Expression profiling of mouse femoral head cultures stimulated with interleukin-1alpha or retinoic acid

Purpose: Cartilage explant cultures are a valuable experimental system for the analysis of the molecular mechanisms cartilage degradation. Catabolic stimulation of explants with the pro-inflammatory cytokine interleukin-1α (IL-1), or with retinoic acid (RetA), leads to degradation of aggrecan and collagen II. To further explore the mechanisms of cartilage degradation in this system, we used whole genome expression profiling to characterize RetA and IL-1 induced changes in gene expression. Methods: Femoral head cartilage of 24-day old mice were cultured in serum-free DMEM for 2 and 4 days with either no treatment or with addition of 10 μM RetA or 10 ng/ml IL-1. Total RNA was extracted and cRNA generated by linear amplification (MessageAmp, Ambion), labeled with Cy3/Cy5 and hybridized to 44k whole genome microarrays (Agilent). Data was validated by quantitative PCR of selected genes. Results: Toluidine blue staining of the explants indicated aggrecan loss in RetA treated explants by 2 days compared with control cartilage, and after 4 days when treated with IL-1. Differential gene expression of gene groups involved in cartilage catabolism, ECM formation or development was analyzed. We found that adamts-4 and -5 were upregulated in both IL-1 and RetA treated cultures, while adamts-9 and -15 were preferentially upregulated by RetA. A number of mmps, including mmp10, were upregulated by both agents, whereas others such as mmp3 were upregulated by IL-1 and downregulated by RetA. Timp-3 was strongly downregulated by both IL-1 and RetA, and timp-1 was upregulated by IL-1, but down-regulated by RetA. Several members of the cathepsin family were also differentially regulated. Cartilage ECM molecules regulated by both IL-1 and RetA, such as collagens II, IX and XI, aggrecan, fibromodulin, link protein and matrilins 1 and 3 were generally most strongly downreguled by RetA, particularly at day-4. Other SLRPs such as decorin, biglycan and prelp were not significantly downregulated. In addition, differential expression of several sox transcription factors, including sox9, and several bmp family members was apparent. Conclusions: The application of microarray expression profiling to mouse explant cultures stimulated with IL-1 or RetA in vitro has revealed the differential expression of many genes. Upregulation of catabolic genes such as several adamts and mmp and cathepsins is new, but unsurprising information, although the differential upregulation by RetA alone, in the case of adamsts-9 and adamts-15 is a novel finding that may provide mechanistic insights. It is important to recognize that gene expression is only part of the equation, and the application of proteomic approaches will be critical in dissecting the molecular pathways of cartilage degradation. However, our data has revealed patterns of gene expression of important candidate gene families, and of novel genes, that will be important targets of further detailed analysis