OBJECTIVE-Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca 2ϩ release channels in the ER stress-associated apoptosis pathway remain unknown. Here, we examined the effects of stimulating or inhibiting the ER-resident inositol trisphosphate receptors (IP 3 Rs) and the ryanodine receptors (RyRs) on the induction of ␤-cell ER stress and apoptosis.RESEARCH DESIGN AND METHODS-Kinetics of ␤-cell death were tracked by imaging propidium iodide incorporation and caspase-3 activity in real time. ER stress and apoptosis were assessed by Western blot. Mitochondrial membrane potential was monitored by flow cytometry. Cytosolic Ca 2ϩ was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)-based probes were used to measure Ca 2ϩ in ER and mitochondria. RESULTS-NeitherRyR nor IP 3 R inhibition, alone or in combination, caused robust death within 24 h. In contrast, blocking sarco/endoplasmic reticulum ATPase (SERCA) pumps depleted ER Ca 2ϩ and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2␣ (eIF2␣), C/EBP homologous protein (CHOP)-associated ER stress, caspase-3 activation, and death. Notably, ER stress following SERCA inhibition was attenuated by blocking IP 3 Rs and RyRs. Conversely, stimulation of ER Ca 2ϩ release channels accelerated thapsigargin-induced ER depletion and apoptosis. SERCA block also activated caspase-9 and induced perturbations of the mitochondrial membrane potential, resulting eventually in the loss of mitochondrial polarization.CONCLUSIONS-This study demonstrates that the activity of ER Ca 2ϩ channels regulates the susceptibility of ␤-cells to ER stress resulting from impaired SERCA function. Our results also suggest the involvement of mitochondria in ␤-cell apoptosis associated with dysfunctional ␤-cell ER Ca 2ϩ homeostasis and ER stress. Diabetes 58:422-432, 2009

There are strong links between obesity, elevated free fatty acids, and type 2 diabetes. Specifically, the saturated fatty acid palmitate has pleiotropic effects on β-cell function and survival. In the present study, we sought to determine the mechanism by which palmitate affects intracellular Ca2+, and in particular the role of the endoplasmic reticulum (ER). In human β-cells and MIN6 cells, palmitate rapidly increased cytosolic Ca2+ through a combination of Ca2+ store release and extracellular Ca2+ influx. Palmitate caused a reversible lowering of ER Ca2+, measured directly with the fluorescent protein-based ER Ca2+ sensor D1ER. Using another genetically encoded indicator, we observed long-lasting oscillations of cytosolic Ca2+ in palmitate-treated cells. In keeping with this observed ER Ca2+ depletion, palmitate induced rapid phosphorylation of the ER Ca2+ sensor protein kinase R-like ER kinase (PERK) and subsequently ER stress and β-cell death. We detected little palmitate-induced insulin secretion, suggesting that these Ca2+ signals are poorly coupled to exocytosis. In summary, we have characterized Ca2+-dependent mechanisms involved in altered β-cell function and survival induced by the free fatty acid palmitate. We present the first direct evidence that free fatty acids reduce ER Ca2+ and shed light on pathways involved in lipotoxicity and the pathogenesis of type 2 diabetes.

Although evolutionarily conserved to expel ectoparasites and aid in the clearance of toxins and noxious environmental stimuli from the host, the type 2 immune response can become pathologic in the setting of a variety of allergic disorders. Itch can be a behavioral extension of type 2 immunity by evoking scratching and, in the setting of disease, can become chronic and thus highly pathologic as well. Classically, our understanding of itch mechanisms has centered around the canonical IgE-mast cell-histamine axis. However, therapies aimed at blocking the histaminergic itch pathway have been largely ineffective, suggesting the existence of nonhistaminergic itch pathways.Indeed, recent advances in itch biology have provided critical new insight into a variety of novel therapeutic avenues for chronic itch in the setting of a number of allergic disorders. Here we highlight how these new developments will likely inform the problem of pruritus in a variety of well-established and emerging conditions in the field of allergy. (J Allergy Clin Immunol 2019;144:353-60.)

Patients with dyskeratosis congenita (DC) suffer from stem cell failure in highly proliferative tissues, including the intestinal epithelium. Few therapeutic options exist for this disorder, and patients are treated primarily with bone marrow transplantation to restore hematopoietic function. Here, we generate isogenic DC patient and disease allele-corrected intestinal tissue using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene correction in induced pluripotent stem cells and directed differentiation. We show that DC tissue has suboptimal Wnt pathway activity causing intestinal stem cell failure and that enhanced expression of the telomere-capping protein TRF2, a Wnt target gene, can alleviate DC phenotypes. Treatment with the clinically relevant Wnt agonists LiCl or CHIR99021 restored TRF2 expression and reversed gastrointestinal DC phenotypes, including organoid formation in vitro, and maturation of intestinal tissue and xenografted organoids in vivo. Thus, the isogenic DC cell model provides a platform for therapeutic discovery and identifies Wnt modulation as a potential strategy for treatment of DC patients.

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