PDX-1 can repress stimulus-induced activation of the INGAP promoter.
Islet neogenesis associated protein (INGAP) promotes the generation of new islet mass in adult animal models. It is not understood what factors control the expression of INGAP. In this study, factors that regulate the expression of INGAP promoter activity are reported. To determine factors that regulate INGAP expression, we previously cloned the promoter region for INGAP. Analysis of the INGAP promoter suggested that candidate regulators of INGAP expression include the transcription factors PDX-1, NeuroD, PAN-1, STAT and AP-1. Using gene addition experiments in the 293 cell line the activity of these transcription factors on an INGAP-promoter construct linked to the beta-galactosidase reporter has been determined. Induction of AP-1 activity or STAT activity using PMA or LIF stimulation respectively, or direct expression of PAN-1 specifically up-regulates INGAP promoter activity. In contrast, co-expression of PDX-1 but not NeuroD inhibits activation of the INGAP-promoter driven by PAN-1, PMA or LIF stimulation. PDX-1 binds directly to the INGAP promoter as determined in electromobility shift and antibody supershift assays. Expression of the INGAP-promoter-reporter construct in the HIT-T15 beta-cell line, a cell line that expresses endogenous PDX-1, did not reveal PMA-mediated stimulation of INGAP promoter activity. HIT-T15 cells however did efficiently transfect (> 68%) and respond (2-fold) to PMA-induced signal transduction to a transfected AP-1-CAT reporter. Partial reduction of PDX-1 expression in HIT-T15 cells was associated with recovery of PMA induced INGAP promoter activity. These data suggest that expression of PDX-1 is associated with a repression of stimulus-induced INGAP promoter activity that appears to be mediated by a direct DNA interaction. These findings implicate PDX-1 in a possible feedback loop to block unbridled islet expansion.
Taylor-Fishwick
David A
DA
Department of Medicine, Strelitz Diabetes Institutes, Eastern Virginia Medical School, Norfolk, Virginia 23510, USA. taylord@evms.edu
Shi
Wenjing
W
Pittenger
Gary L
GL
Vinik
Aaron I
AI
D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics* / Q000378:metabolism; D051792:Basic Helix-Loop-Helix Proteins / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D006224:Cricetinae; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects*; D018398:Homeodomain Proteins / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism*; D015850:Interleukin-6 / Q000494:pharmacology; D037181:Lectins, C-Type / Q000235:genetics* / Q000378:metabolism; D053605:Leukemia Inhibitory Factor; D009419:Nerve Tissue Proteins / Q000494:pharmacology; D000073718:Pancreatitis-Associated Proteins; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D050791:STAT Transcription Factors / Q000378:metabolism; D051820:TCF Transcription Factors / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015534:Trans-Activators / Q000378:metabolism / Q000494:pharmacology*; D058125:Transcription Factor 7-Like 1 Protein; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014162:Transfection / Q000379:methods
D000951:Antigens, Neoplasm; D051792:Basic Helix-Loop-Helix Proteins; D014408:Biomarkers, Tumor; D018398:Homeodomain Proteins; D015850:Interleukin-6; D037181:Lectins, C-Type; D053605:Leukemia Inhibitory Factor; D009419:Nerve Tissue Proteins; D000073718:Pancreatitis-Associated Proteins; D050791:STAT Transcription Factors; D051820:TCF Transcription Factors; D013755:Tetradecanoylphorbol Acetate; D015534:Trans-Activators; D058125:Transcription Factor 7-Like 1 Protein; D018808:Transcription Factor AP-1; C507837:LIF protein, human; C000612141:REG3A protein, human; C544912:TCF7L1 protein, human; C084010:pancreatic and duodenal homeobox 1 protein; C093564:Neurogenic differentiation factor 1