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

Induced Autophagy in SH-SY5Y and HSG cells. (a) HSG and SHSY-5Y cells were treated for 24 h with 1 mM DFO, 1 mM DMOG, and in a serum-free media. Protein was isolated and western blots represent the protein expression of different autophagy marker beclin-1, LC3A, p62, and loading control actin. (b) Corresponding densitometric reading of the autophagy marker protein is shown as a bar diagram. Each bar gives the mean±S.E.M. of four separate experiments. *P<0.05, **P<0.01, and ***P<0.001. Confocal image of HSG and SH-SY5Y cells, respectively, transfected with fluorescent-tagged LC3 and treated for 24 h with 200μM DFO or 200μM DMOG. (c and d) Western blot images showing the expression of autophagy marker LC3A in primary salivary gland cells isolated from normoxia- and hypoxia-induced mice models. (e) Bar diagram representing the densitometric reading of the LC3A in the above-mentioned western blots. Each bar gives the mean±S.E.M. of four separate experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Induced Autophagy in SH-SY5Y and HSG cells. (a) HSG and SHSY-5Y cells were treated for 24 h with 1 mM DFO, 1 mM DMOG, and in a serum-free media. Protein was isolated and western blots represent the protein expression of different autophagy marker beclin-1, LC3A, p62, and loading control actin. (b) Corresponding densitometric reading of the autophagy marker protein is shown as a bar diagram. Each bar gives the mean±S.E.M. of four separate experiments. *P<0.05, **P<0.01, and ***P<0.001. Confocal image of HSG and SH-SY5Y cells, respectively, transfected with fluorescent-tagged LC3 and treated for 24 h with 200μM DFO or 200μM DMOG. (c and d) Western blot images showing the expression of autophagy marker LC3A in primary salivary gland cells isolated from normoxia- and hypoxia-induced mice models. (e) Bar diagram representing the densitometric reading of the LC3A in the above-mentioned western blots. Each bar gives the mean±S.E.M. of four separate experiments

Mentions: The human salivary gland (HSG) cells and neuroblastic SHSY-5Y cells were treated with 1 mM DMOG or DFO or were subjected to serum-free media. After treatment, the cells were lysed and expressions of the autophagic marker proteins were analyzed using western blotting. Both autophagic markers Beclin-1 and LC3A were upregulated in both cells treated with DMOG or DFO or in serum-deprived conditions (Figure 1a, quantification provided in Figure 1b). Similarly, ATG5 levels were also increased under these three conditions in both HSG and SHSY-5Y cells (data not shown). Importantly, it has been previously reported that p62 expression is lost when cells undergo autophagy.23 Thus, we also evaluated the expression of p62, which was significantly decreased in DMOG, DFO, and serum-deprived conditions (Figures 1a and b). To have further evidence, we also evaluated the localization of LC3A as it has been previously reported that LC3B forms a punctate pattern when cells induce autophagy.24 Importantly, the confocal images of cells transfected with fluorescent-tagged LC3B and treated with DMOG or DFO for 24 h, confirm the result that DMOG and DFO treatment in both HSG and SHSY-5Y cells induces autophagy (Figures 1c and d). Although both DMOG and DFO could induce hypoxia, they do not truly represent hypoxic conditions. In addition, to study the physiological responses in animal models, mice were exposed to 10% oxygen for 3 days. Protein were isolated from salivary glands of normoxia- (control) and hypoxia-induced mice, as mentioned previously.25 Similar to our cell culture results, LC3A expression was increased in mice exhibiting hypoxia conditions (Figure 1e, quantification provided as bar graph) suggesting that hypoxia induces autophagy in mouse models.


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)

Induced Autophagy in SH-SY5Y and HSG cells. (a) HSG and SHSY-5Y cells were treated for 24 h with 1 mM DFO, 1 mM DMOG, and in a serum-free media. Protein was isolated and western blots represent the protein expression of different autophagy marker beclin-1, LC3A, p62, and loading control actin. (b) Corresponding densitometric reading of the autophagy marker protein is shown as a bar diagram. Each bar gives the mean±S.E.M. of four separate experiments. *P<0.05, **P<0.01, and ***P<0.001. Confocal image of HSG and SH-SY5Y cells, respectively, transfected with fluorescent-tagged LC3 and treated for 24 h with 200μM DFO or 200μM DMOG. (c and d) Western blot images showing the expression of autophagy marker LC3A in primary salivary gland cells isolated from normoxia- and hypoxia-induced mice models. (e) Bar diagram representing the densitometric reading of the LC3A in the above-mentioned western blots. Each bar gives the mean±S.E.M. of four separate experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Induced Autophagy in SH-SY5Y and HSG cells. (a) HSG and SHSY-5Y cells were treated for 24 h with 1 mM DFO, 1 mM DMOG, and in a serum-free media. Protein was isolated and western blots represent the protein expression of different autophagy marker beclin-1, LC3A, p62, and loading control actin. (b) Corresponding densitometric reading of the autophagy marker protein is shown as a bar diagram. Each bar gives the mean±S.E.M. of four separate experiments. *P<0.05, **P<0.01, and ***P<0.001. Confocal image of HSG and SH-SY5Y cells, respectively, transfected with fluorescent-tagged LC3 and treated for 24 h with 200μM DFO or 200μM DMOG. (c and d) Western blot images showing the expression of autophagy marker LC3A in primary salivary gland cells isolated from normoxia- and hypoxia-induced mice models. (e) Bar diagram representing the densitometric reading of the LC3A in the above-mentioned western blots. Each bar gives the mean±S.E.M. of four separate experiments
Mentions: The human salivary gland (HSG) cells and neuroblastic SHSY-5Y cells were treated with 1 mM DMOG or DFO or were subjected to serum-free media. After treatment, the cells were lysed and expressions of the autophagic marker proteins were analyzed using western blotting. Both autophagic markers Beclin-1 and LC3A were upregulated in both cells treated with DMOG or DFO or in serum-deprived conditions (Figure 1a, quantification provided in Figure 1b). Similarly, ATG5 levels were also increased under these three conditions in both HSG and SHSY-5Y cells (data not shown). Importantly, it has been previously reported that p62 expression is lost when cells undergo autophagy.23 Thus, we also evaluated the expression of p62, which was significantly decreased in DMOG, DFO, and serum-deprived conditions (Figures 1a and b). To have further evidence, we also evaluated the localization of LC3A as it has been previously reported that LC3B forms a punctate pattern when cells induce autophagy.24 Importantly, the confocal images of cells transfected with fluorescent-tagged LC3B and treated with DMOG or DFO for 24 h, confirm the result that DMOG and DFO treatment in both HSG and SHSY-5Y cells induces autophagy (Figures 1c and d). Although both DMOG and DFO could induce hypoxia, they do not truly represent hypoxic conditions. In addition, to study the physiological responses in animal models, mice were exposed to 10% oxygen for 3 days. Protein were isolated from salivary glands of normoxia- (control) and hypoxia-induced mice, as mentioned previously.25 Similar to our cell culture results, LC3A expression was increased in mice exhibiting hypoxia conditions (Figure 1e, quantification provided as bar graph) suggesting that hypoxia induces autophagy in mouse models.

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