It has been reported that hypoxia induced spermatogenesis reduction caused by spermatocyte apoptosis in mice [4, 5, 37]. Continuous autophagic flux play a prosurvival role in spermatocyte cells by directly inhibiting apoptosis of spermatocytes [38]. Previously, we demonstrated that serious hypoxia inhibited autophagic flux by downregulating the expression of V-ATPase in GC-2 cells [20]. In the present study, we determined that V-ATPase deficiency resulted in more severe spermatogenesis deficits after hypoxia exposure in mice. These deficits were associated with exacerbation of spermatocyte apoptosis. These results indicated V-ATPase may exert a protective role against spermatogenesis reduction via the suppression of the apoptosis of spermatocyte. However, the mechanism by which V-ATPase inhibiting apoptosis of spermatocytes is still poorly understood.
So far, studies indicate that the main causeof spermatocyte apoptosis induced by V-ATPase deficiency is the generation of immature autophagosome resultsfrom the decreaseof autophagic flux under hypoxia stimulation, which in turn promotes the caspase-8 and death receptor (DR-) apoptosis pathway [20]. Here,wefoundthat the DR-apoptotic marker DR5 and effector caspase-8 accumulated with prolong hypoxia, which was consistent with gradual reduction of autolysosome marker V-ATPase. We have observed the decrease of lysosomal synthesis in GC-2 cells under hypoxia exposure, including the decrease of the expression of lysosomal marker LAMP2 and the decrease of the ratio of lysosomes to cells. Whether the decreased V-ATPase expression after hypoxia is connected with the observed lysosome biogenesisdefects remains to be determined.
Since TFEB was recently discovered as a major regulator oflysosome biogenesis as well as being a potential therapeutic target torescue myocardial ischemic injury and neurodegeneration[39, 40], we are interested in its role in regulating V-ATPase and spermatocyte apoptosis. Inthepresentstudy,itisnotedthatat 48 hafter hypoxia exposure boththemRNAandtotalproteinlevelofTFEBweresignificantly down-regulated.Additionally,therewasmarked reduction ofTFEBproteininthenuclearsubfraction,suggestingthattherewas reduced translocationofTFEBintothenucleusfrom 24hto 48hafter hypoxia. On the contrary, we conducted LV-mediated GC2-specific overexpression of TFEB to show thatoverexpression of TFEB was effective in reversing the hypoxia-induced reduction of autolysosome marker V-ATPase.The results obtained in this study supporttheprevious suggestion that TFEB might regulate lysosomal biosynthesis.
The V-ATPase was ATP-dependent proton pumps that acidify intracellular compartments and ubiquitously expressed in mammals. The V-ATPase was extensively studied in cancer and neurodegenerative disease. Recent work has supported the concept that the V-ATPase playspro-survival role in cancers [41–43]. A number of studies have shown theoverexpression of V-ATPase subunits in various cancer cell lines and tumor samples, including breast, prostate, pancreatic, colorectal, liver, lung, ovarian, stomach, esophageal cancers and melanoma, where it showed thepro-tumoral activity[44–52]. In contrast, induction of apoptosis by V-ATPase inhibition has beenreported in both human and murine models encompassingmany tumor types.Tumor cells are more sensitive to V-ATPase-inhibition-dependent cancer cell death, and loss of V-ATPase activity reduces cancer cell growth [53]. Additionally, archazolid can induce p53 (tumor suppressor) protein levels by inhibitingV-ATPase expressionin cancer cells [54]. It has also been shown that V-ATPase inhibition differentially affects regulation of AMPK in tumor and nontumor cells, and that this differential regulation contributes to the selectivity of V-ATPase inhibitors for tumor cells[55]. Cancer-associated V-ATPase significantly induced the life span of protumor genic neutrophils by activating the intrinsic pathway of apoptosis[56]. ECDD-S27 retards the autophagy pathway by inhibiting the V-ATPase and restricts cancer cell survival[57]. Some study has found that intracellular acidosis caused by V-ATPase inhibition in breast cancer cells stabilizes the expression of the pro-apoptotic protein Bnip3, resulting in cell death [58]. It has also recently been demonstrated that treatment of breast cancer cells with the V-ATPase inhibitor archazolid disrupts the internalization of the transferrin receptor, leading to iron deprivation and subsequent apoptosis [59]. In our study, using V-ATPase lentivirus for the first time as tool, we found thatV-ATPase overexpression caused the decline of apoptosis in GC-2 cells. Of interest, in contrast to GC-2 cells that deplete V-ATPase, V-ATPase overexpression induces the survival of V-ATPase overexpressed GC-2 cells. Similarly, in vitro studies revealed that V-ATPase reduces the apoptosis of non-cancer cells, including rat proximal tubular cells, neuronal cell and osteoclast[60–62]. Genetic analysis of individuals with X-linked Parkinson Disease with Spasticity (XPDS) yielded a novel candidate gene locus on the X chromosome, and it was later shown that a point mutation (c.345C > T) in exon 4 of the ATP6AP2 gene causes altered splicing of ATP6AP2 in XPDS[63]. It has been reported that in Cln1-/- mice, palmitoyl-protein thioesterase-1 (PPT1)adversely affectedV-ATPase function and dysregulated lysosomal acidification in other lysosomal storage disorders (LSDs) and common neurodegenerative diseases[64].Of note, we used GC-2 cells with V-ATPase silencing or overexpressed under hypoxia treatment to show for the first time that V-ATPase in GC-2 cells regulates apoptosis of spermatocyte by acting on DR-apoptosis pathway.
It has been found that V-ATPase wasclosely associated with multiple signal transduction signaling, such as m-TOR (mammalian Target OfRapamycin), Wnt, TGF-β, Notch,G protein-coupled receptors (GPCRs) and receptortyrosine kinases (RTKs), etc. Several critical pathways of growth,survival and differentiation that are frequently altered incancers rely on the V-ATPase[65]. In Notch signaling, the Notch receptoriscleaved in Golgi and translocated to the plasma membrane, where further cleavage of the receptor occurs in response to Notch ligand binding. Cleaved Notch intracellular domain is translocated to nucleus and activates Notch target genes[66]. TGF-βprotein is glycosylated in theGolgi to form mature TGF-βand secreted into the extracellular space.TGF-βbinds to its receptor (TGF-βR)andleads to the endocytosis andphosphorylation of Smad2, which in turn activates TGF-βtarget genes[67]. During canonical Wnt signaling, the binding of ligands to the Wntreceptor complex inhibits the phosphorylation of β-catenin by GSK-3β and directs the translocation ofβ-catenin into thenucleus, where it activates the transcription of target genes Cyclin D1 and oncogene c-Myc[68]. In addition, it was found that V-ATPase is critical for sensing of amino acids and subsequent activation of mTOR complex 1 (mTORC1). Amino acids stimulate recruitment of mTORC1 to the lysosomal surface, where its direct activator,Ragulator(a family of four GTPases that are related to Ras, RagGTPases) was associatedtightly with the V-ATPase[69, 70].
V-ATPase-mediated acidification can affect signaling in thefollowing ways: (a) Maturation of signaling molecules Notch receptor and TGF-βbyfurin glycosylationin the golgi vesicles. (b) Cleavage and activationof pathway mediators by acid-dependent enzyme like matrix metallo proteinases (MMPs) and γ-secretase. (c) Maintenance of basal signaling byrecycling endocytosis of both ligand and receptor. (d)Degradation of signaling molecules in lysosomes. In our research, by RNA-seq analysis of transcription factors that arepotentially involved in induction of DR5 expression. We found thatV-ATPase overexpression induced down-regulation of phosphorylation/activation of JNK (c-Jun N-terminal kinase, MAPK8) signaling molecules.Instead, phosphorylation of JNK1 andthe nuclear translocation of c-Jun were markedly promotedby V-ATPase deficiency, which was consistent withtheactivation of JNK signaling in glial cells, monocyte-derived macrophages and RAW 264[71–73]. Moreover, some other studies have shown that V-ATPase inhibits JNK pathway in drosophila epithelium, mouse bone marrow macrophages and osteoclast [74–76]. The exact mechanism by which V-ATPase acts on glycosylation, MMPs, γ-secretase, endocytosis and degradation in V-ATPase-mediated JNK signaling still needs further investigation.
The JNK, as a MAPK member, regulates various cell functions, such as proliferation, apoptosis, and differentiation[77]. The mechanism underlying JNK-regulated DR-apoptotic pathway has been largely investigated in cancer cells. It has been demonstrated that erinacine A induces apoptosis by acting JNK, p300, and NFκBp50signaling molecules and an increasingthe cellular transcriptional levels of TNFR, Fas, and FasL[78]. The attenuation of ERK1/2 phosphorylation accompanied by the activation of JNK was detected in D. bulbifera ethyl acetate fraction (DBEAF)-induced activation of death receptor, Fas and HCT116 cell apoptosis [79]. The capsular polysaccharide induced extrinsic cell apoptosis by up-regulating FAS/FASL signaling proteins and cleaved-caspase-8 and promoted a ROS-dependent intrinsic cell apoptosis by activating a JNK and p38 signaling but not ERK1/2 signaling of mitogen-activated protein kinases (MAPK) pathways[80]. The JNK inhibition was validated to block irradiation-induced FasL expression, which was critical in determining non-irradiated hepatocyte injury[81].Short hairpin RNA (shRNA)-mediated knockdown of JNK confirmed its key role in the regulation of sensitivity to this combination as cells with suppressed JNK expression exhibited significantly reduced TRAIL/sunitinib-mediatedcolon cancer apoptosis[82]. Subcutaneous tumor growth analysis revealed that Mucosa-associated lymphoma antigen 1 (a lymphoma oncogene, MALT1) gene silencing significantly increased melanoma apoptosis and forced expression of the c-Jun upstream activator MKK7[83]. Quercetin activated c-Jun N-terminal kinase (JNK) in a dose-dependent manner, which in turn induced the proteasomal degradation of cFLIP, and JNK activation also sensitized pancreatic cancer cells to TRAIL-induced apoptosis[84].Tanshinone IIA (antitumor drug, TIIA) promoted JNK-mediated signaling to up-regulated CHOP and thereby inducing DR5 expression as shown by the ability of a JNK inhibitor to potently suppress the TIIA-mediated activation of CHOP and DR5[36].Saikosaponin D (antitumor drug) alone or in combination with SP600125 (JNK inhibitor) activated caspase-3, -8 in human osteosarcoma U2 cells[85]. Inthe present study,the activation of JNK/c-Jun in spermatocyte was enhanced by V-ATPase deficiency in vitro, while inhibitionof JNK phosphorylation alleviated spermatocyte apoptosis, therebyindicating that V-ATPase attenuates spermatocyte apoptosis,at least in part, via the suppression of the JNK/c-Jun pathwayafter hypoxia exposure.
Emerging studies demonstrated thatJNK signaling regulates non-cancer cell apoptosis induced by ischemic hypoxia, including neuronal, astrocyte and cardiomyocyte [86–88]. Various JNK-related synthetic inhibitors have been reported in ischemic hypoxia injury, such as micromolecules SP600125 and IQ‐1S. It has been reported that SP600125 treatment inhibits JNK activation and provides neuroprotection in ischemia/reperfusion via inhibiting neuronal apoptosis[89]. IQ‐1S releases nitric oxide in the course of redox biological transformation process and improves the results of stroke in a cerebral reperfusion mouse model[90]. In this study, our data provides evidence that V-ATPase deficiency leads to increased phosphorylation of JNK/c-Jun in GC-2 cells under hypoxia exposure. In addition, the V-ATPase deficiency-induced phosphorylation of JNK/c-Jun and the cell apoptosis activity was reduced when c-Jun was inhibited by RNA interference (RNAi). Our data suggest that JNK activation might be a key event in V-ATPase-mediated apoptosis in GC-2 cells.
In conclusion, V-ATPase deficiency aggravates spermatogenesis deficits under hypoxia exposure, which may be due to the exacerbation ofspermatocyte apoptosis. The aggravated spermatocyte damage is associated withenhanced DR-apoptotic pathway activation, which is mediated by V-ATPase via the JNK/c-Jun signal. These results demonstrate the protective role of the V-ATPase against spermatocyteinjury andprovide evidence for the exploration of V-ATPase-based treatmentsfor hypoxia-induced spermatogenesis reduction(Figure 8).