TRPC1-mediated Ca2+ entry is essential for the regulation of hypoxia and nutrient depletion-dependent autophagy

Autophagy is a cellular catabolic process needed for the degradation and recycling of protein aggregates and damaged organelles. Although Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ entry via the TRPC1 channel, which was inhibited by the addition of 2APB and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365. Importantly, TRPC1-mediated Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ entry along with increased expression of autophagic markers. Altogether, we provide evidence for the involvement of Ca²⁺ influx via TRPC1 in regulating autophagy to protect against cell death.Autophagy is a cellular process responsible for the delivery of proteins or organelles to lysosomes for its degradation. Autophagy participates not only in maintaining cellular homeostasis, but also promotes cell survival during cellular stress situations.^{1, 2} The stress conditions including nutrient starvation, hypoxia conditions, invading microbes, and tumor formation, have been shown to induce autophagy that allows cell survival in these stressful or pathological situations.¹ In addition, autophagy also recycles existing cytoplasmic components to generate the molecules that are required to sustain the most vital cellular functions.³ Till date, three forms of autophagy have been identified, which are designated as chaperone-mediated autophagy, microautophagy, and macroautophagy.⁴ Although the precise mechanism as to how autophagy is initiated is not well understood, many of the genes first identified in yeast that are involved in autophagy have orthologs in other eukaryotes including human homologs.^{5, 6} The presence of similar genes in all organisms suggests that autophagy might be a phenomenon that is evolutionally conserved that is essential for cell survival. In addition, since autophagy delivers a fresh pool of amino acids and other essential molecules to the cell, initiation of autophagy is highly beneficial particularly during nutritional stress situations or tissue remodeling during development and embryogenesis.⁶ Consequently, impaired or altered autophagy is often implicated in several pathologies, like neurodegenerative disorders and cancer,^{7, 8, 9} which again highlight its importance.Ca²⁺ has a vital role in the regulation of a large number of cellular processes such as cell proliferation, survival, migration, invasion, motility, and apoptosis.^{10, 11} To perform functions on such a broad spectrum, the cells have evolved multiple mechanisms regulating cellular Ca²⁺ levels, mainly by regulating the function of various Ca²⁺ channels present in different locations. Mitochondrial, ER, lysosomal, and cytosolic Ca²⁺ levels are regulated by Ca²⁺ permeable ion channels localized either on the membranes of the intracellular organelles or on the plasma membrane.¹⁰ The Ca²⁺ permeable channels, including families of TRPCs, Orais, voltage-gated, two-pore, mitochondrial Ca²⁺ uniporter, IP₃, and ryanodine receptors have all been identified to contribute towards changes in intracellular Ca²⁺ (Ca²⁺]_i).^{10, 12, 13, 14} Channels of the TRPCs and Orai families have been related to several Ca²⁺-dependent physiological processes in various cell types, ranging from cell proliferation to contractility, to apoptosis under both physiological and pathological conditions.¹² Moreover, it has been suggested that intracellular Ca²⁺ is one of the key regulators of autophagy;¹⁵ however, the possible role of Ca²⁺ in autophagy is still inconclusive. Many reports also suggest that Ca²⁺ inhibits autophagy,^{16, 17, 18} whereas others have indicated a stimulatory role for Ca²⁺ towards autophagy.^{19, 20, 21} Furthermore, the identity of the major Ca²⁺ channel(s) involved in autophagy is not known. Members of the TRPC family have been suggested as mediators of Ca²⁺ entry into cells. Activation of the G-protein (G_q/11–PLC pathway) leads to the generation of second messenger IP₃.^{10, 22} IP₃ binds to the IP₃R, which initiates Ca²⁺ release from the ER stores, thereby facilitating stromal interacting molecule-1 (STIM1) to rearrange and activate Ca²⁺ entry via the store-operated channels.²² Two families of proteins (TRPCs and Orais) have been identified as potential candidates for SOC-mediated Ca²⁺ entry.^{12, 22} However, their role in autophagy has not yet been determined. Thus, here we investigated the role of Ca²⁺ entry channels (TRPCs and Orais) in autophagy and show that both hypoxia-mimetic and nutrient depression induces autophagy in two different cell lines. Furthermore, our data indicates that autophagy was dependent on TRPC1-mediated increase in intracellular Ca²⁺ levels, suggesting that TRPC1 has an important role in regulating autophagy and inhibiting cell death.