Transforming growth factor-beta inhibits aromatase gene transcription in human trophoblast cells via the Smad2 signaling pathway.
BACKGROUND
Transforming growth factor-beta (TGF-beta) is known to exert multiple regulatory functions in the human placenta, including inhibition of estrodial production. We have previously reported that TGF-beta1 decreased aromatase mRNA levels in human trophoblast cells. The objective of this study was to investigate the molecular mechanisms underlying the regulatory effect of TGF-beta1 on aromatase expression.


METHODS
To determine if TGF-beta regulates aromatase gene transcription, several reporter constructs containing different lengths of the placental specific promoter of the human aromatase gene were generated. JEG-3 cells were transiently transfected with a promoter construct and treated with or without TGF-beta1. The promoter activity was measured by luciferase assays. To examine the downstream signaling molecule mediating the effect of TGF-beta on aromatase transcription, cells were transiently transfected with dominant negative mutants of TGF-beta type II (TbetaRII) and type I receptor (ALK5) receptors before TGF-beta treatment. Smad2 activation was assessed by measuring phophorylated Smad2 protein levels in cytosolic and nuclear fractions. Smad2 expression was silenced using a siRNA expression construct. Finally, aromatase mRNA half-life was determined by treating cells with actinomycin D together with TGF-beta1 and measuring aromatase mRNA levels at various time points after treatment.


CONCLUSIONS
TGF-beta1 inhibited the aromatase promoter activity in a time- and dose-dependent manner. Deletion analysis suggests that the TGF-beta1 response element resides between -422 and -117 nucleotides upstream from the transcription start site where a Smad binding element was found. The inhibitory effect of TGF-beta1 was blocked by dominant negative mutants of TbetaRII and ALK5. TGF-beta1 treatment induced Smad2 phosphorylation and translocation into the nucleus. On the other hand, knockdown of Smad2 expression reversed the inhibitory effect of TGF-beta1 on aroamtase transcription. Furthermore, TGF-beta1 accelerated the degradation of aromatase mRNA.


CONCLUSIONS
Our results demonstrate that TGF-beta1 exerts regulatory effects on aromatase gene at both transcriptional and post-transcriptional levels. The transcriptional regulation of aromatase gene by TGF-beta1 is mediated by the canonical TGF-beta pathway involving TbetaRII, ALK5 and Smad2. These findings further support the role of TGF-beta1 in regulating human placental functions and pregnancy.
Zhou
Hong
H
Department of Biology, York University, Toronto, Ontario M3J1P3, Canada. zhouhongzh@uestc.edu.cn
Fu
Guodong
G
Yu
Hui
H
Peng
Chun
C
D001141:Aromatase / Q000096:biosynthesis* / Q000235:genetics; D015153:Blotting, Western; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D066298:In Vitro Techniques; D008156:Luciferases / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D051899:Smad2 Protein / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D014327:Trophoblasts / Q000201:enzymology*
D012333:RNA, Messenger; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D053773:Transforming Growth Factor beta1; D008156:Luciferases; D001141:Aromatase