Regulatory mechanisms of the store-operated Ca2+ entry through Orai1 and STIM1 by an adaptor protein in non-excitable cells

Store-operated Ca2+ entry (SOCE) represents one of the major Ca2+ entry routes in non-excitable cells. It is involved in a variety of fundamental biological processes and the maintenance of Ca2+ homeostasis. The Ca2+ releaseactivated Ca2+ (CRAC) channel consists of stromal interaction molecule and Orai; however, the role and action of Homer proteins as an adaptor protein to SOCE-mediated Ca2+ signaling through the activation of CRAC channels in non-excitable cells still remain unknown. In the present study, we investigated the role of Homer2 in the process of Ca2+ signaling induced by the interaction between CRACs and Homer2 proteins in non-excitable cells. The response to Ca2+ entry by thapsigargin-mediated Ca2+ store depletion remarkably decreased in pancreatic acinar cells of Homer2–/– mice, as compared to wild-type cells. It also showed critical differences in regulated patterns by the specific blockers of SOCE in pancreatic acinar cells of Homer2–/– mice. The response to Ca2+ entry by the depletion in Ca2+ store markedly increased in the cellular overexpression of Orai1 and STIM1 as compared to the overexpression of Homer2 in cells; however, this response was remarkably inhibited by the overexpression of Orai1, STIM1, and Homer2. These results suggest that Homer2 has a critical role in the regulatory action of SOCE activity and the interactions between CRAC channels.ope

IL-1ra Secreted by ATP-Induced P2Y2 Negatively Regulates MUC5AC Overproduction via PLCβ3 during Airway Inflammation

Mucus secretion is often uncontrolled in many airway inflammatory diseases of humans. Identifying the regulatory pathway(s) of mucus gene expression, mucus overproduction, and hypersecretion is important to alleviate airway inflammation in these diseases. However, the regulatory signaling pathway controlling mucus overproduction has not been fully identified yet. In this study, we report that the ATP/P2Y2 complex secretes many cytokines and chemokines to regulate airway inflammation, among which IL-1 receptor antagonist (IL-1ra) downregulates MUC5AC gene expression via the inhibition of Gαq-induced Ca(2+) signaling. IL-1ra inhibited IL-1α protein expression and secretion, and vice versa. Interestingly, ATP/P2Y2-induced IL-1ra and IL-1α secretion were both mediated by PLCβ3. A dominant-negative mutation in the PDZ-binding domain of PLCβ3 inhibited ATP/P2Y2-induced IL-1ra and IL-1α secretion. IL-1α in the presence of the ATP/P2Y2 complex activated the ERK1/2 pathway in a greater degree and for a longer duration than the ATP/P2Y2 complex itself, which was dramatically inhibited by IL-1ra. These findings suggest that secreted IL-1ra exhibits a regulatory effect on ATP/P2Y2-induced MUC5AC gene expression, through inhibition of IL-1α secretion, to maintain the mucus homeostasis in the airway. Therefore, IL-1ra could be an excellent modality for regulating inflamed airway microenvironments in respiratory diseases.ope

Homer2 regulates amylase secretion via physiological calcium oscillations in mouse parotid gland acinar cells

The salivary glands secrete saliva, which plays a role in the maintenance of a healthy oral environment. Under physiological conditions, saliva secretion within the acinar cells of the gland is regulated by stimulation of specific calcium (Ca2+) signaling mechanisms such as increases in the intracellular Ca2+ concentration ([Ca2+]i) via storeoperated Ca2+ entry, which involves components such as Orai1, transient receptor potential (TRP) canonical 1, stromal interaction molecules, and inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs). Homer proteins are scaffold proteins that bind to G protein-coupled receptors, IP3Rs, ryanodine receptors, and TRP channels. However, their exact role in Ca2+ signaling in the salivary glands remains unknown. In the present study, we investigated the role of Homer2 in Ca2+ signaling and saliva secretion in parotid gland acinar cells under physiological conditions. Deletion of Homer2 (Homer2−/−) markedly decreased the amplitude of [Ca2+]i oscillations via the stimulation of carbachol, which is physiologically concentrated in parotid acinar cells, whereas the frequency of [Ca2+]i oscillations showed no difference between wild-type and Homer2−/− mice. Homer2−/− mice also showed a significant decrease in amylase release by carbachol in the parotid gland in a dose-dependent manner. These results suggest that Homer2 plays a critical role in maintaining [Ca2+]i concentration and secretion of saliva in mouse parotid gland acinar cells.ope

Regulation of the expression and function of TRPCs and Orai1 by Homer2 in mouse pancreatic acinar cells

Under physiological conditions, calcium (Ca2+) regulates essential functions of polarized secretory cells by the stimulation of specific Ca2+ signaling mechanisms, such as increases in intracellular Ca2+ concentration ([Ca2+]i) via the store-operated Ca2+ entry (SOCE) and the receptor-operated Ca2+ entry (ROCE). Homer proteins are scaffold proteins that interact with G protein-coupled receptors, inositol 1,4,5-triphosphate (IP3) receptors, Orai1-stromal interaction molecule 1, and transient receptor potential canonical (TRPC) channels. However, their role in the Ca2+ signaling in exocrine cells remains unknown. In this study, we investigated the role of Homer2 in the Ca2+ signaling and regulatory channels to mediate SOCE and ROCE in pancreatic acinar cells. Deletion of Homer2 (Homer2?/?) markedly increased the expression of TRPC3, TRPC6, and Orai1 in pancreatic acinar cells, whereas these expressions showed no difference in whole brains of wild-type and Homer2?/? mice. Furthermore, the response of Ca2+ entry by carbachol also showed significant changes to the patterns regulated by specific blockers of SOCE and ROCE in pancreatic acinar cells of Homer2?/? mice. Thus, these results suggest that Homer2 plays a critical role in the regulatory action of the [Ca2+]i via SOCE and ROCE in mouse pancreatic acinar cells.ope

Deficiencies of Homer2 and Homer3 accelerate aging-dependent bone loss in mice

Homer proteins are scaffold proteins that regulate calcium (Ca2+) signaling by modulating the activity of multiple Ca2+ signaling proteins. In our previous report, Homer2 and Homer3 regulated NFATc1 function through its interaction with calcineurin, which then acted to regulate receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and bone metabolism. However, to date, the role of Homers in osteoclastogenesis remains unknown. In this study, we investigated the roles of Homer2 and Homer3 in aging-dependent bone remodeling. Deletion of Homer2 /Homer3 (Homer2/3 DKO) markedly decreased the bone density of the femur. The decrease in bone density was not seen in mice with Homer2 (Homer2−/−) and Homer3 (Homer3−/−) deletion. Moreover, RANKL treatment of bone marrow-derived monocytes/macrophages in Homer2/3 DKO mice significantly increased the formation of multinucleated cells and resorption areas. Finally, Homer2/3 DKO mice decreased bone density in an aging-dependent manner. These findings suggest a novel potent mode of bone homeostasis regulation through osteoclasts differentiation during aging by Homer proteins, specifically Homer2 and Homer3.ope

The Role of Ca 2+-NFATc1 Signaling and Its Modulation on Osteoclastogenesis

The increasing of intracellular calcium concentration is a fundamental process for mediating osteoclastogenesis, which is involved in osteoclastic bone resorption. Cytosolic calcium binds to calmodulin and subsequently activates calcineurin, leading to NFATc1 activation, a master transcription factor required for osteoclast differentiation. Targeting the various activation processes in osteoclastogenesis provides various therapeutic strategies for bone loss. Diverse compounds that modulate calcium signaling have been applied to regulate osteoclast differentiation and, subsequently, attenuate bone loss. Thus, in this review, we summarized the modulation of the NFATc1 pathway through various compounds that regulate calcium signaling and the calcium influx machinery. Furthermore, we addressed the involvement of transient receptor potential channels in osteoclastogenesis.ope

Sestrin2 Regulates Osteoclastogenesis via the p62-TRAF6 Interaction

The receptor activator of nuclear factor-kappa B ligand (RANKL) mediates osteoclast differentiation and functions by inducing Ca2+ oscillations, activating mitogen-activated protein kinases (MAPKs), and activating nuclear factor of activated T-cells type c1 (NFATc1) via the RANK and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) interaction. Reactive oxygen species (ROS) also plays an important role during osteoclastogenesis and Sestrin2, an antioxidant, maintains cellular homeostasis upon stress injury via regulation of ROS, autophagy, and inflammation. However, the role of Sestrin2 in osteoclastogenesis remains unknown. In this study, we investigated the role of Sestrin2 in the RANKL-RANK-TRAF6 signaling pathway during osteoclast differentiation. Deletion of Sestrin2 (Sesn2) increased bone mass and reduced the number of multinucleated osteoclasts on bone surfaces. RANKL-induced osteoclast differentiation and function decreased in Sesn2 knockout (KO) bone marrow-derived monocytes/macrophages (BMMs) due to inhibition of NFATc1 expression, but osteoblastogenesis was not affected. mRNA expression of RANKL-induced specific osteoclastogenic genes and MAPK protein expression were lower in Sesn2 KO BMMs than wild-type (WT) BMMs after RANKL treatment. However, the Sesn2 deletion did not affect ROS generation or intracellular Ca2+ oscillations during osteoclastogenesis. In contrast, the interaction between TRAF6 and p62 was reduced during osteoclasts differentiation in Sesn2 KO BMMs. The reduction in the TRAF6/p62 interaction and TRAP activity in osteoclastogenesis in Sesn2 KO BMMs was recovered to the WT level upon expression of Flag-Sesn2 in Sesn2 KO BMMs. These results suggest that Sestrin2 has a novel role in bone homeostasis and osteoclasts differentiation through regulation of NFATc1 and the TRAF6/p62 interaction.ope

Activation of G Proteins by Aluminum Fluoride Enhances RANKLMediated Osteoclastogenesis

Receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis is accompanied by intracellular Ca2+ mobilization in a form of oscillations, which plays essential roles by activating sequentially Ca2+/calmodulin-dependent protein kinase, calcineurin and NFATc1, necessary in the osteoclast differentiation. However, it is not known whether Ca2+ mobilization which is evoked in RANKL-independent way induces to differentiate into osteoclasts. In present study, we investigated Ca2+ mobilization induced by aluminum fluoride (AlF4-), a G-protein activator, with or without RANKL and the effects of AlF4- on the osteoclastogenesis in primary cultured mouse bone marrow-derived macrophages (BMMs). We show here that AlF4- induces intracellular Ca2+ concentration ([Ca2+]i) oscillations, which is dependent on extracellular Ca2+ influx. Notably, co-stimulation of AlF4- with RANKL resulted in enhanced NFATc1 expression and formation of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells. Additionally, we confirmed that mitogen-activated protein kinase (MAPK) is also activated by AlF4-. Taken together, these results demonstrate that G-protein would be a novel modulator responsible for [Ca2+]i oscillations and MAPK activation which lead to enhancement of RANKL-mediated osteoclastogenesis.ope

TRPM7 Is Essential for RANKL-Induced Osteoclastogenesis

The transient receptor potential melastatin type 7 (TRPM7) channel is a widely expressed non-selective cation channel with fusion to the C-terminal alpha kinase domain and regarded as a key regulator of whole body Mg2+ homeostasis in mammals. However, the roles of TRPM7 during osteoclastogenesis in RAW264.7 cells and bone marrow-derived monocyte/macrophage precursor cells (BMMs) are not clear. In the present study, we investigate the roles of TRPM7 in osteoclastogenesis using methods of small interfering RNA (siRNA), RT-PCR, patch-clamp, and calcium imaging. RANKL (receptor activator of NF-κB ligand) stimulation did not affect the TRPM7 expression and TRPM7-mediated current was activated in HEK293, RAW264.7, and BMM cells by the regulation of Mg2+. Knock-down of TRPM7 by siTRPM7 reduced intracellular Ca2+ concentration ([Ca2+]i) increases by 0 mM [Mg2+]e in HEK293 cells and inhibited the generation of RANKL-induced Ca2+ oscillations in RAW264.7 cells. Finally, knock-down of TRPM7 suppressed RANKL-mediated osteoclastogenesis such as activation and translocation of NFATc1, formation of multinucleated cells, and the bone resorptive activity, sequentially. These results suggest that TRPM7 plays an essential role in the RANKL-induced [Ca2+]i oscillations that triggers the late stages of osteoclastogenesis.ope

DA-6034 Induces [Ca2+]i Increase in Epithelial Cells

DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca2+ signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca2+ signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca2+-activated Cl- channels (CaCCs) and increased intracellular calcium concentrations ([Ca2+]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca2+]i in mouse salivary gland cells and human corneal epithelial cells. [Ca2+]i increase of DA-6034 was dependent on the Ca2+ entry from extracellular and Ca2+ release from internal Ca2+ stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca2+ stores. These results suggest that DA-6034 induces Ca2+ signaling via extracellular Ca2+ entry and RyRs-sensitive Ca2+ release from internal Ca2+ stores in epithelial cells.ope