Regulation of the diabetes-associated autoantigen IA-2 in INS-1 pancreatic beta-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 beta-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 micromol/l, 24 h) or 3-isobutyl-1-methylxanthine (100 micromol/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 micromol/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 micromol/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 time- and 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 beta-cell function. These findings may be important to clarify the function of IA-2 in beta-cells and elucidate mechanisms involved in the induction of autoimmunity to IA-2.
Diabetes Research Institute, University of Düsseldorf, Germany. sei@dfi.uni-duesseldorf.de
J
J
Seissler
T B
TB
Nguyen
G
G
Aust
H
H
Steinbrenner
W A
WA
Scherbaum
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D005934:Glucagon / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D007340:Insulinoma; D007515:Islets of Langerhans / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D010190:Pancreatic Neoplasms; D011493:Protein Kinase C / Q000378:metabolism; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013004:Somatostatin / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D001324:Autoantigens; D008565:Membrane Proteins; D012333:RNA, Messenger; D005576:Colforsin; D013004:Somatostatin; D005934:Glucagon; D000242:Cyclic AMP; D011493:Protein Kinase C; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; C516864:Ptprn protein, rat; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D005947:Glucose; D013755:Tetradecanoylphorbol Acetate; D015056:1-Methyl-3-isobutylxanthine