Hedgehog signaling regulation of homeodomain protein islet duodenum homeobox-1 expression in pancreatic beta-cells

Insulin gene expression in pancreatic beta -cells is regulated by signals f rom developmental morphogen proteins known as hedgehogs (Hhs). By analyzing 5'-deletion insulin promoter-reporter constructs in transient transfection s of clonal INS-1 beta -cells, we located activating Hh-responsive regions within the rat insulin I promoter that include the glucose-response element s Far (E2) and Flat (A2/A3). Activation of Hh signaling in INS-1 cells by e ctopic Hh expression increased (and inhibition of Hh signaling with the Hh- specific inhibitor cyclopamine decreased) transcriptional activation of a m ultimerized FarFlat enhancer-reporter construct. In DNA-binding studies, nu clear extracts from INS-1 cells activated by ectopic Hh expression increase d land extracts from INS-1 cells treated with cyclopamine decreased) protei n binding to a radiolabeled FarFlat oligonucleotide probe. An antiserum dir ected against the transcription factor islet duodenum homeobox-l (IDX-1), a regulator of pancreas development and activator of the insulin gene promot er, attenuated the binding activity of Hh-responsive protein complexes. Nuc lear IDX-1 protein levels on Western blots were increased by ectopic Hh exp ression, thereby providing a mechanism for Hh-mediated regulation of the in sulin promoter. Addition of cyclopamine to INS-1 cells decreased IDX-1 mess enger RNA expression. In transient transfections of a -4.5-kb mouse IDX-1 p romoter-reporter construct, ectopic Hh expression increased (and cyclopamin e administration decreased) transcriptional activation of the IDX-1 promote r in a dose-dependent manner. Thus, the IDX-1 gene is a direct regulatory t arget of Hh signaling in insulin-producing pancreatic beta -cells. We propo se that Hh signaling activates the insulin gene promoter indirectly via the direct activation of IDX-1 expression. Because IDX-1 gene expression is es sential for insulin gene expression, pancreatic beta -cell development, and normal glucose homeostasis, our findings that Hh signaling regulates IDX-1 expression in the endocrine pancreas suggest possible novel therapeutic ap proaches for diabetes mellitus.