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BACKGROUND & AIMS: Hepatic ischemia-reperfusion injury (HIRI) often occurs in liver surgery, such as partial hepatectomy and liver transplantation, in which myeloid macrophage-mediated inflammation plays a critical role. Cell division cycle 42 (Cdc42) regulates cell migration, cytoskeleton rearrangement, and cell polarity. In this study, we explore the role of myeloid Cdc42 in HIRI. METHODS: Mouse HIRI models were established with 1-hour ischemia followed by 12-hour reperfusion in myeloid Cdc42 knockout (Cdc42mye) and Cdc42flox mice. Myeloid-derived macrophages were traced with RosamTmG fluorescent reporter under LyzCre-mediated excision. The experiments for serum or hepatic enzymic activities, histologic and immunologic analysis, gene expressions, flow cytometry analysis, and cytokine antibody array were performed. RESULTS: Myeloid deletion of Cdc42 significantly alleviated hepatic damages with the reduction of hepatic necrosis and inflammation, and reserved hepatic functions following HIRI in mice. Myeloid Cdc42 deficiency suppressed the infiltration of myeloid macrophages, reduced the secretion of proinflammatory cytokines, restrained M1 polarization, and promoted M2 polarization of myeloid macrophages in livers. In addition, inactivation of Cdc42 promoted M2 polarization via suppressing the phosphorylation of STAT1 and promoting phosphorylation of STAT3 and STAT6 in myeloid macrophages. Furthermore, pretreatment with Cdc42 inhibitor, ML141, also protected mice from hepatic ischemia-reperfusion injury. CONCLUSIONS: Inhibition or deletion of myeloid Cdc42 protects liver from HIRI via restraining the infiltration of myeloid macrophages, suppressing proinflammatory response, and promoting M2 polarization in macrophages.
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Modelos Animales de Enfermedad , Inflamación , Hígado , Macrófagos , Ratones Noqueados , Daño por Reperfusión , Proteína de Unión al GTP cdc42 , Animales , Daño por Reperfusión/patología , Daño por Reperfusión/inmunología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/genética , Daño por Reperfusión/prevención & control , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP cdc42/genética , Ratones , Macrófagos/metabolismo , Macrófagos/inmunología , Hígado/patología , Hígado/metabolismo , Hígado/inmunología , Inflamación/patología , Inflamación/metabolismo , Células Mieloides/metabolismo , Células Mieloides/patología , Factor de Transcripción STAT3/metabolismo , Masculino , Factor de Transcripción STAT1/metabolismo , Citocinas/metabolismo , Factor de Transcripción STAT6/metabolismo , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/deficiencia , Ratones Endogámicos C57BL , Eliminación de GenRESUMEN
@#[Abstract] Objective: To analyze the expression of miR-185 and cell division cyclin 42 (CDC42) in osteosarcoma tissues and cells, and to preliminarily explore whether miR-185 affects the proliferation and migration of osteosarcoma MG63 cells by regulating CDC42. Methods: The cancer tissues and para-cancerous tissues of 28 patients with osteosarcoma that pathologically confirmed in the Fourth People's Hospital of Hengshui City from January 2020 to January 2021 were collected for this study. Immunohistochemistry was used to detect the expression of CDC42 in osteosarcoma tissues, and qPCR was used to detect the expression of miR-185 in osteosarcoma tissues. Dual-luciferase reporter gene experiment was applied to verify the targeting relationship between CDC42 and miR-185. According to different transfectants, MG63 cells were divided into miR-185 mimic group, miR-NC group, miR-185 inhibitor group, NC-inhibitor group, CDC42 group (transfected with CDC42 over-expression vector), and negative control (NC) group. The effects of miR-185 and CDC42 expression on the migration, proliferation and cell cycle of MG63 cells were detected by scratch healing assay, CCK-8 method and FCM, respectively. A nude mouse xenograft model was constructed by inoculating osteosarcoma MG63 cells. Immunohistochemistry, qPCR and WB methods were used to detect the effects of over-expression or knock-down of miR-185 on the expression of Ki67 and CDC42 in transplanted tumor tissues. Results: Compared with para-cancerous tissues, the expression of miR-185 in osteosarcoma tissues was significantly decreased, while the expression of CDC42 was significantly increased (all P<0.01). CDC42 was verified to be a target gene of miR-185. Compared with the control group, the migration and proliferation of MG63 cells in the miR-185 mimic group were inhibited (all P<0.01), while the migration and proliferation of MG63 cells in the CDC42 group were increased and the cell cycle was arrested in the S phase (all P<0.01). Compared with the miR-185 group, the migration and proliferation abilities of MG63 cells in the miR-185+CDC42 group were promoted, and the proportion of cells in S phase was increased (all P<0.01). Compared with the control group, the expression of Ki67 and CDC42 in the transplanted tumor tissues of miR-185 mimic group was significantly decreased (all P<0.01), while the opposite results were observed in miR-185 inhibitor group (all P<0.01). Conclusion: miR-185 is lowly expressed while CDC42 is highly expressed in osteosarcoma tissues. miR-185 can inhibit the proliferation and migration of osteosarcoma MG63 cells by negatively regulating the expression of CDC42.
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OBJECTIVE: microRNAs (miRNAs) can play a role in a variety of physiological and pathological processes, and their role is achieved by regulating the expression of target genes. Our previous high-throughput sequencing found that ssc-miR-185 plays an important regulatory role in piglet diarrhea, but its specific target genes and functions in intestinal porcine epithelial cell (IPEC-J2) are still unclear. We intended to verify the target relationship between porcine miR-185 and cell division cycle 42 (CDC42) gene in IPEC-J2 and to explore the effect of miR-185 on the proliferation of IPEC-J2 cells. METHODS: The TargetScan, miRDB, and miRanda software were used to predict the target genes of porcine miR-185, and CDC42 was selected as a candidate target gene. The CDC423' UTR-wild type (WT) and CDC42-3'UTR-mutant type (MUT) segments were successfully cloned into pmirGLO luciferase vector, and the luciferase activity was detected after co-transfection with miR-185 mimics and pmirGLO-CDC42-3'UTR. The expression level of CDC42 was analyzed using quantitative polymerase chain reaction and Western blot. The proliferation of IPEC-J2 was detected using cell counting kit-8 (CCK-8), methylthiazolyldiphenyltetrazolium bromide (MTT), and 5-ethynyl-2'-deoxyuridine (EdU) assays. RESULTS: Double enzyme digestion and sequencing confirmed that CDC42-3'UTR-WT and CDC42-3'UTR-MUT were successfully cloned into pmirGLO luciferase reporter vector, and the luciferase activity was significantly reduced after co-transfection with miR-185 mimics and CDC42-3'UTR-WT. Further we found that the mRNA and protein expression level of CDC42 were down-regulated after transfection with miR-185 mimics, while the opposite trend was observed after transfection with miR-185 inhibitor (p<0.01). In addition, the CCK-8, MTT, and EdU results demonstrated that miR-185 promotes IPEC-J2 cells proliferation by targeting CDC42. CONCLUSION: These findings indicate that porcine miR-185 can directly target CDC42 and promote the proliferation of IPEC-J2 cells. However, the detailed regulatory mechanism of miR-185/CDC42 axis in piglets' resistance to diarrhea is yet to be elucidated in further investigation.
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Insulin is a key hormone for maintaining glucose homeostasis in organisms. In general, deficiency of insulin synthesis and secretion results in type I diabetes, whereas insulin resistance leads to type 2 diabetes. Cell division cycle 42 (CDC42), a member of Rho GTPases family, has been shown as an essential regulator in the second phase of glucose-induced insulin secretion in pancreatic islets ß cells in vitro. However, the effect of CDC42 on insulin expression has not been explored. Here we reported that the glucose-induced insulin expression and secretion were significantly inhibited in mice lacking CDC42 gene in pancreatic ß cells (Rip-CDC42cKO) in vivo and in vitro. Deletion of CDC42 gene in pancreatic ß cells did not affect survival or reproduction in mice. However, the Rip-CDC42cKO mice showed the systemic glucose intolerance and the decrease of glucose-induced insulin secretion without apparent alterations of peripheral tissues insulin sensitivity and the morphology of islets. Furthermore, we demonstrated that deletion of CDC42 gene in pancreatic ß cells significantly attenuated the insulin expression through inhibiting the ERK1/2-NeuroD1 signaling pathway. Taken together, our study presents novel evidence that CDC42 is an important modulator in glucose-induced insulin expression as well as insulin secretion in pancreatic ß cells.
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Glucosa/farmacología , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Insulina/genética , Proteína de Unión al GTP cdc42/genética , Animales , Células Cultivadas , Eliminación de Gen , Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Insulina/metabolismo , Resistencia a la Insulina/genética , Secreción de Insulina/efectos de los fármacos , Secreción de Insulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos/genética , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
The Ras-related protein Cell division cycle 42 (Cdc42) is important in cell-signaling processes. Protein interactions involving Cdc42 occur primarily in flexible "Switch" regions that help regulate effector binding. We studied the kinetics of intrinsic GTP hydrolysis reaction in the absence and presence of a biologically active peptide derivative of a p21-activated kinase effector (PBD46) for wt Cdc42 and compared it to the Switch 1 variant Cdc42(T35A). While the binding of PBD46 to wt Cdc42 results in complete inhibition of GTP hydrolysis, this interaction in Cdc42(T35A) does not. Comparison of the crystal structure of wt Cdc42 in the absence of effector (1AN0.pdb), as well as the NMR structure of wt Cdc42 bound to an effector in the Switch 1 region (1CF4.pdb) ( www.rcsb.org ) suggests that the orientation of T(35) with bound Mg(2+) changes in the presence of effector, resulting in movement of GTP away from the catalytic box leading to the inhibition of GTP hydrolysis. For Cdc42(T35A), molecular dynamics simulations and structural analyses suggest that the nucleotide does not undergo the conformational shift observed for the wt Cdc42-effector interaction. Our data suggest that change in dynamics in the Switch 1 region of Cdc42 caused by the T35A mutation (Chandrashekar, et al. 2011, Biochemistry, 50, p. 6196) fosters a conformation for this Cdc42 variant that allows hydrolysis of GTP in the presence of PBD46, and that alteration of the conformational dynamics could potentially modulate Ras-related over-activity.
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Guanosina Trifosfato/química , Péptidos/farmacología , Proteína de Unión al GTP cdc42/química , Proteína de Unión al GTP cdc42/genética , Quinasas p21 Activadas/química , Animales , Sitios de Unión , Dominio Catalítico , Variación Genética , Humanos , Hidrólisis , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
OBJECTIVES: To investigate levels of cell-division cycle 42 (Cdc42) protein, and their relationship with Golgi apparatus function in peripheral lymphocytes, in patients following ischaemic stroke. METHODS: Patients with acute cerebral ischaemic stroke (within 24-72 h of the onset of focal neurological symptoms) and healthy control subjects were enrolled in this prospective case-control study. The cellular location of Cdc42 in peripheral lymphocytes was demonstrated using immunofluorescence. Protein levels of Cdc42 and trans-golgi network protein 2 (TGN46) in peripheral lymphocytes were determined by immunocytochemical staining and Western blotting. RESULTS: A total of 38 patients with stroke and 38 control subjects were studied. The mean ± SD percentage of Cdc42-positive lymphocytes from patients with stroke was significantly lower than that in control subjects (39.53 ± 13.55% versus 66.61 ± 23.30%, respectively). Similar findings were demonstrated for TGN46. Cdc42 levels were positively correlated with TGN46 levels (r = 0.92). CONCLUSIONS: Acute ischaemic stroke was associated with reduced levels of Cdc42 protein. These findings might lead to the development of drugs that could have therapeutic benefits in patients with acute ischaemic stroke.