Interaction of Ihh and BMP/Noggin signaling during cartilage differentiation.

Bone morphogenetic proteins (BMPs) have been implicated in regulating multiple stages of bone development. Recently it has been shown that constitutive activation of the BMP receptor-IA blocks chondrocyte differentiation in a similar manner as misexpression of Indian hedgehog. In this paper we analyze the role of BMPs as possible mediators of Indian hedgehog signaling and use Noggin misexpression to gain insight into additional roles of BMPs during cartilage differentiation. We show by comparative analysis of BMP and Ihh expression domains that the borders of Indian hedgehog expression in the chondrocytes are reflected in changes of the expression level of several BMP genes in the adjacent perichondrium. We further demonstrate that misexpression of Indian hedgehog appears to directly upregulate BMP2 and BMP4 expression, independent of the differentiation state of the flanking chondrocytes. In contrast, changes in BMP5 and BMP7 expression in the perichondrium correspond to altered differentiation states of the flanking chondrocytes. In addition, Noggin and Chordin, which are both expressed in the developing cartilage elements, also change their expression pattern after Ihh misexpression. Finally, we use retroviral misexpression of Noggin, a potent antagonist of BMP signaling, to gain insight into additional roles of BMP signaling during cartilage differentiation. We find that BMP signaling is necessary for the growth and differentiation of the cartilage elements. In addition, this analysis revealed that the members of the BMP/Noggin signaling pathway are linked in a complex autoregulatory network.     

BMP signaling in skeletal development

Development of the vertebrate skeleton, a complex biological event that includes diverse processes such as formation of mesenchymal condensations at the sites of future skeletal elements, osteoblast and chondrocyte differentiation, and three dimensional patterning, is regulated by many growth factors. Bone morphogenetic proteins (BMPs), members of the TGF-beta superfamily, play a pivotal role in the signaling network and are involved in nearly all processes associated with skeletal morphogenesis. BMP signals are transduced from the plasma membrane receptors to the nucleus through both Smad pathway and non-Smad pathways, and regulated by many extracellular and intercellular proteins that interact with BMPs or components of the BMP signaling pathways. To gain a better understanding of the molecular mechanisms underlying the role of BMP in early skeletal development, it is necessary to elucidate the BMP signaling transduction pathways in chondrocytes and osteoblasts. The major objective of this review was to summarize BMP signaling pathways in the context of craniofacial, axial, and limb development. In particular, this discourse will focus on recent advances of the role of different ligands, receptors, Smads, and BMP regulators in osteoblast and chondrocyte differentiation during embryonic development.     

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.     

Bone morphogenetic protein signaling in articular chondrocyte differentiation

Articular chondrocytes progressively undergo dedifferentiation into a spindle-shaped mesenchymal cellular phenotype in monolayers. Chondrocyte dedifferentiation is stimulated by retinoic acid. On the other hand, bone morphogenic proteins (BMPs) stimulate differentiation of chondrocytes. We examined the mechanism of effects of BMP in chondrocyte differentiation with use of a recombinant adenovirus vector system. Constitutively active forms of BMP type I receptors (BMPR-IA and BMPR-IB) and those of activin receptor-like kinase (ALK)-1 and ALK-2 maintained differentiation of chondrocytes in the presence of retinoic acid. The BMP receptor-regulated signaling substrates, Smad1/5, weakly induced chondrocyte differentiation; the effects of Smad1/5 were enhanced by BMP-7 treatment. Inhibitory Smad, Smad6, blocked increase of expression of chondrocyte markers by BMP-7 in a dose-dependent manner. SB202190, a p38 mitogen-activated protein kinase inhibitor, inhibited this effect of BMP-7; however, since SB202190 suppressed phosphorylation of Smad1/5, this may be due to blockade of BMP receptor activation. These results together strongly suggest that induction of chondrocyte differentiation by BMP-7 is regulated by Smad pathways.     

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.     

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.     

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.     

BMPs are required at two steps of limb chondrogenesis: formation of prechondrogenic condensations and their differentiation into chondrocytes

Formation of the long bones requires a cartilage template. Cartilage formation (chondrogenesis) proceeds through determination of cells and their aggregation into prechondrogenic condensations, differentiation into chondrocytes, and later maturation. Several studies indicate that members of the bone morphogenetic protein (BMP) family promote cartilage formation, but the exact step(s) in which BMPs are involved during this process remains undefined. To resolve this issue, we have used a retroviral vector to misexpress the BMP antagonist Noggin in the embryonic chick limb. Unlike previous reports, we have characterized the resulting phenotype in depth, analyzing histological and early chondrogenic markers, as well as the patterns of cell death and proliferation. Misexpression of Noggin prior to the onset of chondrogenesis leads to the total absence of skeletal elements, as previously reported (J. Capdevila and R. L. Johnson, 1998, Dev. Biol. 197, 205-217). Noggin inhibits cartilage formation at two distinct steps. First, we demonstrate that mesenchymal cells do not aggregate into prechondrogenic condensations, and additional results suggest that these cells persist in an undifferentiated state. Second, we show that differentiation of chondroprogenitors into chondrocytes can also be blocked, concurrent with expanded expression of a presumptive joint region marker. In addition, we observed alterations in muscle and tendon morphogenesis, and the potential role of BMPs in these processes will be discussed. Our studies therefore provide in vivo evidence that BMPs are necessary for different steps of chondrogenesis: chondroprogenitor determination and/or condensation and subsequent differentiation into chondrocytes.     

Regulation of osteogenic proteins by chondrocytes

The purpose of this review is to summarize the current scientific knowledge of bone morphogenetic proteins (BMPs) in adult articular cartilage. We specifically focus on adult cartilage, since one of the major potential applications of the members of the BMP family may be a repair of adult tissue after trauma and/or disease. After reviewing cartilage physiology and BMPs, we analyze the data on the role of recombinant BMPs as anabolic agents in tissue formation and restoration in different in vitro and in vivo models following with the endogenous expression of BMPs and factors that regulate their expression. We also discuss recent transgenic modifications of BMP genes and subsequent effect on cartilage matrix synthesis. We found that the most studied BMPs in adult articular cartilage are BMP-7 and BMP-2 as well as transforming growth factor-beta (TGF-beta). There are a number of contradicting reports for some of these growth factors, since different models, animals, doses, time points, culture conditions and devices were used. However, regardless of the experimental conditions, only BMP-7 or osteogenic protein-1 (OP-1) exhibits the most convincing effects. It is the only BMP studied thus far in adult cartilage that demonstrates strong anabolic activity in vitro and in vivo with and without serum. OP-1 stimulates the synthesis of the majority of cartilage extracellular matrix proteins in adult articular chondrocytes derived from different species and of different age. OP-1 counteracts the degenerative effect of numerous catabolic mediators; it is also expressed in adult human, bovine, rabbit and goat articular cartilage. This review reveals the importance of the exploration of the BMPs in the cartilage field and highlights their significance for clinical applications in the treatment of cartilage-related diseases.     

Integrin adhesions

Cell migration requires the coordination of adhesion site assembly and turnover. Canonical models for nascent adhesion formation postulate that integrin binding to extracellular matrix (ECM) proteins results in the rapid recruitment of cytoskeletal proteins such as talin and paxillin to integrin cytoplasmic domains. It is thought that integrin-talin clusters recruit and activate tyrosine kinases such as focal adhesion kinase (FAK). However, the molecular connections of this linkage remain unresolved. Our recent findings support an alternative model whereby FAK recruits talin to new sites of β1 integrin-mediated adhesion in mouse embryonic fibroblasts and human ovarian carcinoma cells. This is dependent on a direct binding interaction between FAK and talin and occurs independently of direct talin binding to β1 integrin. Herein, we discuss differences between nascent and mature adhesions, interactions between FAK, talin and paxillin, possible mechanisms of FAK activation and how this FAK-talin complex may function to promote cell motility through increased adhesion turnover.     

Integrin adhesions: Who’s on first? What's on second?

Cell migration requires the coordination of adhesion site assembly and turnover. Canonical models for nascent adhesion formation postulate that integrin binding to extracellular matrix (ECM) proteins results in the rapid recruitment of cytoskeletal proteins such as talin and paxillin to integrin cytoplasmic domains. It is thought that integrin-talin clusters recruit and activate tyrosine kinases such as focal adhesion kinase (FAK). However, the molecular connections of this linkage remain unresolved. Our recent findings support an alternative model whereby FAK recruits talin to new sites of β1 integrin-mediated adhesion in mouse embryonic fibroblasts and human ovarian carcinoma cells. This is dependent on a direct binding interaction between FAK and talin and occurs independently of direct talin binding to β1 integrin. Herein, we discuss differences between nascent and mature adhesions, interactions between FAK, talin and paxillin, possible mechanisms of FAK activation and how this FAK-talin complex may function to promote cell motility through increased adhesion turnover.     

Development and characterization of a human articular cartilage‐derived chondrocyte cell line that retains chondrocyte phenotype

Chondrocytes, the only cell type present in articular cartilage, regulate tissue homeostasis by a fine balance of metabolism that includes both anabolic and catabolic activities. Therefore, the biology of chondrocytes is critical for understanding cartilage metabolism. One major limitation when studying primary chondrocytes in culture is their loss of phenotype. To overcome this hurdle, limited attempts have been made to develop human chondrocyte cell lines that retain the phenotype for use as a good surrogate model. In this study, we report a novel approach to the establishment and characterization of human articular cartilage‐derived chondrocyte cell lines. Adenoviral infection followed by culture of chondrocytes in 3‐dimensional matrix within 48 h post‐infection maintained the phenotype prior to clonal selection. Cells were then placed in culture either as monolayer, or in 3‐dimensional matrix of alginate or agarose. The clones were characterized by their basal gene expression profile of chondrocyte markers. Based on type II collagen expression, 21 clones were analyzed for gene expression following treatment with IL‐1 or BMP‐7 and compared to similarly stimulated primary chondrocytes. This resulted in selection of two clones that retained the chondrocyte phenotype as evidenced by expression of type II collagen and other extra‐cellular matrix molecules. In addition, one clone (AL‐4‐17) showed similar responses as primary chondrocytes when treated with IL‐1 or BMP‐7. In summary, this report provides a novel procedure to develop human articular cartilage‐derived chondrocyte cell lines, which preserve important characteristics of articular chondrocytes and represent a useful model to study chondrocyte biology. J. Cell. Physiol. 222: 695–702, 2010. © 2009 Wiley‐Liss, Inc.     

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.     

IL-1beta regulates FHL2 and other cytoskeleton-related genes in human chondrocytes

In osteoarthritis (OA), cartilage destruction is associated not only with an imbalance of anabolic and catabolic processes but also with alterations of the cytoskeletal organization in chondrocytes, although their pathogenetic origin is largely unknown so far. Therefore, we have studied possible effects of the proinflammatory cytokine IL-1beta on components of the cytoskeleton in OA chondrocytes on gene expression level. Using a whole genome array, we found that IL-1beta is involved in the regulation of many cytoskeleton-related genes. Apart from well-known cytoskeletal components, the expression and regulation of four genes coding for LIM proteins were shown. These four genes were previously undescribed in the chondrocyte context. Quantitative PCR analysis confirmed significant downregulation of Fhl1, Fhl2, Lasp1, and Pdlim1 as well as Tubb and Vim by IL-1beta. Inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 counteracted the influence of IL-1beta on Fhl2 and Tubb expression, indicating partial involvement of this signaling pathway. Downregulation of the LIM-only protein FHL2 was confirmed additionally on the protein level. In agreement with these results, IL-1beta induced changes in the morphology of chondrocytes, the organization of the cytoskeleton, and the cellular distribution of FHL2. We conclude that L-1beta is involved in the regulation of various cytoskeletal components in human chondrocytes including the multifunctional protein FHL2. This might be relevant for the pathogenesis of OA.     

The interaction of canonical bone morphogenetic protein- and Wnt-signaling pathways may play an important role in regulating cartilage degradation in osteoarthritis

Bone morphogenetic proteins (BMPs) and Wnts are important signaling protein families with key roles in embryologic, patterning, development, and tissue remodeling in growth. BMP and Wnt-β-catenin are highly evolutionarily conserved pathways that, though often regulating similar cellular events, are independent signaling mechanisms that can have complementary or antagonistic effects depending on various factors, including cell type and developmental stage. Although BMP and Wnt-β-catenin have the ability to act entirely independently, there is a developing body of evidence for specific extra- and intra-cellular molecular interactions and crosstalk that occur between BMP and Wnt-β-catenin signaling and that again this may be cell type-specific. In the previous issue of Arthritis Research & Therapy, Papathanasiou and colleagues provide novel insights into the role and direct interaction of BMP2 and canonical Wnt-β-catenin signaling in regulating chondrocyte hypertrophy and matrix metalloproteinase/a disintegrin like and metalloproteinase with thrombospondin type I motif (MMP/ADAMTS) synthesis in osteoarthritis.In the previous issue of Arthritis Research & Therapy, Papathanasiou and colleagues [1] provide novel insights into the role and direct interaction of bone morphogenetic protein 2 (BMP2) and canonical Wnt-β-catenin signaling in regulating chondrocyte hypertrophy and matrix metalloproteinase (MMP)/aggrecanolytic ADAMTS (a disintegrin like and metalloproteinase with thrombospondin type I motif) synthesis in osteoarthritis (OA). OA is the most common cause of joint pain and disability, and with increasing age and obesity of the population, the already major socioeconomic importance will continue to increase. Currently, in most Western cultures, OA afflicts more than 10% of the entire population and over a third of those over 65; an estimated 25 to 30 million people in the US suffer from this disease. The central pathological feature of OA is often considered to be the progressive destruction of articular cartilage that normally provides the load-bearing surface in the joint. Much has been learned in recent years about the mechanisms that drive cartilage matrix breakdown and loss in OA, and chondrocyte-derived metalloproteinases, particularly the ADAMTS and collagenolytic MMPs, have a key role. It is evident that a phenotypic shift in the mature articular chondrocyte to a cell type that displays many characteristics typical of hypertrophic cells in the lower zones of the growth plate is a typical feature of OA and is associated with the progressive cartilage breakdown observed (reviewed in [2]). Less clearly understood are the specific signaling pathways involved in regulating the chondrocyte phenotype, how they interact, and whether this changes in health and in diseases such as OA.BMPs and Wnts are important signaling protein families with key roles in embryologic, patterning, development, and tissue remodeling in growth. BMP and Wnt-β-catenin are highly evolutionarily conserved pathways that, though often regulating similar cellular events, are independent signaling mechanisms that can have complementary or antagonistic effects depending on various factors, including cell type and developmental stage (reviewed in [3]). Although BMP and Wnt-β-catenin have the ability to act entirely independently, there is a developing body of evidence for specific extra-and intra-cellular molecular interactions and crosstalk that occur between BMP and Wnt-β-catenin signaling and that again may be cell type-specific [3]. In addition to having a key role in development, BMPs and Wnts are emerging as critical regulators of bone and cartilage homeostasis in the adult and, importantly, in the onset and progression of musculoskeletal diseases.BMPs are multi-functional growth factors that belong to the transforming growth factor-β super family. Evidence suggests that BMP signaling is mediated primarily through the canonical BMP-Smad pathway in chondrocytes. BMPs bind the type II receptor and phosphorylate type I serine or threonine receptors, which subsequently phosphorylate Smad1, Smad5, and Smad8. BMPs are known to induce human mesenchymal stem cells to differentiate into chondrocytes, and BMP2 is a crucial local factor for chondrocyte proliferation and maturation during endochondral ossification [4,5]. In their report, Papathanasiou and colleagues show not only that human end-stage OA chondrocytes produce BMP2 and BMP4 but also, importantly, that BMP2, but not BMP4, can drive expression of low-density lipoprotein receptor 5 (LRP5). LRP5 is one of the most important co-receptors in the canonical Wnt-β-catenin signaling pathway; binding of Wnt ligands to the frizzled/LRP co-receptor complex leads to β-catenin stabilization, nuclear translocation, and activation of target genes.There is a large body of evidence demonstrating the central role for Wnt signaling in regulating adult bone turnover; increased β-catenin activity inducing bone production and inhibition of soluble antagonists is an emerging therapeutic approach for osteoporotic and inflammatory bone loss [6,7]. In cartilage, Wnt-β-catenin signaling plays a dual role; activity is essential for chondrocyte proliferation and maintenance of their phenotypic characteristics [8], but excessive activity increases chondrocyte hypertrophy and expression of cartilage degrades metalloproteinases [9]. The effect may be cell type- specific, and Wnt-β-catenin activation is essential for maintenance of the superficial zone chondrocyte phenotype and proteoglycan 4 (lubricin) expression [8]. Inhibition of β-catenin rapidly leads to downregulation of lubricin and increased collagen × expression in superficial zone chondrocytes. In chondrocytes from human end-stage OA cartilage, activation of canonical Wnt-β-catenin signaling by Wnt-2B and Wnt-16 can drive MMP and aggrecanase production [9]. Understanding the mechanisms that regulate Wnt signaling in chondrocytes in OA may provide keys to controlling cartilage degradation.One of the most important findings by Papathanasiou and colleagues is the demonstration of a new and unique function of BMP2 in chondrocytes in acting as a regulator of canonical Wnt-β-catenin signaling. Treatment of both normal and OA primary human chondrocytes with BMP2 for 12 hours enhanced total β-catenin expression while diminishing the degradation of β-catenin (phospho-β-catenin). This was accompanied by significant increases in mRNA for key cartilage-degrading enzymes MMP-13 and ADAMTS-5 in concert with a shift toward a hypertrophic chondrocyte phenotype as measured by increased collagen × expression. This effect was absent in LRP5 small interfering RNA (siRNA) pretreated chondrocytes and did not occur with BMP4, suggesting the unique function of BMP2 in specifically upregulating LRP5 and augmenting Wnt-β-catenin signaling. The BMP2-driven increase in LRP5 mRNA was mediated through Smad1/5/8 binding to the LRP5 promoter.The paper by Papathanasiou and colleagues adds to the accumulating evidence that increased or perhaps excessive activation of canonical Wnt-β-catenin signaling in chondrocytes is detrimental and contributes to OA cartilage degradation. Therapeutic approaches to block or suppress canonical Wnt-β-catenin signaling may protect cartilage damage in end-stage OA. There are many naturally occurring Wnt-β-catenin signaling antagonists, including dickkopf 1 (DKK1), secreted frizzled-related proteins (sFRPs), and sclerostin (SOST). Evidence suggests that circulating DKK1 levels negatively correlate with biomarkers of cartilage breakdown in patients with OA [10]; sFRP3 knockout mice have augmented cartilage proteoglycan loss in a collagenase-induced instability model of arthritis [11], and co-treatment of SOST with pro-inflammatory cytokines can attenuate cartilage matrix breakdown [12]. The role of SOST is interesting in light of the interaction between BMP2 and Wnt signaling pathways reported by Papathanasiou and colleagues. It appears that SOST can also function as a BMP antagonist in osteoblast and osteocytes by binding intra-cellularly to BMP7 and targeting the growth factor for proteosomal degradation [13]. This provides yet another mechanism by which BMP and Wnt signaling pathways may directly interact; it will be interesting to see whether this effect of SOST on BMP7 (and possibly other BMPs) also occurs in chondrocytes, particularly in OA, where chondrocyte SOST expression is increased [12].The BMP and Wnt signaling pathways are critical in regulating chondrocytes and maintaining the health and integrity of cartilage matrix. In other cell types/organs such as those in bone, it is the combinatorial integration and complex crosstalk between these two pathways that are emerging as significant regulators of development and tissue homeostasis [3]. The findings by Papathanasiou and colleagues suggest that similar signaling pathway interactions may be important in chondrocytes and could play a role in the development and progression of OA. A better appreciation of chondrocyte regulatory mechanisms may provide new avenues for development of therapeutic approaches for the treatment of OA.     

BMP4 substitutes for loss of BMP7 during kidney development

Functional inactivation of divergent bone morphogenetic proteins (BMPs) causes discrete disturbances during mouse development. BMP4-deficient embryos display mesodermal patterning defects at early post-implantation stages, whereas loss of BMP7 selectively disrupts kidney and eye morphogenesis. Whether these distinct phenotypes simply reflect differences in expression domains, or alternatively intrinsic differences in the signaling properties of these ligands remains unknown. To address this issue, we created embryos exclusively expressing BMP4 under control of the BMP7 locus. Surprisingly, this novel knock-in allele efficiently rescues kidney development. These results demonstrate unequivocally that these structurally divergent BMP family members, sharing only minimal sequence similarity can function interchangeably to activate all the essential signaling pathways for growth and morphogenesis of the kidney. Thus, we conclude that partially overlapping expression patterns of BMPs serve to modulate strength of BMP signaling rather than create discrete fields of ligands with intrinsically different signaling properties.     

Recombinant BMP 4/7 fusion protein induces differentiation of bone marrow stem cells

Bone morphogenetic proteins (BMPs) induce differentiation of mesenchymal cells to cartilage and bone. We cloned BMP4 and BMP7 cDNAs from human placenta and fetal cartilage cells, respectively, and used an Escherichia coli expression system to produce recombinant BMP4 and BMP4/7 proteins. Differentiation of primary cultures of bone marrow stem cells (BMSC) treated with BMP4 or BMP4/7 was evaluated by Von Kossa staining and by determining alkaline phosphatase activity and osteocalcin level. BMP4/7-induced BMSC differentiation more potently than BMP4. We showed that BMP4/7 fusion protein expressed in E. coli is biologically active and is a novel strategy to treat bone injury in a clinical setting.