Gonadotropin-releasing hormone activates the 12-lipoxygenase pathway in the LbetaT2 gonadotrope cell line.
Previous studies have indicated that arachidonic acid and its lipoxygenase (LO) metabolites play a role in the post-receptor effects of gonadotropin-releasing hormone (GnRH) but the exact role and nature of these putative eicosanoids remain unclear. The potential role of arachidonic acid and LO in GnRH receptor-mediated signaling was investigated in the LbetaT2 gonadotrope cell line, which expresses gonadotropins (LH and FSH) and GnRH-receptor mRNAs. Western immunobloting of LbetaT2 cell extracts, performed with a murine leukocyte polyclonal antibody against 12-LO, showed a 70-kD band, suggesting the presence of 12-LO protein in these cells. GnRH nearly doubled the release of 12-hydroeicosatetraenoic acid, a product of the 12-LO enzyme, within 10 min. A specific reverse transcriptase polymerase chain reaction with a set of primers based on the reported sequence of rat brain 12-LO yielded a 170-bp band which showed 100% homology with the expected rat brain 12-LO sequence. Exposure of LbetaT2 cells to pulsatile GnRH treatment (10 nM, 90-min interpulse, one and three pulses) led to a approximately 3-fold increase in 12-LO mRNA levels. In conclusion, we provide evidence for the presence of a 12-LO enzyme in LbetaT2 cells, the expression and activity of which are increased by short-term/pulsatile exposure to GnRH. LbetaT2 cells represent a potential model to further study the involvement of 12-LO in GnRH receptor signaling.
Limor
Rona
R
Institute of Endocrinology, Metabolism and Hypertension, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv, Israel.
Naor
Zvi
Z
Weisinger
Gery
G
Naidich
Michal
M
Knoll
Esther
E
Sharon
Orly
O
Stern
Naftali
N
D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000096:biosynthesis*; D001483:Base Sequence; D015153:Blotting, Western; D004790:Enzyme Induction; D005786:Gene Expression Regulation / Q000502:physiology; D007987:Gonadotropin-Releasing Hormone / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000201:enzymology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D007987:Gonadotropin-Releasing Hormone; D001092:Arachidonate 12-Lipoxygenase