LRP4 induces extracellular matrix productions and facilitates chondrocyte differentiation

Endochondral ossification is an essential step for skeletal development, which requires chondrocyte differentiation in growth cartilage. The low-density lipoprotein receptor-related protein 4 (LRP4), a member of LDLR family, is an inhibitor for Wnt signaling, but its roles in chondrocyte differentiation remain to be investigated. Here we found by laser capture microdissection that LRP4 expression was induced during chondrocyte differentiation in growth plate. In order to address the roles, we overexpressed recombinant human LRP4 or knocked down endogenous LRP4 by lentivirus in mouse ATDC5 chondrocyte cells. We found that LRP4 induced gene expressions of extracellular matrix proteins of type II collagen (Col2a1), aggrecan (Acan), and type X collagen (Col10a1), as well as production of total proteoglycans in ATDC5 cells, whereas LRP4 knockdown had opposite effects. Interestingly, LRP4-knockdown reduced mRNA expression of Sox9, a master regulator for chondrogenesis, as well as Dkk1, an extracellular Wnt inhibitor. Analysis of Wnt signaling revealed that LRP4 blocked the Wnt/β-catenin signaling activity in ATDC5 cells. Finally, the reduction of these extracellular matrix productions by LRP4-knockdown was rescued by a β-catenin/TCF inhibitor, suggesting that LRP4 is an important regulator for extracellular matrix productions and chondrocyte differentiation by suppressing Wnt/β-catenin signaling.     

XBP1S Associates with RUNX2 and Regulates Chondrocyte Hypertrophy

BMP2 (bone morphogenetic protein 2) is known to activate unfolded protein response signaling molecules, including XBP1S and ATF6. However, the influence on XBP1S and ATF6 in BMP2-induced chondrocyte differentiation has not yet been elucidated. In this study, we demonstrate that BMP2 mediates mild endoplasmic reticulum stress-activated ATF6 and directly regulates XBP1S splicing in the course of chondrogenesis. XBP1S is differentially expressed during BMP2-stimulated chondrocyte differentiation and exhibits prominent expression in growth plate chondrocytes. This expression is probably due to the activation of the XBP1 gene by ATF6 and splicing by IRE1a. ATF6 directly binds to the 5′-flanking regulatory region of the XBP1 gene at its consensus binding elements. Overexpression of XBP1S accelerates chondrocyte hypertrophy, as revealed by enhanced expression of type II collagen, type X collagen, and RUNX2; however, knockdown of XBP1S via the RNAi approach abolishes hypertrophic chondrocyte differentiation. In addition, XBP1S associates with RUNX2 and enhances RUNX2-induced chondrocyte hypertrophy. Altered expression of XBP1S in chondrocyte hypertrophy was accompanied by altered levels of IHH (Indian hedgehog) and PTHrP (parathyroid hormone-related peptide). Collectively, XBP1S may be a novel regulator of hypertrophic chondrocyte differentiation by 1) acting as a cofactor of RUNX2 and 2) affecting IHH/PTHrP signaling.     

MSX2 stimulates chondrocyte maturation by controlling Ihh expression

Several studies indicated that a homeobox gene, Msx2, is implicated in regulation of skeletal development by controlling enchondral ossification as well as membranous ossification. However, the molecular basis by which Msx2 conducts chondrogenesis is currently unclear. In this study, we examined the role of Msx2 in chondrocyte differentiation using mouse primary chondrocytes and embryonic metatarsal explants. Treatment with BMP2 up-regulated the expression of Msx2 mRNA along with chondrocyte differentiation in murine primary chondrocytes. Overexpression of wild-type Msx2 stimulated calcification of primary chondrocytes in the presence of BMP2. We also found that constitutively active Msx2 (caMsx2) enhanced BMP2-dependent calcification more efficiently than wild-type Msx2. Consistently, caMsx2 overexpression up-regulated the expression of alkaline phosphatase and collagen type X induced by BMP2. Furthermore, organ culture experiments using mouse embryonic metatarsals indicated that caMsx2 clearly stimulated the maturation of chondrocytes into the prehypertrophic and hypertrophic stages in the presence of BMP2. In contrast, knockdown of Msx2 inhibited maturation of primary chondrocytes. The stimulatory effect of Msx2 on chondrocyte maturation was enhanced by overexpression of Smad1 and Smad4 but inhibited by Smad6, an inhibitory Smad for BMP2 signaling. These data suggest that Msx2 requires BMP2/Smad signaling for its chondrogenic action. In addition, caMsx2 overexpression induced Ihh (Indian hedgehog) expression in mouse primary chondrocytes. Importantly, treatment with cyclopamine, a specific inhibitor for hedgehogs, blocked Msx2-induced chondrogenesis. Collectively, our results indicated that Msx2 promotes the maturation of chondrocytes, at least in part, through up-regulating Ihh expression.     

Dual functions for WNT5A during cartilage development and in disease

Mouse and human genetic data suggests that Wnt5a is required for jaw development but the specific role in facial skeletogenesis is unknown. We mapped expression of WNT5A in the developing chicken skull and found that the highest expression was in early Meckel's cartilage but by stage 35 expression was decreased to background. We focused on chondrogenesis by targeting a retrovirus expressing WNT5A to the mandibular prominence prior to cell differentiation. Unexpectedly, there were no phenotypes in the first 6 days following injection; however later the mandibular bones and Meckel's cartilage were reduced or missing on the treated side. To examine the effects on cartilage differentiation we treated micromass cultures from mandibular mesenchyme with Wnt5a-conditioned media (CM). Similar to in vivo viral data, cartilage differentiates normally, but, after 6 days of culture, nearly all Alcian blue staining is lost. Collagen II and aggrecan were also decreased in treated cultures. The matrix loss was correlated with upregulation of metalloproteinases, MMP1, MMP13, and ADAMTS5 (codes for Aggrecanase). Moreover, Marimastat, an MMP and Aggrecanase inhibitor rescued cartilage matrix in Wnt5a-CM treated cultures. The pathways mediating these cartilage and RNA changes were investigated using luciferase assays. Wnt5a-CM was a potent inhibitor of the canonical pathway and strongly activated JNK/PCP signaling. To determine whether the matrix loss is mediated by repression of canonical signaling or activation of the JNK pathway we treated mandibular cultures with either DKK1, an antagonist of the canonical pathway, or a small molecule that antagonizes JNK signaling (TCS JNK 6o). DKK1 slightly increased cartilage formation and therefore suggested that the endogenous canonical signaling represses chondrogenesis. To test this further we added an excess of Wnt3a-CM and found that far fewer cartilage nodules differentiated. Since DKK1 did not mimic the effects of Wnt5a we excluded the canonical pathway from mediating the matrix loss phenotype. The JNK antagonist partially rescued the Wnt5a phenotype supporting this non-canonical pathway as the main mediator of the cartilage matrix degradation. Our study reveals two new roles for WNT5A in development and disease: 1) to repress canonical Wnt signaling in cartilage blastema in order to promote normal differentiation and 2) in conditions of excess to stimulate degradation of mature cartilage matrix via non-canonical pathways.     

Regulation of chondrocyte differentiation by IRE1α depends on its enzymatic activity

Bone morphogenetic protein 2(BMP2) is known to activate unfolded protein response (UPR) signal molecules in chondrogenesis. Inositol-requiring enzyme-1α (IRE1α),as one of three unfolded protein sensors in UPR signaling pathways, can be activated during ER stress. However, the influence on IRE1α in chondrocyte differentiation has not yet been elucidated. Here we present evidence demonstrating that overexpression of IRE1α inhibits chondrocyte differentiation, as revealed by reduced expression of collagen II (ColII), Sox9, collagen X (ColX), matrix metalloproteinase 13 (MMP-13), Indian hedgehog (IHH), Runx2 and enhanced expression of parathyroid hormone-related peptide (PTHrP). Furthermore, IRE1α-mediated inhibition of chondrogenesis depends on its enzymatic activity, since its point mutant lacking enzymatic activity completely loses this activity. The RNase and Kinase domains of IRE1α C-terminal are necessary for its full enzymatic activity and inhibition of chondrocyte differentiation. Mechanism studies demonstrate that granulin–epithelin precursor(GEP), a growth factor known to stimulate chondrogenesis, induced IRE1α expression in chondrogenesis. The expression of IRE1α is depended on GEP signaling, and IRE1α expression is hardly detectable in GEP^−/− embryos. In addition, IRE1α inhibits GEP-mediated chondrocyte differentiation as a negative regulator. Altered expression of IRE1α in chondrocyte hypertrophy was accompanied by altered levels of IHH and PTHrP. Collectively, IRE1α may be a novel regulator of chondrocyte differentiation by 1) inhibition GEP-mediated chondrocyte differentiation as a negative regulator; 2) promoting IHH/PTHrP signaling.     

The cleavage of N-cadherin is essential for chondrocyte differentiation

The aggregation of chondroprogenitor mesenchymal cells into precartilage condensation represents one of the earliest events in chondrogenesis. N-cadherin is a key cell adhesion molecule implicated in chondrogenic differentiation. Recently, ADAM10-mediated cleavage of N-cadherin has been reported to play an important role in cell adhesion, migration, development and signaling. However, the significance of N-cadherin cleavage in chondrocyte differentiation has not been determined. In the present study, we found that the protein turnover of N-cadherin is accelerated during the early phase of chondrogenic differentiation in ATDC5 cells. Therefore, we generated the subclones of ATDC5 cells overexpressing wild-type N-cadherin, and two types of subclones overexpressing a cleavage-defective N-cadherin mutant, and examined the response of these cells to insulin stimulation. The ATDC5 cells overexpressing cleavage-defective mutants severely prevented the formation of cartilage aggregates, proteoglycan production and the induction of chondrocyte marker gene expression, such as type II collagen, aggrecan and type X collagen. These results suggested that the cleavage of N-cadherin is essential for chondrocyte differentiation.     

Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro

Osteoarthritis is the most prevalent form of arthritis in the world. Certain signaling pathways, such as the wnt pathway, are involved in cartilage pathology. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to chondrocyte de-differentiation. We investigated whether the Wnt pathway is involved in de-differentiation of human articular chondrocytes in vitro. Human articular chondrocytes were cultured for four passages in the presence or absence of IL-1 in monolayer or micromass culture. Changes in cell morphology were monitored by light microscopy. Protein and gene expression of chondrocyte markers and Wnt pathway components were determined by Western blotting and qPCR after culture. After culturing for four passages, chondrocytes exhibited a fibroblast-like morphology. Collagen type II and aggrecan protein and gene expression decreased, while collagen type I, matrix metalloproteinase 13, and nitric oxide synthase expressions increased. Wnt molecule expression profiles changed; Wnt5a protein expression, the Wnt target gene, c-jun, and in Wnt pathway regulator, sFRP4 increased. Treatment with IL-1 caused chondrocyte morphology to become more filament-like. This change in morphology was accompanied by extinction of col II expression and increased col I, MMP13 and eNOS expression. Changes in expression of the Wnt pathway components also were observed. Wnt7a decreased significantly, while Wnt5a, LRP5, β-catenin and c-jun expressions increased. Culture of human articular chondrocytes with or without IL-1 not only induced chondrocyte de-differentiation, but also changed the expression profiles of Wnt components, which suggests that the Wnt pathway is involved in chondrocyte de-differentiation in vitro.     

Association of the Myosin-binding Subunit of Myosin Phosphatase and Moesin: Dual Regulation of Moesin Phosphorylation by Rho-associated Kinase and Myosin Phosphatase

To examine the role of bone morphogenetic protein (BMP) signaling in chondrocytes during endochondral ossification, the dominant negative (DN) forms of BMP receptors were introduced into immature and mature chondrocytes isolated from lower and upper portions of chick embryo sternum, respectively. We found that control sternal chondrocyte populations expressed type IA, IB, and II BMP receptors as well as BMP-4 and -7. Expression of a DN-type II BMP receptor (termed DN-BMPR-II) in immature lower sternal (LS) chondrocytes led to a loss of differentiated functions; compared with control cells, the DN-BMPR- II–expressing LS chondrocytes proliferated more rapidly, acquired a fibroblastic morphology, showed little expression of type II collagen and aggrecan genes, and upregulated type I collagen gene expression. Expression of DN-BMPR-II in mature hypertrophic upper sternal (US) chondrocytes caused similar effects. In addition, the DN-BMPR-II–expressing US cells exhibited little alkaline phosphatase activity and type X collagen gene expression, while the control US cells produced both alkaline phosphatase and type X collagen. Both DN-BMPR-II–expressing US and LS chondrocytes failed to respond to treatment with BMP-2 . When we examined the effects of DN forms of types IA and IB BMP receptors, we found that DN-BMPR-IA had little effect, while DN-BMPR-IB had similar but weaker effects compared with those of DN-BMPR-II. We conclude that BMP signaling, particularly that mediated by the type II BMP receptor, is required for maintenance of the differentiated phenotype, control of cell proliferation, and expression of hypertrophic phenotype.     

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis

Bone marrow mesenchymal stem cells (MSCs) are candidate cells for cartilage tissue engineering. This is due to their ability to undergo chondrogenic differentiation after extensive expansion in vitro and stimulation with various biomaterials in three-dimensional (3-D) systems. Collagen type II is one of the major components of the hyaline cartilage and plays a key role in maintaining chondrocyte function. This study aimed at analyzing the MSC chondrogenic response during culture in different types of extracellular matrix (ECM) with a focus on the influence of collagen type II on MSC chondrogenesis. Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum free medium and medium supplemented with transforming growth factor (TGF) beta1. Chondrogenic differentiation was detected after 3 days of culture in 3-D hydrogels, by examining the presence of glycosaminoglycan and newly synthesized collagen type II in the ECM. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. The expression levels of the of chondrocyte specific genes: sox9, collagen type II, aggrecan, and COMP were measured by quantitative "Real Time" RT-PCR, and genes distribution in the hydrogel beads were localized by in situ hybridization. All genes were upregulated by the presence of collagen, particularly type II, in the ECM. Additionally, the chondrogenic influence of TGF beta1 on MSCs cultured in collagen-incorporated ECM was analyzed. TGF beta1 and dexamethasone treatment in the presence of collagen type II provided more favorable conditions for expression of the chondrogenic phenotype. In this study, we demonstrated that collagen type II alone has the potential to induce and maintain MSC chondrogenesis, and prior interaction with TGF beta1 to enhance the differentiation.     

Identification and characterization of arginase II as a chondrocyte phenotype-specific gene

We have previously shown that activation of extracellular signal-regulated protein kinase-1 and -2 (ERK1/2) causes chondrocyte dedifferentiation, which contributes to the destruction of arthritic cartilage. In the present study, we identified genes involved in the ERK1/2 regulation of chondrocyte dedifferentiation. Several genes were identified by subtractive hybridization, and, of these, arginase II was selected for further functional characterization. Similar to the pattern of type II collagen expression, which is a hallmark of chondrocyte differentiation, arginase II expression was increased during chondrogenesis of mesenchymal cells. The high expression level of arginase II was decreased during dedifferentiation of chondrocytes, whereas its expression was restored during redifferentiation of the dedifferentiated chondrocytes. Inhibition of ERK1/2 signaling in chondrocytes enhanced type II collagen expression with a concomitant increase in expression and activity of arginase II. However, ectopic expression of arginase II or inhibition of its activity did not affect chondrocyte differentiation. The results collectively indicate that expression of arginase II is specific to the chondrocyte phenotype, although the expression of arginase II alone is not sufficient for articular chondrocytes to maintain a differentiated phenotype.