Abstract
The objectives of this study were to perform a quantitative comparison of proteins released from cartilage explants in response to treatment with IL-1beta, TNF-alpha, or mechanical compression injury in vitro and to interpret this release in the context of anabolic-catabolic shifts known to occur in cartilage in response to these insults in vitro and their implications in vivo. Bovine calf cartilage explants from 6-12 animals were subjected to injurious compression, TNF-alpha (100 ng/ml), IL-1beta (10 ng/ml), or no treatment and cultured for 5 days in equal volumes of medium. The pooled medium from each of these four conditions was labeled with one of four iTRAQ labels and subjected to nano-2D-LC/MS/MS on a quadrupole time-of-flight instrument. Data were analysed by ProQuant for peptide identification and quantitation. k-means clustering and biological pathways analysis were used to identify proteins that may correlate with known cartilage phenotypic responses to such treatments. IL-1beta and TNF-alpha treatment caused a decrease in the synthesis of collagen subunits (p < 0.05) as well as increased release of aggrecan G2 and G3 domains to the medium (p < 0.05). MMP-1, MMP-3, MMP-9, and MMP-13 were significantly increased by all treatments compared with untreated samples (p < 0.10). Increased release of proteins involved in innate immunity and immune cell recruitment were noted following IL-1beta and TNF-alpha treatment, whereas increased release of intracellular proteins was seen most dramatically with mechanical compression injury. Proteins involved in insulin-like growth factor and TGF-beta superfamily pathway modulation showed changes in pro-anabolic pathways that may represent early repair signals. At the systems level, two principal components were sufficient to describe 97% of the covariance in the data. A strong correlation was noted between the proteins released in response to IL-1beta and TNF-alpha; in contrast, mechanical injury resulted in both similarities and unique differences in the groups of proteins released compared with cytokine treatment.