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 from developmental morphogen proteins known as hedgehogs (Hhs). By analyzing 5'-deletion insulin promoter-reporter constructs in transient transfections of clonal INS-1 beta-cells, we located activating Hh-responsive regions within the rat insulin I promoter that include the glucose-response elements Far (E2) and Flat (A2/A3). Activation of Hh signaling in INS-1 cells by ectopic Hh expression increased (and inhibition of Hh signaling with the Hh-specific inhibitor cyclopamine decreased) transcriptional activation of a multimerized FarFlat enhancer-reporter construct. In DNA-binding studies, nuclear extracts from INS-1 cells activated by ectopic Hh expression increased (and extracts from INS-1 cells treated with cyclopamine decreased) protein binding to a radiolabeled FarFlat oligonucleotide probe. An antiserum directed against the transcription factor islet duodenum homeobox-1 (IDX-1), a regulator of pancreas development and activator of the insulin gene promoter, attenuated the binding activity of Hh-responsive protein complexes. Nuclear IDX-1 protein levels on Western blots were increased by ectopic Hh expression, thereby providing a mechanism for Hh-mediated regulation of the insulin promoter. Addition of cyclopamine to INS-1 cells decreased IDX-1 messenger RNA expression. In transient transfections of a -4.5-kb mouse IDX-1 promoter-reporter construct, ectopic Hh expression increased (and cyclopamine administration decreased) transcriptional activation of the IDX-1 promoter in a dose-dependent manner. Thus, the IDX-1 gene is a direct regulatory target of Hh signaling in insulin-producing pancreatic beta-cells. We propose that Hh signaling activates the insulin gene promoter indirectly via the direct activation of IDX-1 expression. Because IDX-1 gene expression is essential 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 approaches for diabetes mellitus.
Thomas
M K
MK
Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
Lee
J H
JH
Rastalsky
N
N
Habener
J F
JF
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D005947:Glucose / Q000502:physiology; D053823:Hedgehog Proteins; D018398:Homeodomain Proteins; D007328:Insulin / Q000235:genetics; D007515:Islets of Langerhans / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology; D011506:Proteins / Q000502:physiology*; D051381:Rats; D020218:Response Elements / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D053823:Hedgehog Proteins; D018398:Homeodomain Proteins; D007328:Insulin; D011506:Proteins; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D004247:DNA; D005947:Glucose