Limits...
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.

Show MeSH

Related in: MedlinePlus

TRPC channel inhibitors attenuate the increase intracellular calcium in autophagy induces cells. (a) Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium in the presence of 50 μM 2APB to SHSY-5Y cells pretreated with 1 mM DMOG. Bar diagram shows the [Ca2+]i in nM concentration of the above-mentioned experiment. Each bar gives the mean±S.E.M. of 50 separate experiments. e **P<0.01 and ***P<0.001. (b) Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium in the presence of 10 μM SKF to SHSY-5Y cells pretreated with 1 mM DMOG. Bar diagram shows the [Ca2+]i in nM concentration of the above-mentioned experiment. Each bar gives the mean±S.E.M. of 50 separate experiments. **P<0.01 and ***P<0.001. (c) Bar diagram showing the cell viability assay (MTT assay) in the SHSY-5Y cells, pretreated with 1 mM DMOG in the presence of 10 μM SKF. Each bar gives the mean±S.E.M. of four separate experiments. **P<0.01 and ***P<0.001. (d) Western blot images showing the expression of LC3A, caspase 3 in SHSY-5Y cells pretreated with 1 mM DMOG and 10 μM SKF. Confocal image of HSG (e) and SH-SY5Y (f) cells transfected with GFP-LC3 and treated with 200 μM DFO or 200 μM DMOG and 10 μM SKF
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4385947&req=5

fig4: TRPC channel inhibitors attenuate the increase intracellular calcium in autophagy induces cells. (a) Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium in the presence of 50 μM 2APB to SHSY-5Y cells pretreated with 1 mM DMOG. Bar diagram shows the [Ca2+]i in nM concentration of the above-mentioned experiment. Each bar gives the mean±S.E.M. of 50 separate experiments. e **P<0.01 and ***P<0.001. (b) Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium in the presence of 10 μM SKF to SHSY-5Y cells pretreated with 1 mM DMOG. Bar diagram shows the [Ca2+]i in nM concentration of the above-mentioned experiment. Each bar gives the mean±S.E.M. of 50 separate experiments. **P<0.01 and ***P<0.001. (c) Bar diagram showing the cell viability assay (MTT assay) in the SHSY-5Y cells, pretreated with 1 mM DMOG in the presence of 10 μM SKF. Each bar gives the mean±S.E.M. of four separate experiments. **P<0.01 and ***P<0.001. (d) Western blot images showing the expression of LC3A, caspase 3 in SHSY-5Y cells pretreated with 1 mM DMOG and 10 μM SKF. Confocal image of HSG (e) and SH-SY5Y (f) cells transfected with GFP-LC3 and treated with 200 μM DFO or 200 μM DMOG and 10 μM SKF

Mentions: 2-Aminoethoxydiphenyl borate (2APB) and SKF96365 hydrochloride (SKF) are potent TRPC channel inhibitors;12 we thus investigated their role in autophagy. Importantly, in both SHSY-5Y and HSG cells, the increase in [Ca2+]i observed with DMOG treatment was attenuated in the presence of 2APB (Figure 4a) and SKF (Figure 4b). Similar results were also obtained with DFO or serum starvation where hypoxic-mediated increase in Ca2+ entry was decreased in cells treated with 2APB or SKF (data not shown). The cell viability was also affected when cells were pretreated with DMOG or DFO in the presence of TRPC channel inhibitor SKF, where a significant decrease in cell survival was observed (Figure 4c). Pretreatment of HSG cells with 1 mM DMOG in the presence of SKF, also resulted in a loss of autophagy, where no increase in LC3A expression was observed (Figure 4d). Moreover, an increase in apoptosis marker caspase 3 was observed in HSG cells pretreated with DMOG and TRPC channel blocker SKF (Figure 4d). Similar results were also obtained with DFO and in SHSY-5Y cells (data not shown). To further confirm this, LC3A punctate formation was evaluated in both HSG and SHSY-5Y cells. As shown in Figures 4e and f, no puncta formation of LC3B was observed in cells treated with DMOG or DFO along with SKF96365, further indicating that calcium entry via TRPC channels is essential for the induction of autophagy and inhibition of apoptosis.


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)

TRPC channel inhibitors attenuate the increase intracellular calcium in autophagy induces cells. (a) Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium in the presence of 50 μM 2APB to SHSY-5Y cells pretreated with 1 mM DMOG. Bar diagram shows the [Ca2+]i in nM concentration of the above-mentioned experiment. Each bar gives the mean±S.E.M. of 50 separate experiments. e **P<0.01 and ***P<0.001. (b) Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium in the presence of 10 μM SKF to SHSY-5Y cells pretreated with 1 mM DMOG. Bar diagram shows the [Ca2+]i in nM concentration of the above-mentioned experiment. Each bar gives the mean±S.E.M. of 50 separate experiments. **P<0.01 and ***P<0.001. (c) Bar diagram showing the cell viability assay (MTT assay) in the SHSY-5Y cells, pretreated with 1 mM DMOG in the presence of 10 μM SKF. Each bar gives the mean±S.E.M. of four separate experiments. **P<0.01 and ***P<0.001. (d) Western blot images showing the expression of LC3A, caspase 3 in SHSY-5Y cells pretreated with 1 mM DMOG and 10 μM SKF. Confocal image of HSG (e) and SH-SY5Y (f) cells transfected with GFP-LC3 and treated with 200 μM DFO or 200 μM DMOG and 10 μM SKF
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: TRPC channel inhibitors attenuate the increase intracellular calcium in autophagy induces cells. (a) Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium in the presence of 50 μM 2APB to SHSY-5Y cells pretreated with 1 mM DMOG. Bar diagram shows the [Ca2+]i in nM concentration of the above-mentioned experiment. Each bar gives the mean±S.E.M. of 50 separate experiments. e **P<0.01 and ***P<0.001. (b) Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium in the presence of 10 μM SKF to SHSY-5Y cells pretreated with 1 mM DMOG. Bar diagram shows the [Ca2+]i in nM concentration of the above-mentioned experiment. Each bar gives the mean±S.E.M. of 50 separate experiments. **P<0.01 and ***P<0.001. (c) Bar diagram showing the cell viability assay (MTT assay) in the SHSY-5Y cells, pretreated with 1 mM DMOG in the presence of 10 μM SKF. Each bar gives the mean±S.E.M. of four separate experiments. **P<0.01 and ***P<0.001. (d) Western blot images showing the expression of LC3A, caspase 3 in SHSY-5Y cells pretreated with 1 mM DMOG and 10 μM SKF. Confocal image of HSG (e) and SH-SY5Y (f) cells transfected with GFP-LC3 and treated with 200 μM DFO or 200 μM DMOG and 10 μM SKF
Mentions: 2-Aminoethoxydiphenyl borate (2APB) and SKF96365 hydrochloride (SKF) are potent TRPC channel inhibitors;12 we thus investigated their role in autophagy. Importantly, in both SHSY-5Y and HSG cells, the increase in [Ca2+]i observed with DMOG treatment was attenuated in the presence of 2APB (Figure 4a) and SKF (Figure 4b). Similar results were also obtained with DFO or serum starvation where hypoxic-mediated increase in Ca2+ entry was decreased in cells treated with 2APB or SKF (data not shown). The cell viability was also affected when cells were pretreated with DMOG or DFO in the presence of TRPC channel inhibitor SKF, where a significant decrease in cell survival was observed (Figure 4c). Pretreatment of HSG cells with 1 mM DMOG in the presence of SKF, also resulted in a loss of autophagy, where no increase in LC3A expression was observed (Figure 4d). Moreover, an increase in apoptosis marker caspase 3 was observed in HSG cells pretreated with DMOG and TRPC channel blocker SKF (Figure 4d). Similar results were also obtained with DFO and in SHSY-5Y cells (data not shown). To further confirm this, LC3A punctate formation was evaluated in both HSG and SHSY-5Y cells. As shown in Figures 4e and f, no puncta formation of LC3B was observed in cells treated with DMOG or DFO along with SKF96365, further indicating that calcium entry via TRPC channels is essential for the induction of autophagy and inhibition of apoptosis.

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