Depletion of the phosphatase inhibitor, PPP1R1A, may contribute to beta-cell loss in Type 1 diabetes

Abstract

Increasing evidence implicates a persistent enteroviral infection of β-cells as a potential trigger for the development of Type 1 diabetes (T1D). In support of this, findings presented in this thesis demonstrate that interferon-stimulated genes are upregulated in pancreas samples from T1D donors, but absent from donors without T1D, despite evidence of viral protein in their islets. The reasons for this exaggerated response are unclear but may be related to altered regulation of the viral recognition protein, MDA5. Protein phosphatase 1, regulatory, inhibitory, subunit 1A (PPP1R1A) is a largely unstudied molecule and has a restricted tissue distribution, but is highly expressed in β-cells. PPP1R1A specifically regulates protein phosphatase 1 (PP1) which has a central role in coordinating MDA5 activity. Findings presented in this thesis demonstrate that PPP1R1A is depleted from β-cells in T1D. To explore the impact of PPP1R1A on β-cell function, clonal lines of tetracycline inducible β-cells were developed using the PPP1R1A-deficient 1.1B4 cells as a host line for the Flp-In T-REx system. Two cell lines were generated which express either wild-type (WT) PPP1R1A or a phosphorylation-null (T35A) mutant form, upon addition of tetracycline. During the development of these Flp-In T-REx lines, I made the discovery that the parental 1.1B4 line was contaminated with an unidentified rat cell line. Data are presented on how this contamination was discovered and the steps taken to re-derive and characterise new human 1.1B4 cells lines. These findings have resulted in the withdrawal of 1.1B4 (and other related cell lines) from the European Collection of Authenticated Cell Cultures (ECACC) and a change in international practice for the authentication of cell lines. Despite these difficulties, the Flp-In T-REx PPP1R1A cell lines and other human cell lines available, were used to explore (1) the role of PPP1R1A in cell cycle progression and (2) the role of PPP1R1A in regulation of secretion from β-cells. Cell cycle progression was found to be reliant upon the timing of sequential PPP1R1A phosphorylation and dephosphorylation. Phosphorylation of PPP1R1A is critical for successful completion of the cell cycle and sustained phosphorylation of PPP1R1A resulted in apoptosis. Previous studies had identified PPP1R1A as a critical component necessary for insulin secretion. The studies reported in this thesis demonstrate that PPP1R1A could also play a previously unrecognised role in regulating constitutive secretion of molecules from cells.