RESUMEN
OBJECTIVE: To construct recombinant lentiviral vectors carrying Rheb gene and its mutant Rheb'D60K gene, and examine their expression in human liver cancer cells. METHODS: Rheb gene was amplified by PCR to construct the recombinant plasmid LV31-Rheb-WT and LV31-Rheb-D60K. HEK-293 FT cells were contransfected with the recombinant lentiviral vector together with a lentiviral package plasmid to produce the lentiviral particles. The expression of PS6 protein was detected in the lentivirus-infected MCF-7 cells. The apoptosis of SK-HEP-1 cells transfected with LV31-Rheb-WT or LV31-Rheb-D60K was observed. RESULTS: The recombinant LV31-Rheb-WT and LV31-Rheb-D60K vectors were confirmed by PCR and DNA sequencing. Western blotting showed that PS6 protein expression was increased in LV31-Rheb-WT-transfected cells while decreased in LV31-Rheb-D60K-transfected cells. LV31-Rheb-D60K-transfected SK-HEP-1 cells showed more obvious apoptosis after starvation than LV31-Rheb-WT-transfected cells. CONCLUSION: Lentiviral vectors carrying Rheb gene and its mutant has been successfully constructed, which can be useful in further investigation of the role of Rheb gene in cancer cells.
Asunto(s)
Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Proteínas de Unión al GTP Monoméricas/biosíntesis , Proteínas de Unión al GTP Monoméricas/genética , Proteínas Mutantes/genética , Neuropéptidos/biosíntesis , Neuropéptidos/genética , Apoptosis/genética , Carcinoma Hepatocelular/metabolismo , Vectores Genéticos/genética , Células HEK293 , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Neoplasias Hepáticas/metabolismo , Células MCF-7 , Proteína Homóloga de Ras Enriquecida en el Cerebro , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , TransfecciónRESUMEN
OBJECTIVE: To obtain recombinant N-and C-terminal of FKBP38 and prepare anti-FKBP38 polyclonal antibody for Western blotting (WB), immunohistochemical (IHC) and immunofluorescence (IF) analyses. METHODS: The N-terminal (1-207 aa) and C-terminal (209-387 aa) cDNA of FKBP38 were sub-cloned from the full-length cDNA of FKBP38 and ligated to prokaryotic expression plasmid pGEX-6P-1 for construction of the recombinant vectors pGEX-6P-1-FKBP38-N and pGEX-6P-1-FKBP38-C. After sequencing, the recombinant vectors were transformed into E.coli BL21 and GST-tagged FKBP38-NT and FKBP38-CT were induced by IPTG. The proteins were purified by Glutathione affinity chromatography column and characterized by SDS-PAGE. Rabbits were immunized with the purified recombinant protein to prepare the antiserum, which were analyzed by WB, IHC and IF. RESULTS: The recombinant vectors pGEX-6P-1-FKBP38-N and pGEX-6P-1-FKBP38-C were successfully constructed. After IPTG induction, the E.coli transformed with these plasmids expressed GST-tagged protein, which was successfully purified. Western blotting demonstrated that the purified antibody could specifically bind to FKBP38 in various cell lines. Immunofluorescence assay showed that FKBP38 was located mainly on the mitochondria. Immunohistochemical analysis revealed cytoplasmic location of FKBP38 in breast cells. CONCLUSION: We successfully expressed and purified N- and C-terminal of FKBP38, and FKBP38 polyclonal antibody we prepared can specifically recognize FKBP38 in SB, IF and IHC assays, which facilitates further functional investigation of FKBP38.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Vectores Genéticos/genética , Proteínas de Unión a Tacrolimus/biosíntesis , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/inmunologíaRESUMEN
Reactive oxygen species (ROSs) are involved in osteoporosis by inhibiting osteoblastic differentiation and stimulating osteoclastgenesis. Little is known about the role and how ROS controls proliferation of osteoblasts. Mammalian target of rapamycin, mTOR, is a central regulator of cell growth and proliferation. Here, we report for the first time that 5-200 microM hydrogen peroxide (H(2)O(2)) dose- and time-dependently suppressed cell proliferation without affecting cell viability in mouse osteoblast cell line, MC3T3-E1, and in human osteoblast-like cell line, MG63. Further study revealed that protein level of cyclin B1 decreased markedly and the percentage of the cells in G(2)/M phase increased about 2-4 fold by 200 microM H(2)O(2) treatment for 24-72 hr. A total of 0.5-5 mM of H(2)O(2) but not lower concentrations (5-200 microM) of H(2)O(2) inhibited mTOR signaling, as manifested by dephosphorylation of S6K (T389), 4E-BP1 (T37/46), and S6(S235/236) in MC3T3-E1 and MG63 cells. Rapamycin, which could inhibit mTOR signaling and cell proliferation, however, did not reduce the protein level of cyclin B1. In a summary, H(2)O(2) prevents cell proliferation of osteoblasts by down-regulating cyclin B1 and inducing G(2) cell cycle arrest. Inhibition of mTOR signaling by H(2)O(2) may not be involved in this process.
Asunto(s)
Ciclo Celular , Proliferación Celular/efectos de los fármacos , Fase G2/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Osteoblastos/efectos de los fármacos , Animales , Línea Celular , Ciclina B/metabolismo , Ciclina B1 , Regulación hacia Abajo , Humanos , Ratones , Osteoblastos/citología , Proteínas Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR , Factores de TiempoRESUMEN
It has been established that reactive oxygen species (ROS) such as H2O2 or superoxide anion is involved in bone loss-related diseases by stimulating osteoclast differentiation and bone resorption and that receptor activator of NF-kappaB ligand (RANKL) is a critical osteoclastogenic factor expressed on stromal/osteoblastic cells. However, the roles of ROS in RANKL expression and signaling mechanisms through which ROS regulates RANKL genes are not known. Here we report that increased intracellular ROS levels by H2O2 or xanthine/xanthine oxidase-generated superoxide anion stimulated RANKL mRNA and protein expression in human osteoblast-like MG63 cell line and primary mouse bone marrow stromal cells and calvarial osteoblasts. Further analysis revealed that ROS promoted phosphorylation of cAMP response element-binding protein (CREB)/ATF2 and its binding to CRE-domain in the murine RANKL promoter region. Moreover, the results of protein kinase A (PKA) inhibitor KT5720 and CREB1 RNA interference transfection clearly showed that PKA-CREB signaling pathway was necessary for ROS stimulation of RANKL in mouse osteoblasts. In human MG63 cells, however, we found that ROS promoted heat shock factor 2 (HSF2) binding to heat shock element in human RANKL promoter region and that HSF2, but not PKA, was required for ROS up-regulation of RANKL as revealed by KT5720 and HSF2 RNA interference transfection. We also found that ROS stimulated phosphorylation of extracellular signal-regulated kinases (ERKs) and that PD98059, the inhibitor for ERKs suppressed ROS-induced RANKL expression either in mouse osteoblasts or in MG63 cells. These results demonstrate that ROS stimulates RANKL expression via ERKs and PKA-CREB pathway in mouse osteoblasts and via ERKs and HSF2 in human MG63 cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Glicoproteínas de Membrana/metabolismo , Osteoblastos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Resorción Ósea , Diferenciación Celular , Línea Celular , Línea Celular Tumoral , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cartilla de ADN/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Citometría de Flujo , Células HeLa , Proteínas de Choque Térmico/metabolismo , Calor , Humanos , Peróxido de Hidrógeno/farmacología , Ligandos , Ratones , Osteoclastos/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Ligando RANK , Interferencia de ARN , ARN Mensajero/metabolismo , Receptor Activador del Factor Nuclear kappa-B , Transducción de Señal , Factores de Tiempo , Factores de Transcripción/metabolismo , Transfección , Regulación hacia ArribaRESUMEN
Metallothionein (MT), a cysteine-rich, metal-binding protein, is involved in homeostatic regulation of essential metals and protection of cells against oxidative injury. It has been shown that oxidative stress is associated with pathogenesis of osteoporosis and is capable of inhibiting osteoblastic differentiation of bone cells by nuclear factor-kappaB (NF-kappaB). In this study, the effect of MT on oxidative stress-induced inhibition of osteoblast differentiation was examined. 50-200 microM hydrogen peroxide-induced oxidative stress suppressed the osteoblastic differentiation process of primary mouse bone marrow stromal cells (BMSCs), manifested by a reduction in the differentiation marker alkaline phosphatase (ALP). The presence of exogenous MT (20-500 microM) or induction of endogenous MT by ZnCl2 (50-200 microM) could protect BMSCs against H2O2-induced inhibition of osteoblastic differentiation, manifested by a resumption of H2O2-inhibited ALP activity and ALP positive cells. Furthermore, adding exogenous MT or inducing endogenous MT expression impaired H2O2-stimulated NF-kappaB signaling. These data indicate the ability of MT to protect BMSCs against oxidative stress-induced inhibition of osteoblastic differentiation.
Asunto(s)
Células de la Médula Ósea/metabolismo , Diferenciación Celular/fisiología , Metalotioneína/metabolismo , Osteoblastos/metabolismo , Estrés Oxidativo/fisiología , Células del Estroma/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Biomarcadores/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Cloruros/farmacología , Peróxido de Hidrógeno/farmacología , Ratones , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Osteoblastos/efectos de los fármacos , Osteogénesis/fisiología , Osteoporosis/metabolismo , Osteoporosis/fisiopatología , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células del Estroma/efectos de los fármacos , Compuestos de Zinc/farmacologíaRESUMEN
Phospholipase C-gamma1 (PLC-gamma1) activation has been reported to enhance cell survival during the cellular response to oxidative stress. We studied the role of protein kinase C (PKC) pathways in mediating PLC-gamma1 survival signalling in oxidative stress by using mouse embryonic fibroblasts genetically deficient in PLC-gamma1 (Plcg1(-/-)) and its wild type (Plcg1(+/+)). PLC-gamma1 was activated by H(2)O(2) treatment in a dose- and time-dependent manner. Activation of PKC was also markedly increased in both cell lines treated with H(2)O(2) (1-5 mM), but with low doses (50-200 microM), PKC activation was considerably decreased in Plcg1(-/-) cells. After treatment with H(2)O(2), PKC-dependent phosphorylation of Bcl-2 and cell viability of Plcg1(-/-) cells decreased dramatically and caspase-3-like activity increased significantly compared with that of the wild-type cells. Furthermore, pretreatment of Plcg1(+/+) cells with PKC-specific inhibitor decreased levels of PKC-dependent Bcl-2 phosphorylation, enhanced caspase-3 activity and their sensitivity to H(2)O(2). On the contrary, treatment of Plcg1(-/-) cells with PKC-specific activator increased the Bcl-2 phosphorylation, decreased caspase-3 activity and improved their survival. These results suggest that PLC-gamma1 mediates survival signalling in oxidative-stress response by PKC-dependent phosphorylation of Bcl-2 and inhibition of caspase-3.
Asunto(s)
Supervivencia Celular/fisiología , Isoenzimas/metabolismo , Estrés Oxidativo/fisiología , Proteína Quinasa C/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Caspasa 3 , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Isoenzimas/deficiencia , Isoenzimas/genética , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fosfolipasa C gamma , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Fosfolipasas de Tipo C/deficiencia , Fosfolipasas de Tipo C/genéticaRESUMEN
The consequences of heat-induced phospholipase C-gamma1 (PLC-gamma1) phosphorylation are not known. We investigated the role of PLC-gamma1 activation and its downstream targets during the cellular response to heat stress using mouse embryonic fibroblasts genetically deficient in PLC-gamma1 (Plcg1 null MEF) and its wild type (wt MEF) as models. Treatment of wt MEF with heat resulted in temperature- and heating duration-dependent tyrosine phosphorylation of PLC-gamma1. HSP70 synthesis and the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal protein kinase (JNK) increased equally following heat treatment in both cell lines. However, heat-induced protein kinase C (PKC) activation was dramatically reduced in Plcg1 null MEF compared with wt MEF. Importantly, the mitochondrial localization of PKCalpha, PKC-dependent phosphorylation of Bcl-2, and cell viability in Plcg1 null MEF following heat treatment, were significantly decreased compared with the wild type. Furthermore, pretreatment with bryostatin-1, a PKC activator, enhanced Bcl-2 phosphorylation and cellular resistance to heat-induced apoptosis in Plcg1 null MEF. Taken together, these results suggest that PLC-gamma1 activation enhances cell survival through the PKC-dependent phosphorylation of Bcl-2 during the cellular response to heat stress.