Growth differentiation factor-9 induces Smad2 activation and inhibin B production in cultured human granulosa-luteal cells.
The TGF beta 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 TGF beta and the bone morphogenetic proteins (BMPs). As with TGF beta, GDF-9 treatment caused the phosphorylation of endogenous 53-kDa proteins detected in Western blots with antiphospho-Smad2 antibodies (alpha PS2). However, unlike BMP-2, GDF-9 did not activate the phosphorylation of antiphospho-Smad1 antibody (alphaPS1)-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 alpha PS2-reactive proteins. Furthermore, GDF-9 increased the steady state levels of inhibin beta(B)-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 TGF beta 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.
Kaivo-Oja
Noora
N
Program for Developmental and Reproductive Biology, Biomedicum Helsinki, and Department of Bacteriology, Haartman Institute, University of Helsinki, 00014 Helsinki, Finland.
Bondestam
Jonas
J
Kämäräinen
Meerit
M
Koskimies
Janne
J
Vitt
Ursula
U
Cranfield
Mark
M
Vuojolainen
Kaisa
K
Kallio
Janne P
JP
Olkkonen
Vesa M
VM
Hayashi
Masaru
M
Moustakas
Aristidis
A
Groome
Nigel P
NP
ten Dijke
Peter
P
Hsueh
Aaron J W
AJ
Ritvos
Olli
O
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal; D055430:Bone Morphogenetic Protein 15; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D005260:Female; D015967:Gene Expression Regulation, Viral; D055429:Growth Differentiation Factor 9; D006801:Humans; D007265:Inhibins / Q000737:chemistry / Q000235:genetics*; D036341:Intercellular Signaling Peptides and Proteins / Q000276:immunology / Q000494:pharmacology*; D008184:Luteal Cells / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D051899:Smad2 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; C525901:BMP15 protein, human; C525902:Bmp15 protein, mouse; C525904:Bmp15 protein, rat; D055430:Bone Morphogenetic Protein 15; D004268:DNA-Binding Proteins; C525897:GDF9 protein, human; C525898:Gdf9 protein, mouse; C525899:Gdf9 protein, rat; D055429:Growth Differentiation Factor 9; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C497782:SMAD2 protein, human; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; C497784:Smad2 protein, rat; D015534:Trans-Activators; C422364:inhibin B; D007265:Inhibins