NADPH Oxidase-Generated ROS Participates in GNRH-Stimulated MAP Kinase and Gonadotropin Subunit Gene Promoter Activation

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 134-163-GnRH & Gonadotroph Biology & Signaling
Bench to Bedside
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-157
Taeshin Kim*1 and Mark A Lawson2
1University of CA San Diego, San Diego, CA, 2Univ of California-San Diego, La Jolla, CA
The nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase family is a major source of intracellular Reactive Oxygen Species (ROS), which regulates cell signaling and homeostasis.  However, the role of NADPH oxidase has not been reported in the regulation of FSH and LH production in gonadotropes.  In primary mouse pituitary cultures and in LβT2 cells, GNRH stimulation elevates intracellular ROS via NADPH oxidase as determined by 2-7 dichlorofluorescein diacetate reporter staining. Mouse pituitary and LβT2 cells have highly abundant NOX2 mRNA, which is further increased in response to GnRH stimulation.  Furthermore, the related family members NOX, DUOX1 and DUOX2 mRNA are also expressed in LβT2 cells as are the genes encoding all the components of non-phagocyte NADPH oxidases, p67phox, p47phox, p40phox, NOXO1, Rac 1/2, and p22phox.  Down-regulation of protein kinase C by overnight 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) treatment or by treatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) inhibited ERK activation by GNRH or a second PMA treatment, but treatment with epidermal growth factor was still capable of activating ERK, showing a ROS-dependent pathway of ERK activation by GNRH.  Both  ERK and JNK phosphorylation increases induced by GNRH treatment were significantly diminished by DPI and the antioxidant N-acetyl-L-cysteine (NAC), but p38 was unaffected. Nuclear translocation of phosphorylated ERK by GNRH treatment was also diminished in the presence DPI. Activation of gonadotropin subunit gene Fshb and Lhb promoters by GNRH was also diminished by DPI and NAC. These finding establish that GNRH induces ROS generation through NADPH oxidase, which regulates ERK/JNK activation as well as Fshb and Lhb gene expression. These results strongly suggest that NOX/DUOX-mediated ROS generation potently regulates function of gonadotropes and provides the link between PKC activation and downstream control of MAP kinase signaling.

Nothing to Disclose: TK, MAL

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Sources of Research Support: NIH Grants R01 HD 043758 and U54 HD 012303