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NF-kappaB-mediated induction of mdr1b expression by insulin in rat hepatoma cells
1Department of Molecular Pathology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Abstract:
The expression of P-glycoproteins encoded by the mdr gene family is associated with the emergence of multidrug resistance phenotype in animal cells. However, the mechanisms controlling the expression of these genes have not been well elucidated. Here, we report that the expression of rat mdr1b gene in cultured H-4-II-E hepatoma cells can be induced by insulin. Transient transfection assays using reporter gene constructs containing various 5' mdr1b sequences showed that the sequence located between base pairs -243 and -163 is important for insulin's induction of mdr1b promoter activity. Further analyses revealed that a NF-kappaB-binding site (located between base pairs -167 and -158) is required for insulin-induced promoter activity. Gel mobility shift assay demonstrated that insulin stimulates the binding of nuclear p50/p65 subunits to the mdr1b NF-kappaB sequence. Cotransfection of plasmids expressing either the p50/p65 NF-kappaB subunits or Raf-1 kinase or both resulted in increased expression of the gene containing wild-type but not NF-kappaB site-mutated mdr1b promoter. Finally, expression of either the antisense p65 subunit of NF-kappaB or dominant negative Raf-1 kinase blocked insulin's induction of the mdr1b promoter activity. Taken together, our results suggest that the insulin-induced mdr1b expression is mediated by transcription factor NF-kappaB via the Raf-1 kinase signaling pathway.
Insights
Insulin induces the expression of the rat mdr1b gene in hepatoma cells. This process involves the transcription factor nuclear factor-kappa B (NF-kappaB) and the Raf-1 kinase signaling pathway.
Area of Science:
- Molecular biology
- Cellular signaling
- Gene expression regulation
Background:
- P-glycoprotein expression, encoded by the mdr gene family, is linked to multidrug resistance in animal cells.
- Mechanisms controlling mdr gene expression remain incompletely understood.
- Insulin's role in regulating mdr gene expression has not been previously established.
Purpose of the Study:
- To elucidate the molecular mechanisms by which insulin induces the expression of the rat mdr1b gene in cultured hepatoma cells.
- To identify key regulatory elements and signaling pathways involved in insulin-mediated mdr1b gene induction.
Main Methods:
- Transient transfection assays with reporter gene constructs containing mdr1b promoter sequences.
- Site-directed mutagenesis to investigate the role of the NF-kappaB binding site.
- Gel mobility shift assays to assess protein-DNA interactions.
- Cotransfection experiments with plasmids expressing NF-kappaB subunits and Raf-1 kinase.
- Functional assays using antisense oligonucleotides and dominant-negative constructs.
Main Results:
- Insulin induction of mdr1b promoter activity requires a specific DNA sequence between base pairs -243 and -163.
- A nuclear factor-kappa B (NF-kappaB) binding site within this region is essential for insulin's effect.
- Insulin stimulates the binding of p50/p65 NF-kappaB subunits to the mdr1b promoter.
- Overexpression of NF-kappaB subunits or Raf-1 kinase enhances mdr1b promoter activity.
- Inhibition of NF-kappaB or Raf-1 signaling blocks insulin-induced mdr1b expression.
Conclusions:
- Insulin-induced mdr1b gene expression in hepatoma cells is mediated by the transcription factor NF-kappaB.
- The Raf-1 kinase signaling pathway plays a crucial role in this induction process.
- These findings provide novel insights into the regulation of multidrug resistance genes by hormonal signals.