Supplementary Materials Supplemental Data supp_292_36_14977__index. thapsigargin results in THBS1 and MANF degradation and loss of this prosurvival mechanism. Approaches that sustain intracellular THBS1 and MANF expression in -cells should be explored as a cytoprotective Rabbit Polyclonal to IR (phospho-Thr1375) strategy in type 1 diabetes. to IL-1 and IFN- (4) supports the idea that these cytokines (or other cytokines that elicit similar signal transduction) play a role in the human disease. In the context MKC3946 of T2D, the metabolic stress of chronic exposure to elevated levels of saturated free fatty acids, such as palmitate, and glucose contribute to -cell dysfunction and apoptosis (5, 6). It is of high interest to identify approaches that prevent both immune-mediated and metabolic -cell demise; such an approach would be very useful in the prevention or early treatment of T1D and T2D. This task is made difficult, however, by the fact that proinflammatory cytokines (4) and palmitate (7) induce different gene networks and lead to pancreatic -cell apoptosis by different mechanisms (1). One cellular stress response that is, however, present in -cells in both forms of diabetes is endoplasmic reticulum (ER) stress (8, 9). Pharmacological modulation of the ER stress response might therefore hold promise for -cell therapy (10, 11). The multimeric Ca2+-binding glycoprotein thrombospondin 1 (THBS1) protects cardiomyocytes against ER stress via activation of ATF6 and downstream chaperones (12). We have shown MKC3946 recently that THBS1 protects human and rodent -cells from palmitate-induced apoptosis (13). Different from cardiomyocytes, however, this takes place through activation of the ER stress transducer protein kinase R-like endoplasmic reticulum kinase (PERK) and the downstream transcription factor NRF2, increasing the -cell capacity to withstand oxidative stress induced by saturated fatty MKC3946 acids (13). Here we tested whether THBS1 is equally beneficial to rodent and human -cells exposed to cytokines or chemically induced ER stress. THBS1 was clearly protective, but this was mediated by a different mechanism compared with protection against lipotoxic -cell demise (13) or cardiomyopathy (12); namely, through induction of the mesencephalic astrocyte-derived neutrotrophic factor (MANF). This raises the intriguing possibility that the multifunctional protein THBS1 changes roles and/or partner affinities in a cell- or stress-specific manner, as suggested recently for other complex biological systems (14). Furthermore, these findings indicate that THBS1-inducing agents may represent a novel strategy for -cell protection in both T1D and T2D. Results The cross-talk between THBS1 and ER stress- and proinflammatory cytokineCinduced -cell apoptosis Knockdown of THBS1 in rat INS-1E cells by two independent siRNAs did not affect basal expression of cleaved caspase 9 and 3 and apoptosis (Fig. 1, and and and supplemental Fig. S1) or cytokines (supplemental Fig. S1). Islets isolated from THBS1 knock-out mice were also significantly sensitized to thapsigargin and cytokines MKC3946 (Fig. 1and and = 3C4). = 3C7). = 4). and = 4). and = 3C4). *, 0.05 control ( 0.05 cytokine- or thapsigargin-treated cells transfected with negative siRNA or contaminated with luciferase-expressing adenovirus. As the above results indicate that THBS1 protects human being, rat, and mouse -cells from cytokine- and ER stressCinduced cell loss of life, we examined whether these tensions affect THBS1 manifestation. Exposure of human being islets to two different ER stressors, brefeldin A (which blocks transfer of cargo through the ER towards the Golgi) and thapsigargin, reduced THBS1 mRNA manifestation by almost 80% (Fig. 2and = 3). and = 3). = 3). *, 0.05 control (and = 3). = 3). and = 4). and = 4). *, 0.05 untreated cells; #, 0.05 thapsigargin- or cytokine-treated cells infected with luciferase adenovirus or subjected to ad Luc medium; , 0.05 as indicated. Islet cells from THBS1?/? mice possess improved susceptibility to cell loss of life induced by ER stressors or cytokines (Fig. 1and and and and and and = 3). and = 2C3). = 3). = 3). and and = 3). = 4C5). = 2). These along with other pictures are presented in supplemental Fig also. S4. *, 0.05 control ( 0.05 thapsigargin- or cytokine-treated cells transfected or not with negative siRNA. We following examined the part of MANF using two individual siRNAs MKC3946 directly. Efficient MANF knockdown (Fig. 4and supplemental Fig. S5) or cytokines (supplemental Fig. S5). We.