USF1 and USF2 mediate inhibition of human trophoblast differentiation and CYP19 gene expression by Mash-2 and hypoxia
USF1 and USF2 mediate inhibition of human trophoblast differentiation and CYP19 gene expression by Mash-2 and hypoxia
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DOI：
10.1128/mcb.23.17.6117-6128.2003
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发表时间：
2003-09-01
影响因子：
5.3
通讯作者：
Mendelson, CR
中科院分区：
文献类型：
--
作者：
Jiang, B;Mendelson, CR
在人胎盘合体滋养细胞中，C-19类固醇通过芳香酶P450 （CYP19基因的产物）转化为雌激素。缺乏芳香化酶表达能力的人细胞滋养细胞在20%的O- 2，o -中培养时，会自发融合形成多核合胞滋养细胞，CYP19的表达被显著诱导。另一方面，当细胞滋养层细胞在2% O-2中培养时，合胞滋养层细胞分化和诱导CYP19表达被阻止。我们之前观察到，转录因子Mash-2（哺乳动物无毛细胞/鳞片同源物2）的表达在人类细胞滋养层细胞中升高，并在缺氧条件下维持在较高水平，随着合胞滋养层细胞分化而下降。Mash-2过表达可阻止合体滋养细胞分化并诱导CYP19表达。在本研究中，我们意外地发现免疫反应性的Mash-2蛋白主要定位于人细胞滋养细胞的细胞质中。在2% O-2中培养滋养细胞时，细胞质中mash2的水平保持升高，在20% O-2中培养时下降到检测不到的水平。之前，我们发现mash2通过上游350 bp区域和胎盘特异性外显子I.1内的序列抑制CYP19启动子活性，其中包含三个E盒（E1位于-325 bp， 5'-CACTTG-3‘； E2位于-58 bp， 5’-CACATG-3‘； E3位于+26 bp， 5’-CACGTG-3'）。在本研究中，我们发现，在缺氧诱导下，与这些E盒结合的滋养细胞核蛋白随着20% O-2的合胞滋养细胞分化而下降；然而，Mash-2似乎没有绑定到这些电子邮箱。另一方面，基本螺旋-环-螺旋亮氨酸拉链转录因子上游刺激因子1和2 （USF1和USF2）与E2和E3结合，但不与E1结合。USF1和USF2的核表达水平和dna结合活性随着合胞滋养细胞分化而下降，并通过缺氧和过表达Mash-2维持在较高水平，而USF1 mRNA水平不受影响。最后，培养的人滋养层细胞中USF1过表达显著抑制内源性CYP19表达、培养的人滋养层细胞分化和CYP19启动子活性。这些结果表明，USF1和USF2蛋白水平的升高和DNA结合介导了缺氧和Mash-2对人胎盘CYP19基因表达的抑制作用。
In the human placental syncytiotrophoblast, C-19 steroids are converted to estrogens by aromatase P450, product of the CYP19 gene. When human cytotrophoblasts, which lack the capacity to express aromatase, are cultured in 20% O-2,O- they spontaneously fuse to form a multinuclear syncytiotrophoblast and CYP19 expression is markedly induced. On the other hand, when cytotrophoblasts are cultured in 2% O-2, syncytiotrophoblast differentiation and induction of CYP19 expression are prevented. We previously observed that expression of the transcription factor Mash-2 (mammalian achaete/scute homologue 2), which is elevated in human cytotrophoblasts and maintained at elevated levels by hypoxia, declines with syncytiotrophoblast differentiation. Overexpression of Mash-2 prevents syncytiotrophoblast differentiation and induction of CYP19 expression. In the present study, we observed that unexpectedly immunoreactive Mash-2 protein was localized predominately to the cytoplasm of human cytotrophoblasts. Elevated cytoplasmic levels of Mash-2 were maintained when trophoblasts were cultured in 2% O-2 and declined to undetectable levels upon culture in 20% O-2 Previously, we found that Mash-2 inhibited CYP19 promoter activity through sequences within a 350-bp region upstream and within placenta-specific exon I.1 containing three E boxes (E1 at -325 bp, 5'-CACTTG-3'; E2 at -58 bp, 5'-CACATG-3'; and E3 at +26 bp, 5'-CACGTG-3'). In this study, we found that trophoblast nuclear protein binding to these E boxes declined with syncytiotrophoblast differentiation in 20% O-2 and was induced by hypoxia; however, Mash-2 did not appear to bind to any of these E boxes. On the other hand, the basic helix-loop-helix leucine zipper transcription factors upstream stimulatory factors 1 and 2 (USF1 and USF2) did bind to E2 and E3 but not E1. Nuclear levels of USF1 and USF2 and DNA-binding activity declined with syncytiotrophoblast differentiation and were maintained at elevated levels by hypoxia and overexpression of Mash-2, whereas USF1 mRNA levels were unaffected. Finally, USF1 overexpression in cultured human trophoblasts markedly inhibited endogenous CYP19 expression, differentiation of cultured human trophoblast cells, and CYP19 promoter activity. These findings suggest that increased protein levels and DNA binding of USF1 and USF2 mediate the inhibitory effects of hypoxia and of Mash-2 on CYP19 gene expression in human placenta.