Abstract
BackgroundGAD65 (Glutamic acid decarboxylase 65 KDa isoform) is one of the most important auto-antigens involved in Type 1 diabetes induction. Although it serves as one of the first injury markers of β-islets, the mechanisms governing GAD65 expression remain poorly understood. Since the regulation of GAD65 is crucial for the proper functioning of insulin secreting cells, we investigated the stress induced regulation of GAD65 transcription.ResultsThe present study shows that SMAR1 regulates GAD65 expression at the transcription level. Using a novel protein-DNA pull-down assay, we show that SMAR1 binding is very specific to GAD65 promoter but not to the other isoform, GAD67. We show that Streptozotocin (STZ) mediated DNA damage leads to upregulation of SMAR1 and p53 expression, resulting in elevated levels of GAD65, in both cell lines as well as mouse β-islets. SMAR1 and p53 act synergistically to up-regulate GAD65 expression upon STZ treatment.ConclusionWe propose a novel mechanism of GAD65 regulation by synergistic activities of SMAR1 and p53.
Highlights
GAD65 (Glutamic acid decarboxylase 65 KDa isoform) is one of the most important auto-antigens involved in Type 1 diabetes induction
Further manual alignments of the derived sequences lead to identification of a hexamer (TAATPu/Py Pu) consensus SMAR1 binding sequences where first four nucleotides are conserved while fifth one can be either pyrimidine or purine and the last nucleotide is either of the purines (Figure 1A, sequence shown in red)
In order to confirm that SMAR1 binds to the GAD65 promoter, we performed CNBr pull down assay
Summary
GAD65 (Glutamic acid decarboxylase 65 KDa isoform) is one of the most important auto-antigens involved in Type 1 diabetes induction. It serves as one of the first injury markers of β-islets, the mechanisms governing GAD65 expression remain poorly understood. Type diabetes is an immunologically encountered autoimmune disease characterized by specific destruction of beta cells of islets of langerhans residing in pancreas [1]. This specific response to beta cells is caused due to immune response against self molecules that behave as non-self (known as auto antigens). One study in particular, performed on NOD mice (which are close to humans in their autoimmune manifestation of IDDM), incorporated an antisense GAD transgene into a subset of mice and found that IDDM development was significantly reduced [18]
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