Overexpression of the autoantigen IA-2 puts beta cells into a pre-apoptotic state: autoantigen-induced, but non-autoimmune-mediated, tissue destruction

Abstract

IA-2 is a major autoantigen in type 1 diabetes and autoantibodies to it have become important diagnostic and predictive markers. IA-2 also is an intrinsic transmembrane component of dense core secretory vesicles and knock-out studies showed that IA-2 is a regulator of insulin secretion. Here we show that overexpression of IA-2 puts mouse insulinoma MIN-6 beta cells into a pre-apoptotic state and that exposure to high glucose results in G2/M arrest and apoptosis. Molecular study revealed a decrease in phosphoinositide-dependent kinase (PDK)-1 and Akt/protein kinase B (PKB) phosphorylation. Treatment of IA-2-transfected cells with IA-2 siRNA prevented both G2/M arrest and apoptosis and increased Akt/PKB phosphorylation. A search for IA-2 interacting proteins revealed that IA-2 interacts with sorting nexin (SNX)19 and that SNX19, but not IA-2, inhibits the conversion of PtdIns(4,5)P2 to PtdIns(3,4,5)P3 and thereby suppresses the phosphorylation of proteins in the Akt signalling pathway resulting in apoptosis. We conclude that IA-2 acts through SNX19 to initiate the pre-apoptotic state. Our findings point to the possibility that in autoimmune diseases, tissue destruction may be autoantigen-induced, but not necessarily immunologically mediated.

Fig. 1

IA-2 transfected mouse insulinoma MIN-6 cells undergo growth arrest in high glucose media. (a, b) Growth rate of IA-2 and mock transfected MIN-6 cells as determined by (a) trypan blue staining and (b) bromodeoxyuridine (BrdU) cell proliferation. (c, d) Cell cycle analysis by flow cytometry of mock and IA-2F transfected MIN-6 cells under (c) low and (d) high glucose conditions. (e) Survivin expression in mock and IA-2F transfected MIN-6 cells as determined by Western blot.

Fig. 2

IA-2 transfected mouse insulinoma MIN-6 cells undergo apoptosis in high glucose media. (a, b) Flow cytometry detection of apoptotic cells as determined by uptake of propidium iodide and by staining with anti-annexin V-fluorescein isothiocyanate (FITC) in (a) low and (b) high glucose media in mock and IA-2F transfected cells. (c) DNA fragmentations of cells in low and high glucose media. (d) Caspase-3, 6, 7, 9, 12 and caspase activated DNase (CAD) activity detected by Western blot in low and high glucose media.

Fig. 3

IA-2 siRNA treatment prevents G2/M arrest and apoptosis in IA-2 transfected mouse insulinoma MIN-6 cells. (a) IA-2 expression as determined by Western blot 48 h after transfection with IA-2 siRNA and non-silencing siRNA. (b, c) Cell cycle analysis of IA-2F transfected MIN-6 cells as determined by flow cytometry 48 h after transfection with IA-2 siRNA or non-silencing siRNA in low and high glucose media. (d, e) Detection of apoptotic cells by flow cytometry 72 h after exposure of IA-2F transfected MIN-6 cells to IA-2 siRNA and non-silencing siRNA in low and high glucose media. (f, g) Long-term passage of IA-2F transfected MIN-6 cells kicks out IA-2 as shown by (f) Western blot and (g) when this occurs (e.g. by the 18th passage) glucose-induced G2/M arrest is prevented.

Fig. 4

Phosphorylation of proteins involved in the insulin signalling pathway. (a) Phosphorylation of Akt/protein kinase B (PKB), phosphoinositide-dependent kinase (PDK)-1, mammalian target of rapamycin (mTOR), glycogen synthetase kinase (GSK)-3β, p70S6 kinase (p70S6K), phosphatidylinositol phosphate 3'-phosphatase (PTEN), PI3 kinase (PI3K), insulin receptor substrate (IRS)-1 and insulin receptor (IR) in mock and IA-2F transfected mouse insulinoma MIN-6 cells in high and low glucose media. (b) Phosphorylation of Akt/PKB in IA-2F transfected MIN-6 cells, or (c) non-transfected MIN-6 cells in the presence of high glucose 48 h after exposure to IA-2 siRNA and non-silencing siRNA. (d) Apoptosis as measured by flow cytometry with anti-annexin V and propidium iodine in the presence of tumour necrosis factor (TNF)-α and interferon (IFN)-γ at low or high glucose concentrations in mock and IA-2F transfected MIN-6 cells. Mean ± s.e. of three separate experiments. *P < 0·05, **P < 0·01.

Fig. 5

Sorting nexin (SNX)19 interacts and co-localizes with IA-2 and SNX19 siRNA increases Akt/phosphoinositide-dependent kinase (PDK)-1 phosphorylation and inhibits G2/M arrest and apoptosis. (a) Lysates of NIH3T3 cells co-transfected with anti-flagellar antigen (FLAG)-tagged SNX19 and c-Myc-tagged IA-2IC (a.a. 600–979) were immunoprecipitated with anti-FLAG antibody, anti-c-Myc antibody or anti-IA-2 antibody followed by Western blot analysis. (b) Endogenous interaction of IA-2 and SNX19 in mouse insulinoma MIN-6 cells (non-transfected) detected by immunoprecipitation with anti-IA-2 or anti-SNX19 antibody followed by Western blot. (c) Fractionation of lysates from non-transfected MIN-6 cells followed by Western blot analysis with anti-IA-2, -SNX19 and -insulin antibody. (d) MIN-6 cells were transiently transfected with SNX19 and analysed 48 h later by flow cytometry for apoptosis in the presence of high glucose. (e) PC12 cells were transiently transfected in the presence of high glucose with SNX19 or co-transfected with SNX19 and IA-2 and 48 h later Akt/PKB phosphorylation was determined by Western blot. (f) Non-IA-2 transfected MIN-6 cells were transfected with SNX19 siRNA or non-silencing siRNA and Akt/protein kinase B (PKB) phosphorylation determined by Western blot. (g h) IA-2F transfected MIN-6 cells were transfected with SNX19 siRNA or non-silencing siRNA and the effect on (g) cell cycle and (h) apoptosis was determined by flow cytometry in the presence of high glucose.

Fig. 6

Binding of sorting nexin (SNX)19 to PtdIns. (a) Purified IA-2, SNX19 or a combination of the two was overlaid on a membrane spotted with different PtdIns and binding was determined. (b) Competitive Akt/protein kinase B (PKB) binding to PtdIns(3,4,5)P3 in the presence or absence of IA-2 and/or SNX19. (c) SNX19, but not IA-2, inhibits the conversion of PtdIns(4, 5)P2 to PtdIns(3, 4, 5)P3 by PI3 kinase as determined by enzyme-linked immunosorbent assay (ELISA). Mean ± s.e. of three separate experiments. *P < 0·05. (d) Schematic diagram depicting the proposed mechanism by which overexpression of IA-2 leads to apoptosis. Overexpression of IA-2 increases the number of DCV in mouse insulinoma MIN-6 cells [11]. SNX19 binds to the cytoplasmic domain of IA-2 and in the presence of high glucose dense core vesicles (DCV) are translocated to the plasma membrane. As there are more DCV in IA-2 transfected cells this would result in the delivery of more SNX19 to the plasma membrane, where SNX19 could block the conversion of PI(4,5)P2 to PI (3,4,5)P3 and inhibit the phosphorylation of phosphoinositide-dependent kinase (PDK)-1 and Akt/protein kinase B (PKB). This in turn would down regulate the phosphorylations of proteins in the PDK-1 and Akt/PKB signalling pathway resulting in the activation of caspases and beta cell apoptosis. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine. , increase, , decrease, , inhibition, , translocation.