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Protein kinase C-mediated down-regulation of MDR3 mRNA expression in Chang liver cells
S Wakusawa1, R Ikeda, Y Shiono
1Department of Medicine, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3, Kanagawa-machi, 920-1181, Kanazawa, Japan. s-wakusawa@hokuriku-u.ac.jp
Abstract:
MDR3 is a phospholipid translocator homologous to MDR1 P-glycoprotein. MDR3 localizes to the canalicular membrane and contributes to the secretion of bile. To elucidate the role of protein kinase C in the regulation of MDR3 gene expression, we investigated the effect of phorbol 12-myristate 13-acetate (PMA) on the level of MDR3 mRNA in human Chang liver cells by a reverse transcription-polymerase chain reaction method. The steady-state expression of MDR3 mRNA was decreased by PMA after treatment for 8-20 hr and at concentrations of 1-100 nM. PMA also decreased the doxorubicin-induced expression of MDR3 mRNA. 4alpha-Phorbol 12,13-didecanoate, a negative control compound, did not decrease the expression at these concentrations. The down-regulatory effect of PMA was partially suppressed by the protein kinase C inhibitors 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide (GF109203X) and calphostin C. Furthermore, cycloheximide, a protein synthesis inhibitor, antagonized the effect of PMA. From these results, it was suggested that the level of MDR3 mRNA was negatively regulated by a protein kinase C- and protein synthesis-dependent system and that the system regulated both the stable and inducible expression of MDR3 mRNA.
Insights
Protein kinase C activation by PMA down-regulates MDR3 mRNA levels in liver cells. This regulation of multidrug resistance 3 (MDR3) gene expression is dependent on protein synthesis and affects both stable and inducible expression.
Area of Science:
- Hepatology
- Molecular Biology
- Cell Biology
Background:
- Multidrug resistance protein 3 (MDR3) is a P-glycoprotein homologue crucial for bile secretion.
- MDR3 localizes to the canalicular membrane in liver cells.
- The regulation of MDR3 gene expression by protein kinases is not fully understood.
Purpose of the Study:
- To investigate the role of protein kinase C (PKC) in regulating MDR3 gene expression.
- To determine the effect of phorbol 12-myristate 13-acetate (PMA), a PKC activator, on MDR3 mRNA levels.
- To elucidate the mechanisms underlying PKC-mediated regulation of MDR3 expression.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify MDR3 mRNA levels.
- Human Chang liver cells were treated with PMA and its analogues.
- PKC inhibitors (GF109203X, calphostin C) and a protein synthesis inhibitor (cycloheximide) were employed.
Main Results:
- PMA significantly decreased steady-state MDR3 mRNA levels in a dose- and time-dependent manner.
- PMA also reduced doxorubicin-induced MDR3 mRNA expression.
- The down-regulatory effect of PMA was partially blocked by PKC inhibitors and antagonized by cycloheximide.
Conclusions:
- MDR3 mRNA levels are negatively regulated by a system involving protein kinase C and protein synthesis.
- This regulatory system impacts both the basal and inducible expression of the MDR3 gene.
- PKC activation represents a key mechanism in controlling MDR3 gene expression in liver cells.