Aki Oride

Pulse Frequency-Dependent Gonadotropin Gene Expression by Adenylate Cyclase-Activating Polypeptide 1 in Perifused Mouse Pituitary Gonadotroph LbetaT2 cells

We examined how pulsatile stimulation with adenylate cyclase-activating polypeptide 1 (ADCYAP1) affected gonadotrophs. In static culture, gonadotropin-releasing hormone (GnRH) stimulated transcription of all the gonadotropin subunits. In contrast, ADCYAP1 increased common alpha-glycoprotein subunit gene (Cga) promoter activity but failed to increase luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) promoters. Messenger RNAs for Lhb and Fshb were slightly but significantly increased by ADCYAP1 stimulation. The results of cotreatment of the cells with GnRH and ADCYAP1 was not different from the effects of GnRH alone on Lhb and Fshb transcriptional activities as well as on mRNA expressions. To determine the effect of pulsatile ADCYAP1 stimulation on gonadotropin subunit gene expression, perifused LbetaT2 cells were stimulated either at high frequency (5-min ADCYAP1 pulse every 30 min) or at low frequency (5-min ADCYAP1 pulse every 120 min). High-frequency ADCYAP1 pulses preferentially increased Lhb gene expression 2.29-fold ± 0.15-fold, and low frequency pulses resulted in a 1.55-fold ± 0.16-fold increase. Fshb gene expression was increased 1.87-fold ± 0.3-fold by high-frequency ADCYAP1 pulses and 4.3-fold ± 0.29-fold by low-frequency pulses. These results were similar to the frequency-specific effects of pulsatile GnRH. Follistatin (Fst) gene expression was specifically increased by high-frequency GnRH pulses. High-frequency ADCYAP1 pulses increased Fst to a larger extent (4.7-fold ± 0.57-fold) than did low-frequency pulse (2.72-fold ± 1.09-fold). ADCYAP1 receptor gene (Adcyap1r) expression was increased significantly following pulsatile GnRH regardless of pulse frequency. Low-frequency ADCYAP1 pulses, however, increased Adcyap1r expression (16.49-fold ± 8.41-fold) to a larger extent than high frequency pulses did. In addition, high-frequency ADCYAP1 pulses specifically increased Gnrhr (GnRH receptor) expression by 4.38-fold ± 0.81-fold; however, low-frequency pulses did not result in an increase. These results suggest that ADCYAP1, like GnRH, specifically regulates Lhb and Fshb subunit gene in a pulse frequency-specific manner. This regulation may involve alteration in numbers of GnRH and ADCYAP1 receptors as well as FST expression.

Cyclic Adenosine 3′,5′Monophosphate/Protein Kinase A and Mitogen-Activated Protein Kinase 3/1 Pathways Are Involved in Adenylate Cyclase-Activating Polypeptide 1-Induced Common Alpha-Glycoprotein Subunit Gene (Cga) Expression in Mouse Pituitary Gonadotroph LbetaT2 Cells

Abstract Adenylate cyclase-activating polypeptide 1 (ADCYAP1) binds both Gs- and Gq-coupled receptors and stimulates adenylate cyclase/cAMP and protein kinase C/mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathways in pituitary gonadotrophs. In this study, we investigated the cAMP and MAPK3/1 signaling pathways induced by ADCYAP1 stimulation and examined the effects of ADCYAP1 on the expression of gonadotropin subunit genes using a clonal gonadotroph cell line, LbetaT2. ADCYAP1 increased intracellular cAMP accumulation up to 19-fold in LbetaT2 cells. Common alpha-glycoprotein subunit gene (Cga) promoter activity was strongly activated by both ADCYAP1 and the cyclic-AMP analog, 8-(4-chlorophenylthio) adenosine 3′,5′-cyclic monophosphate (CPT-cAMP). Both had little effect on luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) promoter activities. Cga promoter activity was significantly increased by transfection with constitutively active cAMP-dependent protein kinase (PKA). Activities of the Lhb and Fshb promoters were only modestly increased. Both ADCYAP1 and CPT-cAMP induced MAPK3/1 activation in LbetaT2 cells. The MEK inhibitor, U0126, and the PKA inhibitors, H89 and cAMP-dependent protein kinase peptide inhibitor (PKI), completely inhibited MAPK3/1 activation by either ADCYAP1 or CPT-cAMP. Using luciferase reporter constructs containing cis-elements, the cAMP response element (Cre) promoter was stimulated about 4-fold by ADCYAP1. ADCYAP1-induced Cre promoter activity was completely inhibited by H89, but not by U0126. ADCYAP1 also increased the activity of the serum response element (Sre) promoter, a target for MAPK3/1, and treatment of the cells with U0126 completely inhibited ADCYAP1-induced Sre promoter activity. ADCYAP1-increased Cga promoter activity was inhibited partially by both H89 and U0126. Although combining the inhibitors showed an additive inhibition effect, it did not result in complete inhibition. These results suggest that in LbetaT2 cells, ADCYAP1 mainly increases Cga through activation of PKA and MAPK3/1, as well as through an additional unknown pathway.

Kisspeptin induces expression of gonadotropin-releasing hormone receptor in GnRH-producing GT1-7 cells overexpressing G protein-coupled receptor 54.

Kisspeptin signaling through its receptor is crucial for many reproductive functions. However, the molecular mechanisms and biomedical significance of the regulation of GnRH neurons by kisspeptin have not been adequately elucidated. In the present study, we found that kisspeptin increases GnRH receptor (GnRHR) expression in a GnRH-producing cell line (GT1-7). Because cellular activity of G protein-coupled receptor 54 (GPR54) and GnRHR was limited in GT1-7 cells, we overexpressed these receptors to clarify receptor function. Using luciferase reporter constructs, the activity of both the serum response element (Sre) promoter, a target for extracellular signal-regulated kinase (ERK), and the cyclic AMP (cAMP) response element (Cre) promoter were increased by kisspeptin. Although GnRH increased Sre promoter activity, the Cre promoter was not significantly activated by GnRH. Kisspeptin, but not GnRH, increased cAMP accumulation in these cells. Kisspeptin also increased the transcriptional activity of GnRHR; however, the effect of GnRH on the GnRHR promoter was limited and not significant. Transfection of GT1-7 cells with constitutively active MEK kinase (MEKK) and protein kinase A (PKA) increased GnRHR expression. In addition, GnRHR expression was further increased by co-overexpression of MEKK and PKA. The Cre promoter, but not the Sre promoter, was also further activated by co-overexpression of MEKK and PKA. GnRH significantly increased the activity of the GnRHR promoter in the presence of cAMP. The present findings suggest that kisspeptin is a potent stimulator of GnRHR expression in GnRH-producing neurons in association with ERK and the cAMP/PKA pathways.

Possible involvement of PACAP and PACAP type 1 receptor in GnRH-induced FSH β-subunit gene expression

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor, PACAP type 1 receptor (PAC1-R) play an important role in the induction of pituitary gonadotropins. In this present study, we examined whether the PAC1-R was involved in the action of gonadotropin-releasing hormone (GnRH) on gonadotropin FSHβ subunit expression. In a static culture, GnRH stimulation significantly increased PAC1-R expression as well as PACAP gene expression in the gonadotroph cell line, LβT2. Stimulation with low frequency GnRH pulses, which preferentially increase FSHβ, increased the expression of both the PAC1-R and the PACAP genes to a greater extent than did high frequency pulses. In the determination of transcriptional activity, the GnRH antagonist, cetrotide inhibited GnRH-induced FSHβ promoter activity completely, but PACAP6-38, a PACAP antagonist, had no effect on GnRH-induced FSHβ promoter activity. As expected, PACAP-induced FSHβ promoter activity was significantly prevented by PACAP6-38, but was not affected by cetrotide. PACAP6-38, however, significantly prevented GnRH-increased FSHβ mRNA expression. These observations suggest that GnRH-induced FSHβ gene expression is stimulated partially through PAC1-R by gonadotrophs producing PACAP or PAC1-R.

GnRH-induced PACAP and PAC1 receptor expression in pituitary gonadotrophs: A possible role in the regulation of gonadotropin subunit gene expression

We examined the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and the PACAP type 1 receptor (PAC1-R) mRNA following gonadotropin-releasing hormone (GnRH) stimulation using the gonadotroph cell line LbetaT2. GnRH stimulation increased PACAP and PAC1-R mRNA expression in a static culture. Increase in the cell surface density of the PAC1-R following transfection with PAC1-R expression vectors significantly increased gonadotropin LHbeta and FSHbeta subunit promoter activities following 100 nM PACAP stimulation. In addition, increasing concentrations of PACAP stimulation augmented the promoter activities for both LHbeta and FSHbeta in PAC-1R overexpressing cells. In the cells with PAC1-R, the effect of GnRH was further potentiated in the presence of PACAP from 5.31+/-0.93 to 9.89+/-0.38-fold for LHbeta and for FSHbeta subunit, respectively; from 2.58+/-0.31-fold by GnRH alone to 10.90+/-2.79-fold with PACAP. The combination treatment with GnRH and PACAP did not augment the ERK phosphorylation induced by GnRH alone. PACAP expectedly increased cAMP accumulation and this effect was significantly attenuated in the presence of GnRH. PACAP gene expression was more prominent following lower frequency GnRH pulses (every 120 min) in a perifused culture. Our results suggest that PACAP and PAC1-R are produced locally within the gonadotrophs following GnRH stimulation. They subsequently affect the gonadotrophs in an autocrine manner and modulate the GnRH pulse-dependent specific regulation of gonadotropin subunits.

Therapy‐related myelodysplasia and acute myeloid leukemia following paclitaxel‐ and carboplatin‐based chemotherapy in an ovarian cancer patient: a case report and literature review

Alkylating agents have strong leukemogenic potential. There are a number of recent acute myeloid leukemia (t-AML) cases related to previous paclitaxel exposure. These leukemias tend to be of aggressive subtypes with long-latency periods. Unlike previously reported cases, the present case was of the secondary acute megakaryoblastic myeloid leukemia (AML M7) subtype. Additionally, it did not harbor a translocation in chromosome 19. A 73-year-old woman was diagnosed with t-AML M7 with antecedent myelodysplasia. Leukemia followed a second induction of paclitaxel- and carboplatin-based chemotherapy for recurrent ovarian cancer. Her second induction began 25 months after completion of her first course of chemotherapy. The increased incidence of postpaclitaxel leukemia suggests a probable role for paclitaxel as a leukemogenic agent. It highlights the importance of assessing for leukemia risk factors prior to beginning paclitaxel therapy.

Induction of Dual-Specificity Phosphatase 1 (DUSP1) by Pulsatile Gonadotropin-Releasing Hormone Stimulation: Role for Gonadotropin Subunit Expression in Mouse Pituitary LbetaT2 Cells

In pituitary gonadotrophs, GnRH induces expression of the mitogen-activated protein kinases (MAPK3/1) dephosphorylating enzyme, dual-specificity phosphatase 1 (DUSP1). Here we examined DUSP1 expression levels following pulsatile GnRH stimulation of the LbetaT2 gonadotroph cells. DUSP1 expression was increased more prominently following high-frequency (every 30 min) GnRH pulse stimulation (7.02- ± 1.47-fold) than low-frequency (every 120 min) GnRH pulses (2.68- ± 0.09-fold). With high-frequency GnRH pulses, DUSP1 expression increased by 2.89- ± 0.32-fold 2 h after GnRH pulse initiation (four 5-min pulses). DUSP1 expression was not induced following lower frequency GnRH pulses, even when the GnRH concentration was increased. Under high-frequency conditions, MAPK3/1 phosphorylation was observed 10 min after the GnRH pulse and decreased to basal levels after 25 min. However, MAPK3/1 dephosphorylation did not occur concurrently with DUSP1 expression. Overexpression of MAP3K1, a kinase upstream of MAPK3/1, increased both the Lhb and the Fshb subunit promoter activities, which could be completely inhibited by cotransfection with DUSP1-expressing vectors. Serum response factor (Srf) promoter activities induced by MAP3K1 were also prevented by DUSP1 overexpression, confirming that MAPK3/1 has an important role in gonadotropin subunit gene expression. Both high- and low-frequency GnRH pulse stimulation failed to increase the Lhb and Fshb subunit gonadotropin gene expression levels upon DUSP1 overexpression. Our study demonstrates that DUSP1 is specifically expressed following high-frequency GnRH pulses and that this effect may participate in the differential regulation of gonadotropin subunit expression in association with MAPK3/1 phosphorylation.

Expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) potentiates the effects of GnRH on gonadotropin subunit gene expression

We examined the effect of the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) on gonadotropin-releasing hormone (GnRH)-induced gonadotropin subunit promoter activities using the LβT2 gonadotroph cell line. In mock transfected cells, GnRH-increased LHβ and FSHβ promoters up to 2.74 ± 0.15-fold and 1.6 ± 0.05-fold respectively. When cells were transfected with PAC1R, both LHβ and FSHβ promoter activities were further increased up to 6.1 ± 0.87-fold and 2.22 ± 0.43-fold following GnRH stimulation. ERK phosphorylation, serum response element (SRE) promoters, and cAMP response element (CRE) promoters stimulated by GnRH were also potentiated in the presence of increasing amounts of PAC1R. The EC50 values for LHβ and FSHβ gene transcription by GnRH were significantly decreased by overexpression of PAC1R. PACAP 6-38, a PACAP receptor antagonist, failed to reduce the effect of GnRH on gonadotropin promoter activities in PAC1R overexpressing cells, suggesting that the potentiation of the effects of GnRH by PAC1R expression was not related to an autocrine mechanism of PACAP produced in the gonadotrophs. Our current results show that the action of GnRH in the regulation of gonadotropin subunit expression is enhanced by the presence of PAC1Rs.

The involvement of phosphatidylinositol 3-kinase in gonadotropin-releasing hormone-induced gonadotropin α-and FSHβ-subunit genes expression in clonal gonadotroph LβT2 cells

Akt/protein kinase B (Akt/PKB), which is activated by phosphatidylinositol-3 kinase (PI3-kinase), plays an important role in cell survival and cell proliferation. Using the well differentiated, clonal gonadotroph cell line, LbetaT2, we examined (1) whether Akt/PKB was activated by gonadotropin-releasing hormone (GnRH); (2) the contribution of PI3-kinase-Akt/PKB pathway in each of gonadotropin subunit gene expression; (3) crosstalk between extracellular signal-regulated kinase (ERK) and Akt/PKB pathways. Insulin-like growth factor-1 (IGF-1) was used as Akt/PKBs classic activator. Western blot analyses using antibodies specific for the phosphorylated forms of ERK and Akt/PKB demonstrated that both were rapidly phosphorylated following treatment with GnRH and IGF-1. Akt/PKB activation by GnRH and IGF-1 was completely eliminated in the presence of the PI3-kinase inhibitor, LY 294002, but not in the presence of an Akt/PKB inhibitor. Interestingly, the total amount of Akt/PKB protein was dramatically increased in the presence of LY 294002. Phosphorylation of ERK was significantly increased in the presence of LY 294002 alone, and was further increased when GnRH was used in combination with LY 294002. In experiments using a luciferase reporter construct containing the serum response element (SRE), a known target of the ERK pathway, LY 294002 but not the Akt/PKB inhibitor increased SRE-luciferase activity. GnRH-induced SRE-luciferase activity was significantly increased by LY 294002. GnRH stimulation resulted in gonadotropin LHbeta, FSHbeta, and alpha-subunit promoter activation, while IGF-1 failed to stimulate any of them. GnRH-induced gonadotropin promoter activities were not modulated in the presence of an Akt/PKB inhibitor, but treatment with LY 294002 or Wortmannin resulted in a significant increase in alpha- and FSHbeta-subunit promoter activation, both with and without GnRH. LY 294002, but not the Akt/PKB inhibitor, significantly inhibited cell proliferation. These results suggest that GnRH-induced gonadotropin gene expression is not regulated through the Akt/PKB pathway; however, PI3-kinase may be involved in the negative regulation of alpha- and FSHbeta-subunit gene expression as well as cell proliferation.

Follistatin gene expression by gonadotropin-releasing hormone: A role for cyclic AMP and mitogen-activated protein kinase signaling pathways in clonal gonadotroph LβT2 cells

The purpose of the present study was to examine the signal transduction pathways involved in follistatin gene expression induced by GnRH in the LbetaT2 cell line. The LHbeta-subunit was predominantly increased by high frequency GnRH pulses (30 min interval); whereas low frequency pulses (120 min) increased FSHbeta. In a static culture, follistatin expression was significantly increased at 12 h (2.35 +/- 0.80-fold) after the addition of GnRH. Following pulsatile stimulation, follistatin mRNA was increased by high frequency GnRH pulses, but not by low frequency pulses. In a static culture, GnRH maximally activated extracellular signal-regulated kinase (ERK) 10 min (3.2 +/- 0.55-fold) after treatment. In addition, intracellular cAMP accumulated up to 2.1 +/- 0.76-fold. Follistatin promoter activity was significantly increased following transfection with either a constitutively active cAMP dependent protein kinase (PKA) or a constitutively active MEK kinase (MEKK). The induction of follistatin gene expression by GnRH was completely inhibited by H89, a protein kinase A inhibitor, and U0126, a MEK inhibitor. Follistatin gene expression was also activated by both PACAP and CPT-cAMP under static culture conditions. Maximal ERK activation levels were nearly identical regardless of GnRH pulse frequency; however, high frequency GnRH pulses elevated both the intracellular cAMP level as well as cAMP-response element (Cre) promoter activity. These results suggest that both the PKA and ERK pathways are necessary for the induction of the follistatin promoter. Furthermore, the intracellular cAMP level, but not ERK activity, determined whether follistatin was induced following high frequency GnRH pulses.