Proteomics of chondrocytes with special reference to phosphorylation changes of proteins in stretched human chondrosarcoma cells.
For proteomic analysis, cartilage molecular composition is a challenging mixture of highly glycosylated proteoglycans and triple-helical collagens, which constitute the major part of cartilage macromolecules. Selective separation of these molecules from the minor components is generally needed before mass spectrometry-based identification of lower-abundancy proteins is possible. The cell density of cartilage is also very low, therefore, cell cultures offer an easier approach to study cellular responses of chondrocytic cells, e.g., to mechanical stimuli. In this study, we investigated the phosphorylation events in human chondrosarcoma cells during cellular stretching. Human chondrosarcoma cells were stretched to 8% strain at a frequency of 1 Hz. One set of experiments included cellular stretching which lasted 2 hours, and the other one included experiments of 2 hours daily treatment for up to 3 days. Two-dimensional polyacrylamide gel electrophoresis combined with chromatographic phosphoprotein pre-enrichment and electrospray ionization mass spectrometry-based protein identification was used to reveal changes of phosphoproteins in cells exposed to cyclic stretching. We discovered that 2 hours cyclic stretching increased the phosphorylation of moesin, elongation factor eEF1D and leprecan, while the phosphorylation of elongation factor eEF1B decreased after cellular stretching. Western blot analyses with phospho-specific antibodies suggested that stretching induces phosphorylation of ERK of the MAP kinase pathway, but did not induce phosphorylation of phosphatidylinositol 3-kinase. In conclusion, the proteomic approach revealed that cellular stretching induced specific phosphorylation changes in chondrosarcoma cells.
Department of Biomedicine, Anatomy, University of Kuopio, Kuopio, Finland.
Juha
J
Piltti
Jukka
J
Häyrinen
Hannu M
HM
Karjalainen
Mikko J
MJ
Lammi
D045744:Cell Line, Tumor; D019902:Chondrocytes / Q000378:metabolism*; D002813:Chondrosarcoma / Q000378:metabolism* / Q000473:pathology*; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D020648:Peptide Elongation Factor 1 / Q000378:metabolism; D020652:Peptide Elongation Factor 2 / Q000378:metabolism*; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013314:Stress, Mechanical
D008840:Microfilament Proteins; D020648:Peptide Elongation Factor 1; D020652:Peptide Elongation Factor 2; D010750:Phosphoproteins; C071058:moesin; D058539:Phosphatidylinositol 3-Kinase; D020928:Mitogen-Activated Protein Kinases