CI--Channel Is Essential for LDL-induced Cell Proliferation via the Activation of Erk1/2 and PI3K/Akt and the Upregulation of Egr-1 in Human Aortic Smooth Muscle Cells
Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea. Molecules and Cells
(Impact Factor: 2.09).
Low-density lipoprotein (LDL) induces cell proliferation in human aortic smooth muscle cells (hAoSMCs), which may be involved in atherogenesis and intimal hyperplasia. Recent studies have demonstrated that Cl- channels are related to vessel cell proliferation induced by a variety of stimuli. In this study, we investigated a potential role of Cl-channels in the signaling pathway of LDL effects on hAoSMC proliferation with a focus on the activation of Erk1/2-PI3K/Akt and the subsequent upregulation of Egr-1. Cl- channel blockers, DIDS, but neither NPPB nor Furosemide, completely abolished the LDL-induced DNA synthesis and cell proliferation. Moreover, DIDS, but not NPPB, significantly decreased LDL-stimulated Cl- concentration, as judged by flow cytometry analysis using MQAE as a Cl- -detection dye. DIDS pretreatment completely abolished the activation of Erk1/2 and PI3K/Akt in a dose-dependent manner that is the hallmark of LDL activation, as judged by Western blot and proliferation assays. Moreover, pretreatment with DIDS (Cl- channel blockers) but not LY294002 (PI3K inhibitors) completely abolished the LDL-induced upregulation of Egr-1 to the same extent as PD98059 (MEK inhibitors to inhibit Erk), as judged by Western blot and luciferase reporter assays. This is the first report, to our knowledge, that DIDS-sensitive Cl- channels play a key role in the LDL-induced cell proliferation of hAoSMCs via the activation of Erk1/2 and PI3K/Akt and the upregulation of Egr-1.
Available from: Songhee Jeon
- "Relatively well-known physiological roles of the anion channels include neurotransmission (GABA), epithelial secretion (CFTR, CaCC), and cell volume regulation (VRAC). Apart from these, it is generally thought that anion channels are involved in the regulation of cellular excitability (Wang et al., 2006), proliferation (Heo et al., 2008), apoptosis (Zhang et al., 2006), and differentiation (Yin et al., 2008). "
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ABSTRACT: We have previously shown that in PC12 cells: (1) high extracellular KCl induces moesin phosphorylation, an event which was dependent on chloride channel activation, and (2) NGF induces moesin phosphorylation which is required for neurite outgrowth. These results suggest that NGF-induced intracellular signaling and neurite outgrowth is also mediated by activation of anion channels. Using a patch-clamp technique, we found that NGF treatment increased anionic conductance in PC12 cells, an effect which was completely blocked by NPPB, a chloride channel inhibitor. Also, the NGF-induced moesin phosphorylation was suppressed by NPPB. Additionally, NPPB and SITS, another chloride channel blocker, suppressed NGF-induced TrkA phosphorylation and subsequent PI3K/Akt phosphorylation and Rac1 activation in PC12 cells. Moreover, the chloride channel inhibitors also suppressed the neurite outgrowth and decreased the cell viability in response to long-term treatment of NGF. In summary, our results suggest that chloride ion flux plays an important role in TrkA-mediated signaling pathway during NGF-induced differentiation of PC12 cells.
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We engineered E6.1 Jurkat cells to express CD44. Importantly, these cells lack endogenous CD44 expression. Molecular pathways involved with cell proliferation were studied using RT(2)-PCR array, siRNA, Western blotting and by employing pharmacological inhibitors of ERK1/2, p38 and the PI3K/Akt pathways.
We found that CD44 expression significantly inhibited cell proliferation and down-regulated EGR-1 expression and EGR-1 targets cyclin D1 and cyclin D2. Transfection of control E6.1 Jurkat cells with EGR-1 siRNA also inhibited cell proliferation, confirming its role. Disruption of the PI3K/Akt pathway with pharmacological inhibitors reduced both EGR-1 expression and cell proliferation, recapitulating the properties of CD44 expressing cells. Akt was hypophosphorylated in cells expressing CD44 showing its potential role in negatively regulating Akt activation. Strikingly, constitutively active Akt rescued the proliferation defect showing requirement for active Akt, in our system.
Our results suggest a novel pathway by which CD44 inactivates Akt, down-regulates EGR-1 expression and inhibits cell proliferation.
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