title: Bone morphogenetic protein 2 regulates cell-cell communication by down-regulating connexin43 expression in luteinized human granulosa cells.
abstract: Does bone morphogenetic protein 2 (BMP2) regulate connexin43 (Cx43) and modulate cell-cell communication in luteinized human granulosa cells?
  
  
  
  BMP2 decreases gap junction intercellular communication (GJIC) of luteinized human granulosa cells by down-regulating Cx43 expression through an activin receptor-like kinase (ALK)2/ALK3-mediated Sma- and Mad-related protein (SMAD)-dependent signaling pathway.
  
  
  
  BMP2 and its putative receptors are highly expressed in the human corpus luteum and are involved in the process of luteolysis. Cx43-coupled gap junctions play a critical role in the development and maintenance of corpus luteum.
  
  
  
  This is a laboratory study conducted over a 1-year period. At least three independent experiments with three replicates were conducted and the experimental samples were compared with the appropriate vehicle controls for all of the inhibition-approach, concentration-dependent or time-course studies.
  
  
  
  SVOG cell line (immortalized human granulosa-lutein cells derived from in vitro fertilization patients in an academic research center) was used as the study model. The changes of Cx43 expression and levels of phosphorylated SMAD1/5/8 protein were evaluated after exposure to recombinant human BMP2. Real-time quantitative PCR and Western blot analysis were used to examine the specific mRNA and protein levels, respectively. The BMP/TGF-β type I receptor inhibitors (Dorsomorphin, DMH-1 and SB431542) and target depletion small interfering RNAs (ALK2, ALK3, ALK6 and SMAD4) were used to investigate the underlying molecular mechanisms. A scrape loading and dye transfer assay was used to evaluate the GJIC between the SVOG cells.
  
  
  
  Treatment with BMP2 down-regulated the expression of Cx43 and decreased the GJIC activity, whereas it increased the phosphorylated SMAD1/5/8 protein in SVOG cells (P < 0.05). These biological effects were abolished by pre-treatment with the BMP type I receptor inhibitors, Dorsomorphin and DMH-1 (P < 0.05), but not SB431542. Additionally, the individual or concomitant small interfering RNA-mediated knockdown of ALK2 and ALK3, but not ALK6 attenuated the BMP2-induced increases in phosphorylated SMAD1/5/8 and down-regulation of Cx43 expression (P < 0.05). The knockdown of SMAD4 completely abolished the BMP2-induced down-regulation of Cx43 expression (P < 0.05).
  
  
  
  This experimental study was conducted in an in vitro cell culture system, and may not reflect a realistic intra-ovarian environment.
  
  
  
  Our results suggested that BMP2 may be involved in the local modulation of cell-cell communication in the luteal phase. This study also represents the first comprehensive research of molecular mechanisms of BMP2 in the down-regulation Cx43 in luteinized human granulosa cells. Such data may provide valuable insights into ovarian physiology and benefit the development of potential therapeutic methods for patients suffering from luteal insufficiency.
  
  
  
  N/A.
  
  
  
  This research was supported by an operating grant from the China-Canadian Joint Health Research Initiative Grants Program to P.C.K. Leung and J.Z. Sheng. The authors declare no competing interest with the contents of this article.
authors.affiliation: International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.|Department of Obstetrics and Gynaecology, BC Children's Hospital Research Institute, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4.
authors.forename: Yan-Ting
authors.initials: YT
authors.lastname: Wu
authors.affiliation: Department of Obstetrics and Gynaecology, BC Children's Hospital Research Institute, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4.
authors.forename: Hsun-Ming
authors.initials: HM
authors.lastname: Chang
authors.affiliation: International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: He-Feng
authors.initials: HF
authors.lastname: Huang
authors.affiliation: Department of Pathology and Pathophysiology, School of Medicine, Zhejiang University, Hangzhou, China.
authors.forename: Jian-Zhong
authors.initials: JZ
authors.lastname: Sheng
authors.affiliation: Department of Obstetrics and Gynaecology, BC Children's Hospital Research Institute, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4.
authors.forename: Peter C K
authors.initials: PC
authors.lastname: Leung
mesh_terms: D030201:Activin Receptors, Type I / Q000235:genetics* / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D002450:Cell Communication / Q000187:drug effects*; D002461:Cell Line, Transformed; D018031:Connexin 43 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D005260:Female; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008184:Luteal Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051898:Smad1 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051902:Smad5 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051904:Smad8 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D018031:Connexin 43; C583032:DMH1 compound; D020033:Protein Isoforms; D011720:Pyrazoles; D011743:Pyrimidines; D011804:Quinolines; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C497399:SMAD1 protein, human; C497797:SMAD5 protein, human; C107514:SMAD9 protein, human; D051898:Smad1 Protein; D051902:Smad5 Protein; D051904:Smad8 Protein; C516138:dorsomorphin; C425423:ACVR1 protein, human; D030201:Activin Receptors, Type I
keywords: ALK; BMP2; SMAD; connexin43; luteinized human granulosa cells