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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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Increases in TRPC1 expression and currents in autophagy-induced HSG and SHSY-5Y cells. (a and b) Application of 1 μM Tg in bath solution induced inward currents at −80 mV in control, 1 mM DMOG-, and 1 mM DFO-treated cells. (c and d) Respectively I–V curves under these conditions are shown in c and quantitation (8–10 recordings) of current intensity at −80 mV is shown in d, *P=<0.05. (e) I–V curves of currents induced by the application of 1 μM Tg in standard external Ringer's solution and Na-based DVF external solutions. (f) Bath application of 1 μM Tg in bath solution induced in salivary gland cells and relative I–V curves. (g) Average (8–10 recordings) current intensity at −80 mV under these conditions is shown, *P=<0.05. (h) Represents western blot images showing the expression of SOCE components, STIM1, Orai1, and TRPC1 in HSG and SHSY-5Y cells pretreated with 1 mM DMOG and 1 mM DFO for 24 h. Corresponding densitometric reading of the protein is shown as a bar diagram (i). Each bar gives the mean±S.E.M. of four separate experiments. *P<0.05, **P<0.01, and ***P<0.001. (j) Western blot images showing the expression of TRPC1 in primary salivary gland cells isolated from control and hypoxia-induced mice models. Bar diagram representing the densitometric reading of the TRPC1 in the above-mentioned western blots. Each bar gives the mean±S.E.M. of four separate experiments. (k) Western blot images showing the relative surface expression of TRPC1 obtained from cell surface biotinylation. Bar diagram shows the normalized expression of TRPC1 to the expression of cell surface transferrin receptor (TfR) protein. Each bar gives the mean±S.E.M. of three separate experiments
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fig5: Increases in TRPC1 expression and currents in autophagy-induced HSG and SHSY-5Y cells. (a and b) Application of 1 μM Tg in bath solution induced inward currents at −80 mV in control, 1 mM DMOG-, and 1 mM DFO-treated cells. (c and d) Respectively I–V curves under these conditions are shown in c and quantitation (8–10 recordings) of current intensity at −80 mV is shown in d, *P=<0.05. (e) I–V curves of currents induced by the application of 1 μM Tg in standard external Ringer's solution and Na-based DVF external solutions. (f) Bath application of 1 μM Tg in bath solution induced in salivary gland cells and relative I–V curves. (g) Average (8–10 recordings) current intensity at −80 mV under these conditions is shown, *P=<0.05. (h) Represents western blot images showing the expression of SOCE components, STIM1, Orai1, and TRPC1 in HSG and SHSY-5Y cells pretreated with 1 mM DMOG and 1 mM DFO for 24 h. Corresponding densitometric reading of the protein is shown as a bar diagram (i). Each bar gives the mean±S.E.M. of four separate experiments. *P<0.05, **P<0.01, and ***P<0.001. (j) Western blot images showing the expression of TRPC1 in primary salivary gland cells isolated from control and hypoxia-induced mice models. Bar diagram representing the densitometric reading of the TRPC1 in the above-mentioned western blots. Each bar gives the mean±S.E.M. of four separate experiments. (k) Western blot images showing the relative surface expression of TRPC1 obtained from cell surface biotinylation. Bar diagram shows the normalized expression of TRPC1 to the expression of cell surface transferrin receptor (TfR) protein. Each bar gives the mean±S.E.M. of three separate experiments

Mentions: To establish the identity of the Ca2+ entry channel, electrophysiological recordings were performed in hypoxia conditions. The addition of Tg caused the appearance of an inward current, which reversed between 0 and −5 mV in SHSY-5Y cells (Figures 5a–d) and in HSG cells (results not shown). Perfusion with Na-based DVF solution facilitated the current, which indicated the current is nonselective (Figure 5e). The channel properties were similar to those previously observed with TRPC1 channels,28 which was induced by store deletion (baseline currents without Tg are shown in I–V curves), reversal potential around 0 mV, slightly inward rectifying and nonselective, suggesting that TRPC1 could contribute to the endogenous Ca2+ entry channel in these cells. Importantly, DMOG and DFO treatment significantly facilitated TRPC1-mediated Ca2+ currents without altering the current–voltage (I–V) relationship (Figure 5a–d). Moreover, electrophysiological recordings using dispersed salivary gland cells also showed an inward nonselective current upon addition of Tg, which are consistent with previous results.27, 29 The properties of the current are similar to TRPC1 current and more importantly, hypoxia-treated cells significantly facilitated the Tg-mediated Isoc-currents (Figures 5f and g). To further identify the cellular component of Ca2+ entry channel(s) involved in this process, cell lysates were obtained under these conditions and were used to evaluate the expression of various proteins involved in Ca2+ entry. Importantly, cells pretreated with 1 mM DMOG, or 1 mM DFO, or in serum-free media showed a significant increase in TRPC1 levels (Figure 5h). In contrast, no significant change in either STIM1 or Orai1 expression levels were observed in hypoxia or serum-depletion conditions in both SHSY-5Y and HSG cells (Figures 5h and i). TRPC1 expression was also increased in hypoxia-induced mice models (Figure 5j). To further establish as to how hypoxia-increased calcium entry surface expression of TRPC1 was observed in SHSY-5Y cells, cells were pretreated with 1 mM DMOG or 1 mM DFO. As indicated in Figure 5k, cells treated with DFO or DMOG showed increase in surface expression of TRPC1, but not transferrin receptor (as internal control), which could account for the increase in hypoxia-mediated Ca2+ currents observed above. Consistent with previous reports,30 addition of thapsigargin also induced surface expression of TRPC1 (as positive control). Hereby, our data indicate that autophagy was dependent on TRPC1, suggesting that TRPC1 has an important role in regulating hypoxia inducing autophagy and inhibiting 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)

Increases in TRPC1 expression and currents in autophagy-induced HSG and SHSY-5Y cells. (a and b) Application of 1 μM Tg in bath solution induced inward currents at −80 mV in control, 1 mM DMOG-, and 1 mM DFO-treated cells. (c and d) Respectively I–V curves under these conditions are shown in c and quantitation (8–10 recordings) of current intensity at −80 mV is shown in d, *P=<0.05. (e) I–V curves of currents induced by the application of 1 μM Tg in standard external Ringer's solution and Na-based DVF external solutions. (f) Bath application of 1 μM Tg in bath solution induced in salivary gland cells and relative I–V curves. (g) Average (8–10 recordings) current intensity at −80 mV under these conditions is shown, *P=<0.05. (h) Represents western blot images showing the expression of SOCE components, STIM1, Orai1, and TRPC1 in HSG and SHSY-5Y cells pretreated with 1 mM DMOG and 1 mM DFO for 24 h. Corresponding densitometric reading of the protein is shown as a bar diagram (i). Each bar gives the mean±S.E.M. of four separate experiments. *P<0.05, **P<0.01, and ***P<0.001. (j) Western blot images showing the expression of TRPC1 in primary salivary gland cells isolated from control and hypoxia-induced mice models. Bar diagram representing the densitometric reading of the TRPC1 in the above-mentioned western blots. Each bar gives the mean±S.E.M. of four separate experiments. (k) Western blot images showing the relative surface expression of TRPC1 obtained from cell surface biotinylation. Bar diagram shows the normalized expression of TRPC1 to the expression of cell surface transferrin receptor (TfR) protein. Each bar gives the mean±S.E.M. of three separate experiments
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Related In: Results  -  Collection

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fig5: Increases in TRPC1 expression and currents in autophagy-induced HSG and SHSY-5Y cells. (a and b) Application of 1 μM Tg in bath solution induced inward currents at −80 mV in control, 1 mM DMOG-, and 1 mM DFO-treated cells. (c and d) Respectively I–V curves under these conditions are shown in c and quantitation (8–10 recordings) of current intensity at −80 mV is shown in d, *P=<0.05. (e) I–V curves of currents induced by the application of 1 μM Tg in standard external Ringer's solution and Na-based DVF external solutions. (f) Bath application of 1 μM Tg in bath solution induced in salivary gland cells and relative I–V curves. (g) Average (8–10 recordings) current intensity at −80 mV under these conditions is shown, *P=<0.05. (h) Represents western blot images showing the expression of SOCE components, STIM1, Orai1, and TRPC1 in HSG and SHSY-5Y cells pretreated with 1 mM DMOG and 1 mM DFO for 24 h. Corresponding densitometric reading of the protein is shown as a bar diagram (i). Each bar gives the mean±S.E.M. of four separate experiments. *P<0.05, **P<0.01, and ***P<0.001. (j) Western blot images showing the expression of TRPC1 in primary salivary gland cells isolated from control and hypoxia-induced mice models. Bar diagram representing the densitometric reading of the TRPC1 in the above-mentioned western blots. Each bar gives the mean±S.E.M. of four separate experiments. (k) Western blot images showing the relative surface expression of TRPC1 obtained from cell surface biotinylation. Bar diagram shows the normalized expression of TRPC1 to the expression of cell surface transferrin receptor (TfR) protein. Each bar gives the mean±S.E.M. of three separate experiments
Mentions: To establish the identity of the Ca2+ entry channel, electrophysiological recordings were performed in hypoxia conditions. The addition of Tg caused the appearance of an inward current, which reversed between 0 and −5 mV in SHSY-5Y cells (Figures 5a–d) and in HSG cells (results not shown). Perfusion with Na-based DVF solution facilitated the current, which indicated the current is nonselective (Figure 5e). The channel properties were similar to those previously observed with TRPC1 channels,28 which was induced by store deletion (baseline currents without Tg are shown in I–V curves), reversal potential around 0 mV, slightly inward rectifying and nonselective, suggesting that TRPC1 could contribute to the endogenous Ca2+ entry channel in these cells. Importantly, DMOG and DFO treatment significantly facilitated TRPC1-mediated Ca2+ currents without altering the current–voltage (I–V) relationship (Figure 5a–d). Moreover, electrophysiological recordings using dispersed salivary gland cells also showed an inward nonselective current upon addition of Tg, which are consistent with previous results.27, 29 The properties of the current are similar to TRPC1 current and more importantly, hypoxia-treated cells significantly facilitated the Tg-mediated Isoc-currents (Figures 5f and g). To further identify the cellular component of Ca2+ entry channel(s) involved in this process, cell lysates were obtained under these conditions and were used to evaluate the expression of various proteins involved in Ca2+ entry. Importantly, cells pretreated with 1 mM DMOG, or 1 mM DFO, or in serum-free media showed a significant increase in TRPC1 levels (Figure 5h). In contrast, no significant change in either STIM1 or Orai1 expression levels were observed in hypoxia or serum-depletion conditions in both SHSY-5Y and HSG cells (Figures 5h and i). TRPC1 expression was also increased in hypoxia-induced mice models (Figure 5j). To further establish as to how hypoxia-increased calcium entry surface expression of TRPC1 was observed in SHSY-5Y cells, cells were pretreated with 1 mM DMOG or 1 mM DFO. As indicated in Figure 5k, cells treated with DFO or DMOG showed increase in surface expression of TRPC1, but not transferrin receptor (as internal control), which could account for the increase in hypoxia-mediated Ca2+ currents observed above. Consistent with previous reports,30 addition of thapsigargin also induced surface expression of TRPC1 (as positive control). Hereby, our data indicate that autophagy was dependent on TRPC1, suggesting that TRPC1 has an important role in regulating hypoxia inducing autophagy and inhibiting 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