Regulation of Gene Expression by PI3K in Mouse Growth Plate Chondrocytes
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BACKGROUND: Endochondral ossification, the process through which long bones are formed, involves chondrocyte proliferation and hypertrophic differentiation in the cartilage growth plate. In a previous publication we showed that pharmacological inhibition of the PI3K signaling pathway results in reduced endochondral bone growth, and in particular, shortening of the hypertrophic zone in a tibia organ culture system. In this current study we aimed to investigate targets of the PI3K signaling pathway in hypertrophic chondrocytes.
METHODOLOGY/PRINCIPAL FINDINGS: Through the intersection of two different microarray analyses methods (classical single gene analysis and GSEA) and two different chondrocyte differentiation systems (primary chondrocytes treated with a pharmacological inhibitor of PI3K and microdissected growth plates), we were able to identify a high number of genes grouped in GSEA functional categories regulated by the PI3K signaling pathway. Genes such as Phlda2 and F13a1 were down-regulated upon PI3K inhibition and showed increased expression in the hypertrophic zone compared to the proliferative/resting zone of the growth plate. In contrast, other genes including Nr4a1 and Adamts5 were up-regulated upon PI3K inhibition and showed reduced expression in the hypertrophic zone. Regulation of these genes by PI3K signaling was confirmed by quantitative RT-PCR. We focused on F13a1 as an interesting target because of its known role in chondrocyte hypertrophy and osteoarthritis. Mouse E15.5 tibiae cultured with LY294002 (PI3K inhibitor) for 6 days showed decreased expression of factor XIIIa in the hypertrophic zone compared to control cultures.
CONCLUSIONS/SIGNIFICANCE: Discovering targets of signaling pathways in hypertrophic chondrocytes could lead to targeted therapy in osteoarthritis and a better understanding of the cartilage environment for tissue engineering.