Background on the SLRP and CCN Family of ECM Proteins: The CCN family is named from its founding members (Cyr61, CTGF, Nov) and consists of six members now known as Cyr61/CCN1, CTGF/CCN2, Nov/CCN3 WISP1/CCN4, WISP2/CCN5 and WISP3/CCN6, which have diverse functions including regulation of differentiation, proliferation and cell migration. All six CCN members are found in the skeleton but with unique locations. For example, during normal skeletal development, CCN2 and 3 are highly expressed in cartilage, while CCN4 is largely confined to newly forming bone. The SLRP family is composed of 17 members sub-divided into classes (I-V) based on their amino acid sequence and genomic organization. All members of the SLRP family (excluding asporin) have extensive post-translational glycosylation on a relatively small protein core backbone composed of repeat structures rich in leucine. For years, evidence has been mounting about the importance of SLRPs in skeletal function. We have focused on the SRLP biglycan (BGN) because of its high level of expression in bones and teeth. Taken together our work highlights the fact that BGN is not needed for bone development but, rather, appears to play a role in skeletal aging. This has been demonstrated using mice unable to make bgn that are found to acquire early onset osteoporosis (osteopenia/low bone mass), osteoarthritis and ectopic bone in their tendons. WISP1 Function: CCN4/Wisp1 (here in referred to as Wisp1) was first discovered as a target gene of the Wnt pathway, where it was aberrantly expressed in human colon cancer. Using a combination of in situ hybridization and immunohistochemistry, Wisp1 was subsequently found highly expressed in osteoblasts and in perichondral mesenchyme. Further analysis showed that Wisp1 is up-regulated in healing bone after induced fracture indicating its important role in skeletal homeostasis. Based on these reports, we, and others began to examine Wisp1s effect on osteoprogenitors and found that it could promote osteogenic differentiation when added exogenously to osteoprogenitor cells cultured in vitro. Despite this intriguing foundation, the exact roles of Wisp1 in skeletal tissue in vivo have not been clearly elucidated. To examine the function of Wisp1 in mineralized tissues we generated mice unable to make Wisp1 (Wisp1 KO) by gene targeting. Northern blotting, RT-PCR and western blotting confirmed that Wisp1 was depleted and the bones of the Wisp1 KO were further analyzed by Dual Energy X-Ray Absorptiometry (DEXA) and micro computerized tomography (CT). In one month-old mice there was no significant difference in the total bone mineral density (BMD) in Wisp1 KO compared to normal (wild type/WT); however by three months of age the Wisp1 KO mice had significantly lower BMD compared to WT mice and this decrease persisted until 9 months of age. MicroCT analysis of 3 month-old femurs revealed numerous changes in the Wisp1 KO bones compared to WT bones. In the trabecular region, female mice had lower BMD, lower trabecular numbers and more trabecular separation as well as greater bone surface/bone volume, all indicating a decrease in bone mass. In the cortical bone both males and females had decreased cortical area, decreased cross sectional area and decreased mean thickness, which is likely the basis for the decrease in total BMD noted from the DEXA scans in the KO animals. In addition to the changes in trabecular and cortical character and architecture, microCT analysis showed that the femurs from Wisp1 KO male mice were significantly shorter than WT controls. They also and performed poorly when tested for biomechanical strength. To understand the cell and molecular basis for the decreased bone mass in Wisp1KO mice, we next isolated osteogenic progenitors from the bone marrow (bone marrow stromal cells/BMSC) and tested their ability to differentiate towards osteogenesis. Cells were obtained from 6 week-old female mice, expanded in mineralization media and stained with Alizarin Red to measure calcium accumulation or analyzed by real-time PCR to examine the levels of osteogenic markers. These studies showed that the bone marrow stromal cells (BMSCs) from the Wisp1 KO mice had dramatically reduced levels of alkaline phosphatase, osteocalcin and bone sialoprotein mRNA that was accompanied by a significant reduction in Alizarin Red staining. Considering that the differentiation of osteogenic progenitors was decreased in Wisp1 KO compared to WT cells, it is proposed that Wisp1 could potentially regulate bone mineral density and cortical bone thickness by regulating the differentiation levels within osteogenic precursor pool of multipotent progenitors. Experiments using array technology showed that numerous wnt target genes are dramatically decreased in Wisp1 depleted in osteogenic cells suggesting this pathway an additional molecular basis for WISP-1s mechanism of action in bone which is discussed further below. In addition to a defect in bone forming defects cells in the Wisp1 KO we also tested whether the bone resorbing cells were affected. Our study showed that osteoclast precursors from Wisp1 KO mice developed more tartrate resistant acid phosphatase (TRAP) positive cells in vitro and in transplants, suggesting Wisp1 is a negative regulator of osteoclast differentiation. When bone turnover (formation and resorption) was induced by ovariectomy Wisp1 KO mice had less BMD compared WT mice confirming the potential for multiple roles for Wisp1 in controlling bone homeostasis. To further define the molecular basis for WISP1 action in bone we tested the hypothesis that Wisp1 could regulate the wnt pathway. To test this theory we examined key components of the pathway and found that Wisp1 KO BMSCs had a reduced expression the downstream wnt effector β-catenin as well as its target genes. Because WISP1 is secreted we further theorized it worked outside the cell. We found that Wisp1 inhibited the binding of the wnt inhibitor SOST to its receptor LRP6. This suggests Wisp1 could be both a target and regulator of wnt signaling. Taken together our data shows that the decreased bone mass found in Wisp1 KO mice could, potentially, be caused by an insufficiency in the osteodifferentiation capacity of BMSCs arising from diminished Wnt signaling ultimately leading to altered bone turnover and weaker biomechanically compromised bones. Biglycan Function: This year, non-skeletal functions for BGN were uncovered. This was pursued because BGN was found to be up-regulated in the small vessels in the brain in a rare disease known as Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Its genetic etiology is caused by a mutation in conserved residues of the NOTCH3 gene. BGN was strongly expressed in both small penetrating and leptomeningeal arteries of CADASIL brain being localized to all three layers of arteries (intima, media, and adventitia) with Substantially more immunoreactivity and mRNA for BGN was observed in CADASIL brains compared to controls. Human cerebrovascular smooth muscle cells exposed to purified NOTCH3 ectodomain upregulated BGN, DCN, and COL4A1 through mechanisms that are sensitive to rapamycin, a potent mTOR inhibitor. In addition, BGN protein interacted directly with NOTCH3 protein in cell culture and in direct protein interaction assays. We conclude that BGN is a CADASIL-enriched protein that potentially accumulates in vessels by mTOR-mediated transcriptional activation and/or post-translational accumulation via protein interactions with NOTCH3 and collagen.
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