Search Results (137)

Calcium (Ca²⁺) signaling is a fundamental regulatory mechanism controlling essential processes in the endothelium, vascular smooth muscle cells (VSMCs), and the extracellular matrix (ECM), including maintaining the endothelial barrier, modulation of vascular tone, and vascular remodeling. Cytosolic free Ca²⁺ concentration is tightly regulated by a balance between Ca²⁺ mobilization mechanisms, including Ca²⁺ release from the intracellular stores in the sarcoplasmic/endoplasmic reticulum and Ca²⁺ entry via voltage-dependent, transient-receptor potential, and store-operated Ca²⁺ channels, and Ca²⁺ elimination pathways including Ca²⁺ extrusion by the plasma membrane Ca²⁺-ATPase and Na⁺/Ca²⁺ exchanger and Ca²⁺ re-uptake by the sarco(endo)plasmic reticulum Ca²⁺-ATPase and the mitochondria. Some cell membranes/organelles are multifunctional and have both Ca²⁺ mobilization and Ca²⁺ removal pathways. Also, the individual Ca²⁺ handling pathways could be integrated to function in a regenerative, capacitative, cooperative, bidirectional, or reciprocal feed-forward or feed-back manner. Disruption of these pathways causes dysregulation of the Ca²⁺ signaling dynamics and leads to pathological cardiovascular conditions such as hypertension, coronary artery disease, atherosclerosis, and vascular calcification. In the endothelium, dysregulated Ca²⁺ signaling impairs nitric oxide production, reduces vasodilatory capacity, and increases vascular permeability. In VSMCs, Ca²⁺-dependent phosphorylation of the myosin light chain and Ca²⁺ sensitization by protein kinase-C (PKC) and Rho-kinase (ROCK) increase vascular tone and could lead to increased blood pressure and hypertension. Ca²⁺ activation of matrix metalloproteinases causes collagen/elastin imbalance and promotes vascular remodeling. Ca²⁺-dependent immune cell activation, leukocyte infiltration, and cholesterol accumulation by macrophages promote foam cell formation and atherosclerotic plaque progression. Chronic increases in VSMCs Ca²⁺ promote phenotypic switching to mesenchymal cells and osteogenic transformation and thereby accelerate vascular calcification and plaque instability. Emerging therapeutic strategies targeting these Ca²⁺-dependent mechanisms, including Ca²⁺ channel blockers and PKC and ROCK inhibitors, hold promise for restoring Ca²⁺ homeostasis and mitigating vascular disease progression. Full article

Rap1A and Rap1B are closely related small GTPases that regulate endothelial adhesion, vascular integrity, and signaling pathways via effector domain interactions, with downstream effectors controlling integrins and cadherins. Although both isoforms are essential for vascular development, recent studies using endothelial-specific knockout models have uncovered distinct, non-redundant functions. Rap1B is a key regulator of VEGFR2 signaling, promoting angiogenesis, nitric oxide production, and immune evasion in tumors while restraining proinflammatory signaling in atherosclerosis. In contrast, Rap1A unexpectedly functions as a modulator of endothelial calcium homeostasis by restricting Orai1-mediated store-operated calcium entry, thereby limiting inflammatory responses and vascular permeability. New insights into Rap1 regulation highlight the roles of context-specific guanine nucleotide exchange factors, such as RasGRP3, and non-degradative ubiquitination in effector selection. Emerging data suggest that isoform-specific interactions between the Rap1 hypervariable regions and plasma membrane lipids govern their localization to distinct nanodomains, potentially influencing downstream signaling specificity. Together, these findings redefine the roles of Rap1A and Rap1B in endothelial biology and highlight their relevance in diseases such as tumor angiogenesis, atherosclerosis, and inflammatory lung injury. We discuss the therapeutic implications of targeting Rap1 isoforms in vascular pathologies and cancer, emphasizing the need for isoform-specific strategies that preserve endothelial homeostasis. Full article

The endocannabinoid system (ECS) is known to regulate crucial bodily functions, including healthy muscle activity. However, its precise roles in normal skeletal muscle function and the development of muscle disorders remain unclear. Previously, we developed a tamoxifen-inducible, skeletal muscle-specific CB₁ receptor knockdown (skmCB1-KD) mouse model using the Cre/LoxP system. In this study, we aimed to clarify the mechanisms behind the observed reduction in muscle force generation in these mice. To investigate this, we analyzed calcium dynamics following electrical stimulation-induced muscle fatigue, assessed store-operated calcium entry (SOCE), and performed functional analysis of mitochondrial respiration. Our findings suggest that the reduced muscle performance observed in vivo likely arises from interconnected alterations in ATP production by mitochondria. Moreover, in skmCB1-KD mice, we detected a significant decrease in a component of the respiratory chain (complex IV) and a slowed dissipation of mitochondrial membrane potential upon the addition of an un-coupler (FCCP). Full article

Mechanogated (MG) ion channels play a crucial role in mechano-transduction and immune cell regulation, yet their impact on blood cancers, particularly acute lymphoblastic leukemia (ALL), remains poorly understood. This study investigates the pharmacological effects of GsMTx4, an MG channel inhibitor, in human ALL cells both in vitro and in vivo. Unexpectedly, we found that GsMTx4 remarkably increased basal calcium (Ca²⁺) levels in ALL cells through constitutive Ca²⁺ entry and enhanced store-operated Ca²⁺ influx upon thapsigargin stimulation. This increase in basal Ca²⁺ signaling promoted ALL cell viability and proliferation in vitro. Notably, chelating intracellular Ca²⁺ with BAPTA-AM reduces GsMTx4-mediated leukemia cell viability and proliferation. However, in vivo, GsMTx4 decreases cytosolic Ca²⁺ levels in Nalm-6 GFP⁺ cells isolated from mouse blood, effectively countering leukemia progression and significantly extending survival in NSG mice transplanted with leukemia cells (median survival: GsMTx4 vs. control, 37.5 days vs. 29 days, p = 0.0414). Our results highlight the different properties of GsMTx4 activity in in vitro and in vivo models. They also emphasize that Ca²⁺ signaling is a key vulnerability in leukemia, where its precise modulation dictates disease progression. Thus, targeting Ca²⁺ channels could offer a novel therapeutic strategy for leukemia by exploiting Ca²⁺ homeostasis. Full article

Ryanodine receptors (RyRs) are large intracellular Ca²⁺ release channels primarily found in muscle and nerve cells and also present at low levels in pancreatic islet endocrine cells. This review examines the role of RyRs in islet cell function, focusing on calcium signaling and hormone secretion, while addressing the ongoing debate regarding their significance due to their limited expression. We explore conflicting experimental results and their potential causes, synthesizing current knowledge on RyR isoforms in islet cells, particularly in beta and delta cells. The review discusses how RyR-mediated calcium-induced calcium release enhances, rather than drives, glucose-stimulated insulin secretion. We examine the phosphorylation-dependent regulation of beta-cell RyRs, the concept of “leaky ryanodine receptors”, and the roles of RyRs in endoplasmic reticulum stress, apoptosis, store-operated calcium entry, and beta-cell electrical activity. The relationship between RyR dysfunction and the development of impaired insulin secretion in diabetes is assessed, noting their limited role in human diabetes pathogenesis given the disease’s polygenic nature. We highlight the established role of RyR-mediated CICR in the mechanism of action of common type 2 diabetes treatments, such as glucagon-like peptide-1, which enhances insulin secretion. By integrating findings from electrophysiological, molecular, and clinical studies, this review provides a balanced perspective on RyRs in islet cell physiology and pathology, emphasizing their significance in both normal insulin secretion and current diabetes therapies. Full article

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype lacking estrogen, progesterone, and HER2 receptors, and is associated with poor prognosis and limited targeted therapeutic options. TP53 mutations occur in the majority of TNBC cases, disrupting p53’s role in DNA repair and apoptosis. Beyond gene regulation, p53 also influences calcium signalling through store-operated calcium entry (SOCE), a critical pathway for cell survival and death. However, the impact of different TP53 mutation types on calcium signalling remains unclear. Methods: Calcium channel gene expression was analysed using publicly available TNBC datasets. Calcium channel expression and SOCE activity were assessed in TNBC cell lines with different TP53 mutations using quantitative PCR and calcium imaging (Fura-2AM). Cell proliferation was measured using acid phosphatase assays, while apoptosis was evaluated through caspase 3/7 activation using the Incucyte live-cell fluorescent imager. The p53 reactivator COTI-2 was tested for its ability to restore TP53 function and modulate calcium signalling. Results: Analysis revealed significant downregulation of CACNA1D in TP53-mutant TNBCs. TNBC cell lines harbouring frameshift and stop TP53 mutations exhibited reduced SOCE, lower CACNA1D expression, and resistance to thapsigargin-induced apoptosis compared to wild-type cells. In contrast, cells with the TP53 R273H missense mutation demonstrated similar calcium signalling and proliferation to TP53 wild-type cels. COTI-2 treatment restored CACNA1D expression and SOCE in frameshift and stop mutant cells, enhancing apoptotic sensitivity. Combined treatment with COTI-2 and thapsigargin resulted in a synergistic increase in apoptosis. Conclusions: This study identifies a novel link between TP53 mutation type and calcium signalling in TNBC. Reactivating mutant p53 with COTI-2 restores calcium-mediated apoptosis, supporting combination strategies targeting both TP53 dysfunction and calcium signalling. Full article

Multiple Sclerosis (MS) is a chronic autoimmune disorder characterized by demyelination and neuronal damage in the central nervous system. Dysregulation of calcium homeostasis, particularly through the Store-Operated Calcium Entry-Associated Regulatory Factor (SARAF), has been implicated in MS pathogenesis. This study investigated SARAF, STIM1, and Orai1 expression patterns and their relationship to calcium homeostasis in 45 Bahraini MS patients and 45 matched healthy controls using ELISA and real-time PCR analyses. MS patients showed significantly elevated serum SARAF levels in both early (192.26 ± 47.00 pg/mL) and late MS stages (341.47 ± 96.19 pg/mL) compared to controls (129.82 ± 30.82 pg/mL; p < 0.001. SARAF expressions were markedly increased in MS patients (3.829 ± 0.04422 vs. 1 ± 0; p < 0.0001), while STIM1 (0.4324 ± 0.01471) and ORAI1 (0.2963 ± 0.02156) expressions were significantly reduced compared to the controls (p < 0.0001). Intracellular calcium levels were notably elevated in both early and late MS stages. These findings suggest that the progressive elevation of SARAF, coupled with altered STIM1 and ORAI1 expression, may serve as potential biomarkers for MS progression and represent promising therapeutic targets. Full article

Hepatocellular carcinoma (HCC), a highly aggressive liver malignancy, is often associated with disrupted calcium homeostasis. Store-operated calcium entry (SOCE), involving components such as STIM1, Orai1, and SARAF, plays a critical role in calcium signaling and cancer progression. While STIM1 and Orai1 have been extensively studied, SARAF’s role as a negative regulator of SOCE in HCC remains poorly understood. This preliminary study investigated SARAF’s effects on calcium homeostasis, proliferation, and migration in HepG2 liver cancer cells, providing initial evidence of its tumor-suppressive role. SARAF expression was modulated using siRNA knockdown and overexpression plasmids, with validation by qRT-PCR. Functional assays demonstrated that SARAF silencing increased proliferation by 50% and migration by 40% (p < 0.05), while SARAF overexpression reduced proliferation by 50% and migration by 45% (p < 0.01), highlighting its tumor-suppressive role. Intracellular calcium levels, elevated in HepG2 cells, were partially restored by SARAF overexpression, though SARAF silencing did not further disrupt calcium regulation. These findings suggest that SARAF negatively regulates proliferation and migration in HCC, potentially through its role in maintaining calcium homeostasis. SARAF represents a promising therapeutic target in HCC. Future studies should explore the downstream molecular mechanisms governing SARAF’s effects, investigate its role in other cancers, and assess its clinical potential for liver cancer therapy. Full article

High-risk neuroblastoma (HRNB) is an extracranial solid pediatric cancer. Despite the plethora of treatments available for HRNB, up to 65% of patients are refractory or exhibit an initial response to treatment that transitions to therapy-resistant relapse, which is invariably fatal. A key feature that promotes HRNB progression is aberrant calcium (Ca²⁺) signaling. Ca²⁺ signaling is regulated by several druggable channel proteins, offering tremendous therapeutic potential. Unfortunately, many of the Ca²⁺ channels in HRNB also perform fundamental functions in normal healthy cells, hence targeting them increases the potential for adverse effects. To overcome this challenge, we sought to identify novel Ca²⁺ signaling pathways that are observed in HRNB but not normal non-cancerous cells with the hypothesis that these novel pathways may serve as potential therapeutic targets. One Ca²⁺ signaling pathway that is deregulated in HRNB is store-operated Ca²⁺ entry (SOCE). SOCE relays the release of Ca²⁺ from the endoplasmic reticulum (ER) and Ca²⁺ influx via the plasma membrane and promotes cancer drug resistance by regulating transcriptional programming and the induction of mitochondrial Ca²⁺ (mtCa²⁺)-dependent signaling. mtCa²⁺ signaling is critical for cellular metabolism, reactive oxygen production, cell cycle, and proliferation and has a key role in the regulation of cell death. Therefore, a dynamic interplay between ER, SOCE, and mitochondria tightly regulates cell survival and apoptosis. From a library of synthesized novel molecules, we identified two structurally related compounds that uniquely disrupt the dynamic interplay between SOCE, ER, and mitochondrial signaling pathways and induce cell death in HRNB. Our results revealed that compounds 248 and 249 activate distinct aberrant Ca²⁺ signals that are unique to relapsed HRNB and could be exploited to induce mtCa⁺ overload, a novel calcium influx current, and subsequent cell death. These findings establish a potential new pathway of calcium-mediated cell death; targeting this pathway could be critical for the treatment of refractory and relapsed HRNB. Full article

α-Latrotoxin (αLTX) causes exhaustive release of neurotransmitters from nerve terminals in the absence of extracellular Ca²⁺ (Ca²⁺_e). To investigate the mechanisms underlying this effect, we loaded mouse neuromuscular junctions with BAPTA-AM. This membrane-permeable Ca²⁺-chelator demonstrates that Ca²⁺_e-independent effects of αLTX require an increase in cytosolic Ca²⁺ (Ca²⁺_cyt). We also show that thapsigargin, which depletes Ca²⁺ stores, induces neurotransmitter release, but inhibits the effect of αLTX. We then studied αLTX’s effects on Ca²⁺_cyt using neuroblastoma cells expressing signaling-capable or signaling-incapable variants of latrophilin-1, a G protein-coupled receptor of αLTX. Our results demonstrate that αLTX acts as a cation ionophore and a latrophilin agonist. In model cells at 0 Ca²⁺_e, αLTX forms membrane pores and allows the influx of Na⁺; this reverses the Na⁺-Ca²⁺ exchanger, leading to the release of stored Ca²⁺ and inhibition of its extrusion. Concurrently, αLTX stimulates latrophilin signaling, which depletes a Ca²⁺ store and induces transient opening of Ca²⁺ channels in the plasmalemma that are sensitive to inhibitors of store-operated Ca²⁺ entry. These results indicate that Ca²⁺ release from intracellular stores and that Ca²⁺ influx through latrophilin-activated store-operated Ca²⁺ channels contributes to αLTX actions and may be involved in physiological control of neurotransmitter release at nerve terminals. Full article

Microglia, the unique and motile immune cells of the central nervous system (CNS), function as a security guard in maintaining CNS homeostasis, primarily through calcium signaling. The calcium dynamics in microglia control important functions such as phagocytosis, cytokine release, and migration. Calcium dysregulation in microglia has been linked to several CNS disorders, like Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), and ischemic stroke (IS). Calcium entering through channels such as voltage-gated calcium channels (VGCCs), store-operated calcium entry (SOCE), and transient receptor potential (TRP) channels is essential for microglial activation and pro-inflammatory responses. Under pathological conditions, like the formation of amyloid-β plaques in AD, aggregation of α-synuclein in PD, and oxidative stress in MS, calcium dysregulation exacerbates neuroinflammation, mitochondrial dysfunction, and neurodegeneration. Therapeutic strategies targeting calcium signaling pathways, using calcium channel blockers and antioxidant interventions, show promise for alleviating microglial activation and slowing down disease progression. This review summarizes the underlying mechanisms of microglial calcium dysregulation and potential therapeutic benefits for restoring microglial calcium balance in CNS disorders. Full article

Calcium is an important second messenger that is involved in almost all cellular processes. Disruptions in the regulation of intracellular Ca²⁺ levels ([Ca²⁺]_i) adversely impact normal physiological function and can contribute to various diseased conditions. STIM and Orai proteins play important roles in maintaining [Ca²⁺]_i through store-operated Ca²⁺ entry (SOCE), with STIM being the primary regulatory protein that governs the function of Orai channels. STIM1 and STIM2 are single-pass ER-transmembrane proteins with their N- and C-termini located in the ER lumen and cytoplasm, respectively. The N-terminal EF-SAM domain of STIMs senses [Ca²⁺]_ER changes, while the C-terminus mediates clustering in ER-PM junctions and gating of Orai1. ER-Ca²⁺ store depletion triggers activation of the STIM proteins, which involves their multimerization and clustering in ER-PM junctions, where they recruit and activate Orai1 channels. In this review, we will discuss the structure, organization, and function of EF-hand motifs and the SAM domain of STIM proteins in relation to those of other eukaryotic proteins. Full article

Extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER)-associated proteins that facilitate the tethering of the ER to the plasma membrane (PM), participating in lipid transfer between the membranes and supporting the Orai1–STIM1 interaction at ER–PM junctions. Orai1 and STIM1 are the core proteins of store-operated Ca²⁺ entry (SOCE), a major mechanism for Ca²⁺ influx that regulates a variety of cellular functions. Aberrant modulation of SOCE in cells from different types of cancer has been reported to underlie the development of several tumoral features. Here we show that estrogen receptor-positive (ER+) breast cancer MCF7 and T47D cells and triple-negative breast cancer (TNBC) MDA-MB-231 cells overexpress E-Syt1 and E-Syt2 at the protein level; the latter is also overexpressed in the TNBC BT20 cell line. E-Syt1 and E-Syt2 knockdown was without effect on SOCE in non-tumoral MCF10A breast epithelial cells and ER+ T47D breast cancer cells; however, SOCE was significantly attenuated in ER+ MCF7 cells and TNBC MDA-MB-231 and BT20 cells upon transfection with siRNA E-Syt1 or E-Syt2. Consistent with this, E-Syt1 and E-Syt2 knockdown significantly reduced cell migration and viability in ER+ MCF7 cells and the TNBC cells investigated. To summarize, E-Syt1 and E-Syt2 play a relevant functional role in breast cancer cells. Full article

Sensing the lowering of endoplasmic reticulum (ER) calcium (Ca²⁺), STIM1 mediates a ubiquitous Ca²⁺ influx process called the store-operated Ca²⁺ entry (SOCE). Dysregulated STIM1 function or abnormal SOCE is strongly associated with autoimmune disorders, atherosclerosis, and various forms of cancers. Therefore, uncovering the molecular intricacies of post-translational modifications, such as oxidation, on STIM1 function is of paramount importance. In a recent proteomic screening, we identified three protein disulfide isomerases (PDIs)—Prolyl 4-hydroxylase subunit beta (P4HB), protein disulfide-isomerase A3 (PDIA3), and thioredoxin domain-containing protein 5 (TXNDC5)—as the ER-luminal interactors of STIM1. Here, we demonstrated that these PDIs dynamically associate with STIM1 and STIM2. The mutation of the two conserved cysteine residues of STIM1 (STIM1-2CA) decreased its Ca²⁺ affinity both in cellulo and in situ. Knockdown of PDIA3 or P4HB increased the Ca²⁺ affinity of wild-type STIM1 while showing no impact on the STIM1-2CA mutant, indicating that PDIA3 and P4HB regulate STIM1’s Ca²⁺ affinity by acting on ER-luminal cysteine residues. This modulation of STIM1’s Ca²⁺ sensitivity was further confirmed by Ca²⁺ imaging experiments, which showed that knockdown of these two PDIs does not affect STIM1-mediated SOCE upon full store depletion but leads to enhanced SOCE amplitudes upon partial store depletion. Thus, P4HB and PDIA3 dynamically modulate STIM1 activation by fine-tuning its Ca²⁺ binding affinity, adjusting the level of activated STIM1 in response to physiological cues. The coordination between STIM1-mediated Ca²⁺ signaling and redox responses reported herein may have implications for cell physiology and pathology. Full article

In calcium nephrolithiasis (CaNL), most calcium kidney stones are identified as calcium oxalate (CaOx) with variable amounts of calcium phosphate (CaP), where CaP is found as the core component. The nucleation of CaP could be the first step of CaP+CaOx (mixed) stone formation. High urinary supersaturation of CaP due to hypercalciuria and an elevated urine pH have been described as the two main factors in the nucleation of CaP crystals. Our previous in vivo findings (in mice) show that transient receptor potential canonical type 3 (TRPC3)-mediated Ca²⁺ entry triggers a transepithelial Ca²⁺ flux to regulate proximal tubular (PT) luminal [Ca²⁺], and TRPC3-knockout (KO; -/-) mice exhibited moderate hypercalciuria and microcrystal formation at the loop of Henle (LOH). Therefore, we utilized TRPC3 KO mice and exposed them to both hypercalciuric [2% calcium gluconate (CaG) treatment] and alkalineuric conditions [0.08% acetazolamide (ACZ) treatment] to generate a CaNL phenotype. Our results revealed a significant CaP and mixed crystal formation in those treated KO mice (KOT) compared to their WT counterparts (WTT). Importantly, prolonged exposure to CaG and ACZ resulted in a further increase in crystal size for both treated groups (WTT and KOT), but the KOT mice crystal sizes were markedly larger. Moreover, kidney tissue sections of the KOT mice displayed a greater CaP and mixed microcrystal formation than the kidney sections of the WTT group, specifically in the outer and inner medullary and calyceal region; thus, a higher degree of calcifications and mixed calcium lithiasis in the kidneys of the KOT group was displayed. In our effort to find the Ca²⁺ signaling pathophysiology of PT cells, we found that PT cells from both treated groups (WTT and KOT) elicited a larger Ca²⁺ entry compared to the WT counterparts because of significant inhibition by the store-operated Ca²⁺ entry (SOCE) inhibitor, Pyr6. In the presence of both SOCE (Pyr6) and ROCE (receptor-operated Ca²⁺ entry) inhibitors (Pyr10), Ca²⁺ entry by WTT cells was moderately inhibited, suggesting that the Ca²⁺ and pH levels exerted sensitivity changes in response to ROCE and SOCE. An assessment of the gene expression profiles in the PT cells of WTT and KOT mice revealed a safeguarding effect of TRPC3 against detrimental processes (calcification, fibrosis, inflammation, and apoptosis) in the presence of higher pH and hypercalciuric conditions in mice. Together, these findings show that compromise in both the ROCE and SOCE mechanisms in the absence of TRPC3 under hypercalciuric plus higher tubular pH conditions results in higher CaP and mixed crystal formation and that TRPC3 is protective against those adverse effects. Full article