Transcriptional and post-transcriptional regulation of iNOS expression in human chondrocytes.
Chondrocytes are important for the development and maintenance of articular cartilage. However, both in osteoarthritis (OA) and rheumatoid arthritis (RA) chondrocytes are involved in the process of cartilage degradation and synthesize important immunomodulatory mediators, including nitric oxide (NO) generated by the inducible NO synthase (iNOS). To uncover the role of iNOS in the pathomechanisms of OA and RA, we analyzed the regulation of iNOS expression using immortalized human chondrocytes as a reproducible model. In C-28/I2 chondrocytes, iNOS expression was associated with the expression of the chondrocyte phenotype. Peak induction by a cytokine cocktail occurred between 6 and 8h and declined by 24h. Inhibition of p38MAPK, NF-kappaB and the JAK2-STAT-1alpha pathways resulted in a reduction of iNOS expression. In contrast to other cell types, the cytokine-mediated induction of the human iNOS promoter paralleled the induction rate of the iNOS mRNA expression in C-28/I2 chondrocytes. However, in addition post-transcriptional regulation of iNOS expression by the RNA binding protein KSRP seems to operate in these cells. As seen in other chondrocyte models, glucocorticoids were not able to inhibit cytokine-induced iNOS expression in C-28/I2 cells, due to the lack of the glucocorticoid receptor mRNA expression. In this model of glucocorticoid-resistance, the new fungal anti-inflammatory compound S-curvularin was able to inhibit cytokine-induced iNOS expression and iNOS-dependent NO-production. In summary, we demonstrate for the first time that differentiated human immortalized C-28/I2 chondrocytes are a representative cell culture model to investigate iNOS gene expression in human joint diseases.
Department of Pharmacology, University Medical Center of the Johannes Gutenberg University Mainz, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany.
Nadine
N
Schmidt
Andrea
A
Pautz
Julia
J
Art
Peter
P
Rauschkolb
Matthias
M
Jung
Gerhard
G
Erkel
Mary B
MB
Goldring
Hartmut
H
Kleinert
D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D002358:Cartilage, Articular / Q000187:drug effects / Q000201:enzymology; D002461:Cell Line, Transformed; D019902:Chondrocytes / Q000187:drug effects / Q000201:enzymology*; D016207:Cytokines / Q000494:pharmacology; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D050832:Interferon-Stimulated Gene Factor 3 / Q000037:antagonists & inhibitors; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors; D052002:NF-kappa B p50 Subunit / Q000037:antagonists & inhibitors; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis / Q000235:genetics*; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D015025:Zearalenone / Q000031:analogs & derivatives / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors
D000893:Anti-Inflammatory Agents; D016207:Cytokines; D050832:Interferon-Stimulated Gene Factor 3; C105789:KHSRP protein, human; D052002:NF-kappa B p50 Subunit; C489548:NFKB1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011965:Receptors, Glucocorticoid; D015534:Trans-Activators; C079868:gamma interferon activation factor; D015025:Zearalenone; D052247:Nitric Oxide Synthase Type II; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D048051:p38 Mitogen-Activated Protein Kinases; C013455:curvularin