Motomi Enomoto-Iwamoto

Developmental regulation of Wnt/β-catenin signals is required for growth plate assembly, cartilage integrity, and endochondral ossification

Studies have suggested that continuous Wnt/β-catenin signaling in nascent cartilaginous skeletal elements blocks chondrocyte hypertrophy and endochondral ossification, whereas signaling starting at later stages stimulates hypertrophy and ossification, indicating that Wnt/β-catenin roles are developmentally regulated. To test this conclusion further, we created transgenic mice expressing a fusion mutant protein of β-catenin and LEF (CA-LEF) in nascent chondrocytes. Transgenic mice had severe skeletal defects, particularly in limbs. Growth plates were totally disorganized, lacked maturing chondrocytes expressing Indian hedgehog and collagen X, and failed to undergo endochondral ossification. Interestingly, the transgenic cartilaginous elements were ill defined, intermingled with surrounding connective and vascular tissues, and even displayed abnormal joints. However, when activated β-catenin mutant (Δ-β-catenin) was expressed in chondrocytes already engaged in maturation such as those present in chick limbs, chondrocyte maturation and bone formation were greatly enhanced. Differential responses to Wnt/β-catenin signaling were confirmed in cultured chondrocytes. Activation in immature cells blocked maturation and actually de-stabilized their phenotype, as revealed by reduced expression of chondrocyte markers, abnormal cytoarchitecture, and loss of proteoglycan matrix. Activation in mature cells instead stimulated hypertrophy, matrix mineralization, and expression of terminal markers such as metalloprotease (MMP)-13 and vascular endothelial growth factor. Because proteoglycans are crucial for cartilage function, we tested possible mechanisms for matrix loss. Δ-β-Catenin expression markedly increased expression of MMP-2, MMP-3, MMP-7, MMP-9, MT3-MMP, and ADAMTS5. In conclusion, Wnt/β-catenin signaling regulates chondrocyte phenotype, maturation, and function in a developmentally regulated manner, and regulated action by this pathway is critical for growth plate organization, cartilage boundary definition, and endochondral ossification.

A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis

The origin, roles and fate of progenitor cells forming synovial joints during limb skeletogenesis remain largely unclear. Here we produced prenatal and postnatal genetic cell fate-maps by mating ROSA-LacZ-reporter mice with mice expressing Cre-recombinase at prospective joint sites under the control of Gdf5 regulatory sequences (Gdf5-Cre). Reporter-expressing cells initially constituted the interzone, a compact mesenchymal structure representing the first overt sign of joint formation, and displayed a gradient-like distribution along the ventral-to-dorsal axis. The cells expressed genes such as Wnt9a, Erg and collagen IIA, remained predominant in the joint-forming sites over time, gave rise to articular cartilage, synovial lining and other joint tissues, but contributed little if any to underlying growth plate cartilage and shaft. To study their developmental properties more directly, we isolated the joint-forming cells from prospective autopod joint sites using a novel microsurgical procedure and tested them in vitro. The cells displayed a propensity to undergo chondrogenesis that was enhanced by treatment with exogenous rGdf5 but blocked by Wnt9a over-expression. To test roles for such Wnt-mediated anti-chondrogenic capacity in vivo, we created conditional mutants deficient in Wnt/beta-catenin signaling using Col2-Cre or Gdf5-Cre. Synovial joints did form in both mutants; however, the joints displayed a defective flat cell layer normally abutting the synovial cavity and expressed markedly reduced levels of lubricin. In sum, our data indicate that cells present at prospective joint sites and expressing Gdf5 constitute a distinct cohort of progenitor cells responsible for limb joint formation. The cells appear to be patterned along specific limb symmetry axes and rely on local signaling tools to make distinct contributions to joint formation.

Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-γ agonists

Heterotopic ossification consists of ectopic bone formation within soft tissues after surgery or trauma. It can have debilitating consequences, but there is no definitive cure. Here we show that heterotopic ossification was essentially prevented in mice receiving a nuclear retinoic acid receptor-γ (RAR-γ) agonist. Side effects were minimal, and there was no significant rebound effect. To uncover the mechanisms of these responses, we treated mouse mesenchymal stem cells with an RAR-γ agonist and transplanted them into nude mice. Whereas control cells formed ectopic bone masses, cells that had been pretreated with the RAR-γ agonist did not, suggesting that they had lost their skeletogenic potential. The cells became unresponsive to rBMP-2 treatment in vitro and showed decreases in phosphorylation of Smad1, Smad5 and Smad8 and in overall levels of Smad proteins. In addition, an RAR-γ agonist blocked heterotopic ossification in transgenic mice expressing activin receptor-like kinase-2 (ALK2) Q207D, a constitutively active form of the receptor that is related to ALK2 R206H found in individuals with fibrodysplasia ossificans progressiva. The data indicate that RAR-γ agonists are potent inhibitors of heterotopic ossification in mouse models and, thus, may also be effective against injury-induced and congenital heterotopic ossification in humans.

Fibroblasts expressing Sonic hedgehog induce osteoblast differentiation and ectopic bone formation.

© 1997 Federation of European Biochemical Societies.

Conditional Kif3a ablation causes abnormal hedgehog signaling topography, growth plate dysfunction, and excessive bone and cartilage formation during mouse skeletogenesis.

The motor protein Kif3a and primary cilia regulate important developmental processes, but their roles in skeletogenesis remain ill-defined. Here we created mice deficient in Kif3a in cartilage and focused on the cranial base and synchondroses. Kif3a deficiency caused cranial base growth retardation and dysmorphogenesis, which were evident in neonatal animals by anatomical and micro-computed tomography (μCT) inspection. Kif3a deficiency also changed synchondrosis growth plate organization and function, and the severity of these changes increased over time. By postnatal day (P)7, mutant growth plates lacked typical zones of chondrocyte proliferation and hypertrophy, and were instead composed of chondrocytes with an unusual phenotype characterized by strong collagen II (Col2a1) gene expression but barely detectable expression of Indian hedgehog (Ihh), collagen X (Col10a1), Vegf (Vegfa), MMP-13 (Mmp13) and osterix (Sp7). Concurrently, unexpected developmental events occurred in perichondrial tissues, including excessive intramembranous ossification all along the perichondrial border and the formation of ectopic cartilage masses. Looking for possible culprits for these latter processes, we analyzed hedgehog signalling topography and intensity by monitoring the expression of the hedgehog effectors Patched 1 and Gli1, and of the hedgehog-binding cell-surface component syndecan 3. Compared with controls, hedgehog signaling was quite feeble within mutant growth plates as early as P0, but was actually higher and was widespread all along mutant perichondrial tissues. Lastly, we studied postnatal mice deficient in Ihh in cartilage; their cranial base defects only minimally resembled those in Kif3a-deficient mice. In summary, Kif3a and primary cilia make unique contributions to cranial base development and synchondrosis growth plate function. Their deficiency causes abnormal topography of hedgehog signaling, growth plate dysfunction, and un-physiologic responses and processes in perichondrial tissues, including ectopic cartilage formation and excessive intramembranous ossification.

Wnt/ β -catenin signaling stimulates matrix catabolic genes and activity in articular chondrocytes: its possible role in joint degeneration

A fine balance between anabolic and catabolic mechanisms maintains extracellular matrix homeostasis in articular cartilage, and shifts toward degradation are associated with joint conditions such as osteoarthritis. To test the possible involvement, relevance and significance of the Wnt/β-catenin-signaling pathway in those catabolic shifts, rabbit articular chondrocyte cultures were subjected to experimental activation of β-catenin signaling by Wnt3A treatment or forced expression of constitutive-active β-catenin (CA-β-catenin). Both interventions provoked strong gelatinase activity and stimulated gene expression of matrix metalloprotease-3 and -13 and a disintegrin-like and metalloprotease with thrombospondin motif (ADAMTS)-4 and -5 proteases. Furthermore, Wnt3A treatment additively enhanced the effects of intereukin-1β, a well-known catabolic culprit of proteoglycan matrix loss. To determine whether Wnt/β-catenin signaling is associated with age-associated osteoarthritic changes in articular cartilage in vivo, we analyzed the presence and intracellular distribution of β-catenin in a spontaneous guinea pig osteoarthritis model. Healthy articular chondrocytes in young guinea pig knees contained barely detectable levels of β-catenin. In contrast, the protein was highly abundant in osteoarthritic-like chondrocytes present in older guinea pig joints, and was localized not only in the cytoplasm but also the nucleus, a clear reflection of activated Wnt signaling. These and other data suggest that Wnt/β-catenin signaling is a powerful stimulator of chondrocyte matrix catabolic action and may be part of mechanisms leading to excessive remodeling and degradation of cartilage matrix in age-associated joint pathologies.

Cellular and molecular mechanisms of synovial joint and articular cartilage formation.

Abstract:  Synovial joints and articular cartilage play crucial roles in the skeletal function, but relatively little is actually known about their embryonic development. Here we first focused on the interzone, a thin mesenchymal cell layer forming at future joint sites that is widely thought to be critical for joint and articular cartilage development. To determine interzone cell origin and fate, we microinjected the vital fluorescent dye DiI at several peri‐joint sites in chick limbs and monitored the behavior and fate of labeled cells over time. Peri‐joint mesenchymal cells located immediately adjacent to incipient joints migrated, became part of the interzone, and were eventually found in epiphyseal articular layer and joint capsule. Interzone cells isolated and reared in vitro expressed typical phenotypic markers, including GDF‐5, Wnt‐14, and CD‐44, and differentiated into chondrocytes over time. To determine the molecular mechanisms of articular chondrocyte formation, we carried out additional studies on the ets transcription factor family member ERG and its alternatively spliced variant C‐1‐1 that we previously found to be expressed in developing avian articular chondrocytes. We cloned the human counterpart of avian C‐1‐1 (ERGp55Δ81) and conditionally expressed it in transgenic mice under cartilage‐specific Col2 gene promotor‐enhancer control. The entire transgenic mouse limb chondrocyte population exhibited an immature articular‐like phenotype and a virtual lack of growth plate formation and chondrocyte maturation compared to wild‐type littermate. Together, our studies reveal that peri‐joint mesenchymal cells take part in interzone and articular layer formation, interzone cells can differentiate into chondrocytes, and acquisition of a permanent articular chondrocyte phenotype is aided and perhaps dictated by ets transcription factor ERG.

Involvement of α5β1 Integrin in Matrix Interactions and Proliferation of Chondrocytes

Integrins are cell surface receptors involved in cellular processes including adhesion, migration, and matrix assembly. In the present study, we analyzed the possible involvement of α5β1 integrin in the regulation of chondrocyte adhesion, spreading, and proliferation. We found that rabbit growth plate chondrocytes were able to attach to substrates coated with type I collagen, type II collagen, or fibronectin within 24 h of culture. During this time period, attachment to fibronectin appeared to be dependent on α5β1 integrin, whereas adhesion to collagens was not. By day 3 of culture, chondrocytes spread onto all the substrates tested. We found that regardless of the nature of the substrate, cell spreading was reversed by treatment with RGD peptide or antibodies against α5β1 or fibronectin, indicating that cell spreading involved α5β1 and fibronectin endogenously produced and deposited by the chondrocytes themselves. Colony formation by chondrocytes in soft agar was inhibited by treatment with RGD peptides or BIIG2, an antibody that interferes with α5β1 integrin–ligand interactions. Furthermore, DNA content was decreased by treatment with anti‐fibronectin antibody in micromass culture of chondrocytes. Immunohistochemical analysis on tissue sections revealed that the α5 subunit was particularly abundant in the proliferative and hypertrophic zones of growth plate. The results of the study indicate that α5β1 integrin plays multiple roles in chondrocyte behavior and function and appears to be involved in the regulation of both chondrocyte–matrix interactions and proliferation.

Runx2 deficiency in chondrocytes causes adipogenic changes in vitro

Runx2 (runt-related transcription factor 2) is an important transcription factor for chondrocyte differentiation as well as for osteoblast differentiation. To investigate the function of Runx2 in chondrocytes, we isolated chondrocytes from the rib cartilage of Runx2-deficient (Runx2–/–) mice and examined the effect of Runx2 deficiency on chondrocyte function and behavior in culture for up to 12 days. At the beginning of the culture, Runx2–/– chondrocytes actively proliferated, had a polygonal shape and expressed type II collagen; these are all characteristics of chondrocytes. However, they gradually accumulated lipid droplets that stained with oil red O and resembled adipocytes. Northern blot analysis revealed that the expression of adipocyte-related differentiation marker genes including PPARγ (peroxisome proliferator-activated receptor γ), aP2 and Glut4 increased over time in culture, whereas expression of type II collagen decreased. Furthermore, the expression of Pref-1, an important inhibitory gene of adipogenesis, was remarkably decreased. Adenoviral introduction of Runx2 or treatment with transforming growth factor-β, retinoic acid, interleukin-1β, basic fibroblast growth factor, platelet-derived growth factor or parathyroid hormone inhibited the adipogenic changes in Runx2–/– chondrocytes. Runx2 and transforming growth factor-β synergistically upregulated interleukin-11 expression, and the addition of interleukin-11 to the culture medium reduced adipogenesis in Runx2–/– chondrocytes. These findings indicate that depletion of Runx2 resulted in the loss of the differentiated phenotype in chondrocytes and induced adipogenic differentiation in vitro, and show that Runx2 plays important roles in maintaining the chondrocyte phenotype and in inhibiting adipogenesis. Our findings suggest that these Runx2-dependent functions are mediated, at least in part, by interleukin-11.

Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP‐2

The roles of Sonic hedgehog (Shh) and Bone morphogenetic protein‐2 (Bmp‐2) in osteoblast differentiation were investigated using in vitro cell systems. Recombinant amino‐terminal portion of SHH (rSHH‐N) dose dependently stimulated ALP activity in C3H10T1/2 and MC3T3‐E1 cells. rSHH‐N induced expression of Osteocalcin mRNA in C3H10T1/2 cells. A soluble form of the receptor for type IA BMP receptor antagonized rSHH‐N‐induced ALP activity in C3H10T1/2 and MC3T3‐E1 cells, indicating that BMPs are involved in SHH‐induced osteoblast differentiation. Simultaneous supplement with rSHH‐N and BMP‐2 synergistically induced ALP activity and expression of Osteocalcin mRNA in C3H10T1/2 cells. Pretreatment with rSHH‐N for 6 h enhanced the response to BMP‐2 by increasing ALP activity in C3H10T1/2 and MC3T3‐E1 cells. Stimulatory effects of rSHH‐N and additive effects with rSHH‐N and BMP‐2 on ALP activity were also observed in mouse primary osteoblastic cells. Transplantation of BMP‐2 (1 μg) into muscle of mice induced formation of ectopic bone, whereas transplantation of r‐SHH‐N (1–5 μg) failed to generate it. These results indicate that Shh plays important roles in osteoblast differentiation by cooperating with BMP.