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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.

Molecular cloning and characterization of the human WISP-2/CCN5 gene promoter reveal its upregulation by oestrogens