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.

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.

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.

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.

Increased store-operated Ca²⁺ entry mediated by GNB5 and STIM1

Recent human genetic studies have shown that Gβ5 is related to various clinical symptoms, such as sinus bradycardia, cognitive disability, and attention deficit hyperactivity disorder. Although the calcium signaling cascade is closely associated with a heterotrimeric G-protein, the function of Gβ5 in calcium signaling and its relevance to clinical symptoms remain unknown. In this study, we investigated the in vitro changes of store-operated calcium entry (SOCE) with exogenous expression of Gβ5. The cells expressing Gβ5 had enhanced SOCE after depletion of calcium ion inside the endoplasmic reticulum. Gβ5 also augmented Stim1- and Orai1-dependent SOCE. An ORAI1 loss-of-function mutant did not show inhibition of Gβ5-induced SOCE, and a STIM1-ERM truncation mutant showed no enhancement of SOCE. These results suggested a novel role of GNB5 and Stim1, and provided insight into the regulatory mechanism of SOCE.

Dust particles-induced intracellular Ca²⁺ signaling and reactive oxygen species in lung fibroblast cell line MRC5

Epidemiologic interest in particulate matter (PM) is growing particularly because of its impact of respiratory health. It has been elucidated that PM evoked inflammatory signal in pulmonary epithelia. However, it has not been established Ca²⁺ signaling mechanisms involved in acute PM-derived signaling in pulmonary fibroblasts. In the present study, we explored dust particles PM modulated intracellular Ca²⁺ signaling and sought to provide a therapeutic strategy by antagonizing PM-induced intracellular Ca²⁺ signaling in human lung fibroblasts MRC5 cells. We demonstrated that PM10, less than 10 µm, induced intracellular Ca²⁺ signaling, which was mediated by extracellular Ca²⁺. The PM10-mediated intracellular Ca²⁺ signaling was attenuated by antioxidants, phospholipase blockers, polyADPR polymerase 1 inhibitor, and transient receptor potential melastatin 2 (TRPM2) inhibitors. In addition, PM-mediated increases in reactive oxygen species were attenuated by TRPM2 blockers, clotrimazole (CLZ) and N-(p-amylcinnamoyl) anthranilic acid (ACA). Our results showed that PM10 enhanced reactive oxygen species signal by measuring DCF fluorescence and the DCF signal attenuated by both TRPM2 blockers CLZ and ACA. Here, we suggest functional inhibition of TRPM2 channels as a potential therapeutic strategy for modulation of dust particle-mediated signaling and oxidative stress accompanying lung diseases.