USF1 and USF2 mediate inhibition of human trophoblast differentiation and CYP19 gene expression by Mash-2 and hypoxia

Abstract

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.

FIG. 1.

Mash-2 protein levels, which decline in human trophoblast cells upon differentiation in a 20% O₂ environment and are maintained in trophoblast cells cultured in a hypoxic environment, are primarily localized to the cytoplasm. (A) Immunoblot analysis of hMash-2 protein in nuclear and cytosolic fractions (20 μg/lane) from human cytotrophoblasts before culture (Cyto) and after 3 days of culture in a 20% O₂ environment (Syn) (left panel) or in human trophoblasts cultured in either 2 or 20% O₂ for 3 days (right panel). C, cytoplasmic; N, nuclear. (B) Immunofluorescence staining of hMash-2 protein in freshly isolated cytotrophoblasts (Cyto) or in trophoblast cells cultured for 3 days in either a 2 or a 20% O₂ environment.

FIG. 2.

Binding activity of human trophoblast nuclear proteins for the E1, E2, and E3 boxes upstream and within hCYP19 placenta-specific exon I.1 is elevated in cytotrophoblasts, declines with syncytiotrophoblast differentiation in a 20% O₂ environment, and is maintained at elevated levels by hypoxia (2% O₂). (A) Schematic of three E-box consensus sequences at −325 bp (E1), −58 bp (E2) upstream, and +26 bp (E3) within hCYP19 placenta-specific exon I.1. (B) EMSA of binding of nuclear proteins from freshly isolated human cytotrophoblasts (Cyto) and from syncytiotrophoblast (Syn) after 3 days of culture in a 20% O₂ environment to ³²P-labeled oligonucleotides containing the E1, E2, and E3 boxes in the absence (−) or presence of a 500-fold excess of nonradiolabeled (Cold) E1, E2, or E3 or nonrelated (non-spec) oligonucleotides. (C) EMSA of binding of nuclear proteins from human trophoblast cells after 3 days of culture in a 2 or 20% O₂ environment to a ³²P-labeled oligonucleotide containing the E2 box in the absence (−) or presence of a 500-fold excess of nonradiolabeled (Cold) E2 or nonrelated (non-spec) oligonucleotides. Binding of nuclear proteins to the E1 and E3 boxes was not affected by hypoxia (data not shown).

FIG. 3.

Nuclear levels of USF1 and USF2 proteins decline with syncytiotrophoblast differentiation and are maintained at elevated levels by hypoxia and Mash-2 overexpression, while USF1 mRNA levels are unaffected. (A) Nuclear (N) and cytoplasmic (C) fractions were prepared from human trophoblasts before (Cyto) and after (Syn) culture for 3 days in 20% O₂ or in an independent experiment after 3 days of culture in an atmosphere of 2 or 20% O₂. Twenty micrograms of protein was analyzed for USF1 (43 kDa) and USF2 (44 kDa) by immunoblotting. (B) Nuclear (N) and cytoplasmic (C) fractions were prepared from human trophoblasts before culture (Cyto) or from trophoblasts that were infected with recombinant adenovirus expressing either Mash-2 (CMV-Mash-2; MOI = 5) or β-Gal (CMV-β-Gal; MOI = 5) and analyzed for USF1 and USF2 by immunoblotting (20 μg of protein/lane). (C) Total RNA isolated from human trophoblast cells cultured for 72 h in an atmosphere of 2 or 20% O₂ or from trophoblast cells cultured in 20% O₂ and infected with recombinant adenoviruses containing CMV-β-Gal or CMV-Mash-2 at an MOI of 0.5 to 5.0 was analyzed for hUSF1 mRNA levels by Northern blotting.

FIG. 4.

USF1 and USF2 in trophoblast nuclear extracts bind to the E2 and E3 boxes upstream and within hCYP19 exon I.1, respectively, but not to the E1 box; endogenous USF binding activity declines with syncytiotrophoblast differentiation. Nuclear extracts from human cytotrophoblasts before (Cyto) and after 3 days of culture in a 20% O₂ environment (Syn) were incubated with ³²P-labeled oligonucleotides containing the E1, E2, and E3 boxes in the absence (−) or presence (+) of antibodies specific for USF1 and USF2 and analyzed by EMSA.

FIG. 5.

Mash-2 overexpression increases binding activities of USF1 and USF2 proteins for the E2 and E3 boxes upstream and within hCYP19 exon I.1. Freshly isolated cytotrophoblasts were infected with recombinant adenoviruses expressing either Mash-2 (CMV-Mash-2; MOI = 5) or β-Gal (CMV-β-Gal; MOI = 5) and cultured for 3 days in a 20% O₂ environment. Nuclear proteins isolated from the cultured cells were incubated with ³²P-labeled oligonucleotides containing the E1, E2, and E3 boxes in the absence (−) or presence (+) of antibodies specific for USF1 and USF2 and analyzed by EMSA.

FIG. 6.

USF1 overexpression prevents the induction of endogenous CYP19 gene expression in cultured human trophoblast cells and causes a dose-dependent inhibition of expression of a transfected CYP19I.1₋₅₀₁:hGH fusion gene. (A) Northern blot analysis of endogenous CYP19 mRNA levels in cultured human trophoblast cells infected with recombinant adenoviruses expressing either USF1 (MOI = 5 or 10) or β-Gal (MOI = 10). The cDNA probes used were specific for hCYP19, hUSF1, and β-actin mRNAs. (B) hCYP19 promoter I.1 activity in cultured human trophoblasts coinfected with a recombinant adenovirus containing a CYP19I.1₋₅₀₁:hGH fusion gene and increasing MOI of a recombinant adenovirus expressing hUSF1 (MOI = 0.5, 1, 5, or 10), or β-Gal (MOI = 10). Data are the means ± standard errors of the means of the hGH secreted into the medium between 48 and 72 h of incubation (n = 3).

FIG. 7.

USF1 overexpression prevents trophoblast cell fusion and differentiation. Freshly isolated cytotropholast cells were cultured on coverslips and infected with different amounts of recombinant adenovirus expressing either USF1 (MOI = 5 or 10) or β-Gal (MOI = 10). After 72 h, the cells on the coverslips were stained with hematoxylin and eosin. Magnification, ×200.

FIG. 8.

Possible mechanism for oxygen-mediated induction of human trophoblast differentiation and CYP19 gene expression. During the first trimester of human gestation the placenta exists in a relatively hypoxic environment, resulting in the maintenance of elevated expression of Mash-2 and consequent induction of USF1 and USF2 gene expression. USF1 and USF2, in turn, bind as heterodimers to the E2 and E3 boxes upstream and within hCYP19 placenta-specific exon I.1, respectively, preventing stable assembly of the basal transcription complex and inhibition of CYP19I.1 promoter activity. The elevated levels of USF1 and USF2 also increase cytotrophoblast proliferation and prevent their fusion to form syncytiotrophoblast. Upon cytotrophoblast invasion of the spiral arterioles and the resulting increase in oxygenation, the levels of Mash-2 decline, resulting in decreased USF1 and USF2 expression with increased syncytiotrophoblast differentiation. Decreased binding of USF1 and USF2 to the E2 and E3 boxes, in turn, may facilitate stable assembly of the basal transcription complex and activation of CYP19 gene expression by tissue-specific and general enhancer factors (TSEF and GEF, respectively).