The promoter of the long variant of collagen XVIII, the precursor of endostatin, contains liver-specific regulatory elements.
The endostatin precursor collagen XVIII is expressed at high levels in human livers, the main source being hepatocytes. We have studied the regulatory elements in the promoter 2 of the Col18a1 gene that directs the transcription of the NC1-517 variant of collagen alpha1(XVIII), which is the main form expressed in the liver. The 5'-flanking region of Col18a1 gene was cloned, and a series of 5'-deletions from -3286 bp to +285 bp were linked to the luciferase reporter gene. Transfection experiments in HepG2 cells allowed to identify a silencer-like element containing putative HNF1 and HNF3 sites and activator elements containing stretches of GC-rich sequences. Another putative HNF3 site in close apposition to a NF1/CTF site was localized upstream of the silencer-like element. Cotransfection experiments showed that the Col18a1 promoter 2 was transactivated by Sp1 and HNF3alpha. Gel-shift analyses showed that HNF3, NF1/CTF, and Sp1-like sites specifically recognized nuclear factors. Super-shift experiments indicated that HNF3beta was the major form of HNF3 interacting with the HNF3/NF1 site. The well-differentiated hepatoma cell line mhATFS315 transfected with a truncated form of HNF3beta, which competitively blocks HNF3 transactivating activity, expressed the Col18a1gene at a very low level. Taken together, these data strongly suggest that Col18a1 is a liver-specific gene. Furthermore, gel-shift analyses performed with nuclear factors prepared from well-differentiated hepatocellular carcinomas showed increased HNF3/NF1 binding activity compared with normal livers. Consequently, the precursor of endostatin might be differently expressed according to the differentiated and/or transformed state of hepatocytes.
Liétard
J
J
Detoxication and Tissue Repair Unit, INSERM U-456, Université de Rennes I, France.
Théret
N
N
Rehn
M
M
Musso
O
O
Dargère
D
D
Pihlajaniemi
T
T
Clément
B
B
D001483:Base Sequence / Q000235:genetics; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism*; D043170:Collagen Type XVIII; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D043169:Endostatins; D017353:Gene Deletion; D014644:Genetic Variation; D005822:Genetic Vectors; D051541:Hepatocyte Nuclear Factor 3-alpha; D051542:Hepatocyte Nuclear Factor 3-beta; D006801:Humans; D008099:Liver / Q000502:physiology*; D008113:Liver Neoplasms / Q000378:metabolism; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000502:physiology*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D022762:CCAAT-Enhancer-Binding Proteins; C077305:CTF-1 transcription factor; D043170:Collagen Type XVIII; D004268:DNA-Binding Proteins; D043169:Endostatins; C496499:FOXA1 protein, human; C496502:FOXA2 protein, human; D051541:Hepatocyte Nuclear Factor 3-alpha; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D010446:Peptide Fragments; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051542:Hepatocyte Nuclear Factor 3-beta; D003094:Collagen