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 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.

Human Prolyl-4-hydroxylase α(I) Transcription Is Mediated by Upstream Stimulatory Factors

“…The E-box, the consensus sequence of which is CANNTG, is known as the binding site for USF and regulates the transcription of several genes (37,38). They appear to be involved in the regulation of developmental and tissue-specific or gene-specific expression of a number of genes (39,40), including those for surfactant protein A (41,42), steroidogenic factor 1 (43), and carboxyl ester lipase (44).…”

Hypoxia-Inducible Factors 1α and 2α Regulate Trophoblast Differentiation

“…To further demonstrate that differentiated mutant TS cells produce syncytiotrophoblasts, we analyzed TS cells for additional lineage-specific markers. We examined the expression of Mash2, a bHLH transcription factor essential for spongiotrophoblast formation (25,26). Undifferentiated Arnt ϩ/ϩ and Hif1␣ ϩ/ϩ Hif2␣ ϩ/ϩ TS cells expressed low Mash2 mRNA levels, which increased upon differentiation for 4 days (Fig.…”

Roles of SRF in Endothelial Cells During Hypoxia

“…A number of transcription factors have been reported to respond to oxygen deficiency, including AP-1 [3], FOS [4], JUN [4], CREB/ATF [5], DEC1 [6], EGR1 [7], ETS1 [8], GADD153 [9], GATA2 [10], MASH2 [11], NF-IL-6 [12], NFĸB [13], RTEF-1 [14], SMADs [15], SP1 [16], STAT5 [17], and of course, the most popular ones, HIF [18] and p53 [19].…”