Molecular cloning and characterization of the human WISP-2/CCN5 gene promoter reveal its upregulation by oestrogens.
Wnt-1-induced signalling pathway protein-2 (WISP-2)/connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN)5 is a member of the CCN family of growth factors and was identified as an oestrogen- inducible gene in the MCF-7 cell line. However, the role of WISP-2/CCN5 in breast carcinogenesis remains unclear. In this study, we examined the mechanism by which oestrogens regulate the expression of human (h) Wnt-1 induced signalling pathway protein (WISP-2)/CCN5. Real-time RT-PCR showed that hWISP-2/CCN5 mRNA transcripts level is upregulated by oestrogens in the oestrogen receptor-positive human breast cancer cell lines MCF-7, T47D and ZR-75.1. Cloning of a 1.9 kb fragment of the hWISP-2/CCN5 5'-flanking sequence and subsequent analysis of potential transcription factor-binding sites identified a functional oestrogen response element site located between - 581 and - 569 upstream from the oestrogen-induced transcription start site. Transient transfections of MCF-7 cells with the cloned fragment showed that oestradiol caused an increase in reporter gene activity, which was inhibited by anti-oestrogens ICI 182 780 and 4-hydroxytamoxifen. Chromatin immunoprecipitation analysis revealed an oestradiol-dependent recruitment of the oestrogen receptor alpha to the oestrogen- responsive region of the hWISP-2/CCN5 gene promoter. We also showed that endogenous CREB-binding protein (CBP) and p21(WAF1/CIP1) are recruited to the chromosomal hWISP-2/CCN5 promoter in MCF-7 cells in an oestrogen-dependent manner, suggesting that CBP and p21(WAF1/CIP1) participate in the oestrogen receptor alpha-mediated transcriptional control of the hWISP-2/CCN5 gene.
Fritah
Asmaà
A
Institut National de la Santé et de la Recherche Médicale, U673, Hôpital Saint-Antoine, Université Pierre et Marie Curie (UPMC-Paris 6), Paris, France.
Redeuilh
Gérard
G
Sabbah
Michèle
M
D024506:5' Flanking Region; D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods; D001943:Breast Neoplasms / Q000378:metabolism*; D055511:CCN Intercellular Signaling Proteins; D050882:CREB-Binding Protein / Q000378:metabolism; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000032:analysis / Q000378:metabolism; D004967:Estrogens / Q000378:metabolism*; D005260:Female; D000077267:Fulvestrant; D015870:Gene Expression / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000032:analysis / Q000235:genetics*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods
D055511:CCN Intercellular Signaling Proteins; C413323:CCN5 protein, human; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D004967:Estrogens; D036341:Intercellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D013629:Tamoxifen; C016601:afimoxifene; D000077267:Fulvestrant; D004958:Estradiol; D050882:CREB-Binding Protein; C494363:CREBBP protein, human