Synergistic induction of nitric oxide synthase type II: in vitro effect of leptin and interferon-gamma in human chondrocytes and ATDC5 chondrogenic cells.
OBJECTIVE
To study, in vitro, the effect of leptin (OB), alone or in combination with interferon-gamma (IFNgamma), on inducible nitric oxide synthase (iNOS) and NO production in human primary chondrocytes and in mouse embryonic chondrogenic ATDC5 cells.


METHODS
Leptin receptor expression and iNOS messenger RNA expression were evaluated by reverse transcriptase-polymerase chain reaction. Then, iNOS activity was indirectly studied by measuring nitrite accumulation, using the Griess colorimetric reaction, in culture medium of human primary chondrocytes and ATDC5 cells.


RESULTS
ATDC5 mouse embryonic cells expressed functional OB receptor. Alone, neither OB nor IFNgamma produced nitrite accumulation in culture medium. However, costimulation with OB and IFNgamma resulted in dose-dependent up-regulation of the expression of iNOS and NO production in human primary chondrocytes and ATDC5 cells. Production of NO was blunted by the iNOS-specific inhibitors L-N(G)-nitroarginine methyl ester and aminoguanidine. In addition, the janus-activated kinase 2 (JAK2)-specific inhibitor Tyrphostin AG 490 completely blocked OB + IFNgamma-driven up-regulation of iNOS and NO production.


CONCLUSIONS
Our data show for the first time a putative proinflammatory role of OB via iNOS induction and NO production. This occurs via activation of JAK2.
Otero
Miguel
M
Hospital Clinico Universitario de Santiago, Santiago de Compostela, Spain.
Gomez Reino
Juan Jesús
JJ
Gualillo
Oreste
O
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002358:Cartilage, Articular / Q000166:cytology; D002460:Cell Line; D019902:Chondrocytes / Q000166:cytology / Q000201:enzymology*; D004357:Drug Synergism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000494:pharmacology*; D053614:Janus Kinase 2; D020738:Leptin / Q000494:pharmacology*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins
D000970:Antineoplastic Agents; D020738:Leptin; D011518:Proto-Oncogene Proteins; D009569:Nitric Oxide; D007371:Interferon-gamma; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2