The procatabolic cytokine interleukin-1 alfa induces a dysregulation of o-linked n-acetylglucosamine modified proteins in human osteoarthritic chondrocytes

Abstract

Purpose: Nuclear and cytoplasmic protein glycosylation by the addition of O-linked N-Acetylglucosamine (O-GlcNAc) to serine and threonine residues is a widespread post-translational modification that has been described in degenerative and age-related diseases, such as Alzheimer´s and diabetes. This modification is catalyzed by the O-GlcNAc transferase (OGT) which uses UDP-GlcNAc as a donor, and O-GlcNAc can be removed by the O-GlcNAc-glucosaminidase (OGA). We have seen that O-GlcNAc glycosylation levels in the articular cartilage of patients with OA were increased as well as the expression of the different isoforms of OGT and OGA was dysregulated. The goal of this study was to assess if a procatabolic cytokine, IL-1α, can be responsible of the changes in O-GlcNAc system found in OA human cartilage. Methods: OA knee articular cartilage was obtained during replacement surgery. Twelve OA patients were included in this study (6 woman/6 men; mean age 71±3 years), with a Mankin score for knee specimens between 11 and 14. Human osteoarthritic chondrocytes (HOC) were isolated by protease digestion for primary culture. At confluence, cells were stimulated with different concentrations of IL-1α for different doses and periods of time after they were deprived of serum for 48 h. The level of O-GlcNAcylation, OGT and O-GlcNAcase expression was assessed by western-blot employing specific antibodies. Results: The amount of O-GlcNAcylated proteins was increased in HOC 24 h after stimulation with different doses of IL-1α (1, 10 and 100 ng/ml), showing the highest accumulation with 10 ng/ml. This concentration was chosen for stimulation of HOC during different periods of time (3, 6, and 24 h). The increase in O-GlcNAc proteins amount showed a peak of induction at 6 h. This accumulation occurs especially in proteins between 50 to 150 KDa therefore showing a similar O-GlcNAcylated proteins pattern to that observed in OA cartilage. We also observed an increased expression of the three OGT isoforms, nucleo-cytoplasmic (ncOGT), mitochondrial (mOGT) and small (sOGT) in HOC as early as 3 h, although the peak of induction was at 6 h. Three isoforms were increased in a similar grade, without prevalence of sOGT as found in OA cartilage. IL-1α also increased the presence of both OGA isoforms, long (OGA-L) and short (OGA-S) after 3 and 6 h of stimuli. A clear prevalence of long isoform was found along IL-1α stimulation whereas OGA-S had the highest prevalence in OA cartilage. Conclusions: To our knowledge, this is first evidence of procatabolic cytokines involvement in O-GlcNAc modifications. IL-1α can induce accumulation of O-GlcNAc proteins in human OA chondrocytes. Although both regulatory enzymes of this modification, OGT and OGA, are also increased with IL-1α stimulation, characteristic distribution of the different isoforms in OA cartilage was not reached. IL-1α is in part responsible of a dysregulation in the O-GlcNAc proteins modification during OA although other processes must be involved.