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 6days 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 6days 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.