Abstract. The mechanism by which GnRH stimulates annexin A5 expression was examined with LβT2 gonadotrope cells. Continuous stimulation with GnRH analog (GnRHa, Des-Gly10 [Pro9]-GnRH ethylamide) transiently elevated LHβ mRNA expression while maintaining annexin A5 mRNA at high levels for 24 h. GnRH antagonist blocked the effect of GnRHa on annexin A5. While 12-O-tetradecanoyl-phorbol-13 acetate, a protein kinase C activator, increased the expression of annexin A5 mRNA, bisindolylmaleimide, an inhibitor of protein kinase C, suppressed GnRHa-stimulated expression of annexin A5 and LHβ mRNA. GnRHa stimulation of LHβ mRNA was inhibited to a greater extent than annexin A5 by a calcium chelator BAPTA/AM. Although a calcium ionophore ionomycin stimulated the expression of both genes, only LHβ was down-regulated. The MAPK kinase inhibitor PD98059 inhibited GnRHa induction of annexin A5 but not LHβ mRNA. EGF stimulated the expression of annexin A5 mRNA but caused only a transient effect on LHβ mRNA expression. These results indicate that GnRH stimulation of signaling pathway for annexin A5 mRNA expression is distinct from that of LHβ mRNA and dependent more on MAPK. GnRH is a hypothalamic neuropeptide that regulates pituitary gonadotropin secretion. GnRH neuron secretes GnRH in a pulsatile fashon at the median eminence of the hypothalamus. Portal blood also shows pulsatile fluctuations of GnRH [1]. The frequency of the GnRH pulses seems to influence the different effects on cell function of the gonadotropes [2][3][4]. Recently, we demonstrated that GnRH stimulates the expression of annexin A5 mRNA in gonadotropes [5][6][7][8]. Because ovariectomy enhanced annexin A5 expression in the gonadotropes and estradiol inhibited it, the expression of pituitary annexin A5 was thought to be physiologically regulated by hypothalamic GnRH [6]. We also demonstrated that annexin A5 participates in GnRH control of gonadotropin secretion. Annexin A5 itself stimulates both LH and FSH release, while an annexin A5 antisense oligonucleotide reduces GnRH stimulation of LH [7]. These findings suggest that intracellular annexin A5 is requisite for GnRH stimulation of gonadotropin secretion.In mammals, the annexin family consists of a group of at least 12 Ca 2+ -dependent phospholipid binding proteins [9,10]. Annexin A1 was first identified as a mediator of the anti-inflammatory activity of glucocorticoids [11]. The identification of other annexins followed, including annexin A5 that was discovered as a protein that inhibits phospholipase A 2 and suppresses blood coagulation [12,13]. These activities are thought to be due to its ability to bind phospholipids, although it is not clear whether these activities are relevant to physiological function. However, as we showed in the gonadotropes, annexin A5 may be at least necessary for an intracellular signaling in some cell types [14][15][16].

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