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This Article Full Text Full Text (PDF) Supplementary figure All Versions of this Article: JOE-09-0266v1 204/2/199    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Udelhoven, M. Articles by Schubert, M. PubMed PubMed Citation Articles by Udelhoven, M. Articles by Schubert, M.

Department of Internal Medicine II, Center for Molecular Medicine Cologne (CMMC) and Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), University of Cologne, Kerpener Straße 62, 50937 Cologne, Germany

(Correspondence should be addressed to M Schubert; Email: markus.schubert{at}uni-koeln.de)

Since neuronal insulin receptor substrate 2 (IRS2)-mediated signals coordinate key processes in rodent physiology such as food intake, fertility, longevity, and aging-related behavior, we analyzed the mechanisms of neuronal IRS2 expression in neuroblastoma (SHSY5Y) and hypothalamic (GT1-7) cell lines. Using dual luciferase reporter assays and IRS2 promoter deletion constructs, we identified a regulatory cassette within the IRS2 promoter between –779 and –679 bp from the translational start which is responsible for 50% of neuronal IRS2 promoter activity. Chromatin immunoprecipitation assays and electromobility shift assay revealed four overlapping ZBP89/specificity protein 1 (SP1) binding sites which alternatively bind to ZBP89 (ZNF148 as listed in the HUGO Database) or SP1. Activation of this cassette is inhibited by phosphoinositide-3-kinase (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 IRS2 expression. Mutants containing all the possible combinations of one, two, three, or all the four SP1 binding sites of the IRS2 promoter revealed that SP1 binding to one particular site is most important for promoter activation. Stable downregulation of ZBP89 using siRNA substantially increased IRS2 mRNA and protein expression. Thus, alternative binding of ZBP89 or SP1 to the described region in the IRS2 promoter regulates neuronal IRS2 expression in a PI3K-dependent manner.