WISP1/CCN4 Regulates Osteogenesis by Enhancind BMP-2 Activity- The CCN family is comprised of 6 members (CCN1-6) which are characterized by 4 distinct functional domains 1) an insulin-like growth factor-binding protein-like module (IGFBP), 2) a von Willebrand factor type C repeat (VWC), 3) a thrombospondin type 1 repeat (TSP1), and 4) a cysteine rich C-terminal module (CT). CCN5 is the exception, being a trimodular protein lacking the CT module. Numerous studies have demonstrated a role for the CCN family members in the regulation of osteo/chondrogenic differentiation of musculo-skeletal cells, both in vitro and in vivo. In addition, the CCN family has been implicated in the pathology of a number of fibrotic disorders and several types of malignancies including breast, prostate, and gastric cancer, and have functions identified in numerous biological processes such as embryonic development, angiogenesis, cell differentiation and tissue repair. At the cellular level CCN proteins can act as growth factors to stimulate differentiation and collagen synthesis, or to mediate cell binding, migration and signaling via interactions with integrins. In addition, CCN family members have also been identified as factors that can regulate members of the TGF-beta super-family. BMPs are members of the TGF-beta super-family of polypeptides. A fundamental function of BMPs is to induce the differentiation of mesenchymal cells towards cells of the osteoblastic lineage and then to promote osteoblastic maturation and function. This cellular regulation can, in turn, control the development of bone and cartilage and the acceleration of the healing of fractured bones. While BMPs play essential roles in bone formation and remodeling, there is still a need to modulate their activities. Numerous antagonists of BMPs exist such as noggin, chordin, short gastrulation (Sog) and twisted gastrulation (Tsg) that control BMP activity. The modulation of BMP activity can also be achieved by local feedback mechanisms, involving growth factor binding proteins, and other intracellular factors. Indeed, the CCN proteins, CCN2/CTGF and CCN3/Nov, both bind to BMPs and temper their functions in vitro and in vivo. These studies suggest that the CCN family is involved in regulation of osteoblast differentiation by interacting with members of the TGF-beta family, such as BMP-2, a growth factor long known to be an important modulator of skeletogenesis. With an aging world population the search for more efficient drug targets for age related disorders such as osteoporosis and osteoarthritis has become essential. The use of anti-resorptive drugs has prevailed as the most common treatment of osteoporosis, however in more recent years the use of anabolic agents such as PTH and BMP-2 have become more common. Most recently the search for biological proteins that can increase bone mass has focused on the wnt signaling pathway due to the reports of high bone mass in people with mutations in the wnt receptor, LRP5/6. WISP-1 is a member of the CCN family that is highly expressed in skeletal tissues. Evidence points to a role for WISP-1 in bone formation and maintenance. Expression of WISP-1 has been observed in the developing skeleton align, and later in both pre-osteoblastic and osteoblastic cells, specifically at sites of new bone formation during development or in healing fracture calluses. WISP-1 was initially identified as the principle gene target of the Wnt-1/Frizzled pathway in breast cancer cells, with its expression regulated by beta-catenin. The Wnt/beta-catenin signaling pathway has proved to be essential in the regulation and maintenance of bone mass, and mutations in LRP5/6 which prevent binding to its inhibitor, dickoff 1, cause high bone mass phenotype in humans. Moreover, WISP-1 has been identified as a gene highly up-regulated in osteoarthritis. Previous work from our lab has shown that recombinant WISP-1 promotes the proliferation and differentiation of human bone marrow stromal cells (hBMSC) in vitro via a relationship with members of the TGF-beta super-family. However, the exact skeletal functions of WISP-1 in vivo or the molecular mechanism involved have not yet been elucidated. We have previously shown a co-operative relationship between WISP-1 and TGF-beta in regulating osteoblastic proliferation and differentiation. However, the relationship of WISP-1 to BMP-2 in bone formation has not yet been determined. To deepen our understanding of the function of WISP-1 in osteogenesis, we investigated the effect of WISP-1 on BMP-2 induced osteogenic differentiation and found that WISP-1 potentiates the effects of BMP-2 in progenitor cells from the bone marrow. Evidence points to an essential role of CCN family members in integrin binding and signaling, therefore, we also determined the effects of WISP-1 on integrin production and function and found a functional relationship between WISP-1 and alpha5beta1 integrin which can regulate BMP-2 bio-availability in hBSMC cultures. Finally, to determine the in vivo effects of WISP-1 during osteogenesis we generated transgenic mice that over-express WISP-1 under the control of the 2.3 kb bone-specific Col1A1 promoter and determined their skeletal phenotype. These mice have an increased bone mass phenotype suggesting that WISP-1 may be a potent inducer of bone formation. Taken together, these data present a new paradigm revealing that WISP-1 has enhancing effects on BMP function during osteogenesis that are dependent on the integrin alpha5beta1. Differential effects of fibromodulin deficiency on mouse mandibular bones and teeth - a microCT time course study- Fibromodulin (Fmod) is a keratan sulfate small leucine-rich proteoglycan which is enriched in bones and teeth. In order to determine its functions on bone and tooth mineralization we characterized the phenotype of Fmod-deficient (Fmod-KO) mice using a new-generation microfocus computerized tomography system (micro-CT) and software allowing advanced visualization of 3-D data. Three-week-old and 10- week-old Fmod-KO mandibles and teeth were compared with those of age-matched wild-type (WT) mice. In both young and mature mice, the Fmod-KO mandibles were hypomineralized, especially the posterior (proximal) part of the mandible as it appeared to be the main target of the molecular deficiency, whereas less extensive alterations were found in the alveolar bone. In transverse sections, larger marrow spaces were observed in the Fmod-KO mice compared with age-matched young or mature WT mice. Quantitative evaluation of the pulp volume of the first molar and 3-D reconstructions suggested that dentinogenesis was diminished in 3-week-old Fmod-KO teeth. In contrast, increased dentin formation was found in 10-week-old Fmod-KO mice and was accompanied by a reduced pulp volume. Thus, the differential effects of Fmod deficiency on bones and teeth appear to diverge in adult mice. This may result from the previously reported differences in the molecular weight of Fmod in the 2 tissues or from compensatory mechanisms due to the overexpression of DSP and DMP-1 in the dental pulp of Fmod-KO. It is also possible that a single molecule plays diverging roles in a tissue-specific or region-specific manner.
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