signaling, and the subsequent activation of caspase-3 and the decrease in phosphorylated AKT. CONCLUSIONS: SKLB-163 shows strong anti-tumor activities against NPC and sensitizes NPC to irradiation by affecting the RhoGDI/JNK-1 pathway.


Assuntos
Acetamidas/farmacologia , Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Carcinoma Nasofaríngeo/tratamento farmacológico , Neoplasias Nasofaríngeas/tratamento farmacológico , Radiossensibilizantes/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Nasofaríngeas/patologia , Transplante de Neoplasias/métodos , Fosforilação , Tolerância a Radiação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico
5.
Cancer Lett ; 417: 141-151, 2018 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-29307615

RESUMO

Rho GTPases control a wide range of cellular processes, and their deregulation is associated with promotion of an aggressive and metastatic tumor phenotype in human cancers. Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays a key role in regulating the activity of Rho GTPases. However, the underlying mechanisms are still unclear. In this study, we show that protein phosphatase 1B (PPM1B) interacts with RhoGDI1 and functions as its phosphatase. Ectopic expression of PPM1B results in dephosphorylation of RhoGDI1 and, thereby, abates the activation of RhoA, Rac1 and CDC42 by epidermal growth factor (EGF). PPM1B overexpression in Hs578T and SKBR3 human breast cancer cells decreases their motility and invasiveness in vitro and cancer metastasis in vivo. In contrast, knockdown of PPM1B in MCF-7 and MDA-MB-468 human breast cancer cells that express endogenous PPM1B enhances EGF-induced RhoGDI1 phosphorylation, activation of Rho GTPases, and cancer cell migration and invasion. Knockdown of RhoA or Rac1 by siRNA reverses the enhanced cell migration seen after PP1MB depletion. Collectively, these results indicate that PPM1B negatively regulates cancer cell motility and invasiveness through dephosphorylating RhoGDI1.


Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Proteína Fosfatase 2C/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Fosforilação , Proteína Fosfatase 2C/genética , Interferência de RNA , Transplante Heterólogo
6.
Oncogene ; 37(7): 861-872, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29059157

RESUMO

Eph receptors and their corresponding ephrin ligands have been associated with regulating cell-cell adhesion and motility, and thus have a critical role in various biological processes including tissue morphogenesis and homeostasis, as well as pathogenesis of several diseases. Aberrant regulation of Eph/ephrin signaling pathways is implicated in tumor progression of various human cancers. Here, we show that a Rho family GTPase regulator, Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1), can interact with ephrinB1, and this interaction is enhanced upon binding the extracellular domain of the cognate EphB2 receptor. Deletion mutagenesis revealed that amino acids 327-334 of the ephrinB1 intracellular domain are critical for the interaction with RhoGDI1. Stimulation with an EphB2 extracellular domain-Fc fusion protein (EphB2-Fc) induces RhoA activation and enhances the motility as well as invasiveness of wild-type ephrinB1-expressing cells. These Eph-Fc-induced effects were markedly diminished in cells expressing the mutant ephrinB1 construct (Δ327-334) that is ineffective at interacting with RhoGDI1. Furthermore, ephrinB1 depletion by siRNA suppresses EphB2-Fc-induced RhoA activation, and reduces motility and invasiveness of the SW480 and Hs578T human cancer cell lines. Our study connects the interaction between RhoGDI1 and ephrinB1 to the promotion of cancer cell behavior associated with tumor progression. This interaction may represent a therapeutic target in cancers that express ephrinB1.


Assuntos
Biomarcadores Tumorais/metabolismo , Movimento Celular , Neoplasias/patologia , Receptor EphB2/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Humanos , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Receptor EphB2/genética , Células Tumorais Cultivadas , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/genética , Proteína rhoA de Ligação ao GTP/genética
7.
Elife ; 62017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29231810

RESUMO

Disruption of the sumoylation/desumoylation equilibrium is associated with several disease states such as cancer and infections, however the mechanisms regulating the global SUMO balance remain poorly defined. Here, we show that infection by Shigella flexneri, the causative agent of human bacillary dysentery, switches off host sumoylation during epithelial cell infection in vitro and in vivo and that this effect is mainly mediated by a calcium/calpain-induced cleavage of the SUMO E1 enzyme SAE2, thus leading to sumoylation inhibition. Furthermore, we describe a mechanism by which Shigella promotes its own invasion by altering the sumoylation state of RhoGDIα, a master negative regulator of RhoGTPase activity and actin polymerization. Together, our data suggest that SUMO modification is essential to restrain pathogenic bacterial entry by limiting cytoskeletal rearrangement induced by bacterial effectors. Moreover, these findings identify calcium-activated calpains as powerful modulators of cellular sumoylation levels with potentially broad implications in several physiological and pathological situations.


Assuntos
Cálcio/metabolismo , Calpaína/metabolismo , Disenteria Bacilar/microbiologia , Interações Hospedeiro-Patógeno , Shigella flexneri/patogenicidade , Enzimas Ativadoras de Ubiquitina/metabolismo , Disenteria Bacilar/metabolismo , Disenteria Bacilar/patologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Células HeLa , Humanos , Proteólise , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo
8.
Autophagy ; 13(11): 1841-1854, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29099277

RESUMO

Xenophagy, also known as antibacterial autophagy, functions as a crucial defense system that can utilize intracellular pattern recognition sensors, such as NLRP4, to recognize and selectively eliminate bacterial pathogens. However, little is known about how NLRP4 regulates xenophagy. Here, we report that NLRP4 binds ARHGDIA (Rho GDP dissociation inhibitor α) to regulate Rho GTPase signaling and facilitate actin-mediated xenophagy. Specifically, NLRP4 is recruited to Group A Streptococcus (GAS) and colocalizes with GAS-containing autophagosome-like vacuoles (GcAVs), where it regulates ARHGDIA-Rho GTPase recruitment to promote autophagosome formation. The interaction between NLRP4, ARHGDIA, and Rho GTPases is regulated by ARHGDIA Tyr156 phosphorylation, which acts as a gate to induce Rho-mediated xenophagy. Moreover, ARHGDIA and Rho GTPase are involved in actin-mediated ATG9A recruitment to phagophores, facilitating elongation to form autophagosomes. Collectively, these findings demonstrate that NLRP4 functions as a Rho receptor complex to direct actin dynamics regulating xenophagy.


Assuntos
Autofagossomos/microbiologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/imunologia , Proteínas de Membrana/metabolismo , Proteínas Repressoras/metabolismo , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Proteínas de Transporte Vesicular/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Células HeLa , Humanos , Fosforilação , Transporte Proteico , Infecções Estreptocócicas/microbiologia , Vacúolos/microbiologia
9.
PLoS One ; 12(11): e0187094, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29121646

RESUMO

The small GTPases of the Rho family comprising RhoA, Rac1 and Cdc42 function as molecular switches controlling several essential biochemical pathways in eukaryotic cells. Their activity is cycling between an active GTP-bound and an inactive GDP-bound conformation. The exchange of GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs). Here we report a novel regulatory mechanism of Rac1 activity, which is controlled by a phosphomimetic (Ser179Glu) mutant of syndecan-4 (SDC4). SDC4 is a ubiquitously expressed transmembrane, heparan sulfate proteoglycan. In this study we show that the Ser179Glu mutant binds strongly Tiam1, a Rac1-GEF reducing Rac1-GTP by 3-fold in MCF-7 breast adenocarcinoma cells. Mutational analysis unravels the PDZ interaction between SDC4 and Tiam1 is indispensable for the suppression of the Rac1 activity. Neither of the SDC4 interactions is effective alone to block the Rac1 activity, on the contrary, lack of either of interactions can increase the activity of Rac1, therefore the Rac1 activity is the resultant of the inhibitory and stimulatory effects. In addition, SDC4 can bind and tether RhoGDI1 (GDP-dissociation inhibitor 1) to the membrane. Expression of the phosphomimetic SDC4 results in the accumulation of the Rac1-RhoGDI1 complex. Co-immunoprecipitation assays (co-IP-s) reveal that SDC4 can form complexes with RhoGDI1. Together, the regulation of the basal activity of Rac1 is fine tuned and SDC4 is implicated in multiple ways.


Assuntos
Mutação/genética , Sindecana-4/genética , Sindecana-4/metabolismo , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/química , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Humanos , Células MCF-7 , Modelos Biológicos , Domínios PDZ , Ligação Proteica , Proteína Quinase C-alfa/metabolismo , Sindecana-4/química , Quinases Ativadas por p21/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo
10.
J Am Soc Nephrol ; 28(12): 3545-3562, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28775002

RESUMO

Alteration of podocyte behavior is critically involved in the development and progression of many forms of human glomerular diseases. The molecular mechanisms that control podocyte behavior, however, are not well understood. Here, we investigated the role of Kindlin-2, a component of cell-matrix adhesions, in podocyte behavior in vivo Ablation of Kindlin-2 in podocytes resulted in alteration of actin cytoskeletal organization, reduction of the levels of slit diaphragm proteins, effacement of podocyte foot processes, and ultimately massive proteinuria and death due to kidney failure. Through proteomic analyses and in vitro coimmunoprecipitation experiments, we identified Rho GDP-dissociation inhibitor α (RhoGDIα) as a Kindlin-2-associated protein. Loss of Kindlin-2 in podocytes significantly reduced the expression of RhoGDIα and resulted in the dissociation of Rac1 from RhoGDIα, leading to Rac1 hyperactivation and increased motility of podocytes. Inhibition of Rac1 activation effectively suppressed podocyte motility and alleviated the podocyte defects and proteinuria induced by the loss of Kindlin-2 in vivo Our results identify a novel Kindlin-2-RhoGDIα-Rac1 signaling axis that is critical for regulation of podocyte structure and function in vivo and provide evidence that it may serve as a useful target for therapeutic control of podocyte injury and associated glomerular diseases.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Neuropeptídeos/metabolismo , Podócitos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Albuminúria/metabolismo , Animais , Apoptose , Movimento Celular , Creatinina/análise , Proteínas do Citoesqueleto/genética , Progressão da Doença , Feminino , Fibrose , Genótipo , Humanos , Glomérulos Renais/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Proteínas de Neoplasias/genética , RNA Interferente Pequeno/metabolismo , Insuficiência Renal/patologia , Transdução de Sinais
11.
ChemMedChem ; 12(20): 1703-1714, 2017 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-28776959

RESUMO

RhoGDIα is a key regulator of Rho proteins, coordinating their GTP/GDP and membrane/cytosol cycle. Recently, it was demonstrated by quantitative mass spectrometry that RhoGDIα is heavily targeted by post-translational lysine acetylation. For one site in its N-terminal domain, namely K52, we reported earlier that acetylation completely switches off RhoGDIα function. Herein we show that K52-acetylated RhoGDIα is specifically deacetylated by the sirtuin deacetylase Sirt2. We show that acetylation at K52 decelerates cervical cancer cell proliferation, suggesting RhoGDIα acetylation to be a promising therapeutic target. We demonstrate that treatment of cervical cancer cells with a RhoGDIα-derived K52-trifluoroacetylated, substrate-derived peptidic sirtuin inhibitor severely impairs cell proliferation. Finally, we conclude that the potency of substrate-derived sirtuin inhibitors depends on structural features, the substrate-derived amino acid sequence as a determinant for selectivity, as well as the presence of an acetyl-lysine analogue to increase its potency. These data reveal a prospective therapeutic potential for novel substrate-derived sirtuin inhibitors.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Peptídeos/farmacologia , Sirtuína 2/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Acetilação , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Peptídeos/síntese química , Ligação Proteica , Especificidade por Substrato
12.
Neoplasia ; 19(9): 672-683, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28772241

RESUMO

BACKGROUND: Since invasive bladder cancer (BC) is one of the most lethal urological malignant tumors worldwide, understanding the molecular mechanisms that trigger the migration, invasion, and metastasis of BC has great significance in reducing the mortality of this disease. Although RelA/p65, a member of the NF-kappa B transcription factor family, has been reported to be upregulated in human BCs, its regulation of BC motility and mechanisms have not been explored yet. METHODS: NF-κBp65 expression was evaluated in N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced high invasive BCs by immunohistochemistry staining and in human BC cell lines demonstrated by Western Blot. The effects of NF-κBp65 knockdown on BC cell migration and invasion, as well as its regulated RhoGDIα and FBW7, were also evaluated in T24T cells by using loss- and gain-function approaches. Moreover, the interaction of FBW7 with RhoGDIα was determined with immunoprecipitation assay, while critical role of ubiquitination of RhoGDIα by FBW7 was also demonstrated in the studies. RESULTS: p65 protein was remarkably upregulated in the BBN-induced high invasive BCs and in human BC cell lines. We also observed that p65 overexpression promoted BC cell migration by inhibiting RhoGDIα expression. The regulatory effect of p65 on RhoGDIα expression is mediated by its upregulation of FBW7, which specifically interacted with RhoGDIα and promoted RhoGDIα ubiquitination and degradation. Mechanistic studies revealed that p65 stabilizing the E3 ligase FBW7 protein was mediated by its attenuating pten mRNA transcription. CONCLUSIONS: We demonstrate that p65 overexpression inhibits pten mRNA transcription, which stabilizes the protein expression of ubiquitin E3 ligase FBW7, in turn increasing the ubiquitination and degradation of RhoGDIα protein and finally promoting human BC migration. The novel identification of p65/PTEN/FBW7/RhoGDIα axis provides a significant insight into understanding the nature of BC migration, further offering a new theoretical support for cancer therapy.


Assuntos
Movimento Celular/genética , Proteína 7 com Repetições F-Box-WD/metabolismo , Expressão Gênica , Fator de Transcrição RelA/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Genes Reporter , Xenoenxertos , Humanos , Camundongos , Camundongos Knockout , Ligação Proteica , Estabilidade Proteica , Fator de Transcrição RelA/metabolismo , Neoplasias da Bexiga Urinária/patologia
13.
Cancer Sci ; 108(7): 1293-1302, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28417530

RESUMO

Rho GDP-dissociation inhibitor α (RhoGDIα) is an essential regulator for Rho GTPases. Although RhoGDIα may serve as an oncogene in colorectal cancer (CRC), the underlying mechanism is still unclear. We investigated the function, mechanism, and clinical significance of RhoGDIα in CRC progression. We founded that downregulation of RhoGDIα repressed CRC cell proliferation, motility, and invasion. Overexpression of RhoGDIα increased DNA damage response signals at telomeres, and led to telomere shortening in CRC cells, also being validated in 26 pairs of CRC tissues. Mechanistic studies revealed that RhoGDIα could promote telomeric repeat factor 1 (TRF1) expression through the phosphatidylinositol 3-kinase-protein kinase B signal pathway. Moreover, RhoGDIα protein levels were strongly correlated with TRF1 in CRC tissues. A cohort of 297 CRC samples validated the positive relationship between RhoGDIα and TRF1, and revealed that RhoGDIα and TRF1 levels were negatively associated with CRC patients' survival. Taken together, our results suggest that RhoGDIα regulate TRF1 and telomere length and may be novel prognostic biomarkers in colorectal cancer.


Assuntos
Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Telômero/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/biossíntese , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Biomarcadores Tumorais/análise , Western Blotting , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Invasividade Neoplásica/patologia , Prognóstico , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase em Tempo Real , Encurtamento do Telômero , Análise Serial de Tecidos
14.
Sci Rep ; 7: 43798, 2017 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-28252009

RESUMO

Nicotine can induce the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs). We have previously shown that cytoskeletal proteins and RhoGDIA, a negative regulator of the Rho GTPase pathway, are involved in the nicotine-induced dysfunction of VSMCs. Here, we found that nicotine can activate the Rho GTPase pathway and induce the synthesis of the cytoskeletal proteins in VSMCs through the activation of intracellular downstream signaling pathways, including targets such as MYPT1, PAK1 and PI3K/AKT. Upon nicotine treatment, the mRNA level of RhoGDIA is increased but protein level is decreased both in vitro and in vivo, which suggested a mechanism of post-translational regulation. By the dual luciferase reporter assay, we identified the microRNA-200b (miR-200b) as a modulator of the behavioural changes of VSMCs in response to nicotine through targeting RhoGDIA directly. Introducing miR-200b inhibitors into cultured VSMCs significantly attenuated cell proliferation and migration. Additionally, we found that hypomethylation in the CpG island shore region of miR-200b was responsible for the nicotine-induced miR-200b up-regulation in VSMCs. The study demonstrates that nicotine facilitates VSMC dysfunction through a miR-200b/RhoGDIA/cytoskeleton module through the hypomethylation of miR-200b promoter and suggests that epigenetic modifications may play an important role in the pathological progression.


Assuntos
Citoesqueleto/efeitos dos fármacos , MicroRNAs/genética , Miócitos de Músculo Liso/efeitos dos fármacos , Nicotina/farmacologia , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/genética , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Citoesqueleto/metabolismo , Estimulantes Ganglionares/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo
15.
Acta Histochem ; 119(3): 183-189, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28187905

RESUMO

Cell migration is a vital process for many physiological and pathological events, and Rho GTPases have been confirmed as key factors in its regulation. The most studied negative regulator of Rho GTPases, Rho-specific guanine nucleotide dissociation inhibitor α (RhoGDIα), mediates cell migration through altering the overall expression and spatiotemporal activation of Rho GTPases. The RhoGDIα-Rho GTPases dissociation can be mediated by signal pathways targeting RhoGDIα directly. This review summarizes the research about the regulation of RhoGDIα during cell migration, which can be in a Rho GTPases association independent manner. Non-kinase proteins regulation, phosphorylation, SUMOylation and extracellular environmental factors are classified to discuss their direct signal regulations on RhoGDIα, which provide varied signal pathways for selective activation of Rho GTPases in cell migration.


Assuntos
Movimento Celular/fisiologia , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Linhagem Celular Tumoral , GTP Fosfo-Hidrolases/metabolismo , Humanos , Neoplasias/fisiopatologia , Fosforilação , Transdução de Sinais
16.
Sci Rep ; 6: 36952, 2016 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-27841340

RESUMO

In earlier studies, we showed that ATF4 down-regulation affects post-synaptic development and dendritic spine morphology in neurons through increased turnover of the Rho GTPase Cell Division Cycle 42 (Cdc42) protein. Here, we find that ATF4 down-regulation in both hippocampal and cortical neuron cultures reduces protein and message levels of RhoGDIα, a stabilizer of the Rho GTPases including Cdc42. This effect is rescued by an shATF4-resistant active form of ATF4, but not by a mutant that lacks transcriptional activity. This is, at least in part, due to the fact that Arhgdia, the gene encoding RhoGDIα, is a direct transcriptional target of ATF4 as is shown in ChIP assays. This pathway is not restricted to neurons. This is seen in an impairment of cell migration on ATF4 reduction in non-neuronal cells. In conclusion, we have identified a new cellular pathway in which ATF4 regulates the expression of RhoGDIα that in turn affects Rho GTPase protein levels, and thereby, controls cellular functions as diverse as memory and cell motility.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Córtex Cerebral/citologia , Hipocampo/citologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Animais , Células Cultivadas , Córtex Cerebral/metabolismo , Regulação para Baixo , Células HEK293 , Hipocampo/metabolismo , Humanos , Neurônios/metabolismo , Ratos
17.
PLoS One ; 11(11): e0166715, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27835684

RESUMO

The Rac1 GTPase plays key roles in cytoskeletal organization, cell motility and a variety of physiological and disease-linked responses. Wild type Rac1 signaling entails dissociation of the GTPase from cytosolic Rac1-Rho GDP dissociation inhibitor (GDI) complexes, translocation to membranes, activation by exchange factors, effector binding, and activation of downstream signaling cascades. Out of those steps, membrane translocation is the less understood. Using transfections of a expression cDNA library in cells expressing a Rac1 bioreporter, we previously identified a cytoskeletal feedback loop nucleated by the F-actin binding protein coronin 1A (Coro1A) that promotes Rac1 translocation to the plasma membrane by facilitating the Pak-dependent dissociation of Rac1-Rho GDI complexes. This screening identified other potential regulators of this process, including WDR26, basigin, and TMEM8A. Here, we show that WDR26 promotes Rac1 translocation following a Coro1A-like and Coro1A-dependent mechanism. By contrast, basigin and TMEM8A stabilize Rac1 at the plasma membrane by inhibiting the internalization of caveolin-rich membrane subdomains. This latter pathway is F-actin-dependent but Coro1A-, Pak- and Rho GDI-independent.


Assuntos
Basigina/genética , Membrana Celular/metabolismo , Glicoproteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Proteínas/genética , Proteínas rac1 de Ligação ao GTP/genética , Actinas/genética , Actinas/metabolismo , Animais , Basigina/metabolismo , Células COS , Caveolina 1/genética , Caveolina 1/metabolismo , Fracionamento Celular , Chlorocebus aethiops , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Transporte Proteico , Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/genética , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo
18.
Oncotarget ; 7(38): 61619-61629, 2016 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-27557508

RESUMO

Glioma stem cells (GSCs) are a subset of tumor cells that drive glioma initiation and progression. The molecular mechanisms underlying the maintenance of GSCs are still poorly understood. Here we investigated the role of Rho GDP dissociation inhibitor α (RhoGDIα) in GSCs. RhoGDIα was down-regulated in glioma stem cells. Over-expression of RhoGDIα suppressed the self-renewal and tumorigenesis of GSCs. Further data showed that RhoGDIα inhibited the transcription activity of stem cell marker Oct4. Moreover, inactivation of ROCK1, a downstream effector of RhoGDIα, also decreased the self-renewal and Oct4 transcription activity, and rescued the effects caused by RhoGDIα knockdown. Our results indicate that RhoGDIα is involved in the maintenance of GSCs.


Assuntos
Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Adulto , Idoso , Animais , Neoplasias Encefálicas/terapia , Carcinogênese , Diferenciação Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/patologia , Perfilação da Expressão Gênica , Genes Reporter , Glioma/terapia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Fator 3 de Transcrição de Octâmero/metabolismo , Transcrição Genética , Quinases Associadas a rho/metabolismo
19.
Oncotarget ; 7(34): 55110-55127, 2016 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-27391153

RESUMO

Hepatitis B virus (HBV) X protein (HBx), a trans-regulator, is frequently expressed in truncated form without carboxyl-terminus in hepatocellular carcinoma (HCC), but its functional mechanisms are not fully defined. In this report, we investigated frequency of this natural HBx mutant in HCCs and its functional significance. In 102 HBV-infected patients with HCC, C-terminal truncation of HBx, in contrast to full-length HBx, were more prevalent in tumors (70.6%) rather than adjacent non-tumorous tissues (29.4%) (p = 0.0032). Furthermore, two naturally-occurring HBx variants (HBxΔ31), which have 31 amino acids (aa) deleted (codons 123-125/124-126) at C-terminus were identified in tumors and found that the presence of HBxΔ31 significantly correlated with intrahepatic metastasis. We also show that over-expression of HBxΔ31 enhanced hepatoma cell invasion in vitro and metastasis in vivo compared to full-length HBx. Interestingly, HBxΔ31 exerts this function via down-regulating Maspin, RhoGDIα and CAPZB, a set of putative metastasis-suppressors in HCC, in part, by enhancing the binding of transcriptional repressor, myc-associated zinc finger protein (MAZ) to the promoters through physical association with MAZ. Notably, these HBxΔ31-repressed proteins were also significantly lower expression in a subset of HCC tissues with C-terminal HBx truncation than the adjacent non-tumorous tissues, highlighting the clinical significance of this novel HBxΔ31-driven metastatic molecular cascade. Our data suggest that C-terminal truncation of HBx, particularly breakpoints at 124aa, plays a role in enhancing hepatoma cell invasion and metastasis by deregulating a set of metastasis-suppressors partially through MAZ, thus uncovering a novel mechanism for the progression of HBV-associated hepatocarcinogenesis.


Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Mutação , Transativadores/genética , Adulto , Animais , Proteína de Capeamento de Actina CapZ/genética , Proteína de Capeamento de Actina CapZ/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Feminino , Células Hep G2 , Hepatite B/genética , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Interações Hospedeiro-Patógeno/genética , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Serpinas/genética , Serpinas/metabolismo , Transativadores/química , Transplante Heterólogo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/genética , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo
20.
Artigo em Inglês | MEDLINE | ID: mdl-27302765

RESUMO

Long-term administration of antipsychotic drugs (APDs) has been theorized to effect drug-induced changes in protein expression in the brain. Our previous findings revealed that ADPs can regulate Rho GDP-dissociation inhibitor 1 (RhoGDI1) expression in glial cells. To reveal whether APDs (haloperidol, risperidone, and clozapine) might regulate cell functions in rat brain by affecting RhoGDI1, RhoGDI1 regulation, RhoGDI1-related Rho family protein, and also MLC2 in brain of 7-day APD treatment rat were examined. Increased expression of RhoGDI1 and RhoA and decreased expression of MLC2, p-MLC2 and ARP2/3 were found in the cortex of APD-treated rats. The activation of RhoA in APD-treated rat cortex was also found. The regulation of RhoGDI1-induced protein expression and its relation to intracellular stress filament production and cell migration were further examined in APD-treated C6 and B35 cells. APD-induced RhoA expression and activation in C6 cells and Cdc42 expression and activation in B35 cells were investigated. In C6 cells, ARP2/3, ROCK1, pMLC2, and PFN1 expressions were decreased, and N-WASP expression was increased by any of the three APDs. In B35 cells, haloperidol decreased ROCK1 expression, but risperidone increased ROCK1 expression. MLC2, p-MLC2, and PFN1 expressions were decreased in B35 cells treated with either risperidone or clozapine. N-WASP expression was decreased by haloperidol and clozapine. We also found all three APDs enhance C6 and B35 F-actin condensation and migration ability.


Assuntos
Antipsicóticos/farmacologia , Citoesqueleto/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Animais , Encéfalo/citologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Serina/metabolismo , Transdução de Sinais/genética , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/genética
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