Abstract

Mutations of Filamin genes, which encode actin-binding proteins, cause a wide range of congenital developmental malformations in humans, mainly skeletal abnormalities. However, the molecular mechanisms underlying Filamin functions in skeletal system formation remain elusive. In our screen to identify skeletal development molecules, we found that Cfm (Fam101) genes, Cfm1 (Fam101b) and Cfm2 (Fam101a), are predominantly co-expressed in developing cartilage and intervertebral discs (IVDs). To investigate the functional role of Cfm genes in skeletal development, we generated single knockout mice for Cfm1 and Cfm2, as well as Cfm1/Cfm2 double-knockout (Cfm DKO) mice, by targeted gene disruption. Mice with loss of a single Cfm gene displayed no overt phenotype, whereas Cfm DKO mice showed skeletal malformations including spinal curvatures, vertebral fusions and impairment of bone growth, showing that the phenotypes of Cfm DKO mice resemble those of Filamin B (Flnb)-deficient mice. The number of cartilaginous cells in IVDs is remarkably reduced, and chondrocytes are moderately reduced in Cfm DKO mice. We observed increased apoptosis and decreased proliferation in Cfm DKO cartilaginous cells. In addition to direct interaction between Cfm and Filamin proteins in developing chondrocytes, we showed that Cfm is required for the interaction between Flnb and Smad3, which was reported to regulate Runx2 expression. Furthermore, we found that Cfm DKO primary chondrocytes showed decreased cellular size and fewer actin bundles compared with those of wild-type chondrocytes. These results suggest that Cfms are essential partner molecules of Flnb in regulating differentiation and proliferation of chondryocytes and actin dynamics.

Highlights

  • Chondrocytes provide an essential function in the skeletal system by producing and maintaining the cartilaginous matrix

  • In our screen to identify skeletal development molecules, we found that Cfm (Fam101) genes, Cfm1 (Fam101b) and Cfm2 (Fam101a), are predominantly coexpressed in developing cartilage and intervertebral discs (IVDs)

  • In the course of our microarray screening for genes upregulated upon chondrocyte differentiation [22], we found that mouse Cfm2 transcripts markedly increased in ATDC5 cells upon differentiation

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Summary

Introduction

Chondrocytes provide an essential function in the skeletal system by producing and maintaining the cartilaginous matrix. Chondrocytes play critical roles at several stages of endochondral ossification in skeletal development. Chondrocyte development is regulated by multiple cell-extrinsic and cellintrinsic factors, including growth factors and transcription factors [1,2]. Various studies have shown the importance of actin dynamics for chondrocyte differentiation [3]. 2954 Human Molecular Genetics, 2014, Vol 23, No 11. Inhibitors of actin polymerization stimulate chondrocyte differentiation in cultured mesenchymal cells and murine embryonic stem cells [4,5]. Abnormalities of the actin cytoskeletal system lead to various human chondrodysplasias. Actin cytoskeleton organization, arranged and rearranged by the assembly and disassembly of actins, is regulated by a large number of actin-binding proteins

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