title: Recombinant BMP4 and BMP7 downregulate pentraxin 3 in human granulosa cells.
abstract: BACKGROUND
  Theca cell-derived bone morphogenetic protein 4 (BMP4) and BMP7 are important regulators of folliculogenesis and have been shown to inhibit luteinization. Pentraxin 3 (PTX3) plays a critical role in the assembly of the cumulus oophorus extracellular matrix, which is essential for cumulus expansion during ovulation and may be modulated by BMP4 and BMP7.
  
  
  OBJECTIVE
  The aim of this study was to investigate the effects of BMP4 and BMP7 on the expression of PTX3 in human granulosa cells and to examine their underlying molecular determinants.
  
  
  METHODS
  An established immortalized human granulosa cell line (SVOG), a granulosa cell tumor cell line (KGN), and primary granulosa-lutein cells were used as study models. PTX3 expression and accumulation as well as Smad1/5/8 phosphorylation were examined after exposure to recombinant human BMP4 and BMP7. BMP type I receptor involvement was investigated with inhibitors (dorsomorphin and DMH-1 (4-[6-[4-(1-Methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline)) and small interfering RNAs targeting activin receptor-like kinase (ALK)2, ALK3, and/or ALK6. Small interfering RNAs targeting Smad4 were used to verify the involvement of Smad signaling.
  
  
  METHODS
  The study was conducted at an academic research center.
  
  
  METHODS
  Quantitative RT-PCR and Western blot were used to measure mRNA and protein levels, respectively. Levels of PTX3 and BMP4 were measured by ELISA.
  
  
  RESULTS
  Treatment with BMP4 and BMP7 significantly decreased PTX3 mRNA and protein production. These suppressive effects, along with the induction of Smad1/5/8 phosphorylation, were attenuated by cotreatment with 2 BMP type I receptor inhibitors (dorsomorphin and/or DMH-1). Combined knockdown (ALK3/ALK6 for BMP4 and ALK2/ALK3 for BMP7) reversed the effects of BMP4- and BMP7-induced Smad1/5/8 phosphorylation and PTX3 suppression. Furthermore, Smad4 knockdown reversed the suppressive effects of BMP4 and BMP7 on PTX3 expression. In follicular fluid, concentrations of PTX3 were negatively correlated with concentrations of BMP4.
  
  
  CONCLUSIONS
  BMP4 and BMP7 use differential subsets of BMP type I receptors to downregulate PTX3 expression via Smad-dependent signaling in human granulosa cells.
authors.affiliation: Department of Obstetrics and Gynaecology, Child & Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4.
authors.forename: Hsun-Ming
authors.initials: HM
authors.lastname: Chang
authors.forename: Jung-Chien
authors.initials: JC
authors.lastname: Cheng
authors.forename: Lanlan
authors.initials: L
authors.lastname: Fang
authors.forename: Xin
authors.initials: X
authors.lastname: Qiu
authors.forename: Christian
authors.initials: C
authors.lastname: Klausen
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Taylor
authors.forename: Peter C K
authors.initials: PC
authors.lastname: Leung
mesh_terms: D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology*; D055419:Bone Morphogenetic Protein 7 / Q000494:pharmacology*; D052005:Bone Morphogenetic Protein Receptors, Type I / Q000502:physiology; D002097:C-Reactive Protein / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D034741:RNA, Small Interfering / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D000683:Serum Amyloid P-Component / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D051785:Smad Proteins / Q000378:metabolism; D000099153:Pentraxins
chemical_list: D055415:Bone Morphogenetic Protein 4; D055419:Bone Morphogenetic Protein 7; D052005:Bone Morphogenetic Protein Receptors, Type I; D002097:C-Reactive Protein; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D000683:Serum Amyloid P-Component; D051785:Smad Proteins; D000099153:Pentraxins; C525809:BMP4 protein, human; C525819:BMP7 protein, human