conceptualization; D. demonstrating the presence of an evolutionarily conserved mechanism for IAPP production. In line with these studies, our current data showed that proteasome activity and hIAPP expression are also down-regulated in islets isolated from T2DM Rabbit Polyclonal to EDG4 subjects. Gene Flufenamic acid expression and promoter activity studies demonstrated that the functional Flufenamic acid proteasome complex is required for efficient activation of the promoter and for full expression of promoter region is an important and limiting factor for amylin expression in proteasome-impaired murine cells. This study suggests a novel regulatory pathway in -cells involving proteasome, FOXA2, and and insulin promoters share common glucose-responsive regulatory elements and transcription factors such as PDX1 and ISL1 (13). In addition to these two transcription factors, forkhead box protein A2 (FOXA2) has been also implicated in the regulation of expression in -cells (14, 15). IAPP is first synthesized as an 89-amino acid prepropeptide (16, 17). The prepro-IAPP form, together with nascent insulin, undergoes a series of post-translational and proteolytic processing in the endoplasmic reticulum (ER), Golgi, and secretory vesicles by prohormone convertase 2 (PC2) and 1/3 (PC1/3) and carboxypeptidase E (5). Fully processed IAPP and insulin are then stored in the same secretory granules of pancreatic islet -cells. In normal subjects, upon physiological stimulation (such as elevated glucose/or nutrients), insulin and IAPP are co-secreted to regulate glucose homeostasis in the body (5, 18, 19). However, under conditions Flufenamic acid that favor the development of T2DM, hIAPP misfolds and forms toxic amyloid oligomers and aggregates (5). At present, it is not clear which cellular processes and factors regulate hIAPP-mediated cytotoxicity, but it has been suggested that impaired turnover and cellular processing of hIAPP contribute significantly toward the progressive -cell failure during T2DM (4, 5). Several independent studies have linked impaired ubiquitinCproteasome system (UPS) as a risk factor for age-related diseases such as T2DM (3, 20). The primary component of UPS is the 26S proteasome complex, nonlysosomal protein degradation machinery in eukaryotes. The 26S proteasome is composed of a 20S proteolytic core and 19S regulatory components. The 20S core is a cylinder-like structure, consisting of (1-7) and (1-7) subunits. Within the seven -subunits, 5, 2, and 1 are catalytically active and responsible for chymotrypsin-like, trypsin-like, and post-acidic or caspase-like proteolytic activities, respectively (21). The 26S proteasome complex is responsible for degradation of polyubiquitinated proteins in an ATP-dependent manner. However, recent reports also provide evidence for nonubiquitin and non-ATPCdependent degradation mechanisms of the 20S proteasome (22, 23). Intriguingly, the UPS has also been implicated in transcriptional regulation of several eukaryotic genes. Studies showed that proteolytic and nonproteolytic activities of the 26S proteasome complex regulate the availability, localization, and promoter recruitment of various transcription factors. In this way, UPS controls the key stages of eukaryotic gene expression; transcription initiation, elongation, maturation, and nuclear export of mRNA (24). Although the exact role of UPS in the pathology of T2DM is still emerging, microarray analyses of human pancreatic islets revealed down-regulation of several proteasome subunits in T2DM patients, indicating its possible role in disease onset and progression (25). Studies using pancreatectomy-induced diabetic rat models showed an initial increase followed by gradual down-regulation of rodent IAPP mRNA levels, together with ensuing hyperglycemia (26). Previous studies in our Flufenamic acid laboratory demonstrated the crucial role of the proteasome in the degradation of internalized hIAPP, thereby preventing hIAPP-induced -cell toxicity (27). However, the role of the proteasome in the production, degradation, and secretion, hereafter collectively referred as turnover, of endogenous hIAPP in normal and disease states has yet to be determined. Given the emerging role of the 26S proteasome complex in the regulation of eukaryotic gene transcription and the important pathophysiological roles of hIAPP, in this study, we explored the role of the proteasome in IAPP turnover in rodent and human pancreatic -cells. This study points to the essential and novel role of proteasome complex in IAPP synthesis, secretion, and degradation in -cells. This proteasome-regulated pathway may have important ramifications for amylin-induced amyloid formation in human islets and its pathological role in T2DM. Results Intracellular hIAPP levels and proteasome activity are down-regulated in diabetic human islet cells Previous studies revealed that chronic hyperglycemia may impair proteasome activity and alter insulin and IAPP biosynthesis in human and rodent pancreatic -cells (15, 28, 29). However, causal.