Interleukin-1beta signals through a c-Jun N-terminal kinase-dependent inducible nitric oxide synthase and nitric oxide production pathway in Sertoli epithelial cells.
Our recent Sertoli cell (SC) studies showed that the c-Jun N-terminal kinase (JNK) and inducible cyclooxygenase-2 (COX-2) pathways are key regulatory components of IL (IL-1alpha, IL-1beta, and IL-6) expression and START-domain containing StARD1 and StARD5 proteins. IL-1beta regulates SC autocrine/paracrine activities and subsequently influences developing germ cells and spermatogenesis. This study was designed to evaluate whether IL-1beta mediates high-output inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in these specialized epithelial cells and characterize gonadotropin and cytokine-regulation of NO. Purified SCs were maintained in serum-free cultures and treated with FSH (100 ng-1 microg/ml) or IL-1beta (10 ng/ml) in time-course studies. To determine obligatory intracellular pathways, treatments were conducted with or without activity inhibitors: COX-2 selective (NS-398, 10 microM) or JNK (SP600125, 10 microM) for 1, 3, 6, and 24 h. NOS mRNAs and proteins were evaluated by RT-PCR and Western analysis, respectively. NO and reactive oxygen species were measured by flow cytometry and ELISA. IL-1beta transiently induces intracellular NO (30 min) but not reactive oxygen species. Subsequently, iNOS mRNA and protein expression (3-6 h) significantly increased after IL-1beta but not FSH stimulation, and in time-dependent manner, markedly increased extracellular NO (24 h, 8-fold). No change in the constitutive endothelial NOS isoform was observed. Inhibition of JNK, but not COX-2, activity inhibits IL-1beta-induced iNOS expression and NO production. Such findings suggest that intra- and extracellular NO within the tubule may alert SCs monitoring the microenvironment to an aberrant cytokine, triggering antioxidant and antiinflammatory activities to avoid disruption of spermatogenesis.
Ishikawa
Tomomoto
T
Center for Biomedical Research, Population Council, New York, New York 10021, USA.
Morris
Patricia L
PL
D000818:Animals; D051546:Cyclooxygenase 2 / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism; D053583:Interleukin-1beta / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000502:physiology*; D008297:Male; D016328:NF-kappa B / Q000502:physiology; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D012708:Sertoli Cells / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000502:physiology
D053583:Interleukin-1beta; D016328:NF-kappa B; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases