title: Interaction of the transforming growth factor-β and Notch signaling pathways in the regulation of granulosa cell proliferation.
abstract: The Notch and transforming growth factor (TGF)-β signalling pathways play an important role in granulosa cell proliferation. However, the mechanisms underlying the cross-talk between these two signalling pathways are unknown. Herein we demonstrated a functional synergism between Notch and TGF-β signalling in the regulation of preantral granulosa cell (PAGC) proliferation. Activation of TGF-β signalling increased hairy/enhancer-of-split related with YRPW motif 2 gene (Hey2) expression (one of the target genes of the Notch pathway) in PAGCs, and suppression of TGF-β signalling by Smad3 knockdown reduced Hey2 expression. Inhibition of the proliferation of PAGCs by N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester (DAPT), an inhibitor of Notch signalling, was rescued by both the addition of ActA and overexpression of Smad3, indicating an interaction between the TGF-β and Notch signalling pathways. Co-immunoprecipitation (CoIP) and chromatin immunoprecipitation (ChIP) assays were performed to identify the point of interaction between the two signalling pathways. CoIP showed direct protein-protein interaction between Smad3 and Notch2 intracellular domain (NICD2), whereas ChIP showed that Smad3 could be recruited to the promoter regions of Notch target genes as a transcription factor. Therefore, the findings of the present study support the idea that nuclear Smad3 protein can integrate with NICD2 to form a complex that acts as a transcription factor to bind specific DNA motifs in Notch target genes, such as Hey1 and Hey2, and thus participates in the transcriptional regulation of Notch target genes, as well as regulation of the proliferation of PAGCs.
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Sun
authors.forename: Xing-Hong
authors.initials: XH
authors.lastname: Sun
authors.forename: Shun-Feng
authors.initials: SF
authors.lastname: Cheng
authors.forename: Jun-Jie
authors.initials: JJ
authors.lastname: Wang
authors.forename: Yan-Ni
authors.initials: YN
authors.lastname: Feng
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhao
authors.forename: Shen
authors.initials: S
authors.lastname: Yin
authors.forename: Zhu-Mei
authors.initials: ZM
authors.lastname: Hou
authors.forename: Wei
authors.initials: W
authors.lastname: Shen
authors.forename: Xi-Feng
authors.initials: XF
authors.lastname: Zhang
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D005260:Female; D005786:Gene Expression Regulation; D006107:Granulosa Cells / Q000166:cytology*; D051379:Mice; D011401:Promoter Regions, Genetic; D051880:Receptors, Notch / Q000502:physiology*; D012097:Repressor Proteins / Q000502:physiology; D015398:Signal Transduction; D051900:Smad3 Protein / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000502:physiology*
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D051880:Receptors, Notch; D012097:Repressor Proteins; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; C485778:Hey2 protein, mouse; C497791:Smad3 protein, mouse