| Wnt-1 inducible signaling protein 3 (WISP3/CCN6) is a member of the CCN family of cysteine-rich, multi-functional, growth factors, which includes c&barbelow;onnective tissue growth factor (CTGF/CCN2), c&barbelow;ysteine-rich protein 61 (cyr61/CCN1), n&barbelow;ephroblastoma overexpressed (nov/CCN3), WISP1 (CCN4), and WISP2 (CCN5). Numerous in vitro and in vivo studies performed on CCNs show their involvement in skeletal, vascular, and neural processes. In humans, loss-of-function mutations in WISP3 cause the autosomal recessive skeletal disease Progressive Pseudorheumatoid Dysplasia (PPD; [OMIM 208230]). Functionally, CCN proteins have been implicated in numerous cellular processes, such as cell migration, proliferation, differentiation, and apoptosis, with the exception of WISP3, which has not been thoroughly characterized. A few studies have suggested the involvement of WISP3 in tumorigenesis and the upregulation of cartilage matrix proteins; however, a proper analysis of WISP3 protein and its function(s) has not been performed. This work describes studies that have been performed in order to elucidate the function of WISPS.;To understand the role of WISP3 in the skeleton, we disrupted the Wisp3 gene in mice in order to create an animal model of PPD. We found that homozygous Wisp3-mutant (Wisp3 -/-) mice do not recapitulate any of the features that have been observed in patients with PPD. We also created transgenic mice that over-express human WISP3 in cartilage; these mice are also normal. Remarkably, our results suggest that although WISP3 is critical for normal skeletal function in humans, Wisp3 is not essential in mice. To better characterize WISP3 protein localization, modification, and function we generated WISP3-specific antibodies and cell lines. The lack of reliable WISP3-specific reagents and detectable endogenous expression has limited WISP3 protein studies in the past. Using these tools, in conjunction with our WISP3 transgenic mice, we have been able to show WISP3 is a N-linked glycosylated, secreted protein capable of binding heparin and being cleaved by serine proteases. In vivo protein expression in WISP3 transgenic mice indicates WISP3 undergoes proteolytic processing to allow WISP3 fragments to localize to specific regions of cartilage. We conclude WISP3 is a highly modified protein possibly possessing multiple functions and sites of localization in cartilage. |
