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Molecular & Cellular Proteomics 8:1475-1489, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Mechanical Injury and Cytokines Cause Loss of Cartilage Integrity and Upregulate Proteins Associated with Catabolism, Immunity, Inflammation, and Repair^*,

Anna L. Stevens, John S. Wishnok, Forest M. White, Alan J. Grodzinsky^,,¶,** and Steven R. Tannenbaum^,||

From the Biological Engineering Department and
Departments of Electrical Engineering and Computer Science,
¶Mechanical Engineering, and
||Chemistry Massachusetts Institute of Technology Cambridge, Massachusetts 02139

The objectives of this study were to perform a quantitative comparison of proteins released from cartilage explants in response to treatment with IL-1β, TNF-, 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- (100 ng/ml), IL-1β (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-1β and TNF- 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-1β and TNF- treatment, whereas increased release of intracellular proteins was seen most dramatically with mechanical compression injury. Proteins involved in insulin-like growth factor and TGF-β 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-1β and TNF-; in contrast, mechanical injury resulted in both similarities and unique differences in the groups of proteins released compared with cytokine treatment.

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