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O2 enhancement of human trophoblast differentiation and hCYP19 (aromatase) gene expression are mediated by proteasomal degradation of USF1 and USF2.

ABSTRACT: When cultured in 20% O(2), human cytotrophoblasts fuse to form the syncytiotrophoblast with marked induction of hCYP19 (aromatase) gene expression. When cultured in 2% O(2), cytotrophoblast fusion and induced hCYP19 expression are prevented. These effects of hypoxia are mediated by increased expression of mammalian achaete/scute homologue-2 (Mash-2), which increases levels of upstream stimulatory factors 1 and 2 (USF1/2) and their binding as heterodimers to E-boxes surrounding the hCYP19 promoter. In studies to define mechanisms for O(2) regulation of syncytiotrophoblast differentiation, we found that hypoxia and overexpression of Mash-2 markedly increased cyclin B1 levels in cultured trophoblasts and the proportion of cells at the G(2)/M transition. Unlike USF proteins, USF1/2 mRNA levels are unaffected by O(2) tension. To determine whether increased O(2) might enhance proteasomal degradation of USF1/2, human trophoblasts were cultured in 2% or 20% O(2) with or without proteasome inhibitors. In cells cultured in 20% O(2), proteasome inhibitors increased USF1/2 protein levels and blocked spontaneous induction of hCYP19 expression, cell fusion, and differentiation. Like hypoxia, inhibitory effects of proteasome inhibitors on hCYP19 expression were mediated by increased binding of USF1/2 to the E-boxes. In human trophoblast cells cultured in 20% O(2), increased polyubiquitylation of USF1/2 proteins was observed. Thus, early in gestation when the placenta is relatively hypoxic, increased USF1/2 may block trophoblast differentiation and hCYP19 gene expression. In the second trimester, increased O(2) tension promotes proteasomal degradation of USF1/2, resulting in syncytiotrophoblast differentiation and induction of hCYP19 expression.

SUBMITTER: Jiang B

PROVIDER: S-EPMC1265767 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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O2 enhancement of human trophoblast differentiation and hCYP19 (aromatase) gene expression are mediated by proteasomal degradation of USF1 and USF2.

Jiang Bing B Mendelson Carole R CR

Molecular and cellular biology 20051001 20

When cultured in 20% O(2), human cytotrophoblasts fuse to form the syncytiotrophoblast with marked induction of hCYP19 (aromatase) gene expression. When cultured in 2% O(2), cytotrophoblast fusion and induced hCYP19 expression are prevented. These effects of hypoxia are mediated by increased expression of mammalian achaete/scute homologue-2 (Mash-2), which increases levels of upstream stimulatory factors 1 and 2 (USF1/2) and their binding as heterodimers to E-boxes surrounding the hCYP19 promote ...[more]

PMID: 16199862

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Project description: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), 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 predominantly 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.

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Dataset Information

O2 enhancement of human trophoblast differentiation and hCYP19 (aromatase) gene expression are mediated by proteasomal degradation of USF1 and USF2.

Publications

O2 enhancement of human trophoblast differentiation and hCYP19 (aromatase) gene expression are mediated by proteasomal degradation of USF1 and USF2.

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