Role of Subchondral Bone during Early-Stage Experimental TMJ Osteoarthritis The temporomandibular joint consists of a fibrocartilagenous condyle, disc, synovial membrane and subchondral bone. Temporomandibular joint osteoarthritis (TMJ OA) is a degenerative disease involving all TMJ tissues and leads to anatomical changes and severe pain. While the contribution of the cartilage to the disease pathology is well studied, little is known about the role of subchondral bone. Emerging studies have implicated a potential role of subchondral bone during TMJ OA pathology, where sclerosis and increased bone metabolism is involved during cartilage degeneration. Furthermore, the development of new OA therapeutics that target both cartilage and bone highlight the necessity to better understand the role of subchondral bone turnover during OA pathology. The small leucine-rich repeat proteoglycans (SLRPs) are a family of extracellular matrix proteins highly expressed in both cartilage and bone. A mouse line doubly-deficient in two members of the SLRP family, biglycan and fibromodulin (DKO), is a well-established genetic model for TMJ OA. We have previously reported late-stage osteoarthritic changes in both DKO TMJ cartilage and subchondral bone, including age-dependent cartilage degeneration, osteophyte formation and sclerosis. One advantage of using an experimental, genetic TMJ OA model is the ability to identify early cellular mechanisms that may predispose the animal to late-onset TMJ OA disease phenotype. Specifically, we have shown that Bgn and Fmod modulate cartilage ECM breakdown at an early age, before overt histological cartilage damaged occurred. The goal of this study was to identify new genes in cartilage and bone involved during the early-stage TMJ OA in this genetic model. Based on this data, we aimed to evaluate cellular and tissue defects in the DKO TMJ cartilage and subchondral bone. In order to tackle this problem, microarray was used to identify changes in gene expression levels in the condyles of DKO during early-stages of TMJ-OA. Specifically, differential gene expression analysis was performed using RNA extracted from 3 week-old WT and DKO TMJs with intact cartilage/subchondral bone interface. A total of 22 genes were differentially expressed in DKO TMJs, including 5 genes involved in osteoclast activity/differentiation. TRAP-positive cells were 3-fold more abundant in DKO TMJs than WT. Quantitative RT-PCR showed upregulation of RANKL and OPG with a 128% increase in RANKL/OPG ratio in DKO TMJs. Histology and immunohistochemistry revealed tissue disorganization and reduced type I collagen in DKO TMJ subchondral bone. Early changes in gene expression and tissue defects in young DKO TMJ subchondral bone are likely attributed to increased osteoclast activity. These data show that biglycan and fibromodulin are critical for TMJ subchondral bone integrity and reveal a potential role for TMJ subchondral bone turnover during the initial early stages of TMJ OA disease in this model. A Novel Mechanism for Modulation of Canonical Wnt Signaling by the ECM Component, Biglycan Canonical Wnt signaling regulates diverse biological processes during development and tissue homeostasis and in particular is important for bone biology Mutations in either LRP5 or LRP6 or their downstream signaling targets, have been associated with several bone-related diseases. Wnt signaling has been proposed to control bone mass through diverse mechanisms, including renewal of stem cells, stimulation of preosteoblast replication, and enhancement of osteoblast activity. Wnt signaling in mature osteoblasts has been shown to control bone resorption by regulating the expression and secretion of the osteoclastogenesis inhibitor, osteoprotegerin. Although significant progress has been made over the past decade, our current understanding of the roles of the extracellular microenvironment in modulating Wnt/β-catenin signaling in bone is limited. In this regard, we are particularly interested in one of the members of the small leucine-rich proteoglycan family, biglycan, because of its pronounced expression in bone, where it is concentrated in the pericellular space. Previous studies in our lab have shown that mice deficient in biglycan develop age-related osteopenia resulting from a bone formation defect involving a reduced number of osteoblasts at the bone surface. Additionally, BGN (located on the X chromosome) expression levels may be related to stature in humans as patients with Turner syndrome (XO) have short stature and low levels of BGN, whereas patients with supernumerary sex chromosomes present with increased limb length and high levels of BGN. In vitro studies revealed that biglycans modulation of growth factor activities, including both TGF-βand bone morphogenetic protein (BMP) 2/4 signaling in osteoprogenitor cells, likely contributes to the mechanism whereby biglycan affects bone formation. Although biglycan and canonical Wnt signaling are both associated with skeletal tissues, there was no information whether this proteoglycan plays a direct role in modulating this signaling pathway. In our current study we showed that a member of the small leucine-rich proteoglycan family, biglycan, enhances canonical Wnt signaling by mediating Wnt function via its core protein. Immunoprecipitation analysis revealed that biglycan interacts with both canonical Wnt ligand, Wnt3a, and Wnt co-receptor LRP6. Biglycan-deficient cells treated with exogenous Wnt3a had less Wnt3a retained in cell layers compared to WT cells. Furthermore, the Wnt-induced levels of phosphorylation of LRP6 and the expression of several Wnt target genes was blunted in biglycan-deficient cells. Both recombinant biglycan proteoglycan and biglycan core protein increased Wnt-induced β-catenin/TCF-mediated transcriptional activity and this activity was completely inhibited by Dkk1. Interestingly, biglycan was able to rescue impaired Wnt signaling cause by a previously described missense mutation in the extracellular domain of human LRP6 (R611C). Furthermore, we found that biglycans modulation of canonical Wnt signaling enhanced the functional activities of osteprogenitor cells, including RUNX2- mediated transcriptional activity. In line with this, Wnt-induced calcium deposition was reduced in biglycan-deficient bone marrow stromal cells (BMSCS). Use of a transplant system revealed that expression of Wnt-Induced Secreted Protein (WISP-1) was decreased in bone formed by biglycan-deficient BMSCs further suggesting reduced Wnt signaling in these cells. We propose that biglycan may serve as a reservoir for Wnt in the pericellular space and modulate Wnt availability for activation of the canonical Wnt pathway.
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