Wnt signaling pathway is well known for its roles in embryonic development and oncogenesis. Frizzled related protein (Frp) is a new family of secreted proteins that contain a region homologous to the extracellular cysteine-rich domain (CRD) of the Wnt receptor, the frizzled family proteins. Recent studies indicate that Frps act as Wnt antagonists in regulating or blocking the Wnt signals in the process of development and carcinogenesis. ^val135...[ Read more ]

Wnt signaling pathway is well known for its roles in embryonic development and oncogenesis. Frizzled related protein (Frp) is a new family of secreted proteins that contain a region homologous to the extracellular cysteine-rich domain (CRD) of the Wnt receptor, the frizzled family proteins. Recent studies indicate that Frps act as Wnt antagonists in regulating or blocking the Wnt signals in the process of development and carcinogenesis.

My major research project was to characterize the promoter activity and the expression patterns of secreted frizzled related protein 4 (sfrp4) gene, a homologue of the rat Frp (rFrp) found previously to be activated transcriptionally in a Rat 6 fibroblast cell line overexpressing p53^val135(R6#13-8). In addition, the sfrp4 targeting vector was constructed. This targeting vector would provide the tool for the future development of sfrp4 knockout mice. Data from the early embryonic study showed that the level of sfrp4 mRNA gradually increases from E 9.5 to E 17.5 embryos. Strong signals were detected in otic vesicles, eyes, tail bud and possible other organs in the left abdominal region of the E 9.5, 10.5 and 11.5 embryos using whole mount in-situ hybridization. The results suggest that the sfrp4 gene may play a significant role in the early developmental stage of mouse embryos.

At the same time, the transcriptional regulation of the gene was being explored in my study. A 1.5 kb promoter region of mouse sfrp4 was cloned and sequenced. Sequence alignments among rat, mouse and human reveal the putative transcriptional factor binding elements, some of which are well conserved among these three species. The promoter region of sfrp4 was analyzed using transient reporter assays along with site-directed mutagenesis. The Site 1, STAT3/Lyf-1/MZF1 & Site 2, C/EBP-β/GATA-1/CREB putative transcriptional factor binding sites were located in the mouse sfrp4 promoter spanning from nucleotides -238 to -144 that were found to be essential for the promoter activity of sfrp4. In addition to Sites 1 & 2, putative transcriptional factor binding sites for TFIID, SP1/GC and ATF/CREB exhibited positive, while sites for NRSE exhibited negative regulatory functions, as determined by the alkaline phosphatase activities of the reporter assay. We also demonstrated that the ATF/CREB site might cooperatively interact with the putative NRSF element in regulating sfrp4 activity. The data of this present study, the first promoter analysis of mouse Frp genes, provides the basis toward the understanding the tissue specific functions of frp and its role in regulating Wnt-signals.