ABSTRACT
Granulosa cells (GCs) are essential for proper oocyte, follicular development, and steroidogenesis in the ovary. Transforming growth factor ß (TGF-ß) superfamily members are critical in regulating GCs growth and differentiation. Smad3 is known to serve as a signaling intermediate for the TGF-ß; 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 ß1 was enhanced by Smad3. Kindlin-2, an important cellular mediating molecule of integrin ß 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.
Subject(s)
Cell Proliferation/physiology , Granulosa Cells/physiology , Ovary/cytology , Smad3 Protein/metabolism , Steroids/biosynthesis , Blotting, Western , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Granulosa Cells/metabolism , Humans , Immunohistochemistry , Isoquinolines , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Receptors, FSH/metabolism , Receptors, LH/metabolism , Reverse Transcriptase Polymerase Chain Reaction , SulfonamidesABSTRACT
The function of Smad3, a downstream signaling protein of the transforming growth factor ß (TGFß) pathway, in ovarian follicle development remains to be elucidated. The effects of Smad3 on ovarian granulosa cells (GCs) in rat were studied. Female rats (21 days of age Sprague-Dawley) received i.p. injections of pregnant mare serum gonadotropin, and GCs were harvested for primary culture 48âh later. These cells were engineered to overexpress or knockdown Smad3, which were validated by immunohistochemistry and western blot. The expression of proliferating cell nuclear antigen (PCNA), cyclin D2, TGFß receptor II (TGFßRII), protein kinase A (PKA), and FSH receptor (FSHR) was also detected by western blotting. Cell cycle and apoptosis of GCs were assayed by flow cytometry. The level of estrogen secreted by GCs was detected by ELISA. Smad3 overexpression promoted estrogen production and proliferation while inhibiting apoptosis of GCs. Reduction in Smad3 by RNAi resulted in reduced estrogen production and proliferation and increased apoptosis of GCs. Manipulation of Smad3 expression also resulted in changes in FSHR and PKA expression, suggesting that the effects of Smad3 on follicle development are related to FSHR-mediated cAMP signaling.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , Granulosa Cells/metabolism , Receptors, FSH/metabolism , Second Messenger Systems , Smad3 Protein/metabolism , Animals , Apoptosis , Cell Cycle , Cell Proliferation , Cells, Cultured , Estradiol/metabolism , Female , Gene Silencing , Granulosa Cells/cytology , RNA, Messenger/metabolism , RNA, Small Interfering , Rats , Rats, Sprague-Dawley , Recombinant Fusion Proteins/metabolism , Smad3 Protein/antagonists & inhibitors , Smad3 Protein/genetics , Up-RegulationABSTRACT
Elevated serum homocysteine (Hcys) levels have been suggested to contribute to congenital cardiovascular malformations, neural tube defects, and cardiovascular diseases. To investigate the mechanisms resulting in cardiovascular diseases and birth defects, Kuang-Hueih Chen et al. identified and characterized a novel gene, named rHCY2, whose expression was markedly up-regulated when Hcys was elevated in rat. In vivo, rHCY2 gene could induce chicken embryonic cells apoptosis and embryonic malformations. Its N-terminal kinase domain is apparently similar to human receptor-interacting serine-threonine kinase 3 (hRIP3). In view of this, we hypothesize that a link between the teratogenic effects of Hcys and hRIP3 is theoretically plausible. However, given the lack of data on the topic, it remains to be seen whether an elevated serum Hcys level will increase the expression of hRIP3. Using normal and abnormal human fetal hearts and cultured normal human fetal cardiomyocytes, we show that congenital cardiovascular malformations are associated with the overexpression of hRIP3, and evidence is found for a certain association between overexpression of hRIP3 and homocysteine-induced congenital cardiovascular malformations. Folic acid and anti-hRIP3 antibodies seem to favor maintenance of the shape and ultrastructure of cultured human fetal cardiomyocytes.