1. UNDERSTANDING THE MECHANISMS CONTROLLING PATHOLOGICAL OSSIFICATION IN THE ABSENCE OF BIGLYCAN (BGN) AND FIBROMODULIN (FMOD) (50%)? ? Previous work from the section showed that mice deficient in the SLRPs bgn and fmod have premature osteoarthritis (OA) starting at 3 months of age. Further analysis revealed that the mutants had abnormal tendons that ossified. This pathological feature was proposed to be the foundation of the OA arising from loose joints and mis-alignment of connecting bones. The fact that tendons abnormally ossify (ectopically) when bgn and fmod are absent led us to propose that tendon contains osteogenic stem cells whose fate could be controlled by these SLRPs. In order to determine the possible existence of putative tendon stem/projentior cells (TSPCs), normal tendons were first characterized. This was done using clonogenic and differentiation assays to prove the stem cell character and plastic nature of the cells, coupled with cell surface FACS analysis and RT-PCR to determine their molecular nature. The regenerative capacity and plastic character of the TSPCs were further tested using cells from a GFP mouse that were harvested, expanded and re-implanted in vivo. Tissues developed from the expanded TSPCs were then characterized after in vivo implantation by histology and their multipotent nature demonstrated upon induction in vitro towards fat, bone and cartilage. By analyzing the extent of label retaining cells using BrdU incorporation we showed that TSPCs reside within a unique niche comprised predominantly of extracellular matrix (ECM) and identified bgn and fmod as two critical components that organize the TSPC niche. Depletion of bgn and fmod affected differentiation of TSPCs by modulating bone morphogenetic protein (BMP) signaling and subsequently impairing tendon formation in vivo. In conclusion, isolating TSPCs and identifying their niche provides a new avenue to study tendon biology and offers new strategies to treat tendon diseases. ? ? IDENTIFICATION 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, 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.? ? TGF-beta and WISP-1/CCN-4 C CAN REGULATE EACH OTHERS' ACTIVITY TO COOPERATIVELY CONTROL OSTEOBLAST FUNCTION.? ? Wnt-induced secreted protein 1 (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 hBMSC. 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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