Mitogenic modulation of Ca2+ -activated K+ channels in proliferating A7r5 vascular smooth muscle cells.
Modulation of Ca(2+)-activated K(+) channels (K(Ca)) has been implicated in the control of proliferation in vascular smooth muscle cells (VSMC) and other cell types. In the present study, we investigated the underlying signal transduction mechanisms leading to mitogen-induced alterations in the expression pattern of intermediate-conductance K(Ca) in VSMC. Regulation of expression of IK(Ca)/rK(Ca)3.1 and BK(Ca)/rK(Ca)1.1 in A7r5 cells, a cell line derived from rat aortic VSMC, was investigated by patch-clamp technique, quantitative RT-PCR, immunoblotting procedures, and siRNA strategy.PDGF stimulation for 2 and 48 h induced an 11- and 3.5-fold increase in rK(Ca)3.1 transcript levels resulting in a four- and seven-fold increase in IK(Ca) currents after 4 and 48 h, respectively. Upregulation of rK(Ca)3.1 transcript levels and channel function required phosphorylation of extracellular signal-regulated kinases (ERK1/2) and Ca(2+) mobilization, but not activation of p38-MAP kinase, c-Jun NH(2)-terminal kinase, protein kinase C, calcium-calmodulin kinase II and Src kinases. In contrast to rK(Ca)3.1, mRNA expression and functions of BK(Ca)/rK(Ca)1.1 were decreased by half following mitogenic stimulation. Downregulation of rK(Ca)1.1 did not require ERK1/2 phosphorylation or Ca(2+) mobilization. In an in vitro-proliferation assay, knockdown of rK(Ca)3.1 expression by siRNA completely abolished functional IK(Ca) channels and mitogenesis. Mitogen-induced upregulation of rK(Ca)3.1 expression is mediated via activation of the Raf/MEK- and ERK-signaling cascade in a Ca(2+)-dependent manner. Upregulation of rK(Ca)3.1 promotes VSMC proliferation and may thus represent a pharmacological target in cardiovascular disease states characterized by abnormal cell proliferation.
Si
Han
H
Department of Internal Medicine-Nephrology, Philipps-Universität, Marburg 35033, Germany.
Grgic
Ivica
I
Heyken
Willm-Thomas
WT
Maier
Tanja
T
Hoyer
Joachim
J
Reusch
Hans-Peter
HP
Köhler
Ralf
R
D015153:Blotting, Western; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D020868:Gene Silencing / Q000502:physiology; D006801:Humans; D008934:Mitogens / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000235:genetics; D024681:Potassium Channels, Calcium-Activated / Q000187:drug effects* / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf / Q000502:physiology; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D015854:Up-Regulation / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D008934:Mitogens; D010982:Platelet-Derived Growth Factor; D024681:Potassium Channels, Calcium-Activated; D034741:RNA, Small Interfering; D012313:RNA; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases