Neuronal insulin receptor substrate 2 (IRS2) expression is regulated by ZBP89 and SP1 binding to the IRS2 promoter

Abstract

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 K779 and K679 bp from the translational start which is responsible for w50% 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.

Anti-oncogenic MicroRNA-203 Induces Senescence by Targeting E2F3 Protein in Human Melanoma Cells

“…These findings suggest that the ZBP-89 gene functions as both an oncogene and anti-oncogene. Additionally, ZBP-89 binds to the neuronal IRS2 promoter and inhibits the activation of IRS2 (37). Our data showed that the introduction of siR-ZBP-89 into Mewo cells enhanced their IRS2 and phospho-Akt expression levels.…”

Regulation of Blood Pressure, Appetite, and Glucose by Leptin After Inactivation of Insulin Receptor Substrate 2 Signaling in the Entire Brain or in Proopiomelanocortin Neurons

“…Jak2 tyrosine phosphorylation in the CNS also leads to phosphorylation of IRS2 protein which binds to the regulatory subunit (P85) of phosphatidylinositol 3-kinase (PI3K) and activates the catalytic domain of this intracellular enzyme, initiating a cascade of downstream kinases that regulate ion channel activity, gene transcription, and protein translation 7, 8 .…”

Palmitate increases Nur77 expression by modulating ZBP89 and Sp1 binding to the Nur77 proximal promoter in pancreatic β‐cells

“…ZBP89 inhibits cell growth via the repression of gastrin and ornithine decarboxylase [25,26]. More recently, [28] revealed that neuronal insulin receptor substrate 2 (IRS2) expression is regulated by ZBP89 and Sp1 binding to the IRS2 promoter. A regulatory cassette composed of four ZBP89/SP1 binding sites controlled the IRS2 promoter activity in response to the PI3K pathway activation.…”