Growth Differentiation Factor-9 Induces Smad2 Activation and Inhibin B Production in Cultured Human Granulosa-Luteal Cells

• Published in: The journal of clinical endocrinology and metabolism Vol. 88; no. 2; pp. 755 - 762
• Main Authors: Kaivo-Oja, Noora, Bondestam, Jonas, Kämäräinen, Meerit, Koskimies, Janne, Vitt, Ursula, Cranfield, Mark, Vuojolainen, Kaisa, Kallio, Janne P., Olkkonen, Vesa M., Hayashi, Masaru, Moustakas, Aristidis, Groome, Nigel P., ten Dijke, Peter, Hsueh, Aaron J. W., Ritvos, Olli
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Endocrine Society 01-02-2003
• Subjects: Adenoviridae - genetics; Animals; Antibodies, Monoclonal; Biological and medical sciences; Bone Morphogenetic Protein 15; Cells, Cultured; Dimerization; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; DNA-Binding Proteins/genetics/metabolism; Female; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Viral; Growth Differentiation Factor 9; Hormone metabolism and regulation; Humans; Inhibins - chemistry; Inhibins - genetics; Inhibins/chemistry/genetics; Intercellular Signaling Peptides and Proteins - immunology; Intercellular Signaling Peptides and Proteins - pharmacology; Intercellular Signaling Peptides and Proteins/immunology/pharmacology; Luteal Cells - drug effects; Luteal Cells - metabolism; Luteal Cells/drug effects/metabolism; Mammalian female genital system; Mice; Phosphorylation; Rats; Recombinant Proteins - pharmacology; RNA, Messenger - analysis; Signal Transduction - physiology; Smad2 Protein; Trans-Activators - genetics; Trans-Activators - metabolism; Trans-Activators/genetics/metabolism; Vertebrates: reproduction; Human; Cell culture; Phosphorylation; Transforming growth factor β; Inhibin; Biosynthesis; In vitro; Biological activity; Protein; Granulosa cell; Bone morphogenetic protein; Luteal cell; Female; Comparative study; RNA; Gene Expression Regulation; Viral; Antibodies; Cultured; Monoclonal; Messenger/analysis; Cells
• ISSN: 0021-972X; 1945-7197; 1945-7197
• DOI: 10.1210/jc.2002-021317

The TGFβ family member growth differentiation factor-9 (GDF-9) is an oocyte-derived factor that is essential for mammalian ovarian folliculogenesis. GDF-9 mRNAs have been shown to be expressed in the human ovarian follicle from the primary follicle stage onward, and recombinant GDF-9 has been shown to promote human ovarian follicle growth in vitro. In this study with primary cultures of human granulosa-luteal (hGL) cells, we investigated whether recombinant GDF-9 activates components of the Smad signaling pathways known to be differentially activated by TGFβ and the bone morphogenetic proteins (BMPs). As with TGFβ, GDF-9 treatment caused the phosphorylation of endogenous 53-kDa proteins detected in Western blots with antiphospho-Smad2 antibodies (αPS2). However, unlike BMP-2, GDF-9 did not activate the phosphorylation of antiphospho-Smad1 antibody (αPS1)-immunoreactive proteins in hGL cells. Infection of hGL cells with an adenovirus expressing Smad2 (Ad-Smad2) confirmed that GDF-9 activates specifically phosphorylation of the Smad2 protein. Infection of hGL cells with Ad-Smad7, which expresses the inhibitory Smad7 protein, suppressed the levels of both GDF-9-induced endogenous and adenoviral αPS2-reactive proteins. Furthermore, GDF-9 increased the steady state levels of inhibin βB-subunit mRNAs in hGL cells and strongly stimulated the secretion of dimeric inhibin B. Again, Ad-Smad7 blocked GDF-9-stimulated inhibin B production in a concentration-dependent manner. We identify here for the first time distinct molecular components of the GDF-9 signaling pathway in the human ovary. Our data suggest that GDF-9 mediates its effect through the pathway commonly activated by TGFβ and activin, but not that activated by many BMPs. The results are also consistent with the suggestion that in addition to endocrine control of inhibin production by gonadotropins, a local paracrine control of inhibin production is likely to occur via oocyte-derived factors in the human ovary.