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The cysteine knot protein sclerostin is an osteocyte-derived secreted inhibitor of the Wnt co-receptors LRP5 and LRP6. LRP5 plays a dominant role in bone homeostasis, but we previously reported that Sost/sclerostin suppression significantly increased osteogenesis regardless of Lrp5 presence or absence. Those observations suggested that the bone forming effects of sclerostin inhibition can occur through Lrp6 (when Lrp5 is suppressed), or through other yet undiscovered mechanisms independent of Lrp5/6. To distinguish between these two possibilities, we generated mice with compound deletion of Lrp5 and Lrp6 selectively in bone, and treated them with sclerostin monoclonal antibody (Scl-mAb). All mice were homozygous flox for both Lrp5 and Lrp6 (Lrp5 f/f ; Lrp6 f/f ), and varied only in whether or not they carried the Dmp1-Cre transgene. Positive (Cre+) and negative (Cre-) mice were injected with Scl-mAb or vehicle from 4.5 to 14 weeks of age. Vehicle-treated Cre+ mice exhibited significantly reduced skeletal properties compared to vehicle-treated Cre- mice, as assessed by DXA, μCT, pQCT, and histology, indicating that Lrp5/6 deletions were effective and efficient. Scl-mAb treatment improved nearly every bone-related parameter among Cre- mice, but the same treatment in Cre+ mice resulted in little to no improvement in skeletal properties. For the few endpoints where Cre+ mice responded to Scl-mAb, it is likely that antibody-induced promotion of Wnt signaling occurred in cell types earlier in the mesenchymal/osteoblast differentiation pathway than the Dmp1-expressing stage. This latter conclusion was supported by changes in some histomorphometric parameters. In conclusion, unlike with the deletion of Lrp5 alone, the bone-selective late-stage co-deletion of Lrp5 and Lrp6 significantly impairs or completely nullifies the osteogenic action of Scl-mAb, and highlights a major role for both Lrp5 and Lrp6 in the mechanism of action for the bone-building effects of sclerostin antibody., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Certain missense mutations affecting LRP5 cause high bone mass (HBM) in humans. Based on in vitro evidence, HBM LRP5 receptors are thought to exert their effects by providing resistance to binding/inhibition of secreted LRP5 inhibitors such as sclerostin (SOST) and Dickkopf homolog-1 (DKK1). We previously reported the creation of two Lrp5 HBM knock-in mouse models, in which the human p.A214V or p.G171V missense mutations were knocked into the endogenous Lrp5 locus. To determine whether HBM knock-in mice are resistant to SOST- or DKK1-induced osteopenia, we bred Lrp5 HBM mice with transgenic mice that overexpress human SOST in osteocytes ((8kb) Dmp1-SOST) or mouse DKK1 in osteoblasts and osteocytes ((2.3kb) Col1a1-Dkk1). We observed that the (8kb) Dmp1-SOST transgene significantly lowered whole-body bone mineral density (BMD), bone mineral content (BMC), femoral and vertebral trabecular bone volume fraction (BV/TV), and periosteal bone-formation rate (BFR) in wild-type mice but not in mice with Lrp5 p.G171V and p.A214V alleles. The (2.3kb) Col1a1-Dkk1 transgene significantly lowered whole-body BMD, BMC, and vertebral BV/TV in wild-type mice and affected p.A214V mice more than p.G171V mice. These in vivo data support in vitro studies regarding the mechanism of HBM-causing mutations, and imply that HBM LRP5 receptors differ in their relative sensitivity to inhibition by SOST and DKK1., (© 2015 American Society for Bone and Mineral Research.)

Lubricin, encoded by the gene PRG4, is the principal lubricant in articulating joints. We immunized mice genetically deficient for lubricin (Prg4-/-) with purified human lubricin, and generated several mAbs. We determined each mAb's binding epitope, sensitivity, and specificity using biologic samples and recombinant lubricin sub-domains, and we also developed a competition ELISA assay to measure lubricin in synovial fluid and blood. We found the mAbs all recognized epitopes containing O-linked oligosaccharides conjugated to the peptide motif KEPAPTTT. By western blot, the mAbs detected lubricin in 1 μl of synovial fluid from several animal species, including human. The mAbs were specific for lubricin since they did not cross-react with other synovial fluid constituents from patients with camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP), who genetically lack this protein. The competition ELISA detected lubricin in blood samples from healthy individuals but not from patients with CACP, indicating blood can be used in a diagnostic test for patients suspected of having CACP. Lubricin epitopes in blood do not represent degradation fragments from synovial fluid. Therefore, although blood lubricin levels did not differentiate patients with inflammatory joint disease from healthy controls, epitope-specific anti-lubricin mAbs could be useful for monitoring disease activity in synovial fluid.

Osteoporosis pseudoglioma syndrome (OPPG) is a rare genetic disease that produces debilitating effects in the skeleton. OPPG is caused by mutations in LRP5, a WNT co-receptor that mediates osteoblast activity. WNT signaling through LRP5, and also through the closely related receptor LRP6, is inhibited by the protein sclerostin (SOST). It is unclear whether OPPG patients might benefit from the anabolic action of sclerostin neutralization therapy (an approach currently being pursued in clinical trials for postmenopausal osteoporosis) in light of their LRP5 deficiency and consequent osteoblast impairment. To assess whether loss of sclerostin is anabolic in OPPG, we measured bone properties in a mouse model of OPPG (Lrp5(-/-)), a mouse model of sclerosteosis (Sost(-/-)), and in mice with both genes knocked out (Lrp5(-/-);Sost(-/-)). Lrp5(-/-);Sost(-/-) mice have larger, denser, and stronger bones than do Lrp5(-/-) mice, indicating that SOST deficiency can improve bone properties via pathways that do not require LRP5. Next, we determined whether the anabolic effects of sclerostin depletion in Lrp5(-/-) mice are retained in adult mice by treating 17-week-old Lrp5(-/-) mice with a sclerostin antibody for 3 weeks. Lrp5(+/+) and Lrp5(-/-) mice each exhibited osteoanabolic responses to antibody therapy, as indicated by increased bone mineral density, content, and formation rates. Collectively, our data show that inhibiting sclerostin can improve bone mass whether LRP5 is present or not. In the absence of LRP5, the anabolic effects of SOST depletion can occur via other receptors (such as LRP4/6). Regardless of the mechanism, our results suggest that humans with OPPG might benefit from sclerostin neutralization therapies.

Chordin-like 1 (CHRDL1) is a secreted bone morphogenetic protein (BMP) antagonist expressed in mesenchymal tissues whose function in development of the skeleton has not been examined in detail. Here we show Chrdl1 is dynamically expressed in the early distal limb bud mesenchyme, with expression becoming downregulated as development proceeds. Chrdl1 expression is largely excluded from the critical signaling center of the posterior limb bud, the Zone of Polarizing Activity (ZPA), as has been described for the BMP antagonist Gremlin (GREM1) (Scherz et al., 2004, Science, 305, 396-399). Unlike Grem1, Chrdl1 is expressed in the hindlimb by a small subset of ZPA cells and their descendants suggesting divergent regulation and function between the various BMP antagonists. Ectopic expression of Chrdl1 throughout the avian limb bud using viral misexpression resulted in an oligodactyly phenotype with loss of digits from the anterior limb, although the development of more proximal elements of the zeugopod and stylopod were unaffected. Overgrowths of soft tissue and syndactyly were also observed, resulting from impaired apoptosis and failure of the anterior mesenchyme to undergo SOX9-dependent chondrogenesis, instead persisting as an interdigital-like soft tissue phenotype. Sonic hedgehog (SHH) and fibroblast growth factor (FGF) signaling were upregulated and persisted later in development, however these changes were only detected late in limb development at timepoints when endogenous Grem1 would normally be downregulated and increasing BMP signaling would cause termination of Shh and Fgf expression. Our results suggest that the early stages of the GREM1-SHH-FGF signaling network are resistant to Chrdl1-overexpression, leading to normal formation of proximal limb structures, but that later Bmp expression, impaired by ectopic CHRDL1, is essential for formation of the correct complement of digits., (Published by Elsevier Inc.)

Osteoarthritis is a complex disease involving the mechanical breakdown of articular cartilage in the presence of altered joint mechanics and chondrocyte death, but the connection between these factors is not well established. Lubricin, a mucinous glycoprotein encoded by the PRG4 gene, provides boundary lubrication in articular joints. Joint friction is elevated and accompanied by accelerated cartilage damage in humans and mice that have genetic deficiency of lubricin. Here, we investigated the relationship between coefficient of friction and chondrocyte death using ex vivo and in vitro measurements of friction and apoptosis. We observed increases in whole-joint friction and cellular apoptosis in lubricin knockout mice compared with wild-type mice. When we used an in vitro bovine explant cartilage-on-cartilage bearing system, we observed a direct correlation between coefficient of friction and chondrocyte apoptosis in the superficial layers of cartilage. In the bovine explant system, the addition of lubricin as a test lubricant significantly lowered the static coefficient of friction and number of apoptotic chondrocytes. These results demonstrate a direct connection between lubricin, boundary lubrication, and cell survival and suggest that supplementation of synovial fluid with lubricin may be an effective treatment to prevent cartilage deterioration in patients with genetic or acquired deficiency of lubricin.

Nonsyndromic cleft lip and palate (NSCLP), a common birth defect, affects 4,000 newborns in the US each year. Previously, we described an association between CRISPLD2 and NSCLP and showed Crispld2 expression in the murine palate. These results suggested that a perturbation in CRISPLD2 activity affects craniofacial development. Here, we describe crispld2 expression and the phenotypic consequence of its loss of function in zebrafish. crispld2 was expressed at all stages of zebrafish morphogenesis examined and localized to the rostral end by 1-day postfertilization. Morpholino knockdown of crispld2 resulted in significant jaw and palatal abnormalities in a dose-dependent manner. Loss of crispld2 caused aberrant patterning of neural crest cells (NCC) suggesting that crispld2 is necessary for normal NCC formation. Altogether, we show that crispld2 plays a significant role in the development of the zebrafish craniofacies and alteration of normal protein levels disturbs palate and jaw formation. These data provide support for a role of CRISPLD2 in NSCLP., (Copyright © 2012 Wiley Periodicals, Inc.)

Lubricin, a protein product of the gene PRG4, is a secreted mucin-like proteoglycan that is a major lubricant in articulating joints. Mutations in PRG4 cause the autosomal recessive, human disorder camptodactyly-arthropathy-coxa vara-pericarditis syndrome. We developed rabbit polyclonal antibodies against human lubricin to determine the consequence of disease-causing mutations at the protein level and to study the protein's normal post-translational processing. Antiserum generated against an epitope in the amino-terminal portion of lubricin detected protein in wild-type synovial fluid and in conditioned media from wild-type cultured synoviocytes. However, the antiserum did not detect lubricin in synovial fluid or cultured synoviocytes from several patients with frameshift or nonsense mutations in PRG4. Antiserum generated against an epitope in the protein's carboxyl-terminal, hemopexin-like domain identified a post-translational cleavage event in wild-type lubricin, mediated by a subtilisin-like proprotein convertase (SPC). Interestingly, in contrast to wild-type lubricin, one disease-causing mutation that removes the last 8 amino acids of the protein, including a conserved cysteine residue, was not cleaved within the hemopexin-like domain when expressed in COS-7 cells. This suggests that formation of an intrachain disulfide bond is required for SPC-mediated cleavage and that SPC-mediated cleavage is essential to protein function.

Objective To investigate the effects of lubricin gene dosage and cyclic loading on whole joint coefficient of friction and articular cartilage surface integrity in mouse knee joints. Methods Joints from mice with 2 (Prg4+/+), 1 (Prg4+/−), or no (Prg4−/−) functioning lubricin alleles were subjected to 26 hours of cyclic loading using a custom-built pendulum. Coefficient of friction values were measured at multiple time points. Contralateral control joints were left unloaded. Following testing, joints were examined for histologic evidence of damage and cell viability. Results At baseline, the coefficient of friction values in Prg4−/− mice were significantly higher than those in Prg4+/+ and Prg4+/− mice ( P < 0.001). Cyclic loading continuously increased the coefficient of friction in Prg4−/− mouse joints. In contrast, Prg4+/− and Prg4+/+ mouse joints had no coefficient of friction increases during the first 4 hours of loading. After 26 hours of loading, joints from all genotypes had increased coefficient of friction values compared to baseline and unloaded controls. Significantly greater increases occurred in Prg4−/− and Prg4+/− mouse joints compared to Prg4+/+ mouse joints. The coefficient of friction values were not significantly associated with histologic evidence of damage or loss of cell viability. Conclusion Our findings indicate that mice lacking lubricin have increased baseline coefficient of friction values and are not protected against further increases caused by loading. Prg4+/− mice are indistinguishable from Prg4+/+ mice at baseline, but have significantly greater coefficient of friction values following 26 hours of loading. Lubricin dosage affects joint properties during loading, and may have clinical implications in patients for whom injury or illness alters lubricin abundance. [ABSTRACT FROM AUTHOR]

The article focuses on a study which assessed the role of the glycoprotein proteoglycan 4 (PRG4) in joint lubrication and chondroprotection by measuring friction, stiffness, surface topography, and subsurface histology of the hip joints of PRG4 and wild-type (WT) mice. Staining and grading were made in the histologic sections of each femoral head. Results suggested the significant role played by PRG4 in preserving normal joint structure and function.

The article discusses a study on the association between friction and wear in diarthrodial joints that lack the glycoprotein lubricin. Lubricin is secreted by surface chondrocytes and synovial cells and has been proved to reduce friction in vitro. The research found that synovial fluid (SF) lacking lubricin failed to reduce friction in the boundary mode. It concludes that friction is coupled with wear at the cartilage surface in vivo.

The cell surface receptor, low-density lipoprotein receptor-related protein 5 (LRPS) is a key regulator of bone mass. Loss-of-function mutations in LRP5 cause the human skeletal disease osteoporosis-pseudoglioma syndrome, an autosomal recessive disorder characterized by severely reduced bone mass and strength. We investigated the role of LRP5 on bone strength using mice engineered with a loss-of-function mutation in the gene. We then tested whether the osteogenic response to mechanical loading was affected by the loss of Lrp5 signaling. Lrp5-null (Lrp5-/-) mice exhibited significantly lower bone mineral density and decreased strength. The osteogenic response to mechanical loading of the ulna was reduced by 88 to 99% in Lrp5-/- mice, yet osteoblast recruitment and/or activation at mechanically strained surfaces was normal. Subsequent experiments demonstrated an inability of Lrp5-/- osteoblasts to synthesize the bone matrix protein osteopontin after a mechanical stimulus. We then tested whether Lrp5-/- mice increased bone formation in response to intermittent parathyroid hormone (PTH), a known anabolic treatment. A 4-week course of intermittent PTH (40 µg/kg/day; 5 days/week) enhanced skeletal mass equally in Lrp5-/- and Lrp5+/+ mice, suggesting that the anabolic effects of PTH do not require Lrp5 signaling. We conclude that Lrp5 is critical for mechanotransduction in osteoblasts. Lrp5 is a mediator of mature osteoblast function following loading. Our data suggest an important component of the skeletal fragility phenotype in individuals affected with osteoporosis-pseudoglioma is inadequate processing of signals derived from mechanical stimulation and that PTH might be an effective treatment for improving bone mass in these patients. [ABSTRACT FROM AUTHOR]

The long-term integrity of an articulating joint is dependent upon the nourishment of its cartilage component and the protection of the cartilage surface from friction-induced wear. Loss-of-function mutations in lubricin (a secreted glycoprotein encoded by the gene PRG4) cause the human autosomal recessive disorder camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP). A major feature of CACP is precocious joint failure. In order to delineate the mechanism by which lubricin protects joints, we studied the expression of Prg4 mRNA during mouse joint development, and we created lubricin-mutant mice. Prg4 began to be expressed in surface chondrocytes and synoviocytes after joint cavitation had occurred and remained strongly expressed by these cells postnatally. Mice lacking lubricin were viable and fertile. In the newborn period, their joints appeared normal. As the mice aged, we observed abnormal protein deposits on the cartilage surface and disappearance of underlying superficial zone chondrocytes. In addition to cartilage surface changes and subsequent cartilage deterioration, intimal cells in the synovium surrounding the joint space became hyperplastic, which further contributed to joint failure. Purified or recombinant lubricin inhibited the growth of these synoviocytes in vitro. Tendon and tendon sheath involvement was present in the ankle joints, where morphologic changes and abnormal calcification of these structures were observed. We conclude that lubricin has multiple functions in articulating joints and tendons that include the protection of surfaces and the control of synovial cell growth. [ABSTRACT FROM AUTHOR]