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- Open Access
Changes in the synovial cell proteome induced by hypoxia
Arthritis Res Thervolume 5, Article number: 124 (2003)
Fibroblast-like synovial cells (FLS) have provided considerable insight into the mechanisms underlying joint inflammation and destruction. There is currently an incomplete knowledge of the global FLS proteome and of how this compares with the proteome of other fibroblasts. The rheumatoid arthritis synovial microenvironment is known to be chronically hypoxic. We studied the FLS proteome using two-dimensional gels and mass spectrometry to evaluate the effects of hypoxia on the synovial proteome, comparing this with the expressed genome.
FLS were isolated from rheumatoid arthritis and osteoarthritis synovia obtained at the time of joint arthroplasty. Cells from passages 2–4 were cultured under normoxic and hypoxic conditions for variable time points prior to analysis. Total proteins found in whole-cell FLS lysates were separated on immobilized pH-gradient two-dimensional (2D) PAGE and stained with Coomassie blue. To date, the in-gel digests of > 300 protein spots have been analyzed using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Spectra were analyzed using the Knexus automation client and the Profound search engine (Proteometrics Canada, Winnipeg, Manitoba, Canada). 2D gels from hypoxic and normoxic FLS were superimposed using image analysis software and were compared. Differences were identified, and were related to differences in the expressed genome demonstrated in a microarray experiment using the same cell lines and conditions.
Under normoxic conditions, more than 300 proteins were fully characterized in the FLS lines. Of particular note, FLS intensely expressed uridine diphosphoglucose dehydrogenase, which is an enzyme needed for the synthesis of hyaluronic acid. Other proteins identified fell into the following groups: enzymatic, transcription and cell-cycle regulation, protein binding, cytoskeletal, extracellular matrix, heat shock, protein synthesis and assembly, and membrane channel. In comparing the proteome expressed under hypoxic conditions, most of the apparent differences were quantitative, with shifts in position suggestive of phosphorylation and glycosylation. The microarray data demonstrated a spectrum of known, as well as novel, hypothetical proteins that were regulated by hypoxia. Identification of these proteins in the 2D gels is currently underway.
We demonstrate techniques used in the detailed exploration of the synovial proteome. These techniques are applied to the examination of the effects of hypoxic stimulation on FLS. Comparisons between the expressed proteome and genome provide novel insights into the response of FLS to hypoxia