Our research addresses functional defects in antigen presenting cells (APC) important in immunopathogenesis of Type 1 Diabetes (T1D). We have reported a biochemical component of APC dysfunction in diabetic/at-risk humans and in the nonobese diabetic (NOD) mouse: the aberrant expression of the normally inducible cyclooxygenase, prostaglandin synthase 2 (PGS2/COX2). In our analysis of trans acting factors, we found that autoimmune PGS2/COX2 expression was highly resistant to IL-10 suppression, and that high autocrine production of GM-CSF by at-risk/Type1 diabetic human monocytes and non-obese diabetic (NOD) mouse macrophages promotes a build up of tyrosine phosphorylated STAT5. Moreover, the activated STAT5 isoforms in these autoimmune cells have stage-specific and isoform-specific alterations in their DNA binding capabilities: truncated repressor isoforms are unable to bind DNA in unactivated autoimmune monocytes, and prolonged binding of full-length activator isoforms in autoimmune macrophages. Our preliminary data show that congenic strain allele replacement of a Chromosome 11 lcM locus around the GM-CSF gene, and not the region containing the STAT5A & B genes, can reverse prolonged STAT5 tyrosine phosphorylation in NOD monocytes and macrophages as well as the diminished DNA binding capacity of STAT5 truncated isoforms in NOD monocytes. This region includes the Idd4.2 susceptibility locus and several known genes for cytokines whose effects can be mediated through the activation of STAT5. In addition, we have found that STAT5 is also aberrant in its subcellular translocation after GM-CSF activation of autoimmune human monocytes and NOD monocytes & macrophages. However after activated by GM-CSF, persistent STAT5 phosphorylation and DNA binding in NOD macrophages become independent of GM-CSF and Jak2/3 kinase activity. In our original submission, our goal was to examine STAT5 signal transduction/transcription regulation in Type 1 diabetic patients, their at-risk relatives, and in the NOD mouse, and determine its 1) role in monocyte and macrophage PGS2 expression dysregulation, 2) possible effects on myeloid differentiation, and 3) relationship to APC dysfunction in the immunopathogenesis of Type 1 Diabetes. This competitive renewal proposal outlines our progress on these aims and how these findings and recent advances in the understanding of how STAT5 functions in gene regulation, have led us to a new avenue of investigation of the interplay of autoimmune STAT5 dysfunction, GM-GSF hypersensitivity, and aberrant PGS2 expression. In light of the recent elucidation of the physiological differential induction of STAT5 isoforms,and STAT5's potential role in promoting or suppressing dynamic chromatin, we now propose to expand our phenotypic characterization of GM-CSF-induced STAT5 isoforms to include their interactions with SMRT/NGoR and CBP/gp300 enzymes and chromatin in regulation of histone acetylation at the NOD PGS2/COX2 gene/enhancer-containing Chromosome1 interval and the NOD GM-CSF/Idd4.2-containing Chromosome 11 interval, to see if chromatin modification is involved in the dysregulation of PGS2 transcriptional activation by STAT5 in NOD monocytes and macrophages. In addition, we add a new Specific Aim (4) to investigate STAT5 regulatory mechanisms involving PIAS3, SOCS3, SHP1, SHP2 and IL10 that may be altered in autoimmune monocytes and macrophages, allowing prolonged STAT5 phosphorylation independent of concurrent GM-GSF/Jak activation as well as affecting STAT5 DNA binding capacity.
