Transcriptional regulation of α1H T-type calcium channel under hypoxia

Hassan Sellak, Chun Zhou, Bainan Liu, Hairu Chen, Thomas M. Lincoln, Songwei Wu

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

The low-voltage- activated T-type Ca2+ channels play an important role in mediating the cellular responses to altered oxygen tension. Among three T-type channel isoforms, α1G, α1H, and α1I, only α1H was found to be upregulated under hypoxia. However, mechanisms underlying such hypoxia-dependent isoform-specific gene regulation remain incompletely understood. We, therefore, studied the hypoxia-dependent transcriptional regulation of α1G and α1H gene promoters with the aim to identify the functional hypoxia-response elements (HREs). In rat pulmonary artery smooth muscle cells (PASMCs) and pheochromocytoma (PC12) cells after hypoxia (3% O2) exposure, we observed a prominent increase in α1H mRNA at 12 h along with a significant rise in α1H-mediated T-type current at 24 and 48 h. We then cloned two promoter fragments from the 5'-flanking regions of rat α1G and α1H gene, 2,000 and 3,076 bp, respectively, and inserted these fragments into a luciferase reporter vector. Transient transfection of PASMCs and PC12 cells with these recombinant constructs and subsequent luciferase assay revealed a significant increase in luciferase activity from the reporter containing the α1H, but not α1G, promoter fragment under hypoxia. Using serial deletion and point mutation analysis strategies, we identified a functional HRE at site 1,173CACGC 1,169 within the α1H promoter region. Furthermore, an electrophoretic mobility shift assay using this site as a DNA probe demonstrated an increased binding activity to nuclear protein extracts from the cells after hypoxia exposure. Taken together, these findings indicate that hypoxia-induced α1H upregulation involves binding of hypoxia-inducible factor to an HRE within the α1H promoter region.

Original languageEnglish (US)
Pages (from-to)C648-C656
JournalAmerican Journal of Physiology - Cell Physiology
Volume307
Issue number7
DOIs
StatePublished - Oct 1 2014

Fingerprint

T-Type Calcium Channels
Response Elements
Luciferases
Cell Hypoxia
PC12 Cells
Genetic Promoter Regions
Pulmonary Artery
Smooth Muscle Myocytes
Protein Isoforms
Genes
Hypoxia
Sequence Deletion
5' Flanking Region
DNA Probes
Pheochromocytoma
Electrophoretic Mobility Shift Assay
Nuclear Proteins
Point Mutation
Transfection
Up-Regulation

Keywords

  • Gene expression
  • Hypoxia
  • Hypoxia-response element
  • T-type calcium channel

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

Cite this

Transcriptional regulation of α1H T-type calcium channel under hypoxia. / Sellak, Hassan; Zhou, Chun; Liu, Bainan; Chen, Hairu; Lincoln, Thomas M.; Wu, Songwei.

In: American Journal of Physiology - Cell Physiology, Vol. 307, No. 7, 01.10.2014, p. C648-C656.

Research output: Contribution to journalArticle

Sellak, Hassan ; Zhou, Chun ; Liu, Bainan ; Chen, Hairu ; Lincoln, Thomas M. ; Wu, Songwei. / Transcriptional regulation of α1H T-type calcium channel under hypoxia. In: American Journal of Physiology - Cell Physiology. 2014 ; Vol. 307, No. 7. pp. C648-C656.
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