Regulation of Placental Leptin Expression by Cyclic Adenosine 5′-Monophosphate Involves Cross Talk between Protein Kinase A and Mitogen-Activated Protein Kinase Signaling Pathways
Regulation of Placental Leptin Expression by Cyclic Adenosine 5′-Monophosphate Involves Cross Talk between Protein Kinase A and Mitogen-Activated Protein Kinase Signaling Pathways
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DOI：
10.1210/en.2010-0064
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发表时间：
2010-08-01
期刊：
影响因子：
4.8
通讯作者：
Varone, Cecilia L.
中科院分区：
文献类型：
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作者：
Maymo, Julieta L.;Perez Perez, Antonio;Varone, Cecilia L.
瘦素是一种主要由脂肪组织产生的16 kda蛋白，通过其下丘脑受体参与能量平衡的控制。然而，瘦素在生殖和妊娠中的多效性作用已被确定，特别是在胎盘中，瘦素被发现在胎盘中表达。在本研究中，我们研究了cAMP在滋养细胞中调节瘦素表达的作用。通过Western blot和定量RT-PCR分析，我们发现cAMP类似物二丁基cAMP [(Bu)(2)cAMP]对BeWo和JEG-3细胞系内源性瘦素表达有诱导作用。通过构建报告质粒，瞬时转染瘦素启动子，评估其活性。在人足月胎盘外植体中也得到了类似的结果，从而表明了生理学上的相关性。由于cAMP通常通过激活蛋白激酶A （PKA）信号来发挥作用，因此我们对这一途径进行了分析。我们发现cAMP反应元件结合蛋白（CREB）磷酸化在（Bu） 2cAMP处理下显著增加。此外，与PKA催化亚基和/或转录因子CREB共转染可显著刺激瘦素启动子活性。另一方面，与PKA调控亚基显性负突变体共转染可抑制瘦素启动子活性。在BeWo细胞和人胎盘外植体中，我们发现PKA或腺苷酸环化酶的药理抑制可以阻断cAMP的作用。因此，我们决定研究MAPK/ERK信号通路在cAMP对瘦素诱导作用中的作用。我们发现，50 μ M PD98059，一种MAPK激酶抑制剂，通过Western blot分析和瞬时转染实验检测，部分阻断了cAMP对瘦素的诱导。此外，ERK 1/2磷酸化在（Bu） 2cAMP处理下显著增加，且这种作用是剂量依赖性的。最后，我们观察到50 μ M PD98059抑制了胎盘外植体中camp依赖性CREB的磷酸化。总之，我们提供了一些证据表明cAMP诱导胎盘细胞中瘦素的表达，并且这种作用似乎是由PKA和MAPK信号通路之间的串扰介导的。（中华医学会精神病学分会，2010）
Leptin, a 16-kDa protein mainly produced by adipose tissue, has been involved in the control of energy balance through its hypothalamic receptor. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in placenta, where it was found to be expressed. In the current study, we examined the effect of cAMP in the regulation of leptin expression in trophoblastic cells. We found that dibutyryl cAMP [(Bu)(2)cAMP], a cAMP analog, showed an inducing effect on endogenous leptin expression in BeWo and JEG-3 cell lines when analyzed by Western blot analysis and quantitative RT-PCR. Maximal effect was achieved at 100 mu M. Leptin promoter activity was also stimulated, evaluated by transient transfection with a reporter plasmid construction. Similar results were obtained with human term placental explants, thus indicating physiological relevance. Because cAMP usually exerts its actions through activation of protein kinase A (PKA) signaling, this pathway was analyzed. We found that cAMP response element-binding protein (CREB) phosphorylation was significantly increased with (Bu) 2cAMP treatment. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor CREB caused a significant stimulation on leptin promoter activity. On the other hand, the cotransfection with a dominant negative mutant of the regulatory subunit of PKA inhibited leptin promoter activity. We determined that cAMP effect could be blocked by pharmacologic inhibition of PKA or adenylyl ciclase in BeWo cells and in human placental explants. Thereafter, we decided to investigate the involvement of the MAPK/ERK signaling pathway in the cAMP effect on leptin induction. We found that 50 mu M PD98059, a MAPK kinase inhibitor, partially blocked leptin induction by cAMP, measured both by Western blot analysis and reporter transient transfection assay. Moreover, ERK 1/2 phosphorylation was significantly increased with (Bu) 2cAMP treatment, and this effect was dose dependent. Finally, we observed that 50 mu M PD98059 inhibited cAMP-dependent phosphorylation of CREB in placental explants. In summary, we provide some evidence suggesting that cAMP induces leptin expression in placental cells and that this effect seems to be mediated by a cross talk between PKA and MAPK signaling pathways. (Endocrinology 151: 3738-3751, 2010)