1. UNDERSTANDING THE MECHANISMS CONTROLLING PATHOLOGICAL OSSIFICATION IN THE ABSENCE OF BIGLYCAN (BGN) AND FIBROMODULIN (FMOD) (50%) We characterized in detail the ectopic ossification (EO) found in tendons of biglycan (Bgn), fibromodulin (Fmod) single knockout (KO) and double Bgn/Fmod deficient (DKO) mice with aging. At 3 months, Fmod KO, Bgn KO and DKO displayed torn cruciate ligaments and EO in their quadriceps tendons, menisci and cruciate and patellar ligaments. The phenotype was the least severe in the Fmod KO, intermediate in the Bgn KO and the most severe in the DKO. This condition progressed with age in all 3 mouse strains and resulted in the development of large supernumerary sesmoid bones. To determine the role of exercise on the extent of EO, we subjected normal and DKO mice to treadmill exercise for 3 days a week for 4 weeks. The EO in exercised DKO was decreased compared to unexercised DKO mice. Finally, DKO and Bgn KO mice tested using a rotarod showed they had a reduced ability to maintain their grip on a rotating cylinder compared to WT controls. In summary, we showed: 1) a detailed description of EO formed by Bgn, Fmod or combined depletion, 2) the role of exercise in modulating EO, and 3) that Bgn and Fmod are critical in controlling motor function. IDENTIFICATION AND FUNCTIONAL ANALYSIS OF SLRP ASSOCIATING FACTORS THAT MODULATE OSTEOGENESIS (50%) Our previous studies showed that bgn deficient mice (KO) develop age-dependent osteoporosis (osteopenia). Other experiments demonstrated that bgn binds to WISP-1 (wnt induced secreted protein-1), a CCN family member. The CCN family received it name based on the three founding members: Cysteine-rich angiogenic protein 61, Cyr61;Connective tissue growth factor, CTGF;and nephroblastoma over-expressed gene, NOV. Both bgn and WISP-1 proteins co-localize to sites of new bone formation, leading us to speculate that WISP-1 has functions in bone related to bgn action. The goal of this project was to first determine whether WISP-1 has critical functions in bone, and secondly, to determine if WISP-1 and TGF-beta could affect each others ability to control osteogenic cell function. The studies carried out so far provide the foundation for future work that will test the hypothesis that bgn can modify the effects of WISP-1 and its interaction with growth factors such as TGF-beta. Experiments were carried out to first determine if there is a functional relationship between WISP-1 and bgn. This study showed that WISP-1 could rescue the inhibition of proliferation in BMSCs caused by bgn. We also found that the reduced osteogenesis found in Bgn-deficient osteoblasts could be rescued by addition of WISP-1. These data implicate that bgn may function in cooperation with WISP-1 in controlling bone cell activities. Future work will further define the functional relationship of bgn to TGF-beta1 and WISP-1 during osteogenesis. WISP-1, like other members of the CCN family, is expressed in skeletal tissues. Its mechanism of action remains unknown. Expression of WISP-1 was analyzed in human bone marrow stromal cells (hBMSC) by RT-PCR. We identified two major transcripts corresponding to those of full length WISP-1, and of the splice variant WISP-1va, which lacks a putative BMP/TGF-beta binding site. To investigate the function of WISP-1 in bone, hBMSC cultures were treated with recombinant human (rh) WISP-1 and analyzed for proliferation and osteogenic differentiation. WISP-1 treatment increased both BrdU incorporation and alkaline phosphatase (AP) activity. Considering the known functional synergy found between the TGF-beta super-family and members of the CCN family, we next tested the effect of WISP-1 on TGF-beta activity. We found that rhWISP-1 could reduce rhTGF-beta induced proliferation as judged by BrdU incorporation. Similarly, rhTGF-beta inhibited rhWISP-1 induction of AP activity. To explore functional differences between the WISP-1 variants, WISP-1 or WISP-1va were transfected into hBMSCs. Both variants could strongly induce BrdU incorporation. However, there were no effects of either variant on AP activity without an additional osteogenic stimulus such as TGF-beta. Taken together our results suggest a functional relationship between WISP-1 and TGF-beta. To further define this relationship, we analyzed the effect of WISP-1 on TGF-beta signaling. rhWISP-1 significantly reduced TGF-beta induced phosphorylation of smad-2. Our data indicates that full length WISP-1 and its variant WISP-1va are modulators of proliferation and osteogenic differentiation, and may be a novel regulators of TGF-beta signaling in osteoblast-like cells.
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