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Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes
Published on: January 7, 2013
Protein kinase Cbeta isoform down-regulates the expression of MDR3 P-glycoprotein in human Chang liver cells
Satoshi Suzuki1, Hisao Hayashi, Kenji Takagi
1Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3, Kanagawa-machi, Kanazawa 920-1181, Japan.
Abstract:
The MDR3 protein is a transporter of phosphatidylcholine on the canalicular membrane of human hepatocytes. Previously we showed that the expression of MDR3 mRNA was down-regulated by phorbol 12-myristate 13-acetate (PMA) in human Chang liver cells. In the present study, to elucidate the isoform of protein kinase C (PKC), which influences the level of MDR3 protein, we investigated the effects of PKC-specific inhibitors and antisense oligonucleotides. The level of protein decreased around 50% after treatment for 3-5 days using the dosage of PMA effective against the mRNA expression. The half-life of the MDR3 protein was estimated to be about 5 days. This decrease was antagonized by GF109203X, a non-selective inhibitor of PKCs, and Gö6976, a selective inhibitor for PKCalpha/beta. These inhibitors also suppressed the reduction in MDR3 protein. To specify the isoform of PKC, the cells were treated with antisense oligonucleotide of PKCalpha or PKCbeta. The suppressive effects on MDR3 mRNA of PMA were attenuated in antisense PKCbeta-treated cells, but those in antisense PKCalpha-treated cells were not attenuated. These suggested that PKCbeta plays a regulatory role in the expression of MDR3.
Insights
Phorbol 12-myristate 13-acetate (PMA) reduces MDR3 protein levels in liver cells. Protein kinase C beta (PKCbeta) is identified as a key regulator in this process, influencing MDR3 expression.
Area of Science:
- Hepatology
- Molecular Biology
- Cell Biology
Background:
- MDR3 protein transports phosphatidylcholine on the canalicular membrane of human hepatocytes.
- Previous studies demonstrated phorbol 12-myristate 13-acetate (PMA) down-regulates MDR3 mRNA expression in Chang liver cells.
Purpose of the Study:
- To identify the specific protein kinase C (PKC) isoform involved in regulating MDR3 protein levels.
- To investigate the mechanism by which PMA affects MDR3 protein expression.
Main Methods:
- Treatment of human Chang liver cells with PMA.
- Application of PKC-specific inhibitors (GF109203X, Gö6976) and antisense oligonucleotides for PKCalpha and PKCbeta.
- Quantification of MDR3 protein levels and mRNA expression.
Main Results:
- PMA treatment decreased MDR3 protein levels by approximately 50% over 3-5 days.
- Non-selective PKC inhibitor GF109203X and selective PKCalpha/beta inhibitor Gö6976 antagonized the PMA-induced decrease in MDR3 protein.
- Antisense oligonucleotides targeting PKCbeta, but not PKCalpha, attenuated the suppressive effect of PMA on MDR3 mRNA, suggesting PKCbeta's regulatory role.
Conclusions:
- PKCbeta plays a significant regulatory role in the expression of MDR3 protein in human hepatocytes.
- The findings elucidate a novel signaling pathway involving PKCbeta in the regulation of MDR3, a key transporter in bile formation.
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