Molecular mechanisms involved in the regulation of beta-cell viability by Signal Regulatory Protein-Alpha (SIRPα)

Type 1 diabetes mellitus (T1DM) is characterised by pancreatic β-cell demise, resulting in a requirement for permanent insulin therapy. Pro-inflammatory cytokine pathways, acting via STAT1 and STAT2, contribute to β-cell death whereas anti-inflammatory mechanisms (operating via cytokines such as Interleukin-13 (IL-13)) are downregulated. IL-13 has been shown to protect β-cell viability via STAT6 signalling, and preliminary data suggested that Signal Regulatory Protein-α (SIRPα) also plays a vital part in this protective effect. However, the protective mechanisms by which SIRPα sustains β-cell viability are poorly understood. Recently, it has been proposed that histone deacetylase 6 (HDAC6) binds to SIRPα, and that this binding interaction suppresses the STAT1 mediated pro-apoptotic pathway, thus protecting β-cells against death by apoptosis. It is not known how this interaction is regulated. In this study, the aim was to explore the importance of tyrosine phosphorylation of SIRPα in maintaining the interaction with HDAC6 by replacing the cytoplasmic tyrosine residues with phenylalanine to block the phosphorylation events. Several molecular and cellular approaches were employed, including site-directed mutagenesis to generate single mutants of human SIRPα (Y429F, Y453F, Y470F, Y496F) and a quadruple mutant (FFFF) of rodent SIRPα to block tyrosine phosphorylation; use of INS-1E and HEK293T cells as experimental models, Western blotting, and Co-immunoprecipitation (Co-IP) assays to detect the interaction between SIRPα and HDAC6. The results show that IL-13 stimulation led to STAT6 phosphorylation and upregulation of SIRPα expression in β-cells, linking the STAT6: SIRPα axis to β-cell viability. HDAC6 was found to co-immunoprecipitate with SIRPα, but, surprisingly, this interaction was independent of tyrosine phosphorylation under basal conditions. Overall, this study confirmed that the upregulation of SIRPα expression in pancreatic β-cells is mediated by the IL-13/STAT6 pathway and that this may lead to HDAC6 recruitment and thereby its sequestration. The binding interaction between SIRPα and HDAC6 was not tyrosine phosphorylation dependent. The work also underscores the importance of species-specific differences when using rodent versus human β-cell cells as species-specific structural differences were noted to impact the binding of HDAC6 to SIRPα. Together, these findings establish a framework for further investigation of SIRPα regulatory mechanisms in regulating β-cell viability and their relevance to T1DM pathogenesis.<p></p>

Author dc:creator

• Abdullah Alatawi (21042413)

• Uncategorised value

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• All rights reserved

Identifier

10779/exe.31078924.v1

OAI identifier oai:identifier

oai:figshare.com:article/31078924