Suppression of protein kinase Calpha triggers apoptosis through down-regulation of Bcl-xL in a rat hepatic epithelial cell line.
Inactivation of protein kinase C (PKC)alpha plays an important role in modulating hepatic failure and/or apoptosis during sepsis. To determine whether and how PKCalpha inactivation mediates the apoptosis, PKCalpha was suppressed by antisense treatment or transiently transfection in Clone-9 rat hepatic epithelial cell line. Apoptosis was evaluated by cell survival rate, poly-adenyl ribonuclease polymerase (PARP) cleavage, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling stain. The expressions of PKCalpha and Bcl-xL were quantified by Western blot analysis after antisense treatment. In the transfection studies, cells were co-transfected with green fluorescent protein cDNA as a transfection marker. The expressions of PKCalpha and Bcl-xL were detected by immunohistochemical staining with second antibody conjugated with Texas red. Apoptosis was evaluated by tetramethyl-rhodamine labeling of DNA strand breaks and immunostaining of 85-kDa fragment of PARP. The results showed that cytosolic and membrane-associated PKCalpha were decreased by 54.5% and 41.4%, respectively, after PKCalpha antisense treatment. The apoptotic incidence and percentage of PARP cleavage were significantly increased, whereas protein expression of Bcl-xL was decreased after PKCalpha-antisense treatment. In the transfection studies, the results showed that most of the cells expressing green fluorescent protein revealed less PKCalpha and Bcl-xL protein contents and more in situ PARP cleavage and DNA strand breaks. These findings indicated that decrease of PKCalpha declines the Bcl-xL content and leads to the vulnerability of apoptosis in hepatic epithelial cells. Taken together, our data provide evidence that suppression of PKCalpha plays a critical role in triggering caspase-dependent apoptosis, which may act through modulating the Bcl-xL expression.
Hsieh
Ya-Ching
YC
Department of Physiology, Kaohsiung Medical University, Kaohsiung city, Taiwan.
Jao
Hsiao-Ching
HC
Yang
Rei-Cheng
RC
Hsu
Hseng-Kuang
HK
Hsu
Chin
C
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001483:Base Sequence; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D017931:DNA Primers; D004847:Epithelial Cells / Q000166:cytology* / Q000502:physiology; D008099:Liver / Q000166:cytology* / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics*; D051571:Protein Kinase C-alpha; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D013489:Suppression, Genetic; D014162:Transfection; D051020:bcl-X Protein
C494813:Bcl2l1 protein, rat; D017931:DNA Primers; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D051020:bcl-X Protein; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha