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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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Knockdown of TRPC1 attenuated the autophagy-induced intracellular calcium increase and also affects the cell viability. Western blot images showing the knockdown of TRPC1 using siRNA, HSG cells (a) (70% knockdown, n=3, P<0.01), and SHSY-5Y cells (b) (72% knockdown, n=3, p<0.001). Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium to siRNA TRPC1 knockdown HSG cells (a) and in SHSY-5Y cells (b) pretreated with 1 mM DMOG or in serum-free media. Bar diagram (c and d) shows the [Ca2+]i in nM concentration of the above-mentioned experiment. Each bar gives the mean±S.E.M. of 40 separate cells. *P<0.05 and ***P<0.001. (e) Bar diagram showing the cell viability assay (MTT assay) in the TRPC1 knockdown SHSY-5Y cells, pretreated with 1 mM DMOG or 1 mM DFO or in serum-free media. Each bar gives the mean±S.E.M. of four separate experiments. **P<0.01. (f) Western blot images showing the expression of autophagy marker beclin-1 in HSG and SHSY-5Y cells pretreated with 1 mM DMOG or in serum-free media for 24 h. (g) Western blot image showing the TRPC3 knockdown using siRNA in SHSY-5Y cells (60% knockdown, n=3, P<0.01). Application of 50 μM OAG in bath solution induced inward currents at −80 mV in control and TRPC3 knockout cells. (h) Under DFO treatment, respectively I–V curves of currents induced by the application of 1 μM Tg in control and TRPC3 knockout cells treated with 1 mM DFO. The traces are representative of average (8–10 recordings) of current intensity at −80 mV. (i) Western blot images showing the expression of LC3A in siTRPC3 SHSY-5Y cells with and without 24 h pretreatment with 1 mM DFO (n=3, P<0.05)
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fig6: Knockdown of TRPC1 attenuated the autophagy-induced intracellular calcium increase and also affects the cell viability. Western blot images showing the knockdown of TRPC1 using siRNA, HSG cells (a) (70% knockdown, n=3, P<0.01), and SHSY-5Y cells (b) (72% knockdown, n=3, p<0.001). Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium to siRNA TRPC1 knockdown HSG cells (a) and in SHSY-5Y cells (b) pretreated with 1 mM DMOG or in serum-free media. Bar diagram (c and d) shows the [Ca2+]i in nM concentration of the above-mentioned experiment. Each bar gives the mean±S.E.M. of 40 separate cells. *P<0.05 and ***P<0.001. (e) Bar diagram showing the cell viability assay (MTT assay) in the TRPC1 knockdown SHSY-5Y cells, pretreated with 1 mM DMOG or 1 mM DFO or in serum-free media. Each bar gives the mean±S.E.M. of four separate experiments. **P<0.01. (f) Western blot images showing the expression of autophagy marker beclin-1 in HSG and SHSY-5Y cells pretreated with 1 mM DMOG or in serum-free media for 24 h. (g) Western blot image showing the TRPC3 knockdown using siRNA in SHSY-5Y cells (60% knockdown, n=3, P<0.01). Application of 50 μM OAG in bath solution induced inward currents at −80 mV in control and TRPC3 knockout cells. (h) Under DFO treatment, respectively I–V curves of currents induced by the application of 1 μM Tg in control and TRPC3 knockout cells treated with 1 mM DFO. The traces are representative of average (8–10 recordings) of current intensity at −80 mV. (i) Western blot images showing the expression of LC3A in siTRPC3 SHSY-5Y cells with and without 24 h pretreatment with 1 mM DFO (n=3, P<0.05)

Mentions: Data presented thus far indicate that TRPC1 could be important for hypoxia- and serum starvation induced increase in Ca2+ entry and could modulate autophagy. Thus, to study the importance of TRPC1 channels in hypoxia and serum depletion-induced autophagy, we knocked down TRPC1 in both SHSY-5Y and HSG cells using siRNA (70% knockdown Figures 6a and c shown as insets). Importantly, the DMOG- and serum depletion-induced increase in Ca2+ entry was attenuated in TRPC1 knockdown cells in both SHSY-5Y and HSG cells (Figures 6a–d). Moreover, pretreatment of DMOG, or DFO or serum-free media in TRPC1 knockdown cells also showed an increase in apoptosis (Figure 6e) and autophagy was inhibited as observed by beclin-1 levels (Figure 6f). To further position TRPC1 as the main calcium channel responsible for the currents induced by hypoxia, we knocked down TRPC3 in these cells. We first investigated the TRPC3 channel activity, which was induced by the application of OAG and addition of OAG induced an inward current, which was abolished by the expression of siTRPC3 (Figure 6g). More importantly, siTRPC3 has no significant effect on hypoxia- (1 mM DFO treatment) induced increase in Tg-induced currents (Figure 6h) and LC3A expression (Figure 6i). Collectively, these results suggest that hypoxia and serum starvation-mediated increase in Ca2+ entry is at least in part mediated via the TRPC1 channel, which could lead to the activation of autophagy that together would inhibit cell death in SHSY-5Y and HSG cells.


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)

Knockdown of TRPC1 attenuated the autophagy-induced intracellular calcium increase and also affects the cell viability. Western blot images showing the knockdown of TRPC1 using siRNA, HSG cells (a) (70% knockdown, n=3, P<0.01), and SHSY-5Y cells (b) (72% knockdown, n=3, p<0.001). Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium to siRNA TRPC1 knockdown HSG cells (a) and in SHSY-5Y cells (b) pretreated with 1 mM DMOG or in serum-free media. Bar diagram (c and d) shows the [Ca2+]i in nM concentration of the above-mentioned experiment. Each bar gives the mean±S.E.M. of 40 separate cells. *P<0.05 and ***P<0.001. (e) Bar diagram showing the cell viability assay (MTT assay) in the TRPC1 knockdown SHSY-5Y cells, pretreated with 1 mM DMOG or 1 mM DFO or in serum-free media. Each bar gives the mean±S.E.M. of four separate experiments. **P<0.01. (f) Western blot images showing the expression of autophagy marker beclin-1 in HSG and SHSY-5Y cells pretreated with 1 mM DMOG or in serum-free media for 24 h. (g) Western blot image showing the TRPC3 knockdown using siRNA in SHSY-5Y cells (60% knockdown, n=3, P<0.01). Application of 50 μM OAG in bath solution induced inward currents at −80 mV in control and TRPC3 knockout cells. (h) Under DFO treatment, respectively I–V curves of currents induced by the application of 1 μM Tg in control and TRPC3 knockout cells treated with 1 mM DFO. The traces are representative of average (8–10 recordings) of current intensity at −80 mV. (i) Western blot images showing the expression of LC3A in siTRPC3 SHSY-5Y cells with and without 24 h pretreatment with 1 mM DFO (n=3, P<0.05)
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fig6: Knockdown of TRPC1 attenuated the autophagy-induced intracellular calcium increase and also affects the cell viability. Western blot images showing the knockdown of TRPC1 using siRNA, HSG cells (a) (70% knockdown, n=3, P<0.01), and SHSY-5Y cells (b) (72% knockdown, n=3, p<0.001). Representative traces showing the transient increase in [Ca2+]i after the addition of 1 mM calcium to siRNA TRPC1 knockdown HSG cells (a) and in SHSY-5Y cells (b) pretreated with 1 mM DMOG or in serum-free media. Bar diagram (c and d) shows the [Ca2+]i in nM concentration of the above-mentioned experiment. Each bar gives the mean±S.E.M. of 40 separate cells. *P<0.05 and ***P<0.001. (e) Bar diagram showing the cell viability assay (MTT assay) in the TRPC1 knockdown SHSY-5Y cells, pretreated with 1 mM DMOG or 1 mM DFO or in serum-free media. Each bar gives the mean±S.E.M. of four separate experiments. **P<0.01. (f) Western blot images showing the expression of autophagy marker beclin-1 in HSG and SHSY-5Y cells pretreated with 1 mM DMOG or in serum-free media for 24 h. (g) Western blot image showing the TRPC3 knockdown using siRNA in SHSY-5Y cells (60% knockdown, n=3, P<0.01). Application of 50 μM OAG in bath solution induced inward currents at −80 mV in control and TRPC3 knockout cells. (h) Under DFO treatment, respectively I–V curves of currents induced by the application of 1 μM Tg in control and TRPC3 knockout cells treated with 1 mM DFO. The traces are representative of average (8–10 recordings) of current intensity at −80 mV. (i) Western blot images showing the expression of LC3A in siTRPC3 SHSY-5Y cells with and without 24 h pretreatment with 1 mM DFO (n=3, P<0.05)
Mentions: Data presented thus far indicate that TRPC1 could be important for hypoxia- and serum starvation induced increase in Ca2+ entry and could modulate autophagy. Thus, to study the importance of TRPC1 channels in hypoxia and serum depletion-induced autophagy, we knocked down TRPC1 in both SHSY-5Y and HSG cells using siRNA (70% knockdown Figures 6a and c shown as insets). Importantly, the DMOG- and serum depletion-induced increase in Ca2+ entry was attenuated in TRPC1 knockdown cells in both SHSY-5Y and HSG cells (Figures 6a–d). Moreover, pretreatment of DMOG, or DFO or serum-free media in TRPC1 knockdown cells also showed an increase in apoptosis (Figure 6e) and autophagy was inhibited as observed by beclin-1 levels (Figure 6f). To further position TRPC1 as the main calcium channel responsible for the currents induced by hypoxia, we knocked down TRPC3 in these cells. We first investigated the TRPC3 channel activity, which was induced by the application of OAG and addition of OAG induced an inward current, which was abolished by the expression of siTRPC3 (Figure 6g). More importantly, siTRPC3 has no significant effect on hypoxia- (1 mM DFO treatment) induced increase in Tg-induced currents (Figure 6h) and LC3A expression (Figure 6i). Collectively, these results suggest that hypoxia and serum starvation-mediated increase in Ca2+ entry is at least in part mediated via the TRPC1 channel, which could lead to the activation of autophagy that together would inhibit cell death in SHSY-5Y and HSG cells.

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