Gene expression in articular cartilage - subchondral bone of FRZB knockout mice

Objective : To study molecular changes in the articular cartilage and subchondral bone of the tibial plateau from mice deficient in frizzled related protein (Frzb) compared to wild-type mice by transcriptome analysis. Methods : Gene-expression analysis of the articular cartilage and subchondral bone of 3 wild-type and 3 Frzb-/- mice was performed by microarray. Pathway analysis of differentially expressed genes between 3 wild-type and 2 Frzb-/- samples was explored with PANTHER, DAVID and GSEA bioinformatics tools. Activation of the WNT pathway was analyzed using western blot. The effects of Frzb gain and loss of function on chondrogenesis and cell proliferation was examined using ATDC5 micromasses and mouse ribcage chondrocytes. Results : Extracellular matrix-associated integrin and cadherin pathways, as well as WNT pathway genes were upregulated in Frzb-/- samples. Several WNT receptors, target genes, and other antagonists were upregulated, but no difference in active β-catenin was found. Analysis of ATDC5 cell micromasses overexpressing FRZB indicated an upregulation of aggrecan and Col2a1, and downregulation of molecules related to damage and repair in cartilage, Col3a1 and Col5a1. Silencing of Frzb resulted in downregulation of aggrecan and Col2a1. Pathways associated with cell cycle were downregulated. Ribcage chondrocytes derived from Frzb-/- mice showed decreased proliferation compared to wild-type cells. Conclusions : Our analysis provides evidence for tight regulation of WNT signaling, shifts in extracellular matrix components and effects on cell proliferation and differentiation in the articular cartilage - subchondral bone unit in Frzb-/- mice. These data further support an important role for FRZB in joint homeostasis and highlight the complex biology of WNT signaling in the joint. Gene-expression analysis of the articular cartilage and subchondral bone of 3 wild-type and 3 Frzb-/- mice was performed by microarray. Pathway analysis of differentially expressed genes between 3 wild-type and 2 Frzb-/- samples was explored with PANTHER, DAVID and GSEA bioinformatics tools.研究目的：通过转录组分析，对比野生型小鼠与卷曲相关蛋白（frizzled related protein, Frzb）缺陷小鼠的胫骨平台关节软骨及软骨下骨的分子变化。 研究方法：采用基因芯片技术对3只野生型小鼠及3只Frzb基因敲除（Frzb-/-）小鼠的胫骨平台关节软骨与软骨下骨进行基因表达分析。使用PANTHER、DAVID及GSEA等生物信息学工具，对3份野生型样本与2份Frzb-/-样本之间的差异表达基因进行通路富集分析。采用蛋白质免疫印迹法（Western blot）分析Wnt信号通路的激活情况。通过ATDC5细胞微团培养及小鼠胸廓软骨细胞实验，探究Frzb功能过表达与功能缺失对软骨形成及细胞增殖的影响。 研究结果：Frzb-/-样本中，与细胞外基质相关的整合素、钙粘蛋白通路及Wnt信号通路相关基因均呈现上调表达。多个Wnt受体、靶基因及其他拮抗剂的表达水平升高，但活化型β-连环蛋白未出现显著差异。对过表达FRZB的ATDC5细胞微团进行分析发现，聚集蛋白聚糖与Col2a1的表达上调，而软骨损伤修复相关分子Col3a1及Col5a1的表达下调。Frzb基因沉默可导致聚集蛋白聚糖与Col2a1的表达下调。细胞周期相关通路呈现下调表达。与野生型小鼠来源的软骨细胞相比，Frzb-/-小鼠胸廓软骨细胞的增殖能力下降。 研究结论：本研究通过分析证实，Frzb-/-小鼠的关节软骨-软骨下骨单元中存在Wnt信号通路的精密调控、细胞外基质成分的改变，以及对细胞增殖与分化的影响。上述数据进一步支持FRZB在关节稳态维持中的重要作用，并揭示了关节内Wnt信号通路的复杂生物学机制。采用基因芯片技术对3只野生型小鼠及3只Frzb-/-小鼠的胫骨平台关节软骨与软骨下骨进行基因表达分析。使用PANTHER、DAVID及GSEA等生物信息学工具，对3份野生型样本与2份Frzb-/-样本之间的差异表达基因进行通路富集分析。