Proteomic analysis by two-dimensional electrophoresis to identify the normal human chondrocyte proteome stimulated by tumor necrosis factor α and interleukin-1β
Berta Cillero-Pastor
INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain
Search for more papers by this authorCristina Ruiz-Romero
INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain
Search for more papers by this authorBeatriz Caramés
INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain
Search for more papers by this authorCorresponding Author
María J. López-Armada
INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain
Osteoarticular and Aging Research Laboratory, INIBIC, Complejo Hospitalario Universitario A Coruña, As Xubias, 84; 15006-A Coruña, SpainSearch for more papers by this authorCorresponding Author
Francisco J. Blanco
INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain
Osteoarticular and Aging Research Laboratory, INIBIC, Complejo Hospitalario Universitario A Coruña, As Xubias, 84; 15006-A Coruña, SpainSearch for more papers by this authorBerta Cillero-Pastor
INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain
Search for more papers by this authorCristina Ruiz-Romero
INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain
Search for more papers by this authorBeatriz Caramés
INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain
Search for more papers by this authorCorresponding Author
María J. López-Armada
INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain
Osteoarticular and Aging Research Laboratory, INIBIC, Complejo Hospitalario Universitario A Coruña, As Xubias, 84; 15006-A Coruña, SpainSearch for more papers by this authorCorresponding Author
Francisco J. Blanco
INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain
Osteoarticular and Aging Research Laboratory, INIBIC, Complejo Hospitalario Universitario A Coruña, As Xubias, 84; 15006-A Coruña, SpainSearch for more papers by this authorAbstract
Objective
To determine the intracellular proteome of normal human chondrocytes stimulated with interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) 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-1β (5 ng/ml) or with TNFα (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-1β–stimulated chondrocytes, IL-1β 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-1β included nicotinamide phosphoribosyltransferase, long-chain fatty acid–coenzyme A ligase 4, δ-aminolevulinic acid dehydratase, triosephosphate isomerase, and an isoform of glyceraldehyde-3-phosphate dehydrogenase. In TNFα-stimulated chondrocytes, TNFα 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-1β and TNFα. Of these, 45% were related to metabolism.
Conclusion
IL-1β and TNFα 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.
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