title: Real-time monitoring of cAMP response element binding protein signaling in porcine granulosa cells modulated by ovarian factors.
abstract: The present study was performed to establish a real-time monitoring of the cAMP response element binding protein (CREB) signalling using granulosa cells, and to assess the modulation of CREB activity by potential ovarian autocrine/paracrine and oocyte-derived factors. Granulosa cells were isolated from porcine follicles and cultured for 2 days, and then transfected with CRE-containing pGL3. The cells were directly stimulated or cultured with FSH, LH, forskolin, or a permeable cAMP analog, and/or IGF-I, EGF, bFGF, TGF-beta2 or TNF-alpha, or cumulus-oocyte complex (COCs) for the real-time monitoring of CREB signaling. The activation pattern of CREB signaling consisted of three distinct phases, i.e., burst, attenuation and refractory. In contrast to FSH, LH, and forskolin, a cAMP analog induced the prolonged activation, although three distinct phases were observed at its high concentration. Of all the autocrine/paracrine factors, only IGF-I slightly induced CREB activity. On the other hand, TGF-beta2 and TNF-alpha significantly repressed FSH-stimulated transcriptional activation of CREB by 30% (P < 0.05) and 45% (P < 0.05), respectively. Additionally, coculture with COCs caused a significant suppression of transcriptional activation of CREB signaling stimulated by FSH. These results indicate that ovarian autocrine/paracrine factors such as IGF-I, TGF-beta2, TNF-alpha and oocyte-derived factors modulate the CREB signaling. The present study provides a new approach for direct signaling study on transcription factors under the influences of potential factors.
authors.affiliation: Department of Animal and Marine Bioresource Sciences, Faculty of Agriculture Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Pei Jian
authors.initials: PJ
authors.lastname: He
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Fujimoto
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Yamauchi
authors.forename: Masa-Aki
authors.initials: MA
authors.lastname: Hattori
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D018920:Coculture Techniques; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006062:Gonadotropins / Q000494:pharmacology; D006107:Granulosa Cells / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D007986:Luteinizing Hormone; D010053:Ovary / Q000737:chemistry*; D015398:Signal Transduction; D013552:Swine
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D006062:Gonadotropins; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone