Abstract
Hepatic nuclear factor 1alpha (HNF1alpha) is a key regulator of development and function in pancreatic beta cells and is specifically involved in regulation of glycolysis and glucose-stimulated insulin secretion. Abnormal expression of HNF1alpha leads to development of MODY3 (maturity-onset diabetes of the young 3). We report that NK6 homeodomain 1 (NKX6.1) binds to a cis-regulatory element in the HNF1alpha promoter and is a major regulator of this gene in beta cells. We identified an NKX6.1 recognition sequence in the distal region of the HNF1alpha promoter and demonstrated specific binding of NKX6.1 in beta cells by electrophoretic mobility shift and chromatin immunoprecipitation assays. Site-directed mutagenesis of the NKX6.1 core-binding sequence eliminated NKX6.1-mediated activation and substantially decreased activity of the HNF1alpha promoter in beta cells. Overexpression or small interfering RNA-mediated knockdown of the Nkx6.1 gene resulted in increased or diminished HNF1alpha gene expression, respectively, in beta cells. We conclude that NKX6.1 is a novel regulator of HNF1alpha in pancreatic beta cells. This novel regulatory mechanism for HNF1alpha in beta cells may provide new molecular targets for the diagnosis of MODY3.
Highlights
HNF1␣ is an important gene involved in the developmental regulation of the liver, pancreas, kidneys, stomach, and intestines (10 –14)
HNF1␣ is involved in regulating the transcription of several genes that are involved in glycolysis and GSIS such as insulin [15], the Glut2 glucose transporter [16], pyruvate kinase [17], aldolase B [18], HNF3␥ [19], and HNF4␥ [19]
Huang et al [30] have show that, a Ϫ497-bp proximal HNF1␣-luciferase reporter was activated in hepatocytes, it failed to be activated in rat insulinoma (INS1) beta cells, and this promoter contains the proximal HNF4␣-binding site used for activation in hepatocytes
Summary
Cell Culture—NIH 3T3 mouse fibroblast cells, rodent insulinoma cell lines (TC3, NIT1, and INS1), and Huh human hepatocarcinoma cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 0.1% kanamycin in a 37 °C incubator with 100% humidity and 5% CO2. Mouse Ngn cDNA was cloned into the pcDNA3 vector using the BamHI restriction site. The Xenopus Pax expression plasmid (a generous gift from Dr Marko Horb) was derived by inserting Pax cDNA into the pcDNA3 vector using the HindIII and XhoI restriction sites. The human HNF1 and mouse HNF4␣ expression plasmids (pCMV-Sport6) were purchased from Open Biosystems. Cell lysates were harvested and measured 24 h post-transfection using the Dual Luciferase reporter kit (Promega) according to the manufacturer’s protocol except that only 50 l of each substrate reagent was used (as optimized by our laboratory) to read samples using a Lumat LB 9507 luminometer (Berthold Technologies). Gene Expression and Quantitative Reverse Transcription (RT)-PCR—Cells were cultured as described above and transfected with DNA or small interfering RNA (siRNA) as indicated using Lipofectamine 2000 reagent according to the manufacturer’s protocol.
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