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Sox9 directs hypertrophic maturation and blocks osteoblast differentiation of growth plate chondrocytes.

ABSTRACT: The transcription factor Sox9 is necessary for early chondrogenesis, but its subsequent roles in the cartilage growth plate, a highly specialized structure that drives skeletal growth and endochondral ossification, remain unclear. Using a doxycycline-inducible Cre transgene and Sox9 conditional null alleles in the mouse, we show that Sox9 is required to maintain chondrocyte columnar proliferation and generate cell hypertrophy, two key features of functional growth plates. Sox9 keeps Runx2 expression and ?-catenin signaling in check and thereby inhibits not only progression from proliferation to prehypertrophy, but also subsequent acquisition of an osteoblastic phenotype. Sox9 protein outlives Sox9 RNA in upper hypertrophic chondrocytes, where it contributes with Mef2c to directly activate the major marker of these cells, Col10a1. These findings thus reveal that Sox9 remains a central determinant of the lineage fate and multistep differentiation program of growth plate chondrocytes and thereby illuminate our understanding of key molecular mechanisms underlying skeletogenesis.

SUBMITTER: Dy P

PROVIDER: S-EPMC3306603 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Sox9 directs hypertrophic maturation and blocks osteoblast differentiation of growth plate chondrocytes.

Dy Peter P Wang Weihuan W Bhattaram Pallavi P Wang Qiuqing Q Wang Lai L Ballock R Tracy RT Lefebvre Véronique V

Developmental cell 20120301 3

The transcription factor Sox9 is necessary for early chondrogenesis, but its subsequent roles in the cartilage growth plate, a highly specialized structure that drives skeletal growth and endochondral ossification, remain unclear. Using a doxycycline-inducible Cre transgene and Sox9 conditional null alleles in the mouse, we show that Sox9 is required to maintain chondrocyte columnar proliferation and generate cell hypertrophy, two key features of functional growth plates. Sox9 keeps Runx2 expres ...[more]

PMID: 22421045

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Project description:Cartilage and endochondral bone development require SOX9 activity to regulate chondrogenesis, chondrocyte proliferation, and transition to a non-mitotic hypertrophic state. The restricted and reciprocal expression of the collagen X gene, Col10a1, in hypertrophic chondrocytes and Sox9 in immature chondrocytes epitomise the precise spatiotemporal control of gene expression as chondrocytes progress through phases of differentiation, but how this is achieved is not clear. Here, we have identified a regulatory element upstream of Col10a1 that enhances its expression in hypertrophic chondrocytes in vivo. In immature chondrocytes, where Col10a1 is not expressed, SOX9 interacts with a conserved sequence within this element that is analogous to that within the intronic enhancer of the collagen II gene Col2a1, the known transactivation target of SOX9. By analysing a series of Col10a1 reporter genes in transgenic mice, we show that the SOX9 binding consensus in this element is required to repress expression of the transgene in non-hypertrophic chondrocytes. Forced ectopic Sox9 expression in hypertrophic chondrocytes in vitro and in mice resulted in down-regulation of Col10a1. Mutation of a binding consensus motif for GLI transcription factors, which are the effectors of Indian hedgehog signaling, close to the SOX9 site in the Col10a1 regulatory element, also derepressed transgene expression in non-hypertrophic chondrocytes. GLI2 and GLI3 bound to the Col10a1 regulatory element but not to the enhancer of Col2a1. In addition to Col10a1, paired SOX9-GLI binding motifs are present in the conserved non-coding regions of several genes that are preferentially expressed in hypertrophic chondrocytes and the occurrence of pairing is unlikely to be by chance. We propose a regulatory paradigm whereby direct concomitant positive and negative transcriptional control by SOX9 ensures differentiation phase-specific gene expression in chondrocytes. Discrimination between these opposing modes of transcriptional control by SOX9 may be mediated by cooperation with different partners such as GLI factors.

Project description:Growth plate chondrocytes undergo sequential differentiation to form the resting zone, the proliferative zone (PZ), and the hypertrophic zone (HZ). The important role of microRNAs (miRNAs) in the growth plate was previously revealed by cartilage-specific ablation of Dicer, an enzyme essential for biogenesis of many miRNAs. To identify specific miRNAs that regulate differentiation of PZ chondrocytes to HZ chondrocytes, we microdissected individual growth plate zones from juvenile rats and performed miRNA profiling using a solution hybridization method and miRNA sequencing. Thirty-four miRNAs were differentially expressed between the PZ and the HZ, and we hypothesized that some of the miRNAs that are preferentially expressed in the PZ may promote proliferation and inhibit hypertrophic differentiation. Consistent with this hypothesis, transfection of inhibitors for four of these miRNAs (mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p) decreased proliferation in primary epiphyseal chondrocytes. The inhibitors for three of these miRNAs (mir-374-5p, mir-379-5p, and mir-503-5p) also increased expression of multiple genes that are associated with chondrocyte hypertrophic differentiation. We next hypothesized that preferential expression of these miRNAs in the PZ is driven by the parathyroid hormone-related protein (PTHrP) concentration gradient across the growth plate. Consistent with this hypothesis, treatment of primary chondrocytes with a parathyroid hormone (PTH)/PTHrP receptor agonist, PTH1-34, increased expression of mir-374-5p, mir-379-5p, and mir-503-5p. Taken together, our findings suggest that the PTHrP concentration gradient across the growth plate induces differential expression of mir-374-5p, mir-379-5p, and mir-503-5p between the PZ and the HZ. In the PZ, the higher expression levels of these miRNAs promote proliferation and inhibit hypertrophic differentiation. In the HZ, downregulation of these miRNAs inhibits proliferation and promotes hypertrophic differentiation.

Dataset Information

Sox9 directs hypertrophic maturation and blocks osteoblast differentiation of growth plate chondrocytes.

Publications

Sox9 directs hypertrophic maturation and blocks osteoblast differentiation of growth plate chondrocytes.

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