Real-time monitoring of cAMP response element binding protein signaling in porcine granulosa cells modulated by ovarian factors.
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.
He
Pei Jian
PJ
Department of Animal and Marine Bioresource Sciences, Faculty of Agriculture Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Fujimoto
Yasunori
Y
Yamauchi
Nobuhiko
N
Hattori
Masa-Aki
MA
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
D017362:Cyclic AMP Response Element-Binding Protein; D006062:Gonadotropins; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone