Wnt regulation of limb mesenchymal chondrogenesis is accompanied by altered N‐cadherin‐related functions

Abstract

It has been suggested that Wnt proteins play essential roles in the regulation of limb development and morphogenesis. We have analyzed the effects of RCAS retroviral-mediated misexpression of Wnt-5a and Wnt-7a, two Wnt genes expressed during normal limb development, on chondrogenesis in high-density micromass cultures of chick limb bud mesenchymal cells. Wnt7a misexpression inhibited chondrogenesis in vitro, whereas Wnt-5a did not. Analysis of cellular condensation and precartilage aggregate formation revealed that the inhibitory effect of Wnt-7a is unlikely to act at the cellular condensation step but, instead, influences the actual chondrogenic differentiation process. During early condensation, N-cadherin expression within the cellular aggregates in Wnt-7a cultures was slightly delayed, whereas Wnt-5a cultures appeared similar to control cultures. With the initiation of differentiation, Wnt-5a and control cultures showed downregulation of N-cadherin and up-regulation of collagen type II expression, whereas Wnt-7a cultures showed persistent, high levels of N-cadherin expression both at the mRNA and protein levels, up to 72 h, and nearly no collagen type II expression. Wnt-7a misexpression was also accompanied by an initial increase in the serine/threonine phosphorylation of β-catenin and Ncadherin, events known to affect N-cadherin turnover and stabilization of β-catenin/N-cadherin cell adhesion complex, during chondrogenic differentiation. Wnt regulation of limb mesenchymal chondrogenesis is thus likely to involve modulation of N-cadherin expression and function. In addition, Wnt-7a misexpression stimulates the proliferation of limb mesenchymal cells in vitro, possibly contributing to its chondro-inhibitory effect. Finally, the differences between Wnt-5a and Wnt-7a effects suggest functional diversity within the Wnt family.

Regulation of the chondrocyte phenotype by beta-catenin

“…Because the transforming group of Wnt family, such as Wnt1 and Wnt7a, but not nontransforming Wnts exerts their effects by accumulating cytosolic β-catenin (Shimizu et al, 1997), our current observation that accumulation of β-catenin inhibits chondrogenesis is in agreement with the inhibition of chondrogenesis by the transforming Wnts (Rudnicki and Brown, 1997;Stott et al, 1999;Tufan and Tuan, 2001). Although no direct evidence for the role of β-catenin in chondrogenesis is yet available, Hartmann and Tabin (Hartmann and Tabin, 2000) reported that misexpression of β-catenin in developing chicken wing buds accelerates chondrocyte maturation.…”

“…The expression of N-cadherin is downregulated in the later stage of chondrogenesis, which appears to be required for the progression of precartilage condensation to cartilage nodules (Oberlender and Tuan, 1994;Chang et al, 1998;Oh et al, 2000;Yoon et al, 2000). The involvement of β-catenin in the regulation of chondrogenesis has been suggested from the observation that ectopic expression of members of Wnt genes such as Wnt1, Wnt7a and Wnt14 (Rudnicki and Brown, 1997;Hartmann and Tabin, 2001;Tufan and Tuan, 2001;Tufan et al, 2002a) or frizzled receptor for Wnt (Tufan et al, 2002b) Expression of N-cadherin and α-catenin was determined by western blotting (top). Chondrocytes were untreated (Con) or treated with 1 µM RA or 5 ng/ml IL1β for 72 hours.…”

Wnt-3A Enhances Bone Morphogenetic Protein-2-mediated Chondrogenesis of Murine C3H10T1/2 Mesenchymal Cells

“…This finding also suggests that GSK-3␤ inhibition is more important during early chondrogenesis, supporting our results that introduction of both wild-type and ⌬GSK-3␤ affects younger rather than older cultures during BMP-2-mediated chondrogenesis. Wnt-3A overexpression in C3H10T1/2 micromass cultures also regulates the level of Ncadherin protein, supporting the postulate that Wnts act, at least in part, via regulating cadherin levels (27,37,54). Previously we have observed that overexpression of both wild-type and mutant N-cadherin disrupts BMP-2-induced chondrogenesis (5), indicating that tight regulation of Ncadherin is required for the correct series of event to move mesenchymal cells through condensation to differentiation.…”

“…We have observed that BMP-2-induced chondrogenesis in C3H10T1/2 cells is accompanied by down-regulation of Wnt-7A and up-regulation of Wnt-3A, whereas the level of other Wnts, including Wnt-3 and Wnt-5A, remains unchanged (22). This observation raises the intriguing possibility that BMP-2-mediated mesenchymal chondrogenesis results from the regulated, antagonistic relationship between the chondro-inhibitory Wnts, such as Wnt-7A (26,27), and chondro-enhancing Wnts. Such a scenario in the developing vertebrate limb would thus bear some analogy to the interactive relationship between the actions of decapentaplegic (a BMP homolog) and Wingless (a Wnt homolog), which synergistically regulate a number of developmental and patterning events in Drosophila (39,40,44).…”

N-cadherin in the spotlight of cell-cell adhesion, differentiation, embryogenesis, invasion and signalling

“…Here, N-cadherin has a role in the cellular condensation (Tuan, 2003), being a direct target of SOX9, a transcription factor that is essential for chondrocyte differentiation and cartilage formation (Panda et al,2001). Misexpression of wnt7a (wingless/int, a chondro-inhibitor in vitro) in mesenchymal chondrogenic cultures directly led to prolonged expression of N-cadherin, stabilisation of N-cadherin mediated cell-cell adhesion and eventual inhibition of chondrogenesis (Tufan and Tuan, 2001;Tufan et al, 2002). Ncadherin mRNA levels increase during osteogenic and myogenic differentiation and decrease during adipogenic differentiation.…”