Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: II. Stimulation by bone morphogenetic protein-2 requires modulation of N-cadherin expression and function

Bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta (TGF-beta) superfamily, is characterized by its ability to induce cartilage and bone formation. We have recently demonstrated that the multipotential, murine embryonic mesenchymal cell line, C3H10T1/2, when cultured at high density, is induced by BMP-2 or TGF-beta 1 to undergo chondrogenic differentiation. The high-cell-density requirement suggests that specific cell-cell interactions, such as those mediated by cell adhesion molecules, are important in the chondrogenic response. In view of our recent finding that N-cadherin, a Ca(2+)-dependent cell adhesion molecule, is functionally required in normal embryonic limb mesenchyme cellular condensation and chondrogenesis, we examine here whether N-cadherin is also involved in BMP-2 induction of chondrogenesis in C3H10T1/2 cells. BMP-2 stimulation of chondrogenesis in high-density micromass cultures of C3H10T1/2 cells was evidenced by Alcian blue staining, elevated [35S]sulfate incorporation, and expression of the cartilage matrix markers, collagen type II and cartilage proteoglycan link protein. With BMP-2 treatment, N-cadherin mRNA expression was stimulated 4-fold within 24 h, and by day 5, protein levels were stimulated 8-fold. An N-cadherin peptidomimic containing the His-Ala-Val sequence to abrogate homotypic N-cadherin interactions inhibited chondrogenesis in a concentration-dependent manner. To analyze the functional role of N-cadherin further, C3H10T1/2 cells were stably transfected with expression constructs of either full-length N-cadherin or a dominant negative, N-terminal deletion mutant of N-cadherin. Moderate (2-fold) overexpression of full-length N-cadherin augmented, whereas higher (4-fold) overexpression inhibited the BMP-2-chondrogenic effect. On the other hand, expression of the dominant negative N-cadherin mutant dramatically inhibited BMP-2 stimulated chondrogenesis. These data strongly suggest that upregulation of N-cadherin expression, at defined critical levels, is a candidate mechanistic component of BMP-2 stimulation of mesenchymal chondrogenesis.     

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.     

N-Cadherin expression and signaling in limb mesenchymal chondrogenesis: stimulation by poly-L-lysine

Cellular condensation is a requisite step in the initiation of mesenchymal chondrogenesis in the embryonic limb bud. We have previously shown that cellular condensation of limb chondroprogenitor mesenchymal cells is accompanied by elevated expression of N-cadherin during chondrogenesis both in vivo and in vitro. N-Cadherin-mediated cell-cell interaction is also functionally required for proper mesenchymal chondrogenesis both in vivo and in vitro. In this report, we have further analyzed the functional importance of N-cadherin in the cellular condensation-chondrogenesis pathway by examining N-cadherin expression and related activities in high density micromass cultures of chick limb mesenchymal cells in which chondrogenesis is being stimulated with the cationic polymer, poly-L-lysine (PL). The chondrogenesis-promoting action of PL is thought to involve the clustering of cells via ionic cross-linking, perhaps mimicking the action of an endogenous matrix component. Immunohistochemistry, immunoblotting, and Northern blot analysis all show that PL treatment results in a time-dependent increase in N-cadherin expression at both the protein and mRNA levels. In addition, inhibition of N-cadherin function with a neutralizing monoclonal antibody directed to its extracellular domain inhibits the chondrogenesis-stimulating effect of PL. PL treatment also alters the tyrosine-phosphorylation state of the N-cadherin associated signaling protein, beta-catenin. These results suggest that N-cadherin-mediated cell adhesion is a requisite regulatory component of the limb mesenchymal chondrogenic differentiation program, involving at least in part beta-catenin tyrosine phosphorylation as a signaling step.     

Gap junctional communication during limb cartilage differentiation

The onset of cartilage differentiation in the developing limb bud is characterized by a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely apposed to one another prior to initiating cartilage matrix deposition. During this condensation process intimate cell-cell interactions occur which are necessary to trigger chondrogenic differentiation. In the present study, we demonstrate that extensive cell-cell communication via gap junctions as assayed by the intercellular transfer of lucifer yellow dye occurs during condensation and the onset of overt chondrogenesis in high density micromass cultures prepared from the homogeneous population of chondrogenic precursor cells comprising the distal subridge region of stage 25 embryonic chick wing buds. Furthermore, in heterogeneous micromass cultures prepared from the mesodermal cells of whole stage 23/24 limb buds, extensive gap junctional communication is limited to differentiating cartilage cells, while the nonchondrogenic cells of the cultures that are differentiating into the connective tissue lineage exhibit little or no intercellular communication via gap junctions. These results provide a strong incentive for considering and further investigating the possible involvement of cell-cell communication via gap junctions in the regulation of limb cartilage differentiation.     

Transforming growth factor-beta-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogen-activated protein kinase and Wnt signaling cross-talk

The multilineage differentiation potential of adult tissue-derived mesenchymal progenitor cells (MPCs), such as those from bone marrow and trabecular bone, makes them a useful model to investigate mechanisms regulating tissue development and regeneration, such as cartilage. Treatment with transforming growth factor-beta (TGF-beta) superfamily members is a key requirement for the in vitro chondrogenic differentiation of MPCs. Intracellular signaling cascades, particularly those involving the mitogen-activated protein (MAP) kinases, p38, ERK-1, and JNK, have been shown to be activated by TGF-betas in promoting cartilage-specific gene expression. MPC chondrogenesis in vitro also requires high cell seeding density, reminiscent of the cellular condensation requirements for embryonic mesenchymal chondrogenesis, suggesting common chondro-regulatory mechanisms. Prompted by recent findings of the crucial role of the cell adhesion protein, N-cadherin, and Wnt signaling in condensation and chondrogenesis, we have examined here their involvement, as well as MAP kinase signaling, in TGF-beta1-induced chondrogenesis of trabecular bone-derived MPCs. Our results showed that TGF-beta1 treatment initiates and maintains chondrogenesis of MPCs through the differential chondro-stimulatory activities of p38, ERK-1, and to a lesser extent, JNK. This regulation of MPC chondrogenic differentiation by the MAP kinases involves the modulation of N-cadherin expression levels, thereby likely controlling condensation-like cell-cell interaction and progression to chondrogenic differentiation, by the sequential up-regulation and progressive down-regulation of N-cadherin. TGF-beta1-mediated MAP kinase activation also controls WNT-7A gene expression and Wnt-mediated signaling through the intracellular beta-catenin-TCF pathway, which likely regulates N-cadherin expression and subsequent N-cadherin-mediated cell-adhesion complexes during the early steps of MPC chondrogenesis.     

Successive formative stages of precartilaginous mesenchymal condensations in vitro: Modulation of cell adhesion by Wnt-7A and BMP-2

High-density chick limb bud cell culture is a useful model to study mesenchymal condensatifons and chondrogenesis. Most previous studies have focused on the effects of soluble reagents on terminal chondrogenic differentiation and have not defined the early cellular processes and signaling events. In this study, we defined five successive stages in the differentiation process: 1) dissociated cells, 2) small aggregates, 3) formation of cell clusters, 4) precartilaginous condensations, and 5) cartilage nodule. We used RCAS retrovirus-mediated Wnt-7a gene transduction to test the effect of Wnt-7a on the differentiation process. We found that Wnt-7a suppressed chondrogenic differentiation. Wnt-7a did not inhibit the initiation of condensation formation but blocked the progression of precartilaginous condensations to cartilage nodules. The Wnt-7a-transduced cultures showed characteristics of a less mature culture with persistent expression of NCAM, N-cadherin, wider distribution of integrin β1 and fibronectin, and suppression of tenascin-C. BMP-2 is known to enhance chondrogenic differentiation in these cultures by promoting cell clusters to form continuous sheet-like precartilaginous condensations. However, cultures exposed to both BMP-2 and Wnt-7a showed inhibition of chondrogenic differentiation. Different signaling molecules such as Wnt-7a and BMP-2 may have antagonistic effects on cartilage differentiation and the gradient of the two molecules may be involved in defining the boundaries of the initial precartilaginous condensation. We propose that the shape of the precartilaginous condensations may be modulated by local concentrations of signaling molecules, such as Wnt-7a and BMP-2, which act to alter cell-substrate and cell-cell adhesions. J. Cell. Physiol. 180:314–324, 1999. © 1999 Wiley-Liss, Inc.     

Functional role of growth/differentiation factor 5 in chondrogenesis of limb mesenchymal cells

Growth/Differentiation Factor 5 (GDF5) plays an important role in limb mesenchymal cell condensation and chondrogenesis. Here we demonstrate, using high density cultures of chick embryonic limb mesenchyme, that GDF5 misexpression increased condensation of chondroprogenitor cells and enhanced chondrogenic differentiation. These effects were observed in the absence of altered cellular viability or biosynthetic activity, suggesting that GDF5 action might be directed at the level of cellular adhesion or cell-cell communication. GDF5- enhanced condensation occurred independent of cell density or N-cadherin mediated adhesion and signaling, but was inhibited upon interference of gap junction mediated communication. p38 MAP kinase signaling was required for the GDF5 effect on chondrocyte differentiation, but not for mesenchymal condensation. These findings suggest gap junction involvement in the action of GDF5 in developmental chondrogenesis.     

Signaling Cascades Governing Cdc42-Mediated Chondrogenic Differentiation and Mensenchymal Condensation

Endochondral ossification consists of successive steps of chondrocyte differentiation, including mesenchymal condensation, differentiation of chondrocytes, and hypertrophy followed by mineralization and ossification. Loss-of-function studies have revealed that abnormal growth plate cartilage of the Cdc42 mutant contributes to the defects in endochondral bone formation. Here, we have investigated the roles of Cdc42 in osteogenesis and signaling cascades governing Cdc42-mediated chondrogenic differentiation. Though deletion of Cdc42 in limb mesenchymal progenitors led to severe defects in endochondral ossification, either ablation of Cdc42 in limb preosteoblasts or knockdown of Cdc42 in vitro had no obvious effects on bone formation and osteoblast differentiation. However, in Cdc42 mutant limb buds, loss of Cdc42 in mesenchymal progenitors led to marked inactivation of p38 and Smad1/5, and in micromass cultures, Cdc42 lay on the upstream of p38 to activate Smad1/5 in bone morphogenetic protein-2-induced mesenchymal condensation. Finally, Cdc42 also lay on the upstream of protein kinase B to transactivate Sox9 and subsequently induced the expression of chondrocyte differential marker in transforming growth factor-β1-induced chondrogenesis. Taken together, by using biochemical and genetic approaches, we have demonstrated that Cdc42 is involved not in osteogenesis but in chondrogenesis in which the BMP2/Cdc42/Pak/p38/Smad signaling module promotes mesenchymal condensation and the TGF-β/Cdc42/Pak/Akt/Sox9 signaling module facilitates chondrogenic differentiation.     

C-type natriuretic peptide regulation of limb mesenchymal chondrogenesis is accompanied by altered N-cadherin and collagen type X-related functions

AMDM, a form of osteochondrodysplasia, is due to the loss-of-function mutations in NPR-B gene. This study investigated the functional involvement of CNP-3, chick homolog of human CNP, and its receptor NPR-B in chondrogenesis utilizing the micromass culture of the chick limb mesenchymal cells. Results revealed CNP-3 and NPR-B expression in the chick limb bud making stage-specific peak levels first at Hamburger-Hamilton stage 23-24, and second at stage 30-31, corresponding to pre-chondrogenic mesenchymal condensation and initiation of chondrogenic maturation-hypertrophy in vivo, respectively. CNP-3 and NPR-B expression in vitro increased parallel to collagen type X expression, but not to that of collagen type II. Treatment of cultures with CNP significantly increased N-cadherin, and collagen type X expression, glycosaminoglycan synthesis and chondrogenesis. Collagen type II expression was not significantly affected. Thus, results implicated CNP-3/NPR-B signaling in pre-chondrogenic mesenchymal condensation, glycosaminoglycan synthesis and late differentiation of chondrocytes in the process of endochondral ossification.     

Involvement of Frzb-1 in mesenchymal condensation and cartilage differentiation in the chick limb bud

In developing limb bud, mesenchymal cells form cellular aggregates called "mesenchymal condensations". These condensations show the prepattern of skeletal elements of the limb prior to cartilage differentiation. Roles of various signaling molecules in chondrogenesis in the limb bud have been reported. One group of signaling factors includes the Wnt proteins, which have been shown to have an inhibitory effect on chondrogenesis in the limb bud. Therefore, regulation of Wnt activity may be important in regulating cartilage differentiation. Here we show that Frzb-1, which encodes a secreted frizzled-related protein that can bind to Wnt proteins and can antagonize the activity of some Wnts, is expressed in the developing limb bud. At early stages of limb development, Frzb-1 is expressed in the ventral core mesenchyme of the limb bud, and later Frzb-1 expression becomes restricted to the central core region where mesenchymal condensations occur. At these stages, a chondrogenic marker gene, aggrecan, is not yet expressed. As limb development proceeds, expression of Frzb-1 is detected in cartilage primordial cells, although ultimately Frzb-1 expression is down-regulated. Similar results were obtained in the recombinant limb bud, which was constructed from dissociated and re-aggregated mesenchymal cells and an ectodermal jacket with the apical ectodermal ridge. In addition, Frzb-1 expression preceded aggrecan expression in micromass cultures. These results suggest that Frzb-1 has a role in condensation formation and cartilage differentiation by regulating Wnt activity in the limb bud.     

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.     

Retinoic acid inhibits chondrogenesis of mesenchymal cells by sustaining expression of N-cadherin and its associated proteins

Retinoic acid (RA) is a well-known regulator of chondrocyte phenotype. RA inhibits chondrogenic differentiation of mesenchymal cells and also causes loss of differentiated chondrocyte phenotype. The present study investigated the mechanisms underlying RA regulation of chondrogenesis. RA treatment in chondrifying mesenchymal cells did not affect precartilage condensation, but blocked progression from precartilage condensation to cartilage nodule formation. This inhibitory effect of RA was independent of protein kinase C and extracellular signal-regulated protein kinase, which are positive and negative regulators of cartilage nodule formation, respectively. The progression from precartilage condensation to cartilage nodule requires downregulation of N-cadherin expression. However, RA treatment caused sustained expression of N-cadherin and its associated proteins including alpha- and beta-catenin suggesting that modulation of expression of these molecules is associated with RA-induced inhibition of chondrogenesis. This hypothesis was supported by the observation that disruption of the actin cytoskeleton by cytochalasin D (CD) blocks RA-induced sustained expression of cell adhesion molecules and overcomes RA-induced inhibition of chondrogenesis. Taken together, our results suggest RA inhibits chondrogenesis by stabilizing cell-to-cell interactions at the post-precartilage condensation stage.     

The effect of prostaglandins on in vitro limb cartilage differentiation

A variety of studies indicate that a key event in limb chondrogenic differentiation is a cellular condensation process during which an intimate cell-cell interaction occurs that triggers cartilage differentiation by elevating cAMP levels. It has recently been demonstrated that when limb mesenchymal cells are subjected to high density monolayer culture under conditions conducive to chondrogenesis, they synthesize several prostaglandins, including PGE2 and prostacyclin, which are important local modulators of cAMP formation in a number of cells and tissues. In the present study, we demonstrate that exogenous PGE2 stimulates the in vitro chondrogenic differentiation of the subridge mesoderm of the embryonic chick limb bud. The stimulatory effect of PGE2 is greatly potentiated by the phosphodiesterase inhibitor, theophylline, suggesting its influence on chondrogenesis is mediated by its ability to increase cAMP levels. The stimulatory effect of PGE2 is dose-dependent and can be detected at a concentration as low as 10(-8)M. PGE1 is just as effective as PGE2 in stimulating in vitro chondrogenesis, whereas PGA1 and PGF1 alpha are less than half as effective. Thromboxane B2 has no effect on chondrogenesis. On the basis of our results, the possibility that endogenous prostaglandins might regulate limb cartilage differentiation by acting as local regulators of cAMP content is discussed.     

Effects of transforming growth factor-beta signaling on chondrogenesis in mouse chondrogenic EC cells, ATDC5

Cellular condensation of chondroprogenitors is a distinct cellular event in chondrogenesis. During this process, N-cadherin mediates cell-cell interactions responsible for the initial stage of cellular condensation and subsequently fibronectin contributes to cell-matrix interactions mediating a progression of chondrogenesis. We previously showed that chondrogenesis in mouse chondrogenic EC cells, ATDC5, was induced, at a high incidence in the presence of insulin, through formation of cellular condensation. In this study, we took advantage of the sequential progression of chondrogenesis in ATDC5 cells and evaluated, in vitro in these cells, the role of endogenous transforming growth factor (TGF)-beta in chondrogenesis. ATDC5 cells expressed TGF-beta2 mRNA at a cellular condensation stage. The treatment of undifferentiated ATDC5 cells with anti-TGF-beta32 neutralizing antibody inhibited the accumulation of Alcian blue stainable proteoglycan in a dose-dependent manner. Transfection of a dominant-negative mutant of mouse TGF-beta type II receptor to undifferentiated ATDC5 cells completely inhibited cellular condensation. Moreover, exogenously administered TGF-beta2 upregulated the expression of fibronectin and type II collagen (a phenotypic marker gene of chondrogenesis) mRNAs and downregulated that of N-cadherin mRNA in time- and dose-dependent manners. These results indicate that TGF-beta stimulates chondrogenesis via initiation of cellular condensation by transition from an initial N-cadherin-contributing stage to a fibronectin-contributing stage during processes of chondrogenesis in ATDC5 cells.     

MicroRNA-34a regulates migration of chondroblast and IL-1β-induced degeneration of chondrocytes by targeting EphA5

MicroRNAs function as an endogenous mode of fine gene regulation and have been implicated in multiple differentiation and developmental processes. In the present study, we investigated the role of miRNA-34 during chondrogenic differentiation of chick limb mesenchymal cells. We found that the expression of miR-34a increased upon chondrogenic inhibition. Blockade of miR-34a via PNA-based antisense oligonucleotides (ASOs) recovered the chondro-inhibitory actions of JNK inhibitor on migration of chondrogenic progenitors and the formation of precartilage condensation. Furthermore, we determined that EphA5 is a relevant target of miR-34a during chondrogenesis. MiR-34a was necessary and sufficient to down-regulate EphA5 expression, and up-modulation of EphA5 is sufficient to overcome inhibitory actions of miR-34 inhibition on cell migration and condensation of chick limb mesenchymal cells on collagen substrate. Taken together, our data suggest that miR-34a is a negative modulator of chondrogenesis, particularly in migration of chondroblasts, by targeting EphA5 and resulting inhibition of cellular condensation during chondrogenesis of chick limb mesenchymal cells.     

Bone morphogenetic protein-2 modulation of chondrogenic differentiation in vitro involves gap junction-mediated intercellular communication

Undifferentiated mesenchymal cells in the limb bud integrate a complex array of local and systemic signals during the process of cell condensation and chondrogenic differentiation. To address the relationship between bone morphogenetic protein (BMP) signaling and gap junction-mediated intercellular communication, we examined the effects of BMP-2 and a gap junction blocker 18 alpha glycyrrhetinic acid (18alpha-GCA) on mesenchymal cell condensation and chondrogenic differentiation in an in vitro chondrogenic model. We find that connexin43 protein expression significantly correlates with early mesenchymal cellular condensation and chondrogenesis in high-density limb bud cell culture. The level of connexin43 mRNA is maximally upregulated 48 h after treatment with recombinant human BMP-2 with corresponding changes in protein expression. Inhibition of gap junction-mediated intercellular communication with 2.5 microM 18alpha-GCA decreases chondrogenic differentiation by 50% at 96 h without effects on housekeeping genes. Exposure to 18alpha-GCA for only the first 24-48 h after plating does not affect condensation or later chondrogenic differentiation suggesting that gap junction-mediated intercellular communication is not critical for the initial phase of condensation but is important for the onset of differentiation. 18alpha-GCA can also block the chondrogenic effects of BMP-2 without effects on cell number or connexin43 expression. These observations demonstrate 18alpha-GCA-sensitive regulation of intercellular communication in limb mesenchymal cells undergoing chondrogenic differentiation and suggest that BMP-2 induced chondrogenic differentiation may be mediated in part through the modulation of connexin43 expression and gap junction-mediated intercellular communication.