CIHR Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada.
PLoS One. 2010 Jan 25;5(1):e8866. doi: 10.1371/journal.pone.0008866.
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.
软骨内骨化是长骨形成的过程,涉及软骨细胞在软骨生长板中的增殖和肥大分化。在之前的一篇出版物中,我们表明,PI3K 信号通路的药理学抑制会导致软骨内骨生长减少,特别是在胫骨器官培养系统中,肥大区缩短。在本研究中,我们旨在研究 PI3K 信号通路在肥大软骨细胞中的靶点。
方法/主要发现: 通过两种不同的微阵列分析方法(经典的单基因分析和 GSEA)和两种不同的软骨细胞分化系统(用 PI3K 药理学抑制剂处理的原代软骨细胞和微解剖的生长板)的交叉,我们能够识别出大量的基因,这些基因被归类为 GSEA 功能类别,受 PI3K 信号通路调控。PI3K 抑制后,如 Phlda2 和 F13a1 等基因下调,在生长板的增殖/静止区与肥大区相比,表达增加。相比之下,其他基因,如 Nr4a1 和 Adamts5,在 PI3K 抑制后上调,在肥大区表达减少。这些基因受 PI3K 信号的调控通过定量 RT-PCR 得到了证实。我们之所以关注 F13a1,是因为它在软骨细胞肥大和骨关节炎中具有已知的作用。用 LY294002(PI3K 抑制剂)培养 E15.5 天的小鼠胫骨 6 天后,与对照培养物相比,肥大区的因子 XIIIa 表达减少。
结论/意义: 发现肥大软骨细胞中信号通路的靶点可能导致骨关节炎的靶向治疗,并更好地理解软骨环境,以用于组织工程。