Chemically mimicked hypoxia modulates gene expression and protein levels of the sodium calcium exchanger in HEK 293 cell line via HIF-1α

Abstract

Up to now a little is known about the effect of hypoxia on the sodium calcium exchanger type 1 (NCX1) expression and function. Therefore, we studied how dimethyloxallyl glycine (DMOG), an activator and stabilizer of the hypoxia-inducible factor (HIF)-1α, could affect expression of the NCX1 in HEK 293 cell line. We also tried to determine whether this activation can result in the induction of apoptosis in HEK 293 cells. We have found that DMOG treatment for 3 hours significantly increased gene expression and also protein levels of the NCX1. This increase was accompanied by a decrease in intracellular pH. Wash-out of DMOG did not result in reduction of the NCX1 mRNA and protein to original - control levels, although pH returned to physiological values. Using luciferase reporter assay we observed increase in the NCX1 promoter activity after DMOG treatment and using wild-type mouse embryonic fibroblast (MEF)-HIF-1(+/+) and HIF-1-deficient MEF-HIF-1(-/-) cells we have clearly shown that in the promoter region, HIF-1α is involved in DMOG induced upregulation of the NCX1. Moreover, we also showed that an increase in the NCX1 mRNA due to the apoptosis induction is not regulated by HIF-1α.

Sulphide signalling potentiates apoptosis through the up‐regulation of IP3 receptor types 1 and 2

“…HeLa cells were grown in 6-well plates in DMEM with 10% FBS to 1 9 10 5 . Transfection of siRNAs was performed with DharmaFECT1 (Dharmacon, Thermo Scientific) as described in Hudecova et al (2011). ON-TARGET plus SMART pool ITPR1 and ON-TARGET plus SMART pool ITPR2 (Dharmacon, Thermo Scientific) were applied to the final concentration of 100 pmol per well for 48 h. The same procedure was performed with ON-TARGET plus Non-targeting Pool, which serves for the determination of baseline cellular responses in RNAi experiments.…”

Melatonin-Induced Changes in Cytosolic Calcium Might be Responsible for Apoptosis Induction in Tumour Cells

“…Transfection of siRNAs was performed with DharmaFECT1 transfection reagent (Dharmacon, Thermo Scientific, USA) as described previously Hudecova et al [40]. ONTARGET plus SMART pool human ITPR1 and NCX1 siRNAs (Dharmacon, Thermo Scientific, USA) were applied to the final concentration of 100 pmol per well for 48 h. The same procedure was performed with ON-TARGET plus Non-targeting Pool, which serves for the determination of baseline cellular responses in RNAi experiments.…”

Hyperglycemia Causes Renal Cell Damage via CCN2-Induced Activation of the TrkA Receptor

“…2 A ), but not in the presence of serum unless the cells are otherwise challenged. HK2 cells, grown in 5% serum, were exposed to DMOG, a cell-permeable, competitive inhibitor of HIF-α-prolyl hydroxylase (a stabilizer of HIF1) (34), or DFO, an iron-chelator shown to induce HIF accumulation (35), or DMSO as a negative control. Accumulation of HIF1α with DMOG or DFO led to a significant increase of TrkA protein observed by Western blotting (Fig.…”