TY - JOUR
T1 - CCAAT/enhancer binding protein β regulates aromatase expression via multiple and novel cis-regulatory sequences in uterine leiomyoma
AU - Ishikawa, Hiroshi
AU - Fencki, Veysel
AU - Marsh, Erica E.
AU - Yin, Ping
AU - Chen, Dong
AU - Cheng, You Hong
AU - Reisterd, Scott
AU - Lin, Zhihong
AU - Bulun, Serdar E.
N1 - Funding Information:
This work was supported by National Institutes of Health Grant HD46260, Avon Foundation, Lynn Sage Foundation, and Friends of Prentice.
PY - 2008/3
Y1 - 2008/3
N2 - Context: 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. Objectives: 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/EBPβ. Chromatin immunoprecipitation assays revealed that C/EBPβ binds specifically to the promoter I.3/II region in intact cells. Dibutyryl cAMP significantly induced nuclear C/EBPβ protein levels in LSMCs in a time-dependent manner. Conversely, knockdown of C/EBPβ dramatically suppressed cAMP-induced aromatase mRNA and enzyme activity. Conclusions: C/EBPβ, 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.
AB - Context: 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. Objectives: 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/EBPβ. Chromatin immunoprecipitation assays revealed that C/EBPβ binds specifically to the promoter I.3/II region in intact cells. Dibutyryl cAMP significantly induced nuclear C/EBPβ protein levels in LSMCs in a time-dependent manner. Conversely, knockdown of C/EBPβ dramatically suppressed cAMP-induced aromatase mRNA and enzyme activity. Conclusions: C/EBPβ, 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.
UR - http://www.scopus.com/inward/record.url?scp=40849100077&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=40849100077&partnerID=8YFLogxK
U2 - 10.1210/jc.2007-2507
DO - 10.1210/jc.2007-2507
M3 - Article
C2 - 18182446
AN - SCOPUS:40849100077
VL - 93
SP - 981
EP - 991
JO - Journal of Clinical Endocrinology and Metabolism
JF - Journal of Clinical Endocrinology and Metabolism
SN - 0021-972X
IS - 3
ER -