Harada Takashi, Kanasaki Haruhiko, Mutiara Sandra, Oride Aki, Miyazaki Kohji
Department of Obstetrics and Gynecology, Shimane University School of Medicine, Izumo 693-8501, Japan.
Biol Reprod. 2007 Oct;77(4):707-16. doi: 10.1095/biolreprod.107.060327. Epub 2007 Jun 27.
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.
腺苷酸环化酶激活多肽1（ADCYAP1）可与Gs和Gq偶联受体结合，并刺激垂体促性腺细胞中的腺苷酸环化酶/cAMP和蛋白激酶C/丝裂原活化蛋白激酶3/1（MAPK3/1）信号通路。在本研究中，我们研究了ADCYAP1刺激诱导的cAMP和MAPK3/1信号通路，并使用克隆促性腺细胞系LbetaT2检测了ADCYAP1对促性腺激素亚基基因表达的影响。ADCYAP1可使LbetaT2细胞内的cAMP积累增加至19倍。普通α-糖蛋白亚基基因（Cga）启动子活性受到ADCYAP1和环磷酸腺苷类似物8-（4-氯苯硫基）腺苷3'，5'-环磷酸单酯（CPT-cAMP）的强烈激活。二者对促黄体生成素β（Lhb）和促卵泡激素β（Fshb）启动子活性影响较小。组成型活性环磷酸腺苷依赖性蛋白激酶（PKA）转染可显著增加Cga启动子活性。Lhb和Fshb启动子活性仅适度增加。ADCYAP1和CPT-cAMP均可诱导LbetaT2细胞中的MAPK3/1激活。MEK抑制剂U0126以及PKA抑制剂H89和环磷酸腺苷依赖性蛋白激酶肽抑制剂（PKI）可完全抑制ADCYAP1或CPT-cAMP诱导的MAPK3/1激活。使用含有顺式元件的荧光素酶报告构建体，ADCYAP1可使环磷酸腺苷反应元件（Cre）启动子活性增强约4倍。ADCYAP1诱导的Cre启动子活性被H89完全抑制，但未被U0126抑制。ADCYAP1还可增加血清反应元件（Sre）启动子的活性，Sre启动子是MAPK3/1的作用靶点，用U0126处理细胞可完全抑制ADCYAP1诱导的Sre启动子活性。ADCYAP1增加的Cga启动子活性受到H89和U0126的部分抑制。虽然联合使用抑制剂显示出相加抑制作用，但并未导致完全抑制。这些结果表明，在LbetaT2细胞中，ADCYAP1主要通过激活PKA和MAPK3/1以及一条额外的未知途径来增加Cga。
