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TRPC1-mediated Ca²⁺ entry is essential for the regulation of hypoxia and nutrient depletion-dependent autophagy.

Sukumaran P, Sun Y, Vyas M, Singh BB - Cell Death Dis (2015)

Bottom Line: Importantly, TRPC1-mediated Ca(2+) entry resulted in increased expression of autophagic markers that prevented cell death.Silencing of TRPC1 or inhibition of autophagy by 3-methyladenine, but not TRPC3, attenuated hypoxia-induced increase in intracellular Ca(2+) influx, decreased autophagy, and increased cell death.Altogether, we provide evidence for the involvement of Ca(2+) influx via TRPC1 in regulating autophagy to protect against cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND 58201, USA.

ABSTRACT
Autophagy is a cellular catabolic process needed for the degradation and recycling of protein aggregates and damaged organelles. Although Ca(2+) is suggested to have an important role in cell survival, the ion channel(s) involved in autophagy have not been identified. Here we demonstrate that increase in intracellular Ca(2+) via transient receptor potential canonical channel-1 (TRPC1) regulates autophagy, thereby preventing cell death in two morphologically distinct cells lines. The addition of DMOG or DFO, a cell permeable hypoxia-mimetic agents, or serum starvation, induces autophagy in both epithelial and neuronal cells. The induction of autophagy increases Ca(2+) entry via the TRPC1 channel, which was inhibited by the addition of 2APB and SKF96365. Importantly, TRPC1-mediated Ca(2+) entry resulted in increased expression of autophagic markers that prevented cell death. Furthermore, hypoxia-mediated autophagy also increased TRPC1, but not STIM1 or Orai1, expression. Silencing of TRPC1 or inhibition of autophagy by 3-methyladenine, but not TRPC3, attenuated hypoxia-induced increase in intracellular Ca(2+) influx, decreased autophagy, and increased cell death. Furthermore, the primary salivary gland cells isolated from mice exposed to hypoxic conditions also showed increased expression of TRPC1 as well as increase in Ca(2+) entry along with increased expression of autophagic markers. Altogether, we provide evidence for the involvement of Ca(2+) influx via TRPC1 in regulating autophagy to protect against cell death.

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DMOG and DFO treatment effects on the cell viability or apoptosis in the cells. (a) Bar diagram showing the cell viability assay (MTT assay) in the HSG cells and (b) SHSY-5Y cells pretreated with 1 mM of both DFO and DMOG. Each bar gives the mean±S.E.M. of four separate experiments. NS indicates no significance. (c) Western blot images showing the expression of caspase 3 in SHSY-5Y and HSG cells pretreated with 1 mM DMOG and 1 mM DFO or in serum-free media for 24 h. (d) Bar diagram representing the densitometric reading of the caspase 3 expression in the above-mentioned western blots. Each bar gives the mean±S.E.M. of four separate experiments. NS indicates no significance
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fig2: DMOG and DFO treatment effects on the cell viability or apoptosis in the cells. (a) Bar diagram showing the cell viability assay (MTT assay) in the HSG cells and (b) SHSY-5Y cells pretreated with 1 mM of both DFO and DMOG. Each bar gives the mean±S.E.M. of four separate experiments. NS indicates no significance. (c) Western blot images showing the expression of caspase 3 in SHSY-5Y and HSG cells pretreated with 1 mM DMOG and 1 mM DFO or in serum-free media for 24 h. (d) Bar diagram representing the densitometric reading of the caspase 3 expression in the above-mentioned western blots. Each bar gives the mean±S.E.M. of four separate experiments. NS indicates no significance

Mentions: To confirm that hypoxia-mimetic agents, DMOG and DFO, induce autophagy that inhibits apoptosis, cell viability assays were performed. Treatment with 1 mM DMOG or 1 mM DFO showed no significant decrease in cell viability in both SHSY-5Y and HSG cells (Figures 2a and b). Moreover, the expression of caspase 3, an apoptotic marker protein, was also not altered in cells treated with DMOG or DFO or serum starvation (Figures 2c and d). Overall, these results suggest that DMOG, or DFO, or serum starvation induces autophagy in epithelial and neuronal cells that maintain cell viability perhaps by inhibiting apoptotic-mediated cell death.


TRPC1-mediated Ca²⁺ entry is essential for the regulation of hypoxia and nutrient depletion-dependent autophagy.

Sukumaran P, Sun Y, Vyas M, Singh BB - Cell Death Dis (2015)

DMOG and DFO treatment effects on the cell viability or apoptosis in the cells. (a) Bar diagram showing the cell viability assay (MTT assay) in the HSG cells and (b) SHSY-5Y cells pretreated with 1 mM of both DFO and DMOG. Each bar gives the mean±S.E.M. of four separate experiments. NS indicates no significance. (c) Western blot images showing the expression of caspase 3 in SHSY-5Y and HSG cells pretreated with 1 mM DMOG and 1 mM DFO or in serum-free media for 24 h. (d) Bar diagram representing the densitometric reading of the caspase 3 expression in the above-mentioned western blots. Each bar gives the mean±S.E.M. of four separate experiments. NS indicates no significance
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385947&req=5

fig2: DMOG and DFO treatment effects on the cell viability or apoptosis in the cells. (a) Bar diagram showing the cell viability assay (MTT assay) in the HSG cells and (b) SHSY-5Y cells pretreated with 1 mM of both DFO and DMOG. Each bar gives the mean±S.E.M. of four separate experiments. NS indicates no significance. (c) Western blot images showing the expression of caspase 3 in SHSY-5Y and HSG cells pretreated with 1 mM DMOG and 1 mM DFO or in serum-free media for 24 h. (d) Bar diagram representing the densitometric reading of the caspase 3 expression in the above-mentioned western blots. Each bar gives the mean±S.E.M. of four separate experiments. NS indicates no significance
Mentions: To confirm that hypoxia-mimetic agents, DMOG and DFO, induce autophagy that inhibits apoptosis, cell viability assays were performed. Treatment with 1 mM DMOG or 1 mM DFO showed no significant decrease in cell viability in both SHSY-5Y and HSG cells (Figures 2a and b). Moreover, the expression of caspase 3, an apoptotic marker protein, was also not altered in cells treated with DMOG or DFO or serum starvation (Figures 2c and d). Overall, these results suggest that DMOG, or DFO, or serum starvation induces autophagy in epithelial and neuronal cells that maintain cell viability perhaps by inhibiting apoptotic-mediated cell death.

Bottom Line: Importantly, TRPC1-mediated Ca(2+) entry resulted in increased expression of autophagic markers that prevented cell death.Silencing of TRPC1 or inhibition of autophagy by 3-methyladenine, but not TRPC3, attenuated hypoxia-induced increase in intracellular Ca(2+) influx, decreased autophagy, and increased cell death.Altogether, we provide evidence for the involvement of Ca(2+) influx via TRPC1 in regulating autophagy to protect against cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND 58201, USA.

ABSTRACT
Autophagy is a cellular catabolic process needed for the degradation and recycling of protein aggregates and damaged organelles. Although Ca(2+) is suggested to have an important role in cell survival, the ion channel(s) involved in autophagy have not been identified. Here we demonstrate that increase in intracellular Ca(2+) via transient receptor potential canonical channel-1 (TRPC1) regulates autophagy, thereby preventing cell death in two morphologically distinct cells lines. The addition of DMOG or DFO, a cell permeable hypoxia-mimetic agents, or serum starvation, induces autophagy in both epithelial and neuronal cells. The induction of autophagy increases Ca(2+) entry via the TRPC1 channel, which was inhibited by the addition of 2APB and SKF96365. Importantly, TRPC1-mediated Ca(2+) entry resulted in increased expression of autophagic markers that prevented cell death. Furthermore, hypoxia-mediated autophagy also increased TRPC1, but not STIM1 or Orai1, expression. Silencing of TRPC1 or inhibition of autophagy by 3-methyladenine, but not TRPC3, attenuated hypoxia-induced increase in intracellular Ca(2+) influx, decreased autophagy, and increased cell death. Furthermore, the primary salivary gland cells isolated from mice exposed to hypoxic conditions also showed increased expression of TRPC1 as well as increase in Ca(2+) entry along with increased expression of autophagic markers. Altogether, we provide evidence for the involvement of Ca(2+) influx via TRPC1 in regulating autophagy to protect against cell death.

Show MeSH
Related in: MedlinePlus