Regulation of STAT1 signalling in human pancreatic ß-cells by the lysine deacetylase, HDAC6; a new therapeutic opportunity in type 1 diabetes?

Type 1 diabetes arises from the selective destruction of pancreatic ß-cells by autoimmune mechanisms and intracellular pathways driven by Janus (JAK)-kinase mediated STAT isoforms (especially STAT1 & STAT2) are implicated as mediators of ß-cell demise. Despite this, the molecular mechanisms that regulate JAK-STAT signalling in ß-cells during the autoimmune attack remain only partially disclosed and the factors acting to antagonise pro-inflammatory STAT1 signalling are uncertain. We have recently implicated Signal Regulatory Protein (SIRP)-α in promoting ß-cell viability in the face of ongoing islet autoimmunity and now reveal that this protein controls the availability of a cytosolic lysine deacetylase, HDAC6, whose activity regulates the phosphorylation and activation of STAT1. We provide evidence that STAT1 serves as a substrate for HDAC6 in ß-cells and that sequestration of HDAC6 by SIRPα in response to anti-inflammatory cytokines (such as interleukin-13) leads to increased STAT1 acetylation. This then impairs the ability of STAT1 to promote gene transcription in response to pro-inflammatory cytokines including interferon-gamma (IFNγ). We further find that SIRPα is lost from the ß-cells of subjects with recent-onset type 1 diabetes under conditions when HDAC6 is retained and STAT1 levels are increased. On this basis, we report a previously unrecognised role for cytokine-induced regulation of STAT1 acetylation in the control of ß- cell viability and propose that targeted inhibition of HDAC6 activity may represent a novel therapeutic modality to promote ß-cell viability in the face of active islet autoimmunity.

Temporal regulation of interferon signalling in human EndoC-ßH1 cells.

During the development of type 1 diabetes, interferons (IFN) are elaborated from islet-infiltrating immune cells and/or from virally infected ß-cells. They act via specific receptors to increase, acutely, the phosphorylation of the transcription factors STAT1 and 2. However, the longer-term impacts of chronic IFN stimulation are poorly understood and were investigated in the current study. Human EndoC-ßH1 cells were treated with IFNα, IFNγ or IFNλ either acutely (<2 h) or chronically (≥24 h) and STAT phosphorylation, expression and activity were assessed by Western blotting and transcriptional reporter assays. Exposure of ß-cells to IFNα or IFNλ induced a swift increase in the phosphorylation of both STAT1 and STAT2, whereas IFNγ increased only pSTAT1. Over more extended periods (≥24 h), STAT phosphorylation declined but STAT1 and STAT2 expression were enhanced in a sustained manner. All IFNs stimulated ISRE transcriptional activity (but with different time courses), whereas GAS activity was responsive only to IFNγ. The re-addition of a second bolus of IFNα, 24 h after an initial dose, failed to cause renewed STAT1/2 phosphorylation. By contrast, when IFNγ was added 24 h after exposure to IFNα, rapid STAT1 phosphorylation was re-initiated. Exposure of ß-cells to IFNs leads to rapid, transient, STAT phosphorylation and to slower and more sustained increases in total STAT1/2 levels. The initial phosphorylation response is accompanied by marked desensitisation to the cognate agonist. Together, the results reveal that the response of ß-cells to IFNs is regulated both temporally and quantitatively to achieve effective signal integration.

Expression of CD47 in the pancreatic ß-cells of people with recent-onset type 1 diabetes varies according to disease endotype.

AIMS: We are studying the dialogue between ß-cells and the immune system in type 1 diabetes and have identified a cell surface receptor, signal regulatory protein-alpha (SIRPα) as an important component in the regulation of ß-cell survival. SIRPα interacts with another protein, CD47, to mediate signalling. In the present work, we have studied the expression and role of CD47 in human islet cells in type 1 diabetes. METHODS: Clonal EndoC-ßH1 cells were employed for functional studies. Cells were exposed to pro-inflammatory cytokines and their viability monitored by flow cytometry after staining with propidium iodide. Targeted knockdown of CD47 or SIRPα was achieved with small interference RNA molecules and the expression of relevant proteins studied by Western blotting or immunocytochemistry. Human pancreas sections were selected from the Exeter Archival Diabetes Biobank and used to examine the expression of CD47 by immunofluorescence labelling. Image analysis was employed to quantify expression. RESULTS: CD47 is abundantly expressed in both α and ß cells in human pancreas. In type 1 diabetes, the levels of CD47 are increased in α cells across all age groups, whereas the expression in ß-cells varies according to disease endotype. Knockdown of either CD47 or SIRPα in EndoC-ßH1 cells resulted in a loss of viability. CONCLUSIONS: We conclude that the CD47 plays a previously unrecognised role in the regulation of ß-cell viability. This system is dysregulated in type 1 diabetes suggesting that it may be targeted therapeutically to slow disease progression.