Wolfram syndrome, an autosomal recessive disorder characterized by juvenile-onset diabetes mellitus and optic atrophy, is caused by mutations in the WFS1 gene. In order to gain insight into the pathophysiology of this disease, we disrupted the wfs1 gene in mice. The mutant mice developed glucose intolerance or overt diabetes due to insufficient insulin secretion in vivo. Islets isolated from mutant mice exhibited a decrease in insulin secretion in response to glucose. The defective insulin secretion was accompanied by reduced cellular calcium responses to the secretagogue. Immunohistochemical analyses with morphometry and measurement of whole-pancreas insulin content demonstrated progressive beta-cell loss in mutant mice, while the alpha-cell, which barely expresses WFS1 protein, was preserved. Furthermore, isolated islets from mutant mice exhibited increased apoptosis, as assessed by DNA fragment formation, at high concentration of glucose or with exposure to endoplasmic reticulum-stress inducers. These results strongly suggest that WFS1 protein plays an important role in both stimulus-secretion coupling for insulin exocytosis and maintenance of beta-cell mass, deterioration of which leads to impaired glucose homeostasis. These WFS1 mutant mice provide a valuable tool for understanding better the pathophysiology of Wolfram syndrome as well as WFS1 function.

Wolfram syndrome, an autosomal recessive disorder associated with diabetes mellitus and optic atrophy, is caused by mutations in the WFS1 gene encoding an endoplasmic reticulum (ER) membrane protein. Herein, we report that pancreatic islets of wfs1-deficient mice exhibit increases in phosphorylation of RNA-dependent protein kinase-like ER kinase, chaperone gene expressions and active XBP1 protein levels, indicating an enhanced ER stress response. We established wfs1-deficient MIN6 clonal beta-cells by crossing wfs1-deficient mice with mice expressing simian virus 40 large T antigen in beta-cells. These cells show essentially the same alterations in ER stress responses as wfs1-deficient islets, which were reversed by re-expression of WFS1 protein or overexpression of GRP78, a master regulator of the ER stress response. In contrast, these changes are not observed in heart, skeletal muscle or brown adipose tissues with WFS1-deficiency. The increased ER stress response was accompanied by reduced BrdU incorporation and increased caspase-3 cleavage, indicating impaired cell cycle progression and accelerated apoptotic processes in the mutant islets. These changes are associated with increased expression of the cell cycle regulator p21(CIP1) in wfs1-deficient islets and clonal beta-cells. Treatment of islets with thapsigargin, an ER stress inducer, caused upregulation of p21(CIP1). In addition, forced expression of p21(CIP1) resulted in reduced MIN6 beta-cell numbers, suggesting the ER stress-induced increase in p21(CIP1) expression to be involved in beta-cell loss in the mutant islets. These data indicate that WFS1-deficiency activates the ER stress response specifically in beta-cells, causing beta-cell loss through impaired cell cycle progression and increased apoptosis.

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During endochondral ossification of long bones, the proliferation and differentiation of chondrocytes cause them to be arranged into layered structures constituting the epiphyseal growth plate, where they secrete the cartilage matrix that is subsequently converted into trabecular bone. Ca2+ signaling has been implicated in chondrogenesis in vitro. Through fluorometric imaging of bone slices from embryonic mice, we demonstrated that live growth plate chondrocytes generated small, cell-autonomous Ca2+ fluctuations that were associated with weak and intermittent Ca2+ influx. Several genes encoding Ca2+-permeable channels were expressed in growth plate chondrocytes, but only pharmacological inhibitors of transient receptor potential cation channel subfamily M member 7 (TRPM7) reduced the spontaneous Ca2+ fluctuations. The TRPM7-mediated Ca2+ influx was likely activated downstream of basal phospholipase C activity and was potentiated upon cell hyperpolarization induced by big-conductance Ca2+-dependent K+ channels. Bones from embryos in which Trpm7 was conditionally knocked out during ex vivo culture exhibited reduced outgrowth and displayed histological abnormalities accompanied by insufficient autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the growth plate. The link between TRPM7-mediated Ca2+ fluctuations and CaMKII-dependent chondrogenesis was further supported by experiments with chondrocyte-specific Trpm7 knockout mice. Thus, growth plate chondrocytes generate spontaneous, TRPM7-mediated Ca2+ fluctuations that promote self-maturation and bone development.