Regulation of the Diabetes-Associated Autoantigen IA-2 in INS-1 Pancreatic -Cells

IA-2, a member of the protein tyrosine phosphatase family, represents a major target autoantigen in type 1 diabetes. To study the regulation of IA-2 gene expression, we used INS-1 insulinoma cells to analyze -cell signal transduction pathways as well as the effect of metabolic and hormonal factors involved in the regulation of the insulin secretory pathway. Quantitative competitive reverse transcriptase–polymerase chain reaction revealed that an increase of cellular cAMP mediated by forskolin (10 μmol/l, 24 h) or 3-isobutyl-1-methylxanthine (100 μmol/l, 24 h) induced maximal stimulation of IA-2 mRNA levels (451 ± 85 and 338 ± 86% compared with basal conditions; P < 0.001). In contrast, activation of protein kinase C (PKC) by short-term treatment with phorbol 12-myristate 13-acetate (PMA) (1 μmol/l, 6 h) did not alter IA-2 expression, whereas depletion of PKC by prolonged culturing (24 h) exerted a significant inhibition (57 ± 24%; P < 0.05). cAMP-dependent upregulation was confirmed by the findings that glucagon (10 μmol/l, 24–48 h) increased levels of IA-2 mRNA (190 ± 35%; P < 0.05), whereas short-term incubation with high glucose concentration showed no effect. However, prolonged incubation in high glucose (21 mmol/l) induced a timeand dose-dependent increase of IA-2 mRNA expression, reaching maximal values after 144 h (285 ± 68%; P < 0.05). These studies demonstrate that stimuli of insulin secretion that operate by activation of adenylate cyclase generating cAMP significantly increase IA-2 gene expression. In contrast, activation of PKC by high glucose concentration or PMA exerted no effect, suggesting that IA-2 gene expression is not simply coupled to insulin secretion, but may be involved in the fine regulation of -cell function. These findings may be important to clarify the function of IA-2 in -cells and elucidate mechanisms involved in the induction of autoimmunity to IA-2. Diabetes 49:1137–1141, 2000 Type 1 diabetes results from the destruction of pancreatic -cells mediated by a chronic autoimmune process. Recently, one member of the receptortype protein tyrosine phosphatase (PTP) family, designated as IA-2 or ICA512, has been identified as a major diabetes-specific autoantigen (1–3). Autoantibodies to IA-2 were observed in 50–70% of newly diagnosed patients with type 1 diabetes (4,5) and in 49–64% of prediabetic subjects at high risk for rapid development of the disease (6–8). In addition, a specific T-cell response has been described in 42–60% of diabetic patients, suggesting that IA-2 is a dominant target of humoral and cellular autoimmunity (8–11). IA-2 is predominately expressed in neuroendocrine cells, including the brain, pituitary, and pancreatic -cells, and is highly conserved in evolution. The human IA-2 gene encodes a transmembrane protein of predicted molecular weight (Mr) 105 kDa, which is processed in vivo to a glycosylated mature protein of Mr 65–70 kDa. In the pancreatic -cells, IA-2 is localized in the membrane of insulin secretory vesicles, suggesting that it may be important for maturation or trafficking of insulin granules (12). IA-2 is characterized by an intracytoplasmic domain containing 1 PTP motif, 1 transmembrane region, and an extracytoplasmic part directed to the luminal site of the insulin granula that harbor the receptor-like domain (1,12). Thus far, no enzymatic activity has been detected using common PTP substrates, and no ligand has been identified interacting with the receptor-like domain of IA-2 (13). Therefore, the biological function of IA-2 is still unknown. In the present study, we investigated the regulation of IA-2 gene expression in the glucose-sensitive insulinoma cell line INS-1. By means of quantitative competitive reverse transcriptase–polymerase chain reaction (RT-PCR), we demonstrate that IA-2 gene expression is regulated by the 2 main transduction systems, an increase in cAMP levels, and the activation state of the protein kinase C (PKC), which are both involved in the coordinated -cell function.