CCAAT/enhancer binding protein beta regulates aromatase expression via multiple and novel cis-regulatory sequences in uterine leiomyoma.
BACKGROUND
Control of aromatase expression in uterine leiomyoma has significant clinical implications because aromatase inhibitors reduce tumor growth and associated irregular uterine bleeding. The mechanisms that regulate aromatase expression in leiomyoma are unknown.


OBJECTIVE
We previously demonstrated that the cAMP-responsive proximal promoters I.3 and II regulate aromatase expression in vivo in uterine leiomyoma tissue. Here, we investigated the cellular and molecular mechanisms responsible for promoter I.3/II usage.


RESULTS
In smooth muscle cells isolated from leiomyoma (LSMCs), dibutyryl cAMP significantly induced aromatase mRNA and enzyme activity. Reporter constructs of promoter I.3/II deletion and site-directed mutants with selective disruption of cis-regulatory elements in the -517/-16 bp region revealed that five out of seven elements, including three CCAAT/enhancer binding protein (C/EBP) binding sites and two cAMP response elements, were essential for cAMP-induced promoter activity. EMSAs demonstrated that nuclear extracts from LSMCs contain complexes assembled on four of the five cis-elements, with C/EBP binding sites, including a novel -245/-231 bp sequence, clearly associating with C/EBPbeta. Chromatin immunoprecipitation assays revealed that C/EBPbeta binds specifically to the promoter I.3/II region in intact cells. Dibutyryl cAMP significantly induced nuclear C/EBPbeta protein levels in LSMCs in a time-dependent manner. Conversely, knockdown of C/EBPbeta dramatically suppressed cAMP-induced aromatase mRNA and enzyme activity.


CONCLUSIONS
C/EBPbeta, which binds to multiple cis-regulatory elements in promoter I.3/II, is a key factor in the transcriptional complex controlling aromatase expression in uterine leiomyoma cells. Definition of this mechanism further may assist in designing inhibitors of aromatase specific for leiomyoma tissue.
Ishikawa
Hiroshi
H
Division of Reproductive Biology Research, Feinberg School of Medicine at Northwestern University, 303 Superior Street, Suite 4-123, Chicago, Illinois 60611, USA.
Fenkci
Veysel
V
Fencki
Veysel
V
Marsh
Erica E
EE
Yin
Ping
P
Chen
Dong
D
Cheng
You-Hong
YH
Reisterd
Scott
S
Lin
Zhihong
Z
Bulun
Serdar E
SE
D001141:Aromatase / Q000235:genetics*; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000032:analysis / Q000502:physiology*; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000502:physiology; D003563:Cyclic CMP / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007889:Leiomyoma / Q000201:enzymology* / Q000473:pathology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D014594:Uterine Neoplasms / Q000201:enzymology* / Q000473:pathology
D022782:CCAAT-Enhancer-Binding Protein-beta; D003563:Cyclic CMP; C053441:dibutyryl cyclic-3',5'-cytidine monophosphate; D000242:Cyclic AMP; D001141:Aromatase