OBJECTIVE: SERCA2, one of the P-type pumps encoded by gene ATP2A2, is the only calcium reflux channel of the endoplasmic reticulum (ER) and participates in maintaining calcium homeostasis. The present study was designed to explore SERCA2 expression pattern in auditory hair cells and the possible mechanism underlying the effects of SERCA2 on cisplatin-induced ototoxicity.
METHODS: The SERCA2 expression pattern in cochlea hair cells and HEI-OC1 cells was measured by Western blot (WB) and immunofluorescence staining. The apoptosis and its related factors were detected by TUNEL assay and WB. The expression levels of ER stress-related factors, ATF6, PERK, IRE1α, and GRP78, were measured via WB. As for the determination of SERCA2 overexpression and knockdown, plasmids and lentiviral vectors were constructed, respectively.
RESULTS: We found that SERCA2 was highly expressed in cochlea hair cells and HEI-OC1 cells. Of note, the level of SERCA2 expression in neonatal mice was remarkably higher than that in adult mice. Under the exposure of 30 μM cisplatin, SERCA2 was down-regulated significantly compared with the control group. In addition, cisplatin administration triggered the occurrence of ER stress and apoptosis. Those events were reversed by overexpressing SERCA2. On the contrary, SERCA2 knockdown could aggravate the above processes.
CONCLUSIONS: The findings from the present study disclose, for the first time, that SERCA2 is abundantly expressed in cochlea hair cells, and the suppression of SERCA2 caused by cisplatin could trigger ER homeostasis disruption, thereby implying that SERCA2 might be a promising target to prevent cisplatin-induced cytotoxicity of hair cells.

Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) is critical in maintaining Ca2+ homeostasis. The cysteine 674 (C674) is the key redox regulatory cysteine in regulating SERCA2 activity, which is irreversibly oxidized in the renal cortex of hypertensive mice. We have reported that the substitution of C674 by serine causes SERCA2 dysfunction and increases blood pressure by induction of endoplasmic reticulum stress (ERS). This study is to explore whether the dysfunction of SERCA2 causes hypertension by interrupting mitochondrial homeostasis and inducing oxidative stress.
We used heterozygous SERCA2 C674S gene mutation knock-in (SKI) mice, where one copy of C674 was substituted by serine to represent partial C674 oxidation. In renal proximal tubule (RPT) cells, the substitution of C674 by serine decreased mitochondrial Ca2+ content, increased mitochondrial membrane potential, ATP content, and reactive oxygen species (ROS), which could be reversed by ERS inhibitor 4-phenylbutyric acid or SERCA2 agonist CDN1163. In SKI RPT cells, the redox modulator Tempol alleviated oxidative stress, downregulated the protein expression of ERS markers and soluble epoxide hydrolase, upregulated the protein expression of dopamine D1 receptor, and reduced Na+/K+- ATPase activity. In SKI mice, SERCA2 agonists CDN1163 and [6]-Gingerol, or the redox modulator Tempol increased urine output and lowered blood pressure.
The irreversible oxidation of C674 is not only an indicator of increased ROS, but also further inducing oxidative stress to cause hypertension. Activation of SERCA2 or inhibition of oxidative stress is beneficial to alleviate hypertension caused by SERCA2 dysfunction.

UNASSIGNED: Substitution of Cys674 (C674) in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) causes SERCA2 dysfunction which leads to activated inositol requiring enzyme 1 alpha (IRE1α) and spliced X-box binding protein 1 (XBP1s) pathway accelerating cell proliferation of pulmonary artery smooth muscle cells (PASMCs) followed by significant pulmonary vascular remodeling resembling human pulmonary hypertension. Based on this knowledge, we intend to investigate other potential mechanisms involved in SERCA2 dysfunction-induced pulmonary vascular remodeling.
UNASSIGNED: Heterozygous SERCA2 C674S knock-in (SKI) mice of which half of cysteine in 674 was substituted by serine to mimic the partial irreversible oxidation of C674 were used. The lungs of SKI mice and their littermate wild-type mice were collected for PASMC culture, protein expression, and pulmonary vascular remodeling analysis.
UNASSIGNED: SERCA2 dysfunction increased intracellular Ca2+ levels, which activated Ca2+-dependent calcineurin (CaN) and promoted the nuclear translocation and protein expression of the nuclear factor of activated T-lymphocytes 4 (NFAT4) in an IRE1α/XBP1s pathway-independent manner. In SKI PASMCs, the scavenge of intracellular Ca2+ by BAPTA-AM or inhibition of CaN by cyclosporin A can prevent PASMC phenotypic transition. CDN1163, a SERCA2 agonist, suppressed the activation of CaN/NFAT4 and IRE1α/XBP1s pathways, reversed the protein expression of PASMC phenotypic transition markers and cell cycle-related proteins, and inhibited cell proliferation and migration when given to SKI PASMCs. Furthermore, CDN1163 ameliorated pulmonary vascular remodeling in SKI mice.
UNASSIGNED: SERCA2 dysfunction promotes PASMC phenotypic transition and pulmonary vascular remodeling by multiple mechanisms, which could be improved by SERCA2 agonist CDN1163.
‘What is already known’ l The dysfunction of SERCA2 promotes PASMC hyperproliferation and pulmonary vascular remodeling through activation of the IRE1α/XBP1s pathway.‘What this study adds’ l The dysfunction of SERCA2 activates the Ca2+-dependent CaN-mediated NFAT4 pathway to promote the PASMC phenotypic transition.l Revitalization of SERCA2 suppresses PASMC phenotypic transition and pulmonary vascular remodeling caused by SERCA2 dysfunction.‘Clinical significance’ l SERCA2 dysfunction-induced pulmonary vascular remodeling involves more than one mechanism, implicating that more drugable targets are to be discovered.l SERCA2 is a potential therapeutic target for preventing pulmonary vascular remodeling.

The well-known insulin-like growth factor 1 (IGF1)/IGF-1 receptor (IGF-1R) signaling pathway is overexpressed in many tumors, and is thus an attractive target for cancer treatment. However, results have often been disappointing due to crosstalk with other signals. Here, we report that IGF-1R signaling stimulates the growth of hepatocellular carcinoma (HCC) cells through the translocation of IGF-1R into the ER to enhance sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) activity. In response to ligand binding, IGF-1Rβ is translocated into the ER by β-arrestin2 (β-arr2). Mass spectrometry analysis identified SERCA2 as a target of ER IGF-1Rβ. SERCA2 activity is heavily dependent on the increase in ER IGF-1Rβ levels. ER IGF-1Rβ phosphorylates SERCA2 on Tyr990 to enhance its activity. Mutation of SERCA2-Tyr990 disrupted the interaction of ER IGF-1Rβ with SERCA2, and therefore ER IGF-1Rβ failed to promote SERCA2 activity. The enhancement of SERCA2 activity triggered Ca2+ER perturbation, leading to an increase in autophagy. Thapsigargin blocked the interaction between SERCA2 and ER IGF-1Rβ and therefore SERCA2 activity, resulting in inhibition of HCC growth. In conclusion, the translocation of IGF-1R into the ER triggers Ca2+ER perturbation by enhancing SERCA2 activity through phosphorylating Tyr990 in HCC.

Chemoresistance remains a major obstacle to successful treatment of triple negative breast cancer (TNBC). Identification of druggable vulnerabilities is an important aim for TNBC therapy. Here, we report that SERCA2 expression correlates with TNBC progression in human patients, which promotes TNBC cell proliferation, migration and chemoresistance. Mechanistically, SERCA2 interacts with LC3B via LIR motif, facilitating WIPI2-independent autophagosome formation to induce autophagy. Autophagy-mediated SERCA2 degradation induces SERCA2 transactivation through a Ca2+/CaMKK/CREB-1 feedback. Moreover, we found that SERCA2-targeting small molecule RL71 enhances SERCA2-LC3B interaction and induces excessive autophagic cell death. The increase in SERCA2 expression predisposes TNBC cells to RL71-induced autophagic cell death in vitro and in vivo. This study elucidates a mechanism by which TNBC cells maintain their high autophagy activity to induce chemoresistance, and suggests increased SERCA2 expression as a druggable vulnerability for TNBC.

Lathyrol is a natural product isolated from the traditional Chinese medicine Semen Euphorbiae with unknown anti-tumor effects. We found that lathyrol had significant inhibitory effect on lung cancer cells by inducing apoptosis and inhibiting proliferation. Subsequently, we demonstrated for the first time that endoplasmic reticulum (ER) stress is a key anti-tumor mechanism of lathyrol. Furthermore, we found that lathyrol can induce ER stress in lung cancer cells by upregulating the protein expression levels of GRP78, PERK, p-eIF2α, CHOP, and ATF4, and the inhibitory effect of lathyrol on lung cancer cells was significantly reversed when cells were pretreated with ER stress inhibitor. In addition, we found that inhibition of SERCA2 resulted in depletion of the ER Ca2+ pool followed by a sustained increase in cytoplasmic Ca2+ levels, eventually leading to ER stress induced tumor cell apoptosis and proliferation inhibition. Lathyrol targeted SERCA2 to cause a significant upregulation of Ca2+ levels, and the inhibitory effect of lathyrol on lung cancer cells was significantly reversed after pretreatment with SERCA2 agonist. Taken together, our data suggest that lathyrol exerts its anti-tumor effect primarily by targeting SERCA2. Our findings highlight the potential for lathyrol as a new candidate drug for the treatment of lung cancer.

Background: Given the importance of microvascular injury in infarct formation and expansion, development of therapeutic strategies for microvascular protection against myocardial ischemia/reperfusion injury (IRI) is of great interest. Here, we explored the molecular mechanisms underlying the protective effects of the SGLT2 inhibitor dapagliflozin (DAPA) against cardiac microvascular dysfunction mediated by IRI. Methods: DAPA effects were evaluated both in vivo, in mice subjected to IRI, and in vitro, in human coronary artery endothelial cells (HCAECs) exposed to hypoxia/reoxygenation (H/R). DAPA pretreatment attenuated luminal stenosis, endothelial swelling, and inflammation in cardiac microvessels of IRI-treated mice. Results: In H/R-challenged HCAECs, DAPA treatment improved endothelial barrier function, endothelial nitric oxide synthase (eNOS) activity, and angiogenic capacity, and inhibited H/R-induced apoptosis by preventing cofilin-dependent F-actin depolymerization and cytoskeletal degradation. Inhibition of H/R-induced xanthine oxidase (XO) activation and upregulation, sarco(endo)plasmic reticulum calcium-ATPase 2 (SERCA2) oxidation and inactivation, and cytoplasmic calcium overload was further observed in DAPA-treated HCAECs. DAPA also suppressed calcium/Calmodulin (CaM)-dependent kinase II (CaMKII) activation and cofilin phosphorylation, and preserved cytoskeleton integrity and endothelial cell viability following H/R. Importantly, the beneficial effects of DAPA on cardiac microvascular integrity and endothelial cell survival were largely prevented in IRI-treated SERCA2-knockout mice. Conclusions: These results indicate that DAPA effectively reduces cardiac microvascular damage and endothelial dysfunction during IRI through inhibition of the XO-SERCA2-CaMKII-cofilin pathway.

Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) is vital to maintain intracellular calcium homeostasis, and its redox Cys674 (C674) is the key to regulating activity. Our goal was to investigate whether the redox state of SERCA2 C674 is critical for cardiac fibrosis and the mechanisms involved. Heterozygous SERCA2 C674S knock-in (SKI) mice, in which half of C674 was substituted by serine, were used to mimic the partial loss of the reactive C674 thiol in pathological conditions. In cardiac fibroblasts, the substitution of C674 thiol increased Ca2+ levels in cytoplasm and mitochondria, and intracellular ROS levels, and activated calcineurin/nuclear factor of activated T-lymphocytes (NFAT) pathway, increased the protein expression of profibrotic factors TGF beta 1 (TGF-β1), alpha smooth muscle actin, collagen I and collagen III, and promoted the transformation of cardiac fibroblasts to cardiac myofibroblasts, which could be reversed by calcineurin/NFAT inhibitor, SERCA2 agonist, or ROS scavenger. Activation of SERCA2 or scavenging ROS is beneficial to alleviate cardiac fibrosis caused by the substitution of C674. In conclusion, the partial loss of the reactive C674 thiol in the SERCA2 exacerbates cardiac fibrosis by activating the calcineurin/NFAT/TGF-β1 pathway to promote the transformation of cardiac fibroblasts to cardiac myofibroblasts, which highlights the importance of C674 redox state in maintaining the homeostasis of cardiac fibroblasts. SERCA2 is a potential therapeutic target for the treatment of cardiac fibrosis.

The cysteine674 (C674) thiol of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 is easily and irreversibly oxidized under atherosclerotic conditions. However, the contribution of the C674 thiol redox status in the development of atherosclerosis remains unclear. Our goal was to elucidate the possible mechanism involved.
Heterozygous SERCA2 C674S knock-in mice in which half of the C674 was substituted by serine (S674) were used to mimic the removal of the reactive C674 thiol, which occurs under pathological conditions. Bone marrow-derived macrophages (BMDMs) and cardiac endothelial cells (ECs) were used for intracellular Ca2+ , macrophage adhesion, and protein expression analysis. The whole aorta and aortic root were isolated for histological analysis.
Cell culture studies suggest the partial substitution of SERCA2 C674 increased intracellular Ca2+ levels and induced ER stress in both BMDMs and ECs. The release of proinflammatory factors and macrophage adhesion increased in SKI BMDMs. In ECs, overexpression of S674 induced endothelial inflammation and promoted macrophage recruitment. SKI mice developed more severe atherosclerotic plaque and macrophage accumulation. Additionally, 4-phenyl butyric acid, an ER stress inhibitor, suppressed ER stress and inflammatory responses in BMDMs and ECs, and alleviated atherosclerosis in SKI mice.
The substitution of SERCA2 C674 thiol accelerates the development of atherosclerosis by inducing ER stress and inflammation. Our findings highlight the importance of SERCA2 C674 redox state in the context of atherosclerosis and open up a novel therapeutic strategy to combat atherosclerosis.

Sigma-1 receptor (Sigmar1) is a specific chaperone located in the mitochondria-associated endoplasmic reticulum membrane (MAM) and plays a role in several physiological processes. However, the role of Sigmar1 in bone homeostasis remains unknown. Here, we show that mice lacking Sigmar1 exhibited severe osteoporosis in an ovariectomized model. In contrast, overexpression of Sigmar1 locally alleviated the osteoporosis phenotype. Treatment with Sigmar1 agonists impaired both human and mice osteoclast formation in vitro. Mechanistically, SERCA2 was identified to interact with Sigmar1 based on the immunoprecipitation-mass spectrum (IP-MS) and co-immunoprecipitation (co-IP) assays, and Q615 of SERCA2 was confirmed to be the critical residue for their binding. Furthermore, Sigmar1 promoted SERCA2 degradation through Hrd1/Sel1L-dependent ER-associated degradation (ERAD). Ubiquitination of SERCA2 at K460 and K541 was responsible for its proteasomal degradation. Consequently, inhibition of SERCA2 impeded Sigmar1 deficiency enhanced osteoclastogenesis. Moreover, we found that dimemorfan, an FDA-approved Sigmar1 agonist, effectively rescued bone mass in various established bone-loss models. In conclusion, Sigmar1 is a negative regulator of osteoclastogenesis, and activation of Sigmar1 by dimemorfan may be a potential treatment for osteoporosis in clinical practice.