Matrilin-3 Inhibits Chondrocyte Hypertrophy as a Bone Morphogenetic Protein-2 Antagonist

Increased chondrocyte hypertrophy is often associated with cartilage joint degeneration in human osteoarthritis patients. Matrilin-3 knock-out (Matn3 KO) mice exhibit these features. However, the underlying mechanism is unknown. In this study, we sought a molecular explanation for increased chondrocyte hypertrophy in the mice prone to cartilage degeneration. We analyzed the effects of Matn3 on chondrocyte hypertrophy and bone morphogenetic protein (Bmp) signaling by quantifying the hypertrophic marker collagen type X (Col X) gene expression and Smad1 activity in Matn3 KO mice in vivo and in Matn3-overexpressing chondrocytes in vitro. The effect of Matn3 and its specific domains on BMP activity were quantified by Col X promoter activity containing the Bmp-responsive element. Binding of MATN3 with BMP-2 was determined by immunoprecipitation, solid phase binding, and surface plasmon resonance assays. In Matn3 KO mice, Smad1 activity was increased more in growth plate chondrocytes than in wild-type mice. Conversely, Matn3 overexpression in hypertrophic chondrocytes led to inhibition of Bmp-2-stimulated, BMP-responsive element-dependent Col X expression and Smad1 activity. MATN3 bound BMP-2 in a dose-dependent manner. Multiple epidermal growth factor (EGF)-like domains clustered together by the coiled coil of Matn3 is required for Smad1 inhibition. Hence, as a novel BMP-2-binding protein and antagonist in the cartilage extracellular matrix, MATN3 may have the inherent ability to inhibit premature chondrocyte hypertrophy by suppressing BMP-2/Smad1 activity.     

Smad7 Inhibits chondrocyte differentiation at multiple steps during endochondral bone formation and down-regulates p38 MAPK pathways

Bone morphogenetic proteins (BMPs) play critical roles at various stages in endochondral bone formation. In vitro studies have demonstrated that Smad7 regulates transforming growth factor-beta and BMP signals by inhibiting Smad pathways in chondrocytes. However, the in vivo roles of Smad7 during cartilage development are unknown. To investigate distinct effects of Smad7 at different stages during chondrocyte differentiation, we generated a series of conditional transgenic mice that overexpress Smad7 in chondrocytes at various steps of differentiation by using the Cre/loxP system. We generated Col11a2-lacZ(floxed)-Smad7 transgenic mice and mated them with three types of Cre transgenic mice to obtain Smad7(Prx1), Smad7(11Enh), and Smad7(11Prom) conditional transgenic mice. Smad7(Prx1) mice overexpressing Smad7 in condensing mesenchymal cells showed disturbed mesenchymal condensation associated with decreased Sox9 expression, leading to poor cartilage formation. Smad7(11Enh) mice overexpressing Smad7 in round chondrocytes showed decreased chondrocyte proliferation rates. Smad7(11Prom) mice overexpressing Smad7 in flat chondrocytes showed inhibited maturation of chondrocytes toward hypertrophy. Micromass culture of mesenchymal cells showed that BMP-induced cartilaginous nodule formation was down-regulated by overexpression of Smad7, but not Smad6. Overexpression of Smad7, but not Smad6, down-regulated the phosphorylation of p38 MAPKs. Our data provide in vivo evidence for distinct effects of Smad7 at different stages during chondrocyte differentiation and suggest that Smad7 in prechondrogenic cells inhibits chondrocyte differentiation possibly by down-regulating BMP-activated p38 MAPK pathways.     

SnoN Suppresses Maturation of Chondrocytes by Mediating Signal Cross-talk between Transforming Growth Factor-β and Bone Morphogenetic Protein Pathways

Hypertrophic maturation of chondrocytes is a crucial step in endochondral ossification, whereas abnormally accelerated differentiation of hypertrophic chondrocytes in articular cartilage is linked to pathogenesis of osteoarthritis. This cellular process is promoted or inhibited by bone morphogenetic protein (BMP) or transforming growth factor-β (TGF-β) signaling, respectively, suggesting that these signaling pathways cross-talk during chondrocyte maturation. Here, we demonstrated that expression of Tgfb1 was increased, followed by phosphorylation of Smad2, during BMP-2-induced hypertrophic maturation of ATDC5 chondrocytes. Application of a TGF-β type I receptor inhibitor compound, SB431542, increased the expression of Id1, without affecting the phosphorylation status of Smad1/5/8, indicating that the activated endogenous TGF-β pathway inhibited BMP signaling downstream of the Smad activation step. We searched for TGF-β-inducible effectors that are able to inhibit BMP signaling in ATDC5 cells and identified SnoN. Overexpression of SnoN suppressed the activity of a BMP-responsive luciferase reporter in COS-7 cells as well as expression of Id1 in ATDC5 cells and, subsequently, the expression of Col10a1, a hallmark of hypertrophic chondrocyte maturation. siRNA-mediated loss of SnoN showed opposite effects in BMP-treated ATDC5 cells. In adult mice, we found the highest level of SnoN expression in articular cartilage. Importantly, SnoN was expressed, in combination with phosphorylated Smad2/3, in prehypertrophic chondrocytes in the growth plate of mouse embryo bones and in chondrocytes around the ectopically existing hypertrophic chondrocytes of human osteoarthritis cartilage. Our results indicate that SnoN mediates a negative feedback mechanism evoked by TGF-β to inhibit BMP signaling and, subsequently, hypertrophic maturation of chondrocytes.     

骨形成蛋白4条件性RNA干扰小鼠的建立

为研究骨形成蛋白4(Bone morphogenetic protein 4, BMP4)在骨骼发育中的作用, 我们以含有LoxPneo的pBSK/U6载体为骨架, 构建小鼠BMP4条件性RNAi(conditional RNA interference), CRNA; 载体(BMP4CRNAi), 经KpnⅠ和AflⅢ双酶切获取针对bmp4并含neo基因的目的干扰片段, 纯化后的目的片段显微注射入0.5 d的FVB/NJ小鼠受精卵, 并植入同期发情的假孕母鼠中, 获取G0代转基因小鼠; 利用PCR对G0代转基因小鼠基因型进行鉴定, PCR阳性的小鼠与FVB/NJ小鼠交配, 最终获取稳定传代的BMP4CRNAi小鼠。稳定传代的BMP4^CRNAi小鼠与成骨和软骨前体细胞表达Cre的转基因小鼠(Col2a1-Cre)交配, 获取BMP4^Col2a1-CRNAi小鼠。分离BMP4Col2a1-CRNAi小鼠原代软骨细胞, 提取总RNA, 利用半定量RT-PCR检测RNA干扰效率。小鼠基因型鉴定结果表明：成功获得条件性RNAi转基因小鼠; BMP4干扰效率检测结果表明：在软骨细胞中BMP4的干扰效率为81％。以上结果表明, 我们成功制备了BMP4^CRNAi小鼠和BMP4^Col2a1-CRNAi小鼠, BMP4^CRNAi小鼠与不同Cre转基因小鼠交配, 可以研究BMP4在不同细胞、组织和器官的功能, BMP4^Col2a1-CRNAi小鼠的获得为研究BMP4在软骨发育中的作用提供了合适的动物模型。     

Bone morphogenetic protein (BMP) localization in developing human and rat growth plate, metaphysis, epiphysis, and articular cartilage.

We assessed the distribution and relative staining intensity of bone morphogenetic protein (BMP)-1-7 by immunohistochemistry in tibial growth plates, epiphyses, metaphyses, and articular cartilage in one 21-week and one 22-week human fetus and in five 10-week-old Sprague-Dawley rats. In the rats, articular cartilage was also examined. BMP proteins were mostly cytoplasmic, with negligible matrix staining. Highest BMP levels were seen in (a) hypertrophic and calcifying zone chondrocytes of growth plate (BMP-1-7), (b) osteoblasts and/or osteoprogenitor fibroblasts and vascular cells of the metaphyseal cortex and medulla (BMP-1-6), (c) osteoclasts of the metaphysis and epiphysis (BMP-1,-4,-5, and -6), and (d) mid to deep zone articular chondrocytes of weanling rats (BMP-1-7). BMP staining in osteoclasts, an unexpected finding, was consistently strong with BMP-4, -5, and -6 but was variable and dependent on osteoclast location with BMP-2,-3, and -7. BMP-1-7 were moderately to intensely stained in vascular canals of human fetal epiphyseal cartilage by endothelial cells and pericytes. BMP-1,-3,-5,-6, and -7 were localized in hypertrophic chondrocytes adjacent to cartilage canals. We conclude that BMP expression is associated with maturing chondrocytes of growth plate and articular cartilage, and may play a role in chondrocyte differentiation and/or apoptosis. BMP appears to be expressed by osteoclasts and might be involved in the intercellular "cross-talk" between osteoclasts and neighboring osteoprogenitor cells at sites of bone remodeling.     

Site-1 protease is essential to growth plate maintenance and is a critical regulator of chondrocyte hypertrophic differentiation in postnatal mice

Site-1 protease (S1P) is a proprotein convertase with essential functions in lipid homeostasis and unfolded protein response pathways. We previously studied a mouse model of cartilage-specific knock-out of S1P in chondroprogenitor cells. These mice exhibited a defective cartilage matrix devoid of type II collagen protein (Col II) and displayed chondrodysplasia with no endochondral bone formation even though the molecular program for endochondral bone development appeared intact. To gain insights into S1P function, we generated and studied a mouse model in which S1P is ablated in postnatal chondrocytes. Postnatal ablation of S1P results in chondrodysplasia. However, unlike early embryonic ablations, the growth plates of these mice exhibit a lack of Ihh, PTHrP-R, and Col10 expression indicating a loss of chondrocyte hypertrophic differentiation and thus disruption of the molecular program required for endochondral bone development. S1P ablation results in rapid growth plate disruption due to intracellular Col II entrapment concomitant with loss of chondrocyte hypertrophy suggesting that these two processes are related. Entrapment of Col II in the chondrocytes of the prospective secondary ossification center precludes its development. Trabecular bone formation is dramatically diminished in the primary spongiosa and is eventually lost. The primary growth plate is eradicated by apoptosis but is gradually replaced by a fully functional new growth plate from progenitor stem cells capable of supporting new bone growth. Our study thus demonstrates that S1P has fundamental roles in the preservation of postnatal growth plate through chondrocyte differentiation and Col II deposition and functions to couple growth plate maturation to trabecular bone development in growing mice.     

Role of Runx genes in chondrocyte differentiation

Runx2/Cbfa1 plays a central role in skeletal development as demonstrated by the absence of osteoblasts/bone in mice with inactivated Runx2/Cbfa1 alleles. To further investigate the role of Runx2 in cartilage differentiation and to assess the potential of Runx2 to induce bone formation, we cloned chicken Runx2 and overexpressed it in chick embryos using a retroviral system. Infected chick wings showed multiple phenotypes consisting of (1) joint fusions, (2) expansion of carpal elements, and (3) shortening of skeletal elements. In contrast, bone formation was not affected. To investigate the function of Runx2/Cbfa1 during cartilage development, we have generated transgenic mice that express a dominant negative form of Runx2 in cartilage. The selective inactivation of Runx2 in chondrocytes results in a severe shortening of the limbs due to a disturbance in chondrocyte differentiation, vascular invasion, osteoclast differentiation, and periosteal bone formation. Analysis of the growth plates in transgenic mice and in chick limbs shows that Runx2 is a positive regulator of chondrocyte differentiation and vascular invasion. The results further indicate that Runx2 promotes chondrogenesis either by maintaining or by initiating early chondrocyte differentiation. Furthermore, Runx2 is essential but not sufficient to induce osteoblast differentiation. To analyze the role of runx genes in skeletal development, we performed in situ hybridization with Runx2- and Runx3-specific probes. Both genes were coexpressed in cartilaginous condensations, indicating a cooperative role in the regulation of early chondrocyte differentiation and thus explaining the expansion/maintenance of cartilage in the carpus and joints of infected chick limbs.     

Smad6/Smurf1 overexpression in cartilage delays chondrocyte hypertrophy and causes dwarfism with osteopenia

Biochemical experiments have shown that Smad6 and Smad ubiquitin regulatory factor 1 (Smurf1) block the signal transduction of bone morphogenetic proteins (BMPs). However, their in vivo functions are largely unknown. Here, we generated transgenic mice overexpressing Smad6 in chondrocytes. Smad6 transgenic mice showed postnatal dwarfism with osteopenia and inhibition of Smad1/5/8 phosphorylation in chondrocytes. Endochondral ossification during development in these mice was associated with almost normal chondrocyte proliferation, significantly delayed chondrocyte hypertrophy, and thin trabecular bone. The reduced population of hypertrophic chondrocytes after birth seemed to be related to impaired bone growth and formation. Organ culture of cartilage rudiments showed that chondrocyte hypertrophy induced by BMP2 was inhibited in cartilage prepared from Smad6 transgenic mice. We then generated transgenic mice overexpressing Smurf1 in chondrocytes. Abnormalities were undetectable in Smurf1 transgenic mice. Mating Smad6 and Smurf1 transgenic mice produced double-transgenic pups with more delayed endochondral ossification than Smad6 transgenic mice. These results provided evidence that Smurf1 supports Smad6 function in vivo.     

BMP-5 expression increases during chondrocyte differentiation in vivo and in vitro and promotes proliferation and cartilage matrix synthesis in primary chondrocyte cultures

Bone morphogenetic proteins (BMPs) play pivotal roles in bone and cartilage growth and repair. Through phenotypes of short-ear (se) mice, which have BMP-5 mutations, a role for BMP-5 in some specific aspects of skeletogenesis and cartilage growth is known. This report examines BMP-5 expression in the growth plate and in differentiating cultures of primary chondrocytes, and the effects of addition of BMP-5 or its inhibition by anti-BMP-5 antibody in chondrocyte cultures. By laser capture microdissection and immunohistochemistry, we found that BMP-5 is expressed in proliferating zone (PZ) chondrocytes and that the expression increases sharply with hypertrophic differentiation. A similar pattern was observed in differentiating cultures of primary chondrocytes, with BMP-5 expression increasing as cells differentiated, in contrast to other BMPs. BMP-5 added to cultures increased cell proliferation early in the culture period and also stimulated cartilage matrix synthesis. Also, BMP-5 addition to the cultures activated phosphorylation of Smad 1/5/8 and p38 MAP kinase and caused increased nuclear accumulation of phospho-Smads. Anti-BMP-5 antibody inhibited the endogenous BMP-5, reducing cell proliferation and phospho-Smad nuclear accumulation. Together, the results demonstrate that BMP-5 is normally an important regulator of chondrocyte proliferation and differentiation. Whether other BMPs may compensate in BMP-5 loss-of-function mutations is discussed.     

Transgenic mice that express Cre recombinase in hypertrophic chondrocytes

In order to investigate the physiological control of hypertrophic chondrocytes which present the terminally differentiated form of chondrocytes, we generated a mouse line expressing the Cre recombinase under the control of the mouse type X collagen (Col10a1) promoter. In situ hybridization analysis demonstrated the expression of Col10a1-Cre transgene in hypertrophic chondrocytes of femur at postnatal day 2 (P2). In order to test the excision activity of the Cre recombinase, the Col10a1-Cre transgenic line was crossed with the mouse strain carrying the Smad4 conditional alleles (Smad4co/co) and the reporter line ROSA26. Multiple tissue PCR of Col10a1-Cre;Smad4co/+ mice revealed the restricted Cre activity in tissues containing hypertrophic chondrocytes. LacZ staining revealed that the Cre activity was observed in the cartilage primordia of ribs at E14.5 and only detected in the lower hypertrophic region of ribs at P1. These data suggest that the Col10a1-Cre mouse line described here could be used to achieve conditional gene targeting in hypertrophic chondrocytes.     

Conditional expression of constitutively active estrogen receptor α in chondrocytes impairs longitudinal bone growth in mice

Estrogen plays important roles in the regulation of chondrocyte proliferation and differentiation, which are essential steps for longitudinal bone growth; however, the mechanisms of estrogen action on chondrocytes have not been fully elucidated. In the present study, we generated conditional transgenic mice, designated as caERα(ColII), expressing constitutively active mutant estrogen receptor (ER) α in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caERα(ColII) mice showed retardation in longitudinal growth, with short bone lengths. BrdU labeling showed reduced proliferation of hypertrophic chondrocytes in the proliferating layer of the growth plate of tibia in caERα(ColII) mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caERα(ColII) mice. These results suggest that ERα is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.     

BMP and Ihh/PTHrP signaling interact to coordinate chondrocyte proliferation and differentiation.

During endochondral ossification, two secreted signals, Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP), have been shown to form a negative feedback loop regulating the onset of hypertrophic differentiation of chondrocytes. Bone morphogenetic proteins (BMPs), another family of secreted factors regulating bone formation, have been implicated as potential interactors of the Ihh/PTHrP feedback loop. To analyze the relationship between the two signaling pathways, we used an organ culture system for limb explants of mouse and chick embryos. We manipulated chondrocyte differentiation by supplementing these cultures either with BMP2, PTHrP and Sonic hedgehog as activators or with Noggin and cyclopamine as inhibitors of the BMP and Ihh/PTHrP signaling systems. Overexpression of Ihh in the cartilage elements of transgenic mice results in an upregulation of PTHrP expression and a delayed onset of hypertrophic differentiation. Noggin treatment of limbs from these mice did not antagonize the effects of Ihh overexpression. Conversely, the promotion of chondrocyte maturation induced by cyclopamine, which blocks Ihh signaling, could not be rescued with BMP2. Thus BMP signaling does not act as a secondary signal of Ihh to induce PTHrP expression or to delay the onset of hypertrophic differentiation. Similar results were obtained using cultures of chick limbs. We further investigated the role of BMP signaling in regulating proliferation and hypertrophic differentiation of chondrocytes and identified three functions of BMP signaling in this process. First we found that maintaining a normal proliferation rate requires BMP and Ihh signaling acting in parallel. We further identified a role for BMP signaling in modulating the expression of IHH: Finally, the application of Noggin to mouse limb explants resulted in advanced differentiation of terminally hypertrophic cells, implicating BMP signaling in delaying the process of hypertrophic differentiation itself. This role of BMP signaling is independent of the Ihh/PTHrP pathway.     

Immunohistochemical Localization of Bone Morphogenetic Proteins (BMPs) and their Receptors in Solitary and Multiple Human Osteochondromas

The expression of bone morphogenetic proteins (BMPs) and their cognate receptors (BMPRs) in osteochondromas has not been investigated. We determined the immunohistochemical localization and distribution of BMP-2/4, -6 and -7; BMP receptors BMPR-1A, BMPR-1B and BMPR-2; signal transducing proteins phosphorylated Smad1/5/8; and BMP antagonist noggin in the cartilaginous cap of solitary (SO) and multiple (MO) human osteochondromas and compared these with bovine growth plate and articular cartilage. The distribution and localization patterns for BMP-6, BMP-7, BMPR-1A and BMPR-2 were similar between the cartilaginous cap and the growth plate. BMP-2/4 and BMPR-1B were present throughout the growth plate. However, BMP-2/4 and phosphorylated Smad1/5/8 were mainly detected in proliferating chondrocytes of the cartilaginous cap. Also, BMPR-1B was found in hypertrophic chondrocytes of SO and proliferating chondrocytes of MO. Noggin was observed in resting chondrocytes and, to a lesser extent, in clustered proliferating chondrocytes in SO. On the other hand, noggin in MO was observed in proliferating chondrocytes. Since BMPs can stimulate proliferation and hypertrophic differentiation of chondrocytes, these findings suggest that there is an imbalance of BMP-2/4 and noggin interactions that may lead to abnormal regulation of chondrocyte proliferation and differentiation in the cartilaginous cap of human osteochondromas.     

Distinct functions of BMP4 and GDF5 in the regulation of chondrogenesis

Bone morphogenetic protein 4 (BMP4) and growth/differentiation factor 5 (GDF5) are closely related protein family members and regulate early cartilage patterning and differentiation. In this study, we compared the functional outcome of their actions systematically at various stages of chondrogenesis in mouse embryonic limb bud mesenchyme grown in micromass cultures. Overall, both growth factors enhanced cartilage growth and differentiation in these cultures. Uniquely, BMP4 not only accelerated the formation and maturation of cartilaginous nodules, but also induced internodular mesenchymal cells to express cartilage differentiation markers. On the other hand, GDF5 increased the number of prechondrogenic mesenchymal cell condensation and cartilaginous nodules, without altering the overall pattern of differentiation. In addition, GDF5 caused a more sustained elevated expression level of Sox9 relative to that associated with BMP4. BMP4 accelerated chondrocyte maturation throughout the cultures and sustained an elevated level of Col10 expression, whereas GDF5 caused a transient increase in Col10 expression. Taken together, we conclude that BMP4 is instructive to chondrogenesis and induces mesenchymal cells toward the chondrogenic lineage. Furthermore, BMP4 accelerates the progression of cartilage differentiation to maturation. GDF5 enhances cartilage formation by promoting chondroprogenitor cell aggregation, and amplifying the responses of cartilage differentiation markers. These differences may serve to fine-tune the normal cartilage differentiation program, and can be exploited for the molecular manipulation in biomimetics.     

Indian hedgehog in the late-phase differentiation in mouse chondrogenic EC cells, ATDC5: upregulation of type X collagen and osteoprotegerin ligand mRNAs

Endochondral bone formation includes a cascade of cellular events such as proliferation, maturation, hypertrophic conversion and calcification of chondrocytes and the cartilage replacement by bone. During these processes, hypertrophic conversion and calcification of chondrocytes (the late-phase differentiation) is a crucial process of chondrogenic differentiation. Indian hedgehog (Ihh), a secreted protein expressed in early hypertrophic chondrocytes, is thought to be involved in regulation of hypertrophic conversion via a feedback loop through the perichondrium. In the present study, we showed by Northern analysis and in situ hybridization that Smoothened (Smo), a key component in hedgehog signal transduction, was expressed in chondrocytes in both adult mice and mouse embryos at 16 days post-coitum in vivo, suggesting that Ihh directly acts on chondrocytes. We previously reported that Ihh, Patched and Smo were all expressed in differentiated ATDC5 cells. Exogenously administered mouse recombinant N-terminal protein of Ihh (mrIhh-N) upregulated the gene expression of type X collagen, a phenotypic marker of hypertrophic chondrocytes, as well as osteoprotegerin ligand (OPGL), a potent stimulator of osteoclastogenesis and osteoclast activity, while it did not modulate the expression of Ihh itself, bone morphogenetic protein (BMP)-4, BMP-6, transforming growth factor (TGF)-beta1 and TGF-beta2 in differentiated ATDC5 cells. Moreover, when added to the osteoclast cultures, mrIhh-N markedly stimulated the formation of resorption pits on dentine slices. Our data support the hypothesis that Ihh stimulated the late-phase chondrogenic differentiation in differentiated ATDC5 cells and upregulated the gene expression of OPGL in these cells.