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Format:
Print
Author:
Pulver-Kaste, Renee Alissa
Dept./Program:
Pharmacology
Year:
2005
Degree:
PhD
Abstract:
Altered Ca²⁺ handling has detrimental effects on vascular smooth muscle function and transcriptional regulation leading to arterial pathologies. Ca²⁺/cAMP response element binding protein (CREB), a transcription factor activated by Ca²⁺ -induced phosphorylation, regulates expression of many genes. Multiple Ca2+ entry pathways may contribute to CREB activation and provide a mechanism for differential induction of gene transcription in vascular smooth muscle. Previously we have shown that elevation of cytoplasmic Ca²⁺ through voltage-dependent Ca²⁺ channels (VOCCs) results in phosphorylation of CREB in cerebral arteries. To investigate a role for store-operated Ca²⁺ entry (SOCE) in CREB activation, we measured CREB phosphorylation using immunofluorescence after depletion of intracellular Ca²⁺ stores with thapsigargin. We report that SOCE increased nuclear phospho-CREB levels in both rat cultured smooth muscle cells and intact arteries. To determine whether Ca²⁺ elevation induced by distinct stimuli can differentially regulate gene transcription, we measured changes in RNA expression levels in human cerebral vascular smooth muscle cells using oligonucleotide array analysis after depletion of intracellular Ca²⁺ stores with thapsigargin and membrane depolarization with potassium. Array results for selected CRE-regulated genes were confirmed by quantitative RT-PCR and corresponding changes in protein expression. We report that the stimulation of SOCE and Ca²⁺ influx through VDCCs results in transcriptional activation of a distinct, yet overlapping set of genes, and that the induction of selected CRE-regulated genes is prevented by the addition of corresponding Ca²⁺ channel blockers. These data indicate that SOCE and Ca²⁺ entry through VOCCs can differentially regulate gene expression patterns, suggesting a novel mechanism by which the temporal and spatial distribution of Ca²⁺ can modulate proliferation in vascular smooth muscle.