Differential Signaling by Regulatory Subunits of Phosphoinositide-3-kinase Influences Cell Survival in INS-1E Insulinoma Cells

Abstract

Class 1A phosphoinositide 3-kinase (PI3K) is essential for beta-cell growth and survival. Although PI3K has been studied extensively in diabetes the effect of alternatively spliced isoforms of the catalytic subunit p85α on beta cell proliferation and survival remains to be defined.

We examined expression and signaling of alternatively spliced PI3K regulatory subunits p85α, p55α and p50α in insulinoma cells (INS-1E), an insulin-producing beta cell line. PI3K regulatory isoforms were knocked down by siRNA transfection or overexpressed by adenoviral gene delivery.

Knockdown of p85α elevated PI3K activation determined by Akt phosphorylation at baseline and after stimulation with growth factors. In contrast, Akt phosphorylation was inhibited by overexpression of all isoforms of p85α. Correspondingly, p55α and p85α overexpression decreased downstream kinase GSK-3 phosphorylation as well, whereas p50α overexpression resulted in an activation of GSK-3. Moreover, overexpression of p50α and p85α lead to retinoblastoma protein hyperphosphorylation and S-phase entry. Upon challenge of INS-1E cells with a cytotoxic cytokine cocktail, levels of p85α were reduced and p50α was upregulated. Selective overexpression of p50α prevented cytokine induced apoptosis in INS-1E cells.

In conclusion, signalling of p50α, p55α and p85α is similar at the level of Akt, but differentially influence downstream GSK-3 activation and cell cycle entry. PI3K isoform p50α induction by cytokines provides a link between regeneration and cell survival under cytotoxic stress in insulin-producing pancreatic beta-cells.

Key words

apoptosis - cell cycle regulation - diabetes - GSK-3

• References

• 1 Hirsch E, Costa C, Ciraolo E. Phosphoinositide 3-kinases as a common platform for multi-hormone signaling. J Endocrinol 2007; 194: 243-256
• 2 Jia S, Liu Z, Zhang S et al. Essential roles of PI(3)K-p110beta in cell growth, metabolism and tumorigenesis. Nature 2008; 454: 776-779
• 3 Mauvais-Jarvis F, Ueki K, Fruman DA et al. Reduced expression of the murine p85alpha subunit of phosphoinositide 3-kinase improves insulin signaling and ameliorates diabetes. J Clin Invest 2002; 109: 141-149
• 4 Ueki K, Fruman DA, Brachmann SM et al. Molecular balance between the regulatory and catalytic subunits of phosphoinositide 3-kinase regulates cell signaling and survival. Mol Cell Biol 2002; 22: 965-977
• 5 Ueki K, Fruman DA, Yballe CM et al. Positive and negative roles of p85 alpha and p85 beta regulatory subunits of phosphoinositide 3-kinase in insulin signalling. J Biol Chem 2003; 278: 48453-48466
• 6 Hallmann D, Trümper K, Trusheim H et al. Altered signaling and cell cycle regulation in embryonal stem cells with a disruption of the gene for phosphoinositide 3-kinase regulatory subunit p85alpha. J Biol Chem 2003; 278: 5099-5108
• 7 Brachmann SM, Ueki K, Engelman JA et al. Phosphoinositide 3-kinase catalytic subunit deletion and regulatory subunit deletion have opposite effects on insulin sensitivity in mice. Mol Cell Biol 2005; 25: 1596-1607
• 8 Barber DF, Alvarado-Kristensson M, González-García A et al. PTEN regulation, a novel function for the p85 subunit of phosphoinositide 3-kinase. Sci STKE 2006; 21 pe49
• 9 Geering B, Cutillas PR, Nock G et al. Class IA phosphoinositide 3-kinases are obligate p85-p110 heterodimers. Proc Natl Acad Sci USA 2007; 104: 7809-7814
• 10 Geerin B, Cutillas PR, Vanhaesebroeck B. Regulation of class IA PI3Ks: is there a role for monomeric PI3K subunits?. Biochem Soc Trans 2007; 35: 199-203
• 11 Kaneko K, Ueki K, Takahashi N et al. Class IA phosphatidylinositol 3-kinase in pancreatic β cells controls insulin secretion by multiple mechanisms. Cell Metab 2010; 12: 619-632
• 12 Kolic J, Spigelman AF, Plummer G et al. Distinct and opposing roles for the phosphatidylinositol 3-OH kinase catalytic subunits p110α and p110β in the regulation of insulin secretion from rodent and human beta cells. Diabetologia 2013; 56: 1339-1349
• 13 Bernal-Mizrachi E, Wen W, Stahlhut S et al. Islet beta cell expression of constitutively active Akt1/PKB alpha induces striking hypertrophy, hyperplasia, and hyperinsulinemia. J Clin Invest 2001; 108: 1631-1638
• 14 Tuttle RL, Gill NS, Pugh W et al. Regulation of pancreatic beta-cell growth and survival by the serine/threonine protein kinase Akt1/PKBalpha. Nat Med 2001; 7: 1133-1137
• 15 Elghazi L, Rachdi L, Weiss AJ et al. Regulation of beta-cell mass and function by the Akt/protein kinase B signalling pathway. Diabetes Obes Metab 2007; 9 (Suppl. 02) 147-157
• 16 Liu Y, Tanabe K, Baronnier D et al. Conditional ablation of Gsk-3β in islet beta cells results in expanded mass and resistance to fat feeding-induced diabetes in mice. Diabetologia 2010; 53: 2600-2610
• 17 Kushner JA, Simpson L, Wartschow LM et al. Phosphatase and tensin homolog regulation of islet growth and glucose homeostasis. J Biol Chem 2005; 280: 39388-39393
• 18 Stiles BL, Kuralwalla-Martinez C, Guo W et al. Selective deletion of Pten in pancreatic beta cells leads to increased islet mass and resistance to STZ-induced diabetes. Mol Cell Biol 2006; 26: 2772-2781
• 19 Nguyen KT, Tajmir P, Lin CH et al. Essential role of Pten in body size determination and pancreatic beta-cell homeostasis in vivo. Mol Cell Biol 2006; 26: 4511-4518
• 20 Trümper A, Trümper K, Trusheim H et al. Glucose-dependent insulinotropic polypeptide is a growth factor for beta (INS-1) cells by pleiotropic signaling. Mol Endocrinol 2001; 5: 1559-1570
• 21 Xu G, Stoffers DA, Habener JF et al. Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats. Diabetes 1999; 48: 2270-2276
• 22 Casellas A, Salavert A, Agudo J et al. Expression of IGF-I in pancreatic islets prevents lymphocytic infiltration and protects mice from type 1 diabetes. Diabetes 2006; 55: 3246-3255
• 23 Chagpar RB, Links PH, Pastor MC et al. Direct positive regulation of PTEN by the p85 subunit of phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA 2010; 107: 5471-5476
• 24 Taniguchi CM, Aleman JO, Ueki K et al. The p85alpha regulatory subunit of phosphoinositide 3-kinase potentiates c-Jun N-terminal kinase-mediated insulin resistance. Mol Cell Biol 2007; 27: 2830-2840
• 25 García Z, Silio V, Marqués M et al. A PI3K activity-independent function of p85 regulatory subunit in control of mammalian cytokinesis. EMBO J 2006; 25: 4740-4751
• 26 Meier JJ, Bhushan A, Butler AE et al. Sustained beta cell apoptosis in patients with long-standing type 1 diabetes: indirect evidence for islet regeneration?. Diabetologia 2005; 48: 2221-2228
• 27 Kauri LM, Wang GS, Patrick C et al. Increased islet neogenesis without increased islet mass precedes autoimmune attack in diabetes-prone rats. Lab Invest 2007; 87: 1240-1251
• 28 Schrader J, Rennekamp W, Niebergall U et al. Cytokine-induced osteoprotegerin expression protects pancreatic beta cells through p38 mitogen-activated protein kinase signalling against cell death. Diabetologia 2007; 50: 1243-1247
• 29 Abell K, Bilancio A, Clarkson RW et al. Stat3-induced apoptosis requires a molecular switch in PI(3)K subunit composition. Nat Cell Biol 2005; 7: 392-398
• 30 Nir T, Melton DA, Dor Y. Recovery from diabetes in mice by beta cell regeneration. J Clin Invest 2007; 117: 2553-2561
• 31 Mussmann R, Geese M, Harder F et al. Inhibition of GSK3 promotes replication and survival of pancreatic beta cells. J Biol Chem 2007; 282: 12030-12037
• 32 George M, Ayuso E, Casellas A et al. Beta cell expression of IGF-I leads to recovery from type 1 diabetes. J Clin Invest 2002; 109: 1153-1163
• 33 Yi P, Park JS, Melton DA. Betatrophin: A Hormone that Controls Pancreatic β Cell Proliferation. Cell 2013; 153: 747-745