In most cells, the mitochondrion is an important organelle, because it plays a role in energy production and is a target and a generator of free radicals, as well as being a sensor of apoptotic/survival signals (1, 2). The chondrocyte is the only cell type present in mature cartilage and is responsible for cartilage repair and maintenance (3, 4). In recent years, links between mitochondria and chondrocyte function have been identified. The activity of the mitochondrial complexes succinate dehydrogenase (complex II) and ubiquinol cytochrome c reductase (complex III) is known to be down-regulated in osteoarthritis (OA) (5). In addition, nitric oxide (NO) seems to inhibit the activity of cytochrome c oxidase (complex IV) (6). Johnson et al have reported that a substantial decrease in mitochondrial ATP generation contributes to the pathogenesis of OA (7). Inflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα), inhibit mitochondrial complex I, affecting the oxidative phosphorylation process and mitochondrial membrane potential (8). The catabolic program induced by these proinflammatory stimuli is characterized by secretion of proteinases, suppression of matrix synthesis, and reduction in the number of chondrocytes (9, 10). Consequently, there are recently recognized important dimensions to the role of inflammation in OA progression (11).
In a previous report, we described the total proteomic profile of chondrocytes stimulated with IL-1β and TNFα, showing a clear up-regulation of metabolic pathways related to TNFα in IL-1β–stimulated cells (12). We also demonstrated that some proteins regulated by IL-1β had mitochondrial locations. There are, however, no previous reported studies of the exact effects of IL-1β on the mitochondrial proteome of normal human chondrocytes. The present study identifies, for the first time, the role of IL-1β in regulation of the mitochondrial proteome profile of normal human chondrocytes. Of particular interest is the finding that all of the proteins identified were up-regulated by IL-1β. We also identified a protein that was not previously recognized in chondrocytes, dimethylarginine dimethylaminohydrolase 2 (DDAH-2), and were able to show that IL-1β induced the translocation of DDAH-2 to the mitochondria and to demonstrate the role of DDAH-2 as a regulator of NO production. These findings may provide a better understanding of the participation of chondrocyte mitochondria in stress signaling and NO pathways and their relationship to the development of rheumatic diseases such as OA.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
The present study is, to our knowledge, the first to compare the proteome of mitochondrial proteins in unstimulated and IL-1β–stimulated chondrocytes. We found 6 proteins, AKR1C2, ANXA2, SAP18, SODMn, SYW, and DDAH-2, that were up-regulated after 48 hours of treatment with IL-1β. After identifying these proteins we observed that most of them are active in metabolic and stress-related pathways, indicating that IL-1β induced a high level of activity in the cell. Using Pathway Studio 7.0 software, we studied the role of some of these proteins in cellular pathways, as well as in certain disease states (additional information available from the corresponding author upon request).
AKR1C2 has a key role in the metabolism of steroid hormones and prostaglandin E synthesis, and it confers resistance to certain tumor cells in lung, prostate, and breast cancer, regulating survival cascades (13–15). ANXA2 is important in the remodeling of the cytoskeleton and in the cell proliferation process, because of its role as a vesicular transporter in membrane–membrane or membrane–cytoskeleton connections. It can bind CD44, one of the principal cellular receptors of hyaluronic acid, in the formation of compartments rich in cholesterol (16). Histone deacetylases, such as SAP18, have been shown to regulate matrix metalloproteinases and aggrecanases in cartilage, and inhibitors of histone deacetylases could block degradation of the extracellular matrix and inhibit the expression of these enzymes mediated by proinflammatory enzymes (17).
SODMn is a protein related to stress signals; it eliminates O2− and releases H2O2 (18). It is important to note that the mitochondrion is one of the principal producers of reactive oxygen species (ROS). This is particularly relevant with regard to diseases related to aging, in which accumulation of ROS is very high (19, 20). Superoxide can damage DNA, inducing mutations, and also reacts with such molecules as NO, forming peroxynitrite (ONOO−), which initiates DNA strand breakage and modifications of purines and pyrimidines (21, 22). A decrease in the expression of extracellular SODMn has been observed in OA chondrocytes (23, 24), and our group has obtained similar results using mitochondrial fractions of OA and normal chondrocytes in culture (25). In contrast, other groups have found increased levels of SODMn in synovial fluid from patients with rheumatoid arthritis (26), and, in accordance with those results, we showed in a previous study that SODMn in chondrocytes was increased after IL-1β incubation (12). It seems that in a short-term model of acute inflammation, an increased level of SODMn is a key factor for moderating ROS levels. SYW regulates ERK, Akt, and endothelial NOS (eNOS) activation pathways that are associated with angiogenesis, cytoskeletal reorganization, and sheer stress–responsive gene expression.
From all identified proteins we found DDAH-2, a stress-related protein, to be increased by IL-1β; this protein has not been previously identified in chondrocytes. In other cell types, DDAH-2 has the ability to inhibit asymmetric dimethylarginine (ADMA), a natural inhibitor of NO synthase (NOS). The DDAH family of proteins is formed by two isoforms, DDAH-1 and DDAH-2, both of which utilize ADMA as their substrate and increase NOS activity; however, their regulation, cellular location, and tissue specificity are very different (27). DDAH-2 is the most prevalent isoform in blood vessels and endothelium and is important for embryonic development (27). NOS enzymes convert L-arginine to L-citrulline, producing NO. Thus, the increase in NOS activity leads to an increase in NO levels, a characteristic phenomenon observed after exposure to IL-1β (28, 29) and in cartilage and synovial fluid in many articular diseases. DDAH-2 has also been linked to other diseases, such as hypertension, cerebral hemorrhage, cardiac disease, and diabetes (27, 30–32).
A surprising finding of the present study was the demonstration of the effect of IL-1β in transport of DDAH-2 to the mitochondria, a localization not previously described in any cell type. In cultured human endothelial cells, DDAH-1 has been associated with the cytosol and the nucleus, but DDAH-2 had been found only in the cytosol (33). However, in a recently reported study, DDAH-2 was found in the nuclei of rat vascular smooth muscle cells (27). In Western blot analyses of total cell extracts as well as mRNA analyses (results not shown), we found no difference between control and IL-1β–exposed conditions. However, assays using purified mitochondrial proteins and conventional and confocal microscopy confirmed the results from 2-D electrophoresis in mitochondrial extracts, showing that DDAH-2 was found only in the mitochondria of IL-1β–treated cells. This effect was also observed in cartilage explants from human donors. Therefore, we tested whether DDAH-2 is translocated from the cytosol to the mitochondria by the action of IL-1β. We used an inhibitor of general protein transport, monensin (34, 35), to block the transport of DDAH-2 with IL-1β treatment. Interestingly, results similar to those obtained in IL-1–stimulated normal cartilage were found in OA cartilage, supporting the notion that NO and DDAH-2 have a role in OA.
After we ascertained that the protein migrated to the mitochondria, we examined the role of DDAH-2 in regulating NO production in chondrocytes, as it does in other cell types. To test this, we reduced the expression of DDAH-2 using homocysteine, as previously described (36, 37). As expected, treatment with 10 μM homocysteine reduced DDAH-2 mRNA expression in chondrocytes, but it also inhibited the production of NO induced by the cytokine. This finding demonstrates how DDAH-2 relates to NO production in IL-1β–stimulated cells. The family of NOS enzymes represents a complex group of proteins with two described isoforms in chondrocytes: a constitutive enzyme (eNOS) and an inducible isoform (inducible NOS [iNOS]) (38). DDAH-2 has been reported to interact with eNOS in the cytosol of endothelial cells and with iNOS in vascular smooth muscle cells (29). The isoform that is up-regulated by lipopolysaccharide, IL-1, or TNF in chondrocytes is iNOS, suggesting that DDAH-2 may directly or indirectly increase iNOS activity. We tested whether inhibition of the translocation of DDAH-2 into mitochondria also had an effect on NO production. We used monensin to block this transport, as described above, and found that levels of NO released to the supernatants were also reduced, indicating the important actions of DDAH-2 in chondrocyte mitochondria.
Further evidence of the relationship of DDAH-2 and mitochondria comes from studies showing that DDAH-2 gene expression is down-regulated by coupling factor 6, a mitochondrial protein of the ATP synthase complex, again establishing the relationship among the pathways of mitochondria, ROS, and NO (39, 40). It is well known that NO reduces the activity of mitochondrial complex IV in chondrocytes and that many other factors affect the mitochondrial respiratory chain, inducing inflammation processes (6, 41). NO can also interact with other ROS, producing ONOO− and inducing a cascade of negative ROS-mediated mitochondrial and cellular effects, including DNA damage, inflammatory pathway activation, induction of the expression of apoptotic proteins, and cellular senescence. These combined results indicate the importance of DDAH-2 in the regulation of NO-related pathways, particularly in relation to chondrocyte mitochondria.
In conclusion, in this study of normal human knee chondrocytes we have identified some mitochondrial proteins that are linked to the inflammatory response and up-regulated by IL-1β exposure. In particular, DDAH-2 could represent a new focus in the study of NO production in chondrocytes, particularly in relation to the mitochondrial ROS-related events that could be involved in aging-related rheumatic diseases such as OA.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Blanco had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Cillero-Pastor, Mateos, Fernández-López, Oreiro, Ruiz-Romero, Blanco.
Acquisition of data. Cillero-Pastor, Mateos, Fernández-López, Oreiro, Ruiz-Romero, Blanco.
Analysis and interpretation of data. Cillero-Pastor, Blanco.