Profiling Eph receptor expression in cells and tissues

Addendum to: Diarra D, Stolina M, Polzer K, Zwerina J, Ominsky MS, Dwyer D, Korb A, Smolen J, Hoffmann M, Scheinecker C, van der Heijde D, Landewe R, Lacey D, Richards WG and Schett G. Dickkopf-1 Is a Master Regulator of Joint Remodeling. Nat Med. 2007; 13:156-63. Remodeling of joints is a key feature of inflammatory and degenerative joint disease. Bone erosion, cartilage degeneration and growth of bony spurs termed osteophytes are key features of structural joint pathology in the course of arthritis, which lead to impairment of joint function. Understanding their molecular mechanisms is essential to tailor targeted therapeutic approaches to protect joint architecture from inflammatory and mechanical stress. This addendum summarizes the new insights in the molecular regulation of bone formation in the joint and its relation to bone resorption. It describes how inflammatory cytokines impair bone formation and block the repair response of joints towards inflammatory stimuli. It particularly points out the key role of Dickkopf-1 protein, a regulator of the Wingless signaling and inhibitor of bone formation. This new link between inflammation and bone formation is also crucial for explaining the generation of osteophytes, bony spurs along joints, which are characterized by new bone and cartilage formation. This mechanism is largely dependent on an activation of wingless protein signaling and can lead to complete joint fusion. This addendum summarized the current concepts of joint remodeling in the limelight of these new findings.     

Mesenchymal stem cells from development to postnatal joint homeostasis, aging, and disease

Joint morphogenesis involves signaling pathways and growth factors that recur in the adult life with less redundancy to safeguard joint homeostasis. Loss of such homeostasis due to abnormal signaling networks as in aging could lead to diseases such as osteoarthritis. Stem cells are the cellular counterpart and targets of the morphogenetic signals, and they function to maintain the tissues by ensuring replacement of cells lost to physiological turnover, injury, aging, and disease. Mesenchymal stem cells (MSCs) are key players in regenerative medicine for their ability to differentiate toward multiple lineages such as cartilage and bone, but they age along the host body and senesce when serially passaged in culture. Understanding correlations between aging and its effects on MSCs is of the utmost importance to explain how aging happens and unravel the underlying mechanisms. The investigation of the MSC senescence in culture will help in developing more efficient and standardized cell culture methods for cellular therapies in skeletal regenerative medicine. An important area to explore in biomedical sciences is the role of endogenous stem cell niches in joint homeostasis, remodeling, and disease. It is anticipated that an understanding of the stem cell niches and related remodeling signals will allow the development of pharmacological interventions to support effective joint tissue regeneration, to restore joint homeostasis, and to prevent osteoarthritis.     

Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy

Chondrocyte hypertrophy is an essential process required for endochondral bone formation. Proper regulation of chondrocyte hypertrophy is also required in postnatal cartilage homeostasis. Indian hedgehog (Ihh) and PTHrP signaling play crucial roles in regulating the onset of chondrocyte hypertrophy by forming a negative feedback loop, in which Ihh signaling regulates chondrocyte hypertrophy by controlling PTHrP expression. To understand whether there is a PTHrP-independent role of Ihh signaling in regulating chondrocyte hypertrophy, we have both activated and inactivated Ihh signaling in the absence of PTHrP during endochondral skeletal development. We found that upregulating Ihh signaling in the developing cartilage by treating PTHrP(-/-) limb explants with sonic hedgehog (Shh) protein in vitro, or overexpressing Ihh in the cartilage of PTHrP(-/-) embryos or inactivating patched 1 (Ptch1), a negative regulator of hedgehog (Hh) signaling, accelerated chondrocyte hypertrophy in the PTHrP(-/-) embryos. Conversely, when Hh signaling was blocked by cyclopamine or by removing Smoothened (Smo), a positive regulator of Hh signaling, chondrocyte hypertrophy was delayed in the PTHrP(-/-) embryo. Furthermore, we show that upregulated Hh signaling in the postnatal cartilage led to accelerated chondrocyte hypertrophy during secondary ossification, which in turn caused reduction of joint cartilage. Our results revealed a novel role of Ihh signaling in promoting chondrocyte hypertrophy independently of PTHrP, which is particularly important in postnatal cartilage development and homeostasis. In addition, we found that bone morphogenetic protein (Bmp) and Wnt/beta-catenin signaling in the cartilage may both mediate the effect of upregulated Ihh signaling in promoting chondrocyte hypertrophy.     

Crosstalk between cartilage and bone: when bone cytokines matter

The cartilage damage which characterizes osteoarthritis is often accompanied by bone lesions. Joint integrity results from the balance in the physiological interactions between bone and cartilage. Several local factors regulate the physiological remodeling of cartilage, the disequilibrium of these leading to a higher cartilage catabolism. Several cytokines secreted by bone cells can induce chondrocyte differentiation, which suggests their role in the dialogue between both cells. Accumulative in vivo evidence shows that increased bone resorption occurs at an early stage in the development of osteoarthritis and that blocking bone-resorbing cytokines prevents cartilage damage, confirming the role of bone factors in the crosstalk of both tissues. Recently, molecules of the Wnt pathway have emerged as key regulators of bone and cartilage. Activation of Wnt/βcatenin induces an imbalance in cartilage homeostasis, and agonists/antagonists of Wnt are potential candidates for this interaction. This review will summarize what is known about the contribution of bone cytokines to the physiological remodeling of cartilage and in the pathophysiology of osteoarthritis.     

Wnt signaling pathway in rheumatoid arthritis,with special emphasis on the different roles in synovial inflammation and bone remodeling

Rheumatoid arthritis (RA) is a chronic symmetrical autoimmune disease of unknown etiology that affects primarily the diarthrodial joints. Characteristic features of RA pathogenesis are synovial inflammation and proliferation accompanied by cartilage erosion and bone loss. Fibroblast-like synoviocytes (FLS) display an important role in the pathogenesis of RA. Several lines of evidence show that the Wnt signaling pathway significantly participates in the RA pathogenesis. The Wnt proteins are glycoproteins that bind to the Fz receptors on the cell surface, which leads to several important biological functions, such as cell differentiation, embryonic development, limb development and joint formation. Accumulated evidence has suggested that this signaling pathway plays a key role in the FLS activation, bone resorption and joint destruction during RA development. Greater knowledge of the role of the Wnt signaling pathway in RA could improve understanding of the RA pathogenesis and the differences in RA clinical presentation and prognosis. In this review, new advances of the Wnt signaling pathway in RA pathogenesis are discussed, with special emphasis on its different roles in synovial inflammation and bone remodeling. Further studies are needed to reveal the important role of the members of the Wnt signaling pathway in the RA pathogenesis and treatment.     

The Biology of Small Leucine-rich Proteoglycans in Bone Pathophysiology

The class of small leucine-rich proteoglycans (SLRPs) is a family of homologous proteoglycans harboring relatively small (36–42 kDa) protein cores compared with the larger cartilage and mesenchymal proteoglycans. SLRPs have been localized to most skeletal regions, with specific roles designated during all phases of bone formation, including periods relating to cell proliferation, organic matrix deposition, remodeling, and mineral deposition. This is mediated by key signaling pathways regulating the osteogenic program, including the activities of TGF-β, bone morphogenetic protein, Wnt, and NF-κB, which influence both the number of available osteogenic precursors and their subsequent development, differentiation, and function. On the other hand, SLRP depletion is correlated with degenerative diseases such as osteoporosis and ectopic bone formation. This minireview will focus on the SLRP roles in bone physiology and pathology.     

Transglutaminase-2 differently regulates cartilage destruction and osteophyte formation in a surgical model of osteoarthritis

Osteoarthritis is a progressive joint disease characterized by cartilage degradation and bone remodeling. Transglutaminases catalyze a calcium-dependent transamidation reaction that produces covalent cross-linking of available substrate glutamine residues and modifies the extracellular matrix. Increased transglutaminases-mediated activity is reported in osteoarthritis, but the relative contribution of transglutaminases-2 (TG2) is uncertain. We describe TG2 expression in human femoral osteoarthritis and in wild-type and homozygous TG2 knockout mice after surgically-induced knee joint instability. Increased TG2 levels were observed in human and wild-type murine osteoarthritic cartilage compared to the respective controls. Histomorphometrical but not X-ray investigation documented in osteoarthritic TG2 knockout mice reduced cartilage destruction and an increased osteophyte formation compared to wild-type mice. These differences were associated with increased TGFβ-1 expression. In addition to confirming its important role in osteoarthritis development, our results demonstrated that TG2 expression differently influences cartilage destruction and bone remodeling, suggesting new targeted TG2-related therapeutic strategies.     

Perlecan modulates VEGF signaling and is essential for vascularization in endochondral bone formation

Perlecan (Hspg2) is a heparan sulfate proteoglycan expressed in basement membranes and cartilage. Perlecan deficiency (Hspg2(-/-)) in mice and humans causes lethal chondrodysplasia, which indicates that perlecan is essential for cartilage development. However, the function of perlecan in endochondral ossification is not clear. Here, we report the critical role of perlecan in VEGF signaling and angiogenesis in growth plate formation. The Hspg2(-/-) growth plate was significantly wider but shorter due to severely impaired endochondral bone formation. Hypertrophic chondrocytes were differentiated in Hspg2(-/-) growth plates; however, removal of the hypertrophic matrix and calcified cartilage was inhibited. Although the expression of MMP-13, CTGF, and VEGFA was significantly upregulated in Hspg2(-/-) growth plates, vascular invasion into the hypertrophic zone was impaired, which resulted in an almost complete lack of bone marrow and trabecular bone. We demonstrated that cartilage perlecan promoted activation of VEGF/VEGFR by binding to the VEGFR of endothelial cells. Expression of the perlecan transgene specific to the cartilage of Hspg2(-/-) mice rescued their perinatal lethality and growth plate abnormalities, and vascularization into the growth plate was restored, indicating that perlecan in the growth plate, not in endothelial cells, is critical in this process. These results suggest that perlecan in cartilage is required for activating VEGFR signaling of endothelial cells for vascular invasion and for osteoblast migration into the growth plate. Thus, perlecan in cartilage plays a critical role in endochondral bone formation by promoting angiogenesis essential for cartilage matrix remodeling and subsequent endochondral bone formation.     

GDF5 coordinates bone and joint formation during digit development

A functional skeletal system requires the coordinated development of many different tissue types, including cartilage, bones, joints, and tendons. Members of the Bone morphogenetic protein (BMP) family of secreted signaling molecules have been implicated as endogenous regulators of skeletal development. This is based on their expression during bone and joint formation, their ability to induce ectopic bone and cartilage, and the skeletal abnormalities present in animals with mutations in BMP family members. One member of this family, Growth/differentiation factor 5 (GDF5), is encoded by the mouse brachypodism locus. Mice with mutations in this gene show reductions in the length of bones in the limbs, altered formation of bones and joints in the sternum, and a reduction in the number of bones in the digits. The expression pattern of Gdf5 during normal development and the phenotypes seen in mice with single or double mutations in Gdf5 and Bmp5 suggested that Gdf5 has multiple functions in skeletogenesis, including roles in joint and cartilage development. To further understand the function of GDF5 in skeletal development, we assayed the response of developing chick and mouse limbs to recombinant GDF5 protein. The results from these assays, coupled with an analysis of the development of brachypodism digits, indicate that GDF5 is necessary and sufficient for both cartilage development and the restriction of joint formation to the appropriate location. Thus, GDF5 function in the digits demonstrates a link between cartilage development and joint development and is an important determinant of the pattern of bones and articulations in the digits.     

Specific inhibition of FAK signaling attenuates subchondral bone deterioration and articular cartilage degeneration during osteoarthritis pathogenesis

Osteoarthritis (OA), a disease of the entire joint, is characterized by abnormal bone remodeling and coalescent degradation of articular cartilage. We have previously found that elevated levels of H-type vessels in subchondral bone correlate with OA and that focal adhesion kinase (FAK) is critical for H-type vessel formation in osteoporosis. However, the potential role of FAK in OA remains unexplored. Here, we demonstrate that the p-FAK level was dramatically elevated in subchondral bone following anterior cruciate ligament transection (ACLT) in rats. Specific inhibition of FAK signaling with Y15 in subchondral bone resulted in the suppression of subchondral bone deterioration and this effect was mediated by H-type vessel-induced ectopic bone formation. Further, articular cartilage degeneration was also alleviated after Y15 treatment. In vitro, the p-FAK level was significantly elevated in mesenchymal stem cells (MSCs) from vehicle-treated ACLT rats as compared to that in MSCs from sham controls and Y15-treated ACLT rats. Elevated p-FAK level in MSCs promoted vascular endothelial growth factor (VEGF) expression, as demonstrated from the high VEGF level in the blood, subchondral bone, and conditioned medium (CM) of MSCs from vehicle-treated ACLT rats. The CM of MSCs from vehicle-treated ACLT rats might promote the angiogenesis of endothelial cells and the catabolic response of chondrocytes through the FAK-growth factor receptor-bound protein 2-mitogen-activated protein kinase-mediated expression of VEGF. The effect of the CM from MSCs of Y15-treated ACLT rats or that treated with a VEGF-neutralizing antibody on vessel formation and the catabolic response was lowered. Thus, the specific inhibition of FAK signaling may be a promising avenue for the prevention or early treatment of OA.     

The BMP signaling and in vivo bone formation

Bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor beta (TGFbeta) superfamily. The roles of BMPs in embryonic development and cellular functions in postnatal and adult animals have been extensively studied in recent years. Signal transduction studies have revealed that Smads 1, 5 and 8 are the immediate downstream molecules of BMP receptors and play a central role in BMP signal transduction. Studies from transgenic and knockout mice and from animals and humans with naturally occurring mutations in BMPs and their signaling molecules have shown that BMP signaling plays critical roles in bone and cartilage development and postnatal bone formation. BMP activities are regulated at different molecular levels. Tissue-specific knockout of a specific BMP ligand, a subtype of BMP receptors or a specific signaling molecule is required to further determine the specific role of a BMP ligand, receptor or signaling molecule in a particular tissue.     

Collagen IX is indispensable for timely maturation of cartilage during fracture repair in mice.

Fracture repair recapitulates in adult organisms the sequence of cell biological events of endochondral ossification during skeletal development and growth. After initial inflammation and deposition of granulation tissue, a cartilaginous callus is formed which, subsequently, is remodeled into bone. In part, bone formation is influenced also by the properties of the extracellular matrix of the cartilaginous callus. Deletion of individual macromolecular components can alter extracellular matrix suprastructures, and hence stability and organization of mesenchymal tissues. Here, we took advantage of the collagen IX knockout mouse model to better understand the role of this collagen for organization, differentiation and maturation of a cartilaginous template during formation of new bone. Although a seemingly crucial component of cartilage fibrils is missing, collagen IX-deficient mice develop normally, but are predisposed to premature joint cartilage degeneration. However, we show here that lack of collagen IX alters the time course of callus differentiation during bone fracture healing. The maturation of cartilage matrix was delayed in collagen IX-deficient mice calli as judged by collagen X expression during the repair phase and the total amount of cartilage matrix was reduced. Entering the remodeling phase of fracture healing, Col9a1(-/-) calli retained a larger percentage of cartilage matrix than in wild type indicating also a delayed formation of new bone. We concluded that endochondral bone formation can occur in collagen IX knockout mice but is impaired under conditions of stress, such as the repair of an unfixed fractured long bone.     

Subchondral bone in osteoarthritis: insight into risk factors and microstructural changes

Osteoarthritis (OA) is a major cause of disability in the adult population. As a progressive degenerative joint disorder, OA is characterized by cartilage damage, changes in the subchondral bone, osteophyte formation, muscle weakness, and inflammation of the synovium tissue and tendon. Although OA has long been viewed as a primary disorder of articular cartilage, subchondral bone is attracting increasing attention. It is commonly reported to play a vital role in the pathogenesis of OA. Subchondral bone sclerosis, together with progressive cartilage degradation, is widely considered as a hallmark of OA. Despite the increase in bone volume fraction, subchondral bone is hypomineralized, due to abnormal bone remodeling. Some histopathological changes in the subchondral bone have also been detected, including microdamage, bone marrow edema-like lesions and bone cysts. This review summarizes basic features of the osteochondral junction, which comprises subchondral bone and articular cartilage. Importantly, we discuss risk factors influencing subchondral bone integrity. We also focus on the microarchitectural and histopathological changes of subchondral bone in OA, and provide an overview of their potential contribution to the progression of OA. A hypothetical model for the pathogenesis of OA is proposed.     

How Does Joint Remodeling Work?: New Insights in the Molecular Regulation of the Architecture of Joints

Remodeling of joints is a key feature of inflammatory and degenerative joint disease. Bone erosion, cartilage degeneration and growth of bony spurs termed osteophytes are key features of structural joint pathology in the course of arthritis, which lead to impairment of joint function. Understanding their molecular mechanisms is essential to tailor targeted therapeutic approaches to protect joint architecture from inflammatory and mechanical stress. This addendum summarizes the new insights in the molecular regulation of bone formation in the joint and its relation to bone resorption. It describes how inflammatory cytokines impair bone formation and block the repair response of joints towards inflammatory stimuli. It particularly points out the key role of Dickkopf-1 protein, a regulator of the Wingless signaling and inhibitor of bone formation. This new link between inflammation and bone formation is also crucial for explaining the generation of osteophytes, bony spurs along joints, which are characterized by new bone and cartilage formation. This mechanism is largely dependent on an activation of wingless protein signaling and can lead to complete joint fusion. This addendum summarized the current concepts of joint remodeling in the limelight of these new findings.Key words: joint remodeling, arthritis, bone formation, bone erosion, osteoblasts, osteoclasts, Dickkopf, wingless proteinsJoints face profound remodeling in the course of arthritis. In humans, pathologic joint remodeling manifests as (i) destruction of joints due to bone erosion (rheumatoid arthritis), (ii) fusion of joints due to formation of bony spurs such as osteophytes, spondylophytes and syndesmophytes (ankylosing spondylitis) or (iii) a mixture of both changes (psoriatic arthritis). The molecular mechanisms determining these different forms of joint remodeling are not fully clarified, Insights in these mechanisms however are a clue to a deeper understanding of the architectural changes of human joints.Similar to systemic bone turnover, which most is most prominent in the trabecular bone compartment of the spine and long bones, joints are hot spots of bone remodeling during inflammatory disease. Cytokines expressed by inflammatory cells in the synovial membrane regulate local bone homeostasis and enable to remodel joints during disease—a process which can either lead to crippling and functional loss or to fusion and stabilization of the affected joint. Rheumatoid arthritis is characterized by bone erosions, which are the result of an enhanced bone resorption. In rheumatoid arthritis osteoclasts, the primary bone resorbing cells, accumulate and degrade the periarticular bone as well as the mineralized cartilage.¹ Molecularly increased osteoclast formation is based on the expression of macrophage colony-stimulating factor (MCSF) and receptor-antagonist of NFκB ligand (RANKL) in the synovial tissue, which both drive the differentiation of osteoclasts from monocytic precursors.^2–4 Osteoclasts are specialized cells to resorb bone and their local accumulation in the joint leads to a catabolic state, which by far outweighs bone formation resulting in a negative net effect of bone remodeling. Inflammatory cytokines, such as TNF, IL-1, IL-6 and IL-17 induce osteoclast formation by enhancing the expression of RANKL and promoting differentiation of osteoclast precursor cells to mature osteoclasts.^5–8 Abundance of proinflammatory cytokines in the synovial membrane of patients with RA, their induction of molecules involved in osteoclast formation and the influx of monocytes/macrophages serving as osteoclast precursor cells represent ideal prerequisites for osteoclast formation in joints.⁹The fact that appropriate repair strategies are virtually absent in patients with RA and that bone is hardly rebuilt when bone erosions have emerged, suggests activation of molecular signals, which blunt bone formation. Bone formation itself is regulated by growth factors and hormones, which stimulate differentiation and activity of osteoblasts. Typical regulators of bone formation constitute parathyroid hormone, prostaglandins, bone morphogenic proteins (BMPs) and wingless proteins (Wnt). Particularly the role of Wnt proteins in bone formation have achieved growing interest during the past few years, leading to identification of the LRP5/6 receptor as a key molecule for anabolic skeletal responses. Wnt proteins bind to the LRP5/6 receptor and lead to activation of a signal pathway involving GSK3 and β-catenin, which drive differentiation of mesenchymal cells into osteoblastogenesis.¹⁰ Regulators of Wnt- induced bone formation are Dickkopf (DKK) proteins, which competitively bind to LRP5/6 and prevent signaling activation by additionally engaging a negative coreceptor termed Kremen-1.^11,12 DKK proteins thus regulate bone homeostasis by interference with Wnt signaling.¹³We recently showed that inflammatory cytokines such as TNF induce DKK-1, a member of the DKK- family, which inhibits Wnt signaling. DKK-1 is highly expressed in inflammatory lesions of experimental arthritis and human rheumatoid arthritis.¹⁴ Moreover, increased levels can be detected in the serum of patients with RA, which depend on TNF. This is supported by the normalization of elevated DKK-1 levels in RA patients upon initiation of systemic TNF- blockade. Inhibition of DKK-1 in mice completely abolishes bone erosions in different models of experimental arthritis and leads to increased bone growth, which manifests as osteophyte formation in the joint.DKK-1 links the inflammation with bone formation as RANKL links inflammation with bone resorption. The fact that TNF and presumably also other inflammatory mediators induce both proteins explains the profound negative effect of inflammation on bone. Inflammation uncouples the balance between bone resorption and formation, enhancing the former by inducing RANKL and by repressing the latter by DKK-1. Also appears to be a tight cross talk between the Wnt- and RANKL-pathways.¹⁵ Inhibition of DKK-1 in arthritic mice lead to protection from bone erosions and osteoclasts did not appropriately form. This effect is based on the induction of osteoprotegerin (OPG) a natural decoy receptor for RANKL, which blocks RANKL and thus osteoclast formation. OPG is induced by Wnt proteins and shifts the balance from bone resorption to bone formation.In contrast to rheumatoid arthritis joints in ankylosing spondylitis and also in degenerative joint disease (osteoarthritis) show an attempt towards joint fusion rather than joint destruction. These bony spurs are the result of endochondral bone formation starting from the periosteum close to the joints, where osteoblasts differentiate build up bone matrix. We could demonstrate that Wnt proteins are crucially involved in this process since inhibition of DKK-1 lead to emergence of osteophytes and even complete fusion of joints. Taken together these data suggest that the balance of the Wnt/DKK system determines the remodeling of joints by governing bone destruction as well as osteophyte formation in joints (Fig. 1).Open in a separate windowFigure 1Patterns of joint remodeling.