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pp90RSK- and protein kinase C-dependent pathway regulates p42/44MAPK-induced LDL receptor transcription in HepG2
Gurpreet S Kapoor1, Carmen Golden, Brett Atkins
1Department of Molecular and Cellular Biochemistry, The Ohio State University College of Medicine and Public Health, 464 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210, USA. mehta.80@osu.edu
Abstract:
We have previously shown that different extracellular stimuli require signaling through the Raf/MEK/p42/44MAPK cascade to induce LDL receptor expression. The present studies were designed to delineate the molecular mechanisms underlying p42/44MAPK-induced LDL receptor transcription in HepG2-Delta Raf-1:ER cells, a modified HepG2 cell line in which the Raf-1/MEK/p42/44MAPK cascade can be specifically activated by anti-estradiol ICI182,780 in an agonist-specific manner. Using these cells, we show that: a) LDL receptor induction was reduced in reporter constructs containing mutation in either Sp1 or sterol-regulatory element-1 (SRE-1) sites, whereas inactivation of both sites abolished the induction; b) E1A, which inhibits CREB binding protein (CBP), a common activator of SRE-1 binding protein and Sp1, strongly repressed the induction; c) intracellular inhibition of the 90 kDa ribosomal S6 kinase (pp90RSK) cascade reduced LDL receptor induction; d) highly selective protein kinase C (PKC) inhibitors effectively abrogated the induction without affecting activation of pp90RSK; and e) overexpression of PKC beta significantly induced LDL receptor promoter activity. Taken together, these results demonstrate that pp90RSK and PKC beta are downstream effectors and Sp1, SRE-1 binding protein, and CBP are part of the transcriptional complex resulting in induction of LDL receptor expression in response to activation of the Raf/MEK/p42/44MAPK cascade. These findings identify for the first time a role for PKC beta in determining the specificity of p42/44MAPK signaling by participating with pp90RSK in regulating gene expression.
Insights
This study reveals how the Raf/MEK/p42/44MAPK pathway regulates LDL receptor expression. It identifies protein kinase C beta (PKC beta) and pp90RSK as key downstream effectors in this process.
Area of Science:
- Molecular Biology
- Cell Signaling
- Gene Regulation
Background:
- Extracellular stimuli activate the Raf/MEK/p42/44MAPK cascade to induce LDL receptor expression.
- HepG2-Delta Raf-1:ER cells allow specific activation of this cascade for mechanistic studies.
Purpose of the Study:
- To delineate the molecular mechanisms of p42/44MAPK-induced LDL receptor transcription.
- To identify downstream effectors and transcriptional complex components involved in LDL receptor gene regulation.
Main Methods:
- Utilized reporter constructs with mutations in Sp1 and sterol-regulatory element-1 (SRE-1) sites.
- Employed E1A to inhibit CREB binding protein (CBP) and pp90RSK inhibitors.
- Investigated the role of protein kinase C (PKC) using selective inhibitors and overexpression of PKC beta.
Main Results:
- Mutations in Sp1 or SRE-1 sites reduced, while inactivation of both abolished, LDL receptor induction.
- E1A strongly repressed induction, indicating CBP's role.
- Inhibition of pp90RSK reduced induction; PKC inhibitors abrogated induction without affecting pp90RSK activation.
- Overexpression of PKC beta significantly induced LDL receptor promoter activity.
Conclusions:
- pp90RSK and PKC beta are downstream effectors of the Raf/MEK/p42/44MAPK cascade in LDL receptor gene induction.
- Sp1, SRE-1 binding protein, and CBP form part of the transcriptional complex.
- PKC beta plays a novel role in specifying p42/44MAPK signaling by collaborating with pp90RSK in gene regulation.