Abstract
Endoplasmic reticulum (ER) stress regulates a wide range of cellular responses including apoptosis, proliferation, inflammation, and differentiation in mammalian cells. In this study, we observed the role of 2-deoxy-D-glucose (2DG) on inflammation of chondrocytes. 2DG is well known as an inducer of ER stress, via inhibition of glycolysis and glycosylation. Treatment of 2DG in chondrocytes considerably induced ER stress in a dose- and time-dependent manner, which was demonstrated by a reduction of glucose regulated protein of 94 kDa (grp94), an ER stress-inducible protein, as determined by a Western blot analysis. In addition, induction of ER stress by 2DG led to the expression of COX-2 protein with an apparent molecular mass of 66-70kDa as compared with the normally expressed 72-74 kDa protein. The suppression of ER stress with salubrinal (Salub), a selective inhibitor of eif2-alpha dephosphorylation, successfully prevented grp94 induction and efficiently recovered 2DG- modified COX-2 molecular mass and COX-2 activity might be associated with COX-2 N-glycosylation. Also, treatment of 2DG increased phosphorylation of Src in chondrocytes. The inhibition of the Src signaling pathway with PP2 (Src tyrosine kinase inhibitor) suppressed grp94 expression and restored COX-2 expression, N-glycosylation, and PGE2 production, as determined by a Western blot analysis and PGE2 assay. Taken together, our results indicate that the ER stress induced by 2DG results in a decrease of the transcription level, the molecular mass, and the activity of COX-2 in rabbit articular chondrocytes via a Src kinase-dependent pathway.
Highlights
Chondrocytes in joint cartilage are differentiated from mesenchymal cells during embryo development (Sandell and Adler, 1999; DeLise et al, 2000; Singh Khillan, 2007)
The increase in grp94 was apparent ∼3 h after treatment of 2DG (Figure 1A). These data indicated that 2DG induces Endoplasmic reticulum (ER) stress in rabbit articular chondrocytes (Figure 1)
Expression of COX-2 was detected by a Western blot analysis and expression of β-actin was used as a loading control. (B) Articular chondrocytes were untreated or treated with 5 mM 2DG for the indicated time periods or with the specificied concentrations of 2DG for 24 h
Summary
Chondrocytes in joint cartilage are differentiated from mesenchymal cells during embryo development (Sandell and Adler, 1999; DeLise et al, 2000; Singh Khillan, 2007). They are comprised of a single-cell population responsible for the biosynthesis of matrix molecular components. Balance of anabolic and catabolic processes within the tissue is important for homeostasis maintenance of cartilage. This homeostasis is destroyed in degenerative diseases, such as rheumatoid arthritis.
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