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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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Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) attenuated the intracellular calcium influx and induces apoptosis. (a) Western blot images showing the expression of SOCE components STIM1, TRPC1, and Orai1, autophagy marker beclin-1 and loading control actin in HSG and SHSY-5Y cells pretreated with 1 mM DMOG or in serum-free media in the presence of 1 mM autophagy marker 3-MA for 24 h. (b) Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium in the presence of 1 mM 3-MA to SHSY-5Y cells pretreated with 1 mM DMOG or in serum-free media. (c) 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<0.05, **P<0.01. (d) Bar diagram showing the cell viability assay (MTT assay) in the SH-SY5Y cells, pretreated with 1 mM DMOG in the presence of 1 mM. Each bar gives the mean±S.E.M. of four separate experiments. ***P<0.001. (e) Application of 1 μM Tg in bath solution induced inward currents at −80 mV in control, cells treated in serum-free media and autophagy inhibitor 3-MA-treated cells. (f) Average (8–10) recordings current intensity at −80 mV are shown, *P<0.05
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fig7: Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) attenuated the intracellular calcium influx and induces apoptosis. (a) Western blot images showing the expression of SOCE components STIM1, TRPC1, and Orai1, autophagy marker beclin-1 and loading control actin in HSG and SHSY-5Y cells pretreated with 1 mM DMOG or in serum-free media in the presence of 1 mM autophagy marker 3-MA for 24 h. (b) Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium in the presence of 1 mM 3-MA to SHSY-5Y cells pretreated with 1 mM DMOG or in serum-free media. (c) 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<0.05, **P<0.01. (d) Bar diagram showing the cell viability assay (MTT assay) in the SH-SY5Y cells, pretreated with 1 mM DMOG in the presence of 1 mM. Each bar gives the mean±S.E.M. of four separate experiments. ***P<0.001. (e) Application of 1 μM Tg in bath solution induced inward currents at −80 mV in control, cells treated in serum-free media and autophagy inhibitor 3-MA-treated cells. (f) Average (8–10) recordings current intensity at −80 mV are shown, *P<0.05

Mentions: To further understand the link between Ca2+ entry via the TRPC1 channel and autophagy, 3-methyladenine (3-MA) a known autophagy inhibitor,4 was used. The cells were pretreated with 1 mM 3-MA, along with DMOG, or DFO, or serum-free media and protein expression of STIM1, Orai1, TRPC1, and autophagy markers were analyzed. Importantly, cells treated with 3-MA showed no increase in DMOG or serum-deprived increase in TRPC1 expression (Figure 7a). Furthermore, no increase in beclin-1 an autophagic marker was observed in cells pretreated with 3-MA along with DMOG- or serum-deprived conditions (Figure 7a). Pretreatment with 3-MA also attenuated the increase in Ca2+ entry that was induced by DMOG and serum starvation in both SHSY-5Y (Figures 7b and c) and HSG cells (result not shown). Consistent with these results, pretreatment with 3-MA also attenuated cell viability (Figure 7d) and serum starvation induced increase in Tg-induced Ca2+ currents (Figures 7e and f). Altogether, these data suggest that Ca2+ entry via the TRPC1 channel is essential for autophagy that leads to the inhibition of cell death and loss of TRPC1 function or autophagy could lead to decrease in cell survival.


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)

Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) attenuated the intracellular calcium influx and induces apoptosis. (a) Western blot images showing the expression of SOCE components STIM1, TRPC1, and Orai1, autophagy marker beclin-1 and loading control actin in HSG and SHSY-5Y cells pretreated with 1 mM DMOG or in serum-free media in the presence of 1 mM autophagy marker 3-MA for 24 h. (b) Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium in the presence of 1 mM 3-MA to SHSY-5Y cells pretreated with 1 mM DMOG or in serum-free media. (c) 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<0.05, **P<0.01. (d) Bar diagram showing the cell viability assay (MTT assay) in the SH-SY5Y cells, pretreated with 1 mM DMOG in the presence of 1 mM. Each bar gives the mean±S.E.M. of four separate experiments. ***P<0.001. (e) Application of 1 μM Tg in bath solution induced inward currents at −80 mV in control, cells treated in serum-free media and autophagy inhibitor 3-MA-treated cells. (f) Average (8–10) recordings current intensity at −80 mV are shown, *P<0.05
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fig7: Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) attenuated the intracellular calcium influx and induces apoptosis. (a) Western blot images showing the expression of SOCE components STIM1, TRPC1, and Orai1, autophagy marker beclin-1 and loading control actin in HSG and SHSY-5Y cells pretreated with 1 mM DMOG or in serum-free media in the presence of 1 mM autophagy marker 3-MA for 24 h. (b) Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium in the presence of 1 mM 3-MA to SHSY-5Y cells pretreated with 1 mM DMOG or in serum-free media. (c) 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<0.05, **P<0.01. (d) Bar diagram showing the cell viability assay (MTT assay) in the SH-SY5Y cells, pretreated with 1 mM DMOG in the presence of 1 mM. Each bar gives the mean±S.E.M. of four separate experiments. ***P<0.001. (e) Application of 1 μM Tg in bath solution induced inward currents at −80 mV in control, cells treated in serum-free media and autophagy inhibitor 3-MA-treated cells. (f) Average (8–10) recordings current intensity at −80 mV are shown, *P<0.05
Mentions: To further understand the link between Ca2+ entry via the TRPC1 channel and autophagy, 3-methyladenine (3-MA) a known autophagy inhibitor,4 was used. The cells were pretreated with 1 mM 3-MA, along with DMOG, or DFO, or serum-free media and protein expression of STIM1, Orai1, TRPC1, and autophagy markers were analyzed. Importantly, cells treated with 3-MA showed no increase in DMOG or serum-deprived increase in TRPC1 expression (Figure 7a). Furthermore, no increase in beclin-1 an autophagic marker was observed in cells pretreated with 3-MA along with DMOG- or serum-deprived conditions (Figure 7a). Pretreatment with 3-MA also attenuated the increase in Ca2+ entry that was induced by DMOG and serum starvation in both SHSY-5Y (Figures 7b and c) and HSG cells (result not shown). Consistent with these results, pretreatment with 3-MA also attenuated cell viability (Figure 7d) and serum starvation induced increase in Tg-induced Ca2+ currents (Figures 7e and f). Altogether, these data suggest that Ca2+ entry via the TRPC1 channel is essential for autophagy that leads to the inhibition of cell death and loss of TRPC1 function or autophagy could lead to decrease in cell survival.

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