Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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AP-1 family members act at DNA targets in conjunction with Sox9 to promote chondrocyte hypertrophy

ABSTRACT: A comprehensive analysis of Sox9 binding profiles in developing chondrocytes identified marked enrichment of an AP-1-like motif (Ohba et al. 2015). Here, we have explored the functional interplay between Sox9 and AP-1 in mammalian chondrocyte development. Among AP-1 family members, Jun and Fosl2 were highly expressed within prehypertrophic and early hypertrophic chondrocytes. Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) showed a striking overlap in Jun- and Sox9-bound regions throughout the chondrocyte genome, a reflection of direct binding of each factor to target motifs in shared enhancers, and physical interactions of AP-1 with Sox9. In vitro expression analysis indicates that direct co-binding of Sox9 and AP-1 at target motifs enhanced target gene expression, while protein-protein interactions suppressed AP-1- and Sox9-driven transcription. Analysis of prehypertrophic chondrocyte removal of Sox9 demonstrated Sox9 was essential for hypertrophic chondrocyte development, while in vitro and ex vivo analyses showed AP-1 promotes chondrocyte hypertrophy. Sox9 and Jun co-bound and co-activated a Col10a1 enhancer in Sox9 and AP-1 motif-dependent manners consistent with their combined action promoting hypertrophic gene expression. Together, the data support a model where AP-1-family members promote Sox9-action in the transition of chondrocytes to a terminal hypertrophic program. Intersection of ChIP-seq data from Sox9 and AP-1 factor Jun, RNA-seq data from developing rib chondrocytes and Col10a1mCherry positive hypertrophic chondrocytes in neonatal mice to uncover regulation of Sox9 by AP-1 factors during chondrocyte hypertrophy.

ORGANISM(S): Mus musculus

SUBMITTER: Xinjun He

PROVIDER: E-GEOD-73372 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA. Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score >3, AhlbÃ¤ck Score >2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using quantitative RT-PCR. Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (COL2A1, COMP, aggrecan, CRTL1, SOX9) and genes involved in matrix synthesis (biglycan, COL9A2, COL11A1, TIMP4, CILP2) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (COL10A1, RUNX2, periostin, ALP, PTHR1, MMP13, COL1A1, COL3A1) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, were differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated. Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The risk of differentiation into hypertrophic cartilage does not seem to be increased for OA chondrocytes. Our findings suggest that the chondrogenic capacity is not significantly affected by OA and OA chondrocytes fulfill the requirements for matrix-associated ACT. Experiment Overall Design: Gene expression profiles of monolayer cultures (ML; passage 2) and Hyaff-11 scaffold cultures (3D; 14 days in vitro) of chondrocytes from 3 normal donors (ND; underwent ACT treatment) and 3 donors suffering from Osteoarthritis (OA; underwent knee replacement surgery) were determined. Comparative analyses between 3D and ML cultures (3D vs. ML) were performed to assess differentiation capacity of ND and OA chondrocytes. Furthermore, OA-related differences were determined comparing OA and ND monolayers as well as scaffold cultures (each OA vs. ND).

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AP-1 family members act at DNA targets in conjunction with Sox9 to promote chondrocyte hypertrophy

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