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Program for Developmental and Reproductive Biology, Biomedicum Helsinki and Departments of Bacteriology and Immunology, Haartman Institute, University of Helsinki (D.G.M., N.K.-O., S.My., S.D., O.R.), 00014 Helsinki, Finland; Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine (S.Ma., A.J.H.), Palo Alto, California 94305-5317; Research Center for Reproductive Health, Department of Obstetrics and Gynecology, University of Adelaide (R.B.G.), Woodville, South Australia 5011, Australia; and School of Biological and Molecular Sciences, Oxford Brookes University (N.P.G.), Headington, Oxford, United Kingdom OX3 0BP
Address all correspondence and requests for reprints to: Dr. Olli Ritvos, Biomedicum Helsinki, Room C502b, P.O. Box 63, University of Helsinki, Haartmaninkatu 8, 00014 Helsinki, Finland. E-mail: olli.ritvos{at}helsinki.fi.
In the human ovary, cell growth and differentiation are regulated by members of the TGF-ß superfamily, including growth differentiation factor-9 (GDF9), TGF-ß, and activin. TGF-ß and activin are known to signal via Smad3 activation, and we have recently shown the involvement of Smad3 in cellular responses to GDF9. Recent studies with Smad3-deficient mice have also indicated a key role for this signaling mediator in ovarian folliculogenesis. We now demonstrate the use of a Smad3 reporter (CAGA-luciferase) adenovirus in primary cultures of human granulosa-luteal (hGL) cells to detect GDF9, TGF-ß, and activin responses. In rodent granulosa cells, TGF-ß and GDF9 signal through the TGF-ß type I receptor or activin receptor-like kinase 5 (Alk5), whereas the effect of activin is mediated though the activin type IB receptor, also known as Alk4. We now show that the GDF9 response in hGL cells is markedly potentiated upon overexpression of Alk5 by adenoviral gene transduction, as measured by the CAGA-luciferase reporter activity. A similar response to Alk5 overexpression was observed for TGF-ß, but not for activin. Adenoviral overexpression of the activin type IB receptor Alk4 in hGL cells specifically potentiated activin signaling, but not GDF9 or TGF-ß signaling. Alk5 overexpression in hGL cells also potentiated the GDF9 response when inhibin B production was used as the read-out. These results indicate that the CAGA-luciferase adenovirus can be used to study Smad3 signaling in primary cultures of human cells, and that adenoviral overexpression of wild-type receptors of the TGF-ß superfamily can be used to amplify the cellular response to ligands such as GDF9, TGF-ß, and activin. Furthermore, these studies indicate the involvement of Alk5 in GDF9 signaling in human cells and therefore, along with other recent studies, highlight how a limited number of type I and II receptors cooperate to generate specificity of action within the TGF-ß superfamily.
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