title: CCAAT/enhancer binding protein beta regulates aromatase expression via multiple and novel cis-regulatory sequences in uterine leiomyoma.
abstract: 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.
authors.affiliation: Division of Reproductive Biology Research, Feinberg School of Medicine at Northwestern University, 303 Superior Street, Suite 4-123, Chicago, Illinois 60611, USA.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ishikawa
authors.forename: Veysel
authors.initials: V
authors.lastname: Fenkci
authors.forename: Veysel
authors.initials: V
authors.lastname: Fencki
authors.forename: Erica E
authors.initials: EE
authors.lastname: Marsh
authors.forename: Ping
authors.initials: P
authors.lastname: Yin
authors.forename: Dong
authors.initials: D
authors.lastname: Chen
authors.forename: You-Hong
authors.initials: YH
authors.lastname: Cheng
authors.forename: Scott
authors.initials: S
authors.lastname: Reisterd
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Lin
authors.forename: Serdar E
authors.initials: SE
authors.lastname: Bulun
mesh_terms: 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
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D003563:Cyclic CMP; C053441:dibutyryl cyclic-3',5'-cytidine monophosphate; D000242:Cyclic AMP; D001141:Aromatase