Development of comprehensive functional genomic screens to identify novel mediators of osteoarthritis.

OBJECTIVE: The aim of this study was to develop high-throughput assays for the analysis of major chondrocyte functions that are important in osteoarthritis (OA) pathogenesis and methods for high-level gene expression and analysis in primary human chondrocytes. METHODS: In the first approach, complementary DNA (cDNA) libraries were constructed from OA cartilage RNA and full-length clones were selected. These cDNAs were transferred into a retroviral vector using Gateway Technology. Full-length clones were over-expressed in human articular chondrocytes (HAC) by retroviral-mediated gene transfer. The induction of OA-associated markers, including aggrecanase-1 (Agg-1), matrix metalloproteinase-13 (MMP-13), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), collagen IIA and collagen X was measured by quantitative real-time polymerase chain reaction (QPCR). Induction of a marker gene was verified by independent isolation of 2-3 clones per gene, re-transfection followed by QPCR as well as nucleotide sequencing. In the second approach, whole cDNA libraries were transduced into chondrocytes and screened for chondrocyte cluster formation in three-dimensional agarose cultures. RESULTS: Using green fluorescent protein (eGFP) as a marker gene, it was shown that the retroviral method has a transduction efficiency of >90%. A total of 40 verified hits were identified in the QPCR screen. The first set of 19 hits coordinately induced iNOS, COX-2, Agg-1 and MMP-13. The most potent of these genes were the tyrosine kinases Axl and Tyro-3, receptor interacting kinase-2 (RIPK2), tumor necrosis factor receptor 1A (TNFR1A), fibroblast growth factor (FGF) and its receptor FGFR, MUS81 endonuclease and Sentrin/SUMO-specific protease 3. The second set of seven hits induced both Agg-1 and MMP-13 but none of the other markers. Five of these seven genes regulate the phosphoinositide-3-kinase pathway. The most potently induced OA marker was iNOS. This marker was induced 20-500 fold by seven genes. Collagen IIA was also induced by seven genes, the most potent being transforming growth factor beta (TGFbeta)-stimulated protein TSC22, vascular endothelial growth factor (VEGF) and splicing factor 3a. This screening assay did not identify inducers of collagen X. The second chondrocyte cluster formation screen identified 14 verified hits. Most of the genes inducing cluster formation were kinases. Additional genes had not been previously known to regulate chondrocyte cluster formation or any other chondrocyte function. CONCLUSIONS: The methods developed in this study can be applied to screen for genes capable of inducing an OA-like phenotype in chondrocytes on a genome-wide scale and identify novel mediators of OA pathogenesis. Thus, coordinated functional genomic approaches can be used to delineate key genes and pathways activated in complex human diseases such as OA.

Synthesis of 27-hydroxycholesterol in rat liver mitochondria: HPLC assay and marked activation by exogenous cholesterol.

Sterol 27-hydroxylase, the mitochondrial enzyme that catalyzes the first step in oxidation of the sterol side chain in hepatic bile acid synthesis, also catalyzes the synthesis of 27-hydroxycholesterol from cholesterol. We have developed a high performance liquid chromatography (HPLC) assay for this enzyme, using either endogenous or exogenous cholesterol as substrate and cholesterol oxidase to convert 27-hydroxycholesterol to 4-cholesten-27-hydroxy-3-one. The alpha,beta-unsaturated ketone product was separated by normal phase HPLC and quantitated via absorption at 240 nm. Addition of cholesterol dissolved in 2-hydroxypropyl-beta-cyclodextrin to the assay mixture raised the enzyme activity of rat liver mitochondria more than 10-fold. 2-Hydroxypropyl-beta-cyclodextrin itself was partially effective, apparently by making more endogenous cholesterol accessible to the enzyme. Availability of cholesterol to the enzyme limits synthesis of 27-hydroxycholesterol in rat liver. Using our assay to simultaneously determine the activities of cholesterol 7 alpha-hydroxylase and cholesterol 27-hydroxylase in rat liver homogenates, we demonstrated that the two enzymes are separately regulated.