Adenoviral gene transfer allows Smad-responsive gene promoter analyses and delineation of type I receptor usage of transforming growth factor-beta family ligands in cultured human granulosa luteal cells.
In the human ovary, cell growth and differentiation are regulated by members of the TGF-beta superfamily, including growth differentiation factor-9 (GDF9), TGF-beta, and activin. TGF-beta 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-beta, and activin responses. In rodent granulosa cells, TGF-beta and GDF9 signal through the TGF-beta 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-beta, 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-beta 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-beta superfamily can be used to amplify the cellular response to ligands such as GDF9, TGF-beta, 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-beta superfamily.
Program for Developmental and Reproductive Biology, Biomedicum Helsinki and Department of Bacteriology, Haartman Institute, University of Helsinki, Haartmaninkatu 8, 00014 Helsinki, Finland.
Noora
N
Kaivo-Oja
David G
DG
Mottershead
Sabine
S
Mazerbourg
Samu
S
Myllymaa
Sébastien
S
Duprat
Robert B
RB
Gilchrist
Nigel P
NP
Groome
Aaron J
AJ
Hsueh
Olli
O
Ritvos
D030201:Activin Receptors, Type I / Q000502:physiology*; D028341:Activins / Q000494:pharmacology; D000256:Adenoviridae / Q000235:genetics*; D055430:Bone Morphogenetic Protein 15; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D022761:Gene Transfer, Horizontal; D055429:Growth Differentiation Factor 9; D006801:Humans; D007265:Inhibins / Q000096:biosynthesis; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D008024:Ligands; D008184:Luteal Cells / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000502:physiology*; D051900:Smad3 Protein; D015534:Trans-Activators / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
C525901:BMP15 protein, human; C525902:Bmp15 protein, mouse; D055430:Bone Morphogenetic Protein 15; D004268:DNA-Binding Proteins; C525897:GDF9 protein, human; C525898:Gdf9 protein, mouse; D055429:Growth Differentiation Factor 9; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D018125:Receptors, Transforming Growth Factor beta; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; C422364:inhibin B; D028341:Activins; D007265:Inhibins; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625841:TGFBR1 protein, human; C000625842:Tgfbr1 protein, mouse