title: Knockdown of CREB3/Luman by shRNA in Mouse Granulosa Cells Results in Decreased Estradiol and Progesterone Synthesis and Promotes Cell Proliferation.
abstract: Luman (also known as LZIP or CREB3) is a transcription factor and a member of the cAMP responsive element-binding (CREB) family proteins. Although Luman has been detected in apoptotic granulosa cells and disorganized atretic bodies, the physiological function of Luman in follicular development has not been reported. Our objective is to determine the role of Luman in folliculogenesis by knocking down Luman expression in mouse GCs (granulosa cells) using shRNA. Luman expression was successfully knocked down in mouse GCs at the mRNA and protein level, as confirmed by real-time quantitative PCR, western blot and immunofluorescence staining, respectively. Knockdown of Luman significantly decreased the concentrations of estradiol (E2) and progesterone (P4) in cell culture medium. Furthermore, Luman knockdown promoted cell proliferation but had no effect on cell apoptosis. To elucidate the regulatory mechanism underlying the effects of Luman knockdown on steroid synthesis and cell cycle, we measured the mRNA and protein expression levels of several related genes. The expression of Star, Cyp19a1, and Cyp1b1, which encode steroidogenic enzymes, was down-regulated, while that of Cyp11a1 and Runx2, which also encode steroidogenic enzymes, was up-regulated. The expression of the cell cycle factors Cyclin A1, Cyclin B1, Cyclin D2, and Cyclin E was significantly up-regulated. Among apoptosis-related genes, only Bcl-2 was down-regulated, while Caspase 3, Bax and p53 were not significantly affected, suggesting that Luman knockdown may regulate cell cycle activity and hormone secretion at the transcriptional and translational level in mouse GCs. The expression of two important genes associated with folliculogenesis in mouse GCs, Has2 and Ptgs2, were also significantly altered by Luman knockdown. In conclusion, the findings of this study indicate that Luman regulates mouse GCs modulation of steroid synthesis, cell cycle activity and other regulators of folliculogenesis.
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, China.|College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, China.|College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Yanglei
authors.initials: Y
authors.lastname: Yi
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, China.|College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Lin
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, China.|College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Keqiong
authors.initials: K
authors.lastname: Tang
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Aihua
authors.initials: A
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, China.|College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Yaping
authors.identifier: http://orcid.org/0000-0001-5708-3159
authors.initials: Y
authors.lastname: Jin
mesh_terms: D000818:Animals; D017209:Apoptosis; D002453:Cell Cycle; D049109:Cell Proliferation; D002469:Cell Separation; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics*; D065633:Cytochrome P-450 CYP1B1 / Q000378:metabolism; D000072496:Cytochrome P450 Family 19 / Q000378:metabolism; D004958:Estradiol / Q000096:biosynthesis*; D005260:Female; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D006107:Granulosa Cells / Q000166:cytology* / Q000378:metabolism; D016086:Lentivirus / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D010750:Phosphoproteins / Q000378:metabolism; D011374:Progesterone / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: C116472:Creb3 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D010750:Phosphoproteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000098682:Steroidogenic Acute Regulatory Protein; D011374:Progesterone; D004958:Estradiol; C585498:Cyp1b1 protein, mouse; D065633:Cytochrome P-450 CYP1B1; D000072496:Cytochrome P450 Family 19