Control of differentiation-induced calbindin-D9k gene expression in Caco-2 cells by cdx-2 and HNF-1alpha.
Calbindin D9k (CaBP) is critical for intestinal calcium absorption; its in vivo expression is restricted to differentiated enterocytes of the small intestine. Our goal was to identify factors controlling the transcriptional regulation of this gene in the human intestine. Both the natural gene and a 4600-bp promoter construct were strongly regulated by differentiation (>100-fold) but not by treatment with 1,25(OH)2 vitamin D (<2-fold) in the Caco-2 clone TC7. Deletion-mutation studies revealed that conserved promoter sequences for cdx-2 (at -3158 bp) and hepatocyte nuclear factor (HNF)-1 (at -3131 and at -98 bp) combined to control CaBP expression during differentiation. Other putative response elements were not important for CaBP regulation in TC7 cells (CCAAT enhancer binding protein, pancreatic duodenal homebox-1 (pdx-1), a proximal cdx-2 element). Mutation of the distal HNF-1 site had the greatest impact on CaBP gene expression through disruption of HNF-1alpha binding; both basal and differentiation-mediated CaBP expression was reduced by 80%. In contrast, mutation of the distal cdx-2 element reduced only basal CaBP expression. Whereas a 60% reduction of CaBP mRNA in the duodenum of HNF-1alpha null mice confirmed the physiological importance of HNF-1alpha for CaBP gene regulation, additional studies showed that maximal CaBP expression requires the presence of both HNF-1alpha and cdx-2. Our data suggest that cdx-2 is a permissive factor that influences basal CaBP expression in enterocytes and that HNF-1alpha modulates CaBP gene expression during cellular differentiation.
Wang
Liyong
L
Interdepartmental Nutrition Program, Purdue University, West Lafayette, Indiana 47907, USA.
Klopot
Anna
A
Freund
Jean-Noel
JN
Dowling
Lauren N
LN
Krasinski
Stephen D
SD
Fleet
James C
JC
D000818:Animals; D000071616:CDX2 Transcription Factor; D018938:Caco-2 Cells; D064026:Calbindins; D002454:Cell Differentiation / Q000502:physiology; D002999:Clone Cells; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000502:physiology*; D004586:Electrophoresis / Q000379:methods; D015870:Gene Expression / Q000187:drug effects; D005786:Gene Expression Regulation / Q000502:physiology; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D018398:Homeodomain Proteins / Q000502:physiology*; D006801:Humans; D007422:Intestines / Q000166:cytology*; D051379:Mice; D018345:Mice, Knockout; D005810:Multigene Family; D009687:Nuclear Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D020218:Response Elements; D064030:S100 Calcium Binding Protein G / Q000235:genetics / Q000378:metabolism*; D013237:Stereoisomerism; D015534:Trans-Activators; D014157:Transcription Factors / Q000502:physiology*; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology
D000071616:CDX2 Transcription Factor; D064026:Calbindins; D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; C496488:Hnf1a protein, mouse; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D064030:S100 Calcium Binding Protein G; C576506:S100G protein, human; C576507:S100g protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D014807:Vitamin D; C097949:1,25-dihydroxyvitamin D