title: Effects of Smad3 on the proliferation and steroidogenesis in human ovarian luteinized granulosa cells.
abstract: Granulosa cells (GCs) are essential for proper oocyte, follicular development, and steroidogenesis in the ovary. Transforming growth factor β (TGF-β) superfamily members are critical in regulating GCs growth and differentiation. Smad3 is known to serve as a signaling intermediate for the TGF-β; however, the functions of Smad3 in the human GCs remain unidentified. In this study, the luteinized GCs collected from follicular aspirates from patients undergoing in vitro fertilization were cultured and engineered to overexpress and knockdown Smad3, which were validated by RT-PCR and Western blotting. Immunocytochemistry showed that Smad3 protein was strongly expressed in human ovarian luteinized GCs. EdU incorporation demonstrated that Smad3 promoted the proliferation of GCs, and the expression of PCNA was also enhanced by Smad3. ELISA analysis indicated that the secretion of both estradiol and progesterone was stimulated by Smad3. In addition, Smad3 upregulated the level of follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and protein kinase A (PKA) proteins. We subsequently added special PKA inhibitor H89 into the GCs and found that the stimulating effect on the growth of GCs by Smad3 was blocked partly. The morphology of cultured GCs was changed by Smad3, and the expression level of integrin β1 was enhanced by Smad3. Kindlin-2, an important cellular mediating molecule of integrin β signaling, was expressed in human ovarian luteinized GCs and was upregulated by Smad3. Our results indicated that Smad3 promoted the proliferation and steroidogenesis of human ovarian luteinized GCs, and these effects may be mediated by the FSHR/LHR-PKA signaling pathway.
authors: Department of Scientific Research, Peking Union Medical Collage Hospital, Beijing, China; Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Peking University, Beijing, China.
authors: Yuxia
authors: Y
authors: Liu
authors: Xi
authors: X
authors: Chen
authors: Xinying
authors: X
authors: Xue
authors: Chunyan
authors: C
authors: Shen
authors: Cheng
authors: C
authors: Shi
authors: Jingxia
authors: J
authors: Dong
authors: Hongquan
authors: H
authors: Zhang
authors: Rong
authors: R
authors: Liang
authors: Sen
authors: S
authors: Li
authors: Jian
authors: J
authors: Xu
mesh_terms: D015153:Blotting, Western; D049109:Cell Proliferation / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006107:Granulosa Cells / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D007546:Isoquinolines; D008565:Membrane Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D010053:Ovary / Q000166:cytology*; D011962:Receptors, FSH / Q000378:metabolism; D011974:Receptors, LH / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051900:Smad3 Protein / Q000378:metabolism*; D013256:Steroids / Q000096:biosynthesis*; D013449:Sulfonamides
chemical_list: C477322:FERMT3 protein, human; D007546:Isoquinolines; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011962:Receptors, FSH; D011974:Receptors, LH; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D013256:Steroids; D013449:Sulfonamides; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide
keywords: Smad3; human luteinized granulosa cells; proliferation; steroidogenesis