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M Udelhoven, Cologne, Germany
M Pasieka, Cologne, Germany
U Leeser, Cologne, Germany
W Krone, Cologne, Germany
M Schubert, Department of Internal Medicine II, University of Cologne, Cologne, 50937, Germany

Correspondence: Markus Schubert, Email: markus.schubert{at}uni-koeln.de

Since neuronal IRS-2 mediated signals coordinate key processes in rodent physiology like food intake, fertility, longevity, as well as aging related behaviour we analyzed mechanisms of neuronal IRS-2 expression in neuroblastoma (SHSY5Y) and hypothalamic (GT1-7) cell lines. Using dual luciferase reporter assays and IRS-2 promoter deletion constructs we identified a regulatory cassette within the IRS-2 promoter between -779 bp and -679 bp from the translational start which is responsible for ~50 % of neuronal IRS-2 promoter activity. ChIP assays and EMSA revealed four overlapping ZBP89/SP1 binding sites which alternatively bind ZBP89 or SP1. Activation of this cassette is inhibited by PI3K via increased ZBP89 binding to the promoter. Serum starvation caused increased SP1 binding at one specific SP1 site and decreased binding to another proving a regulatory interaction between the different binding sites within this promoter cassette to tightly control IRS-2 expression. Mutants containing all possible combinations of one, two, three or all four SP1 binding sites of the IRS-2 promoter revealed that SP1 binding to one particular site is most important for promoter activation. Stable downregulation of ZBP89 using siRNA substantially increased IRS-2 mRNA and protein expression. Thus, alternative binding of ZBP89 or SP1 to the described region in the IRS-2 promoter regulates neuronal IRS-2 expression in a PI3K dependant manner.