Mechanical injury and cytokines cause loss of cartilage integrity and upregulate proteins associated with catabolism, immunity, inflammation, and repair.
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.
Stevens
Anna L
AL
Biological Engineering Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Wishnok
John S
JS
White
Forest M
FM
Grodzinsky
Alan J
AJ
Tannenbaum
Steven R
SR
D000818:Animals; D002356:Cartilage / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology* / Q000637:transplantation; D002417:Cattle; D016000:Cluster Analysis; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D020007:Implants, Experimental; D007249:Inflammation / Q000378:metabolism*; D053583:Interleukin-1beta / Q000276:immunology / Q000494:pharmacology*; D025341:Principal Component Analysis; D012038:Regeneration / Q000502:physiology*; D013314:Stress, Mechanical; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000494:pharmacology*
D016326:Extracellular Matrix Proteins; D053583:Interleukin-1beta; D014409:Tumor Necrosis Factor-alpha