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Phorbol ester induced MDR1 expression in K562 cells occurs independently of mitogen-activated protein kinase
M T Osborn1, A Berry, M S Ruberu
1Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Abstract:
The MDR1 gene encoding the multidrug pump P-glycoprotein is transcriptionally activated in response to diverse extracellular stimuli, including the tumor promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the signal transduction pathway responsible is unknown. Downstream of protein kinase C (PKC), the effects of TPA are often mediated by the Raf-1/MEK/ERK mitogen-activated protein kinase (MAPK) cascade, and Raf-1 has been implicated in MDR1 induction by serum and mitogens. Therefore, we examined the potential role of MAPK activation in TPA-mediated MDR1 induction in human leukemia K562 cells. MDR1 mRNA expression was significantly increased by TPA in the concentration range of 4 - 100 nM, with a maximal response 5 - 10 h after TPA addition. TPA-mediated MDR1 induction was inhibited by several PKC inhibitors including staurosporine, H7 and calphostin C. TPA stimulated the subcellular translocation of PKCalpha from the cytosol to the membrane and nucleus but did not affect other PKC isozymes. TPA also activated the Raf1/MEK/ERK cascade and activated another MAPK member, p38, but not JNK. In order to determine the potential role of MAPKs in MDR1 induction by TPA, specific inhibitors were utilized. The MEK inhibitor PD 098059, as well as the PKC inhibitors, completely blocked TPA-mediated ERK activation. However, under identical conditions, MDR1 induction by TPA was completely unaffected by PD 098059. Furthermore, SB 202190, which effectively inhibited TPA-mediated p38 activation, failed to inhibit TPA-induced MDR1 mRNA expression. These data demonstrate that MDR1 induction by TPA occurs via a PKC-dependent mechanism that operates independently of ERK, p38 or JNK pathways, and thus have important implications for understanding the mechanisms of MDR1 induction by extracellular stimuli.
Insights
The study shows that tumor promoter TPA activates the MDR1 gene via protein kinase C (PKC), independent of MAPK pathways like ERK and p38. This finding clarifies MDR1 gene regulation mechanisms.
Area of Science:
- Molecular Biology
- Cell Signaling
- Cancer Research
Background:
- The MDR1 gene, encoding P-glycoprotein, is activated by stimuli like TPA.
- The specific signaling pathway for TPA-induced MDR1 activation remains unclear.
- Mitogen-activated protein kinase (MAPK) cascades, including Raf-1/MEK/ERK, are often downstream of protein kinase C (PKC).
Purpose of the Study:
- To investigate the role of MAPK activation in TPA-mediated MDR1 gene induction in human leukemia K562 cells.
- To elucidate the signal transduction pathway linking TPA to MDR1 expression.
Main Methods:
- TPA treatment of K562 cells to measure MDR1 mRNA expression.
- Use of PKC and specific MAPK inhibitors (PD 098059, SB 202190) to assess pathway involvement.
- Analysis of PKC isozyme translocation and MAPK activation (ERK, p38, JNK).
Main Results:
- TPA significantly increased MDR1 mRNA expression in a dose- and time-dependent manner.
- TPA-induced MDR1 expression was inhibited by PKC inhibitors and involved PKCalpha translocation.
- While TPA activated ERK and p38 MAPKs, inhibitors of these pathways (PD 098059, SB 202190) did not affect TPA-mediated MDR1 induction.
- MDR1 induction occurred independently of ERK, p38, or JNK activation.
Conclusions:
- TPA induces MDR1 gene expression through a PKC-dependent pathway.
- This induction mechanism operates independently of the ERK, p38, and JNK MAPK signaling cascades.
- The findings provide critical insights into the regulation of MDR1 by extracellular signals.