Downregulation of Fas activity rescues early onset of diabetes inc-Kit^Wv/+mice

Abstract

Wv/ϩ mouse islets, suggesting that there is a critical balance between c-Kit and Fas activation in ␤-cells. In the present study, we investigated the interrelationship between c-Kit and Fas activation that mediates ␤-cell survival and function. We generated double mutant, c-Kit Wv/ϩ ;Fas lpr/lpr (Wv Ϫ/Ϫ ), mice to study the physiological and functional role of Fas with respect to ␤-cell function in c-Kit Wv/ϩ mice. Isolated islets from these mice and the INS-1 cell line were used. We observed that islets in c-Kit Wv/ϩ mice showed a significant increase in ␤-cell apoptosis along with upregulated p53 and Fas expression. These results were verified in vitro in INS-1 cells treated with SCF or c-Kit siRNA combined with a p53 inhibitor and Fas siRNA. In vivo, Wv Ϫ/Ϫ mice displayed improved ␤-cell function, with significantly enhanced insulin secretion and increased ␤-cell mass and proliferation compared with Wv ϩ/ϩ mice. This improvement was associated with downregulation of the Fas-mediated caspase-dependent apoptotic pathway and upregulation of the cFlip/NF-B pathway. These findings demonstrate that a balance between the c-Kit and Fas signaling pathways is critical in the regulation of ␤-cell survival and function. c-Kit; Fas; ␤-cell apoptosis; insulin secretion; diabetes THE PREVALENCE OF DIABETES MELLITUS has been increasing at an alarming rate. During the progression of diabetes, pancreatic ␤-cells are often lost because the delicate balance between ␤-cell proliferation and death is disrupted (4, 5, 45). Recent accumulating evidence has suggested that c-Kit, with its ligand stem cell factor (SCF), not only plays an essential role in hematopoiesis, melanogenesis, and gametogenesis (1), but also in ␤-cell differentiation, maturation, and function (19,21,34,35,37,46,48,49). c-Kit is a receptor tyrosine kinase that belongs to the platelet-derived growth factor receptor family and affects multiple pro-survival downstream signaling pathways, including the phosphoinositide 3-kinase (PI3K) pathway (1). Our previous studies showed that c-Kit Wv/ϩ mice, which contain a point mutation in the kinase region (Wv) of c-Kit, exhibit severe ␤-cell mass loss (16) that was associated with a significant downregulation of phospho-Akt/glycogen synthase kinase-3␤ (Gsk3␤)/cyclin D1 signaling (9). However, the underlying mechanisms involved in c-Kit-mediated effects on ␤-cell survival have yet to be determined.The Fas receptor (Fas) belongs to the tumor necrosis factor family and requires the Fas ligand (FasL) for activation. Fas/FasL interactions result in activation of Fas-associated death domains (FADD) and cleavage of caspase 8, which triggers apoptosis. In diabetes, ␤-cells constitutively express Fas (24,26). Fas/FasL interactions were suggested to be one of the major mechanisms leading to ␤-cell apoptosis in T cellmediated autoimmune diabetes (7,10,40). In addition, cytokine-induced upregulation of the Fas apoptotic pathway is also involved in glucotoxicity and subsequent increases in ␤-cell death (25); meanwhile, deletion of Fa...

A survival Kit for pancreatic beta cells: stem cell factor and c-Kit receptor tyrosine kinase

“…Lack of functional FasR in c-Kit Wv/+ mice reduced the extent of beta cell apoptosis via downregulation of the caspase 8-mediated extrinsic apoptotic pathway, and enhanced FLICE-like inhibitor protein/nuclear factor κB pro-survival signalling [51]. These in vivo results were further supported by in vitro observations using INS-1 cells, whereby c-Kit activation negatively modulated both p53 and FasR in a PI3K-dependent manner.…”

“…Fas ligand binding and subsequent activation of FasR leads to programmed cell death in many systems via activation of the caspase 8-mediated downstream cell death machinery. We recently reported that deficient c-Kit signalling resulted in increased beta cell death in c-Kit Wv/+ mice, which was associated with upregulation of p53 levels and induction of FasR activity in c-Kit Wv/+ mouse islets [51] (Fig. 3).…”

“…These in vivo results were further supported by in vitro observations using INS-1 cells, whereby c-Kit activation negatively modulated both p53 and FasR in a PI3K-dependent manner. In particular, knockdown of c-Kit in INS-1 cells led to increased p53 and FasR levels, reversed by treatment with a p53 inhibitor or Fas small interfering (si)RNA, ultimately resulting in a reduction in apoptosis [51]. These data imply that a balance between c-Kit and FasR signalling is instrumental in maintaining proper beta cell mass turnover.…”

“…Furthermore, W and Sl mutant mice display increased germ cell apoptosis, with a corresponding degree of sterility [41]. More importantly, accumulating evidence suggests that c-Kit expression is also linked to the development of the endocrine pancreas, as well as to beta cell survival and function [11,12,[42][43][44][45][46][47][48][49][50][51][52][53].…”

“…Stimulation of c-Kit activity promoted gene transcription and proliferation in INS-1 rat insulinoma cells [12,46,51]. Furthermore, treatment by exogenous SCF induced differentiation of human pancreatic carcinoma, epithelial-like Fetal islet-epithelium (human)…”