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Distinct transcriptional regulation of human large conductance voltage- and calcium-activated K+ channel gene (hSlo1) by activated estrogen receptor alpha and c-Src tyrosine kinase.

ABSTRACT: Estrogen receptor ? (ER?) regulates gene transcription via "genomic" (binding directly or indirectly, typically via Sp1 or AP-1 sites, to target genes) and/or "nongenomic" (signaling) mechanisms. ER? activation by estrogen up-regulates the murine Ca(2+)-activated K(+) channel ? subunit gene (mSlo1) via genomic mechanisms. Here, we investigated whether ER? also drives transcription of the human (hSlo1) gene. Consistent with this view, estrogen increased hSlo1 transcript levels in primary human smooth muscle cells. Promoter studies revealed that estrogen/hER?-mediated hSlo1 transcription was nearly 6-fold more efficient than for mSlo1 (EC(50), 0.07 versus 0.4 nM). Unlike the genomic transcriptional mechanism employed by mSlo1, hSlo1 exhibits a nongenomic hER?-mediated regulatory mechanism. This is supported by the following: 1) efficient hSlo1 transcription after disruption of the DNA-binding domain of hER? or knockdown of Sp1, and 2) lack of AP-1 sites in the hSlo1 promoter. Three nongenomic signaling pathways were explored: Src, Rho, and PI3K. Inhibition of Src with 10 ?M PP2, and reported downstream ERK with 25 ?M PD98059 did not prevent estrogen action but caused an increase in hSlo1 basal transcription; conversely, constitutively active c-Src (Y527F) decreased hSlo1 basal transcription even preventing its estrogen/hER?-mediated transcriptional activation. Rho inhibition by coexpressed Clostridium botulinum C3 transferase did not alter estrogen action. In contrast, inhibition of PI3K activity with 10 ?M LY294002 decreased estrogen-stimulated hSlo1 transcription by ?40%. These results indicate that the nongenomic PI3K signaling pathway plays a role in estrogen/hER?-stimulated hSlo1 gene expression; whereas c-Src activity leads to hSlo1 gene tonic repression independently of estrogen, likely through ERK activation.

SUBMITTER: Danesh SM

PROVIDER: S-EPMC3173123 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Distinct transcriptional regulation of human large conductance voltage- and calcium-activated K+ channel gene (hSlo1) by activated estrogen receptor alpha and c-Src tyrosine kinase.

Danesh Shahab M SM Kundu Pallob P Lu Rong R Stefani Enrico E Toro Ligia L

The Journal of biological chemistry 20110711 36

Estrogen receptor α (ERα) regulates gene transcription via "genomic" (binding directly or indirectly, typically via Sp1 or AP-1 sites, to target genes) and/or "nongenomic" (signaling) mechanisms. ERα activation by estrogen up-regulates the murine Ca(2+)-activated K(+) channel α subunit gene (mSlo1) via genomic mechanisms. Here, we investigated whether ERα also drives transcription of the human (hSlo1) gene. Consistent with this view, estrogen increased hSlo1 transcript levels in primary human sm ...[more]

PMID: 21757754

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Project description:Large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channels hyperpolarize coronary artery smooth muscle cells, causing vasorelaxation. Dopamine activates BK(Ca) channels by stimulating D(1)-like receptor-mediated increases in cAMP in porcine coronary artery myocytes. There are two D(1)-like receptors (R), D(1)R and D(5)R. We hypothesize that the specific D(1)-like receptor involved in BK(Ca) channel activation in human coronary artery smooth muscle cells (HCASMCs) is the D(5)R and that activation occurs via cAMP cross-activation of cGMP-dependent protein kinase (PKG), rather than cAMP-dependent protein kinase (PKA). The effects of D(1)-like receptor agonists and antagonists on BK(Ca) channel opening in HCASMCs were examined in the presence and absence of PKG/PKA inhibition by cell-attached patch clamp. In the absence of commercially available ligands specific for D(1)R or D(5)R, D(1)R or D(5)R protein was down-regulated by transfecting HCASMCs with human D(1)R or D(5)R antisense oligonucleotides, respectively: cells transfected with scrambled oligonucleotides and nontransfected HCASMCs served as controls. The predominant ion channel conducting outward currents in nontransfected HCASMCs was identified as the large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel, which was activated by D(1)-like receptor agonists despite PKA inhibition with (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid (KT 5720) (300 nM), but was abolished by inhibiting PKG with 9-methoxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b-11a-triazadibenzo(a,g) cycloocta(cde)-trinden-1-one (KT 5823) (300 nM). D(1)-like receptor agonists activated BK(Ca) channels in all transfected cells except those transfected with D(5)R antisense oligonucleotides. Thus, the dopamine (D(1)-like) receptor mediates activation of BK(Ca) channels in HCASMCs by D(5)R, not D(1)R, and via PKG, not PKA. This is the first report of differential D(1)-like receptor regulation of vascular smooth muscle function in human cells.

Dataset Information

Distinct transcriptional regulation of human large conductance voltage- and calcium-activated K+ channel gene (hSlo1) by activated estrogen receptor alpha and c-Src tyrosine kinase.

Publications

Distinct transcriptional regulation of human large conductance voltage- and calcium-activated K+ channel gene (hSlo1) by activated estrogen receptor alpha and c-Src tyrosine kinase.

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