The Wnt Antagonist Frzb-1 Regulates Chondrocyte Maturation and Long Bone Development during Limb Skeletogenesis

Abstract

The Wnt antagonist Frzb-1 is expressed during limb skeletogenesis, but its roles in this complex multistep process are not fully understood. To address this issue, we determined Frzb-1 gene expression patterns during chick long bone development and carried out gain- and loss-of-function studies by misexpression of Frzb-1, Wnt-8 (a known Frzb-1 target), or different forms of the intracellular Wnt mediator LEF-1 in developing limbs and cultured chondrocytes. Frzb-1 expression was quite strong in mesenchymal prechondrogenic condensations and then characterized epiphyseal articular chondrocytes and prehypertrophic chondrocytes in growth plates. Virally driven Frzb-1 misexpression caused shortening of skeletal elements, joint fusion, and delayed chondrocyte maturation, with consequent inhibition of matrix mineralization, metalloprotease expression, and marrow/bone formation. In good agreement, misexpression of Frzb-1 or a dominant-negative form of LEF-1 in cultured chondrocytes maintained the cells at an immature stage. Instead, misexpression of Wnt-8 or a constitutively active LEF-1 strongly promoted chondrocyte maturation, hypertrophy, and calcification. Immunostaining revealed that the distribution of endogenous Wnt mediator beta-catenin changes dramatically in vivo and in vitro, from largely cytoplasmic in immature proliferating and prehypertrophic chondrocytes to nuclear in hypertrophic mineralizing chondrocytes. Misexpression of Frzb-1 prevented beta-catenin nuclear relocalization in chondrocytes in vivo or in vitro. The data demonstrate that Frzb-1 exerts a strong influence on limb skeletogenesis and is a powerful and direct modulator of chondrocyte maturation, phenotype, and function. Phases of skeletogenesis, such as terminal chondrocyte maturation and joint formation, appear to be particularly dependent on Wnt signaling and thus very sensitive to Frzb-1 antagonistic action.

Functional variants within the secreted frizzled-related protein 3 gene are associated with hip osteoarthritis in females

“…This interaction may be required for the efficient localization and functional activity of sFRP3 in the joints. Inhibition of wnt signaling, mediated by sFRP3 may be required to retain chondrocytes in their immature prehypertrophic state and maintain the integrity of the cartilage-bone junction (26,34). FRZB is expressed by developing and mature chondrocytes (27).…”

“…sFRP3 is expressed by human chondrocytes during skeletal development (26,27), but it had not been analyzed in adults. However, immunoreactive sFRP3 was easily detectable in OA articular cartilage by immunohistochemistry (Fig.…”

Small GTPase protein Rac-1 is activated with maturation and regulates cell morphology and function in chondrocytes

“…The Wnt/β-catenin pathway has previously been shown to be necessary for growth plate development and endochondral ossification. [42] Activation of Wnt signaling in mature chondrocytes induces hypertrophy, matrix mineralization and MMP-7, -9 and -13 production. [31] In our study, treatment of chondrocytes expressing GFP-Rac-1 with Wnt3A, a Wnt/β-catenin signaling inducing factor, stimulates flattened cell morphology and Rac-1 localization to the cell membrane and cell-cell contacts.…”

Sex and ethnic differences in the association of ASPN, CALM1, COL2A1, COMP, and FRZB with genetic susceptibility to osteoarthritis of the knee

“…Wnt/␤-catenin signaling regulates chondrocyte phenotype, maturation, and function (5). Through its influence on Wnt signaling, FRZB is a powerful and direct modulator of chondrocyte maturation (6). Accelerated cartilage breakdown has been shown to develop in knockout mice deficient in this gene (7).…”