Transcriptional regulation of α1H T-type calcium channel under hypoxia

Abstract

The low-voltage-activated T-type Ca(2+) 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.

Keywords: T-type calcium channel; gene expression; hypoxia; hypoxia-response element.

Fig. 1.

Selective upregulation of α_1H under hypoxia. A: rat pulmonary artery smooth muscle cells (PASMCs), pulmonary microvascular endothelial cells (PMVECs), PC12 cells, and A7r5 cells were subjected to hypoxia (3% O₂) or maintained under normoxia (21% O₂) for 12 h. RT-PCR amplification was performed using total RNA isolated from each of the cells at the end of the exposure with the primer sets indicated in materials and methods. B: immunofluorescence labeling of α_1G and α_1H in PASMCs and PC12 cells. Representative projected experimental images of all parts of z-stacks taken from apical to basal cell aspects at 0.2-μm intervals. Immunofluorescence labeling was performed with an anti-α_1G or anti-α_1H antibody followed by an Alexa Fluor 488- or 594-conjugated secondary antibody (green or red) and nuclei visualized with DAPI (blue). Note the increase in Alexa Fluor 488 and 594 fluorescence in PC12 cells and PASMCs after 48-h exposure to hypoxia (3% O₂) (denoted by arrows or arrowheads). Images acquired at 21°C on a Nikon A1 confocal laser microscope with a ×60, 1.20 numerical aperture water immersion objective (replicated 3 times).

Fig. 2.

Functional expression of the α_1H T-type Ca²⁺ channel is elevated under hypoxia. A: representative deactivating tail current traces from PASMCs and PC12 cells after 24-h or 48-h exposure to hypoxia (3% O₂) (gray, ■) or under normoxia (black, □), elicited by repolarizing the cell to −120 mV following a short depolarizing pulse at +80 mV (3.25 ms) from a holding potential of −90 mV, as schematically illustrated. B: scatter plot of individual data points of maximally elicited T-type tail currents (I_tail) from PASMCs and PC12 cells after 24-h or 48-h exposure to hypoxia or under normoxia (hypoxia: ■, normoxia: □; median with range: red lines). Note I_tail median in PASMCs after hypoxia: −7.1 pA/pF vs. −2.3 pA/pF under normoxia at 24 h (n = 12 each, P < 0.05), −3.9 pA/pF vs. −2.2 pA/pF under normoxia at 48 h (n = 20 and 22, P < 0.001); in PC12 cells after hypoxia: 25.9 pA/pF vs. −16.3 pA/pF under normoxia at 24 h (n = 15 each, P < 0.05), −34.5 pA/pF vs. −18.4 pA/pF under normoxia at 48 h (n = 15 each, P < 0.05). C and D: comparison of the T-type currents in PASMCs after 24-h exposure to hypoxia (3% O₂) to those in PASMCs maintained under normoxia (21% O₂) for the same period of time (hypoxia: ■, normoxia: □). C: representative macroscopic current traces recorded a depolarization to −30 mV from a holding potential of −90 mV, as schematically illustrated. D: current-voltage (I–V) relationships of peak currents recorded by step depolarizations to varying test potentials (−70 to +50 mV with 10-mV increments) from a holding potential of −90 mV. Data represent the means ± SE, n = 12 each. Ca²⁺ (10 mmol/l) was used as a charge carrier. Nifedipine (10 μmol/l) was present in the extracellular solution. Temperature was 22–25°C.

Fig. 3.

The α_1G promoter activity was unchanged under hypoxia. Serial deletion constructs of the 5′ flaking region of α_1G were generated as described in materials and methods. PC12 cells were transiently cotransfected with different α_1G serial deletion constructs (250 ng) and 50 ng of β-galactosidase using Fugene 6 HD (1 μl) as transfecting reagent. Cells were then exposed to hypoxia (3% O₂) (H) or normoxia (21% O₂) (N) for 48 h. Luciferase assays were performed as described in materials and methods, and values were normalized to β-galactosidase serving as internal control for transfection efficiency and expressed as relative light units (RLU). Representative of 3 independent experiments performed in triplicate. Left: potential hypoxia-response elements (HRE) on the 2,000-bp 5′ franking sequence of α_1G: −1901gcgtg−1895 and −73gcgtg−69 on forward strand.

Fig. 4.

Hypoxia increases α_1H promoter activity. PC12 cells were transiently cotransfected with different α_1H serial deletion constructs (250 ng) and β-galactosidase (50 ng). Cells were then exposed to hypoxia (3% O₂) for 48 h. Luciferase assays were performed as described in materials and methods. The luciferase values were normalized to β-galactosidase activity, which served as internal control for transfection efficiency. Representative of 3 independent experiments performed in triplicate. *P < 0.05, **P < 0.005. Left: potential HRE on the 3,076-bp 5′-flanking sequence of α_1H: −3,025acgtg−3,021, −2,933acgtg−2,929, and −1,090gcgtg−1,086 on forward strand; −3,026cacgt−3,022, −2,934cacgt−2,930, −2,315cacgc−2,311, −1,182cacgt−1,178, −1,173cacgc−1,169, −1,132cacgc−1,128, −1,114cacgc−1,110, −1,079cacgc−1,075, and −1,079cacgc−1,075 on reverse strand. Arrow denotes the HRE site responsive to hypoxia.

Fig. 5.

HRE-5 within α_1H promoter mediates hypoxia response. A: hypoxia increases hypoxia-inducible factor (HIF)-1α binding to HRE-5. PC12 cells and PASMCs were subjected to hypoxia (3% O₂) for 6 h. Nuclear protein extracts were prepared as described in materials and methods and incubated with different radio-labeled probes consensus sequence (I), HRE-5 (II) within α_1H promoter, mutated HRE-5 (III) for 30 min before resolving DNA-protein complex on gel. In the immune competition assay, the anti-HIF-1α antibody was incubated for 30 min with nuclear extracts prepared from PC12 subjected to hypoxia (3% O₂ for 6 h) before addition of the probe. F0, free probe; N, normoxia (21% O₂₎; H, hypoxia (3% O₂). B: HRE-5 mutation abolishes hypoxia response. Site-directed mutagenesis was performed as described in materials and methods to generate constructs F1 and F5 harboring mutated HRE-5 named mF1 and mF5, respectively. PC12 cells were cotransfected with wild-type (F1, F5) or mutated HRE-5 (mF1, mF5) luciferase reporter constructs (250 ng) and β-galactosidase (50 ng). Cells were then exposed to hypoxia (3% O₂ for 24 h). The luciferase values (RLU) were normalized to β-galactosidase activity serving as internal control. Representative of 3 independent experiments performed in triplicate. CACGC, wild-type HRE-5; CAAAC, mutated HRE; F0, empty vector. *P < 0.05, **P < 0.005.