Adenoviral Gene Transfer Allows Smad-Responsive Gene Promoter Analyses and Delineation of Type I Receptor Usage of Transforming Growth Factor-β Family Ligands in Cultured Human Granulosa Luteal Cells

Abstract

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.

The activin receptor-like kinase 6 Booroola mutation enhances suppressive effects of bone morphogenetic protein 2 (BMP2), BMP4, BMP6 and growth and differentiation factor-9 on FSH release from ovine primary pituitary cell cultures

“…This suggests that GDF9 is acting like a BMP rather than a TGFb, as TGFb1 had no significant effect on FSH secretion in either BB or WT genotypes. GDF9 is expressed in sheep pituitary tissue (Faure et al 2005) and the present theory is that GDF9 acts through the BMPRII receptor and TGFb type I receptors (Alk5) to activate the Smad 3 pathway (Kaivo-Oja et al 2005). BMP2 and BMP4 both bind to BMPRII and ALK3/6 (BMPRIA/B) activating the Smad 1/5/8 pathway, but BMP6 and BMP7 are thought to bind to BMPRII as well as the activin receptors ActRII/IIB and ALK2 (ActRIA) in addition to ALK6 (see Shimasaki et al 2004 for a review).…”

Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells

“…In these initial studies, Smad3 activation was monitored by transducing human granulosa-luteal (hGL) cells with an adenovirus encoding the CAGAluciferase reporter (Kaivo-Oja et al 2005), which contains repeats of the Smad3/4 response element (GTCT/AGAC) in front of the luciferase cDNA (Dennler et al 1998). This lack of bioactivity in the case of hGDF9 was not due to a problem in the production of the protein or in processing of the precursor (Fig 1C).…”

“…Bioactivity of the conditioned media and the purified GDF9 mature regions was assayed on primary cultures of hGL cells (Kaivo-Oja et al 2005). The hGL cells were treated with either purified mouse (Fig.…”

“…We have shown that mGDF9 induces Smad2 phosphorylation and inhibin production in rat diethylstilbestrol-treated granulosa cells (Roh et al 2003) and in human granulosa-luteal (hGL) cells , not requiring any purification steps to reveal these activities. Further, we have shown that the downstream signaling actions of mGDF9 are mediated by the type I receptor, ALK5, initiating the subsequent activation of Smad2 and Smad3, both in cultured rat granulosa cells (Mazerbourg et al 2004) and in hGL cells Kaivo-Oja et al 2005). It has also been shown that mGDF9 uses the BMP type II receptor (BMPRII) as its other signaling receptor in rat granulosa cells (Vitt et al 2002).…”

Growth and differentiation factor 9 promotes oocyte growth at the primary but not the early secondary stage in three-dimensional follicle culture

“…Whether GDF9 acts directly on the oocyte to induce follicle growth, indirectly through the granulosa cell or both, is not clear, given that both granulosa cells and oocytes in primary follicles express ALK5 [57][58][59][60][61] and BMPR2 [58,62], the reported type I [63,64] and II receptors for GDF9 [65], respectively. However, it is controversial whether ALK5 is the only type I receptor for GDF9 as Alk5 conditional knockout mice have no defects in follicular development and cumulus expansion [62], which is a distinct phenotype from Gdf9 null mice [17].…”