RESUMO
Phospholipase D (PLD) plays a crucial role in the regulation of some cellular processes, including autophagy and apoptosis. Accumulation of protein in the endoplasmic reticulum (ER) lumen causes ER stress. Although ER stress is a principal cause of apoptosis and autophagy, the relationship between PLD activity and ER stress remains unclear. Protein transport from the ER to the Golgi apparatus is conducted by coat complex II (COPII) transport vesicles. Here, we demonstrated that inhibition of PLD1 activity or PLD1 knockdown suppressed COPII vesicle transport in normal rat kidney (NRK) cells. COPII vesicle coat proteins are composed of Sar1 as well as Sec23/24 and Sec13/31 complexes. For COPII vesicle formation on the ER membrane, Sar1, Sec23/24, and Sec13/31 are sequentially recruited from the cytosol to the ER membrane. Using a cell-free COPII coat protein recruitment assay, we demonstrated that inhibition of PLD1 activity suppressed Sec13/31 recruitment from the cytosol to the ER membrane in COPII vesicle formation. PLD1 knockdown in NRK cells was associated with increased expression of the ER stress marker GRP78 and apoptosis. Taken together, these results suggest that PLD1 activity regulates COPII vesicle transport from the ER to the Golgi apparatus by regulating Sec13/31 recruitment from the cytosol to the ER membrane during COPII vesicle formation.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Estresse do Retículo Endoplasmático , Fosfolipase D/metabolismo , Animais , Linhagem Celular , Citosol/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Masculino , Fosfolipase D/genética , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos Sprague-Dawley , Proteínas de Transporte Vesicular/metabolismoRESUMO
We reported previously that treatment of the pig kidney proximal tubular epithelial cell line LLC-PK(1) with cephaloridine (CLD) decreased the activity of cytochrome c oxidase in the mitochondria of the cells followed by increases in lipid peroxidation and cell necrosis. In this study, we investigated the effects of CLD on the activity of cytochrome c oxidase in mitochondria isolated from LLC-PK(1) cells and purified the enzyme from mitochondria of the rat renal cortex. The activity of cytochrome c oxidase in the isolated mitochondria from LLC-PK(1) cells was significantly decreased from 1 h after addition of 1 mM CLD. Other cephalosporin antibiotics, cefazolin and cefalotin, also decreased the activity of cytochrome c oxidase in the isolated mitochondria. The activity of cytochrome c oxidase purified from the mitochondria of the rat renal cortex was also decreased from 2 h after addition of 1 mM CLD in a non-competitive manner. These results suggest that the direct inhibition of cytochrome c oxidase activity in the mitochondrial electron transport chain by cephlosporins may result from the observed nephrotoxicity.
Assuntos
Antibacterianos/farmacologia , Cefaloridina/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Rim/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Linhagem Celular , Cefalosporinas/farmacologia , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Rim/citologia , Rim/enzimologia , Córtex Renal/enzimologia , Cinética , Masculino , Mitocôndrias/química , Mitocôndrias/enzimologia , Proteínas Mitocondriais/análise , Ratos , Ratos WistarRESUMO
To clarify the role of neurotrophin receptors in the development of Ruffini endings, periodontal ligaments and trigeminal ganglia of trkA, trkB, and trkC knockout mice were immunostained for protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP), parvalbumin (PV), and calretinin (CR). Innervation patterns of PGP 9.5- and CGRP-immunoreactive fibers were examined in the periodontal ligament of the knockout mice. PGP 9.5-positive fibers in the incisal periodontal ligaments of trkA and trkC knockout mice form Ruffini endings distinguished by dendritic ramifications and branches. However, Ruffini endings were not present in the periodontal ligament of trkB knockout mice. Only free nerve endings were observed in tissue of trkB knockout mice. Compared with trkA and trkC knockouts, the proportion of CR-positive neurons in mandibular and maxillary regions of the trigeminal ganglion of trkB knockout mice is decreased. These findings indicate that the development of periodontal Ruffini endings is regulated by trkB-dependent and CR-coexpressing neurons.
Assuntos
Mecanorreceptores/fisiologia , Ligamento Periodontal/inervação , Receptor trkB/fisiologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Mecanorreceptores/anormalidades , Mecanorreceptores/ultraestrutura , Camundongos , Camundongos Knockout/genética , Terminações Nervosas/ultraestrutura , Fibras Nervosas/fisiologia , Ligamento Periodontal/metabolismo , Receptor trkA/deficiência , Receptor trkA/genética , Receptor trkB/deficiência , Receptor trkB/genética , Receptor trkC/deficiência , Receptor trkC/genética , Tioléster Hidrolases/metabolismo , Ubiquitina TiolesteraseRESUMO
4'-O-tetrahydropyranyladriamycin (THP) showed an approximately 10-fold greater inhibitory effect on DNA synthesis in L1210 mouse leukemia cells than adriamycin (ADM). The intracellular transfer rate and nuclear accumulation of THP were approximately 5-fold higher than those of ADM. The intensity of in vitro inhibition of topoisomerase II activity by ADM was almost the same as that by THP. There were no significant differences between the uptake of either of these agents by the isolated nuclei of L1210 cells. The nuclear uptake of both agents in the presence of the cytosolic fraction of L1210 cells consisted of both simple diffusion and carrier-mediated components, and the carrier-mediated component of THP was approximately 2-fold higher than that of ADM. THP showed approximately 5-fold higher affinity to the proteasome than ADM, and interfered with ADM binding in a competitive manner. These results suggest that the binding affinity of these anticancer agents to the proteasome is an important factor in their transport to the nucleus and determines their specificity of action for the nuclear DNA.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Núcleo Celular/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacologia , Complexos Multienzimáticos/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , DNA/efeitos dos fármacos , Dano ao DNA , Doxorrubicina/metabolismo , Leucemia L1210/metabolismo , Leucemia L1210/patologia , Camundongos , Complexo de Endopeptidases do Proteassoma , Timidina/metabolismoRESUMO
To elucidate the involvement of proteasome inhibition in apoptosis induced by anthracycline anticancer agents, we investigated the interaction between the proteasome and anthracycline anticancer agents, and the function of the proteasome in apoptosis. Exposure of L1210 mouse lymphocytic leukemia cells to adriamycin (ADM) or 4'-O-tetrahydropyranyladriamycin (THP) resulted in apoptosis in a dose-dependent manner: 5 microM ADM and 0.5 microM THP induced apoptosis efficiently, but the effects of 10 microM ADM and 5 microM THP were markedly decreased or completely absent. Carbobenzoxy-leucyl-leucyl-leucinal (Z-LLLal), a specific proteasome inhibitor, also induced dose-dependent apoptosis of the cells. The inhibitory effect of THP on chymotrypsin-like protease activity of proteasomes purified from the cytosol of L1210 cells was stronger than that of ADM. Both of these agents showed reversible non-competitive inhibitory effects. The intracellular content of ubiquitinated protein increased in ADM-, THP- or Z-LLLal-treated L1210 cells during apoptosis. These results suggested that anthracycline anticancer agents induce apoptosis by interacting, at least in part, with proteasomes.