Proteomic analysis by two-dimensional electrophoresis to identify the normal human chondrocyte proteome stimulated by tumor necrosis factor alpha and interleukin-1beta

Abstract

Objective: To determine the intracellular proteome of normal human chondrocytes stimulated with interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) and to ascertain differences in the protein expression patterns of these 2 cytokines.

Methods: Normal human knee cartilage chondrocytes were incubated for 48 hours without stimulation or stimulated with IL-1beta (5 ng/ml) or with TNFalpha (10 ng/ml). For each culture condition, protein extracts from 4 normal subjects were pooled and resolved using 2-dimensional electrophoresis. Protein spots were visualized with Sypro stain, and qualitative and quantitative analyses were performed using PDQuest software. Protein spots were then identified by mass spectrometry, using matrix-assisted laser desorption ionization-time-of-flight/time-of-flight technology.

Results: We identified 37 spots by mass spectrometry (MS) or by MS/MS, corresponding to 35 different proteins. In IL-1beta-stimulated chondrocytes, IL-1beta was found to modulate 22 proteins, as compared with unstimulated chondrocytes. All of these proteins except connective tissue growth factor (CCND2) were up-regulated. Proteins involved in cellular metabolism and energy (23%) that were up-regulated or induced by IL-1beta included nicotinamide phosphoribosyltransferase, long-chain fatty acid-coenzyme A ligase 4, delta-aminolevulinic acid dehydratase, triosephosphate isomerase, and an isoform of glyceraldehyde-3-phosphate dehydrogenase. In TNFalpha-stimulated chondrocytes, TNFalpha was found to modulate 20 proteins, as compared with unstimulated chondrocytes. All of these except chitinase 3-like 1 (cartilage glycoprotein 39), proteasome activator complex subunit 2, and G3PDH, were up-regulated. Eighteen proteins were differently modulated by IL-1beta and TNFalpha. Of these, 45% were related to metabolism.

Conclusion: IL-1beta and TNFalpha induce different profiles of intracellular protein expression in healthy human chondrocytes. Most of the proteins that are differently regulated are proteins that are implicated in the generation of cellular energy and in glycolysis.