Abstract
BackgroundConserved Wnt ligands are critical for signalling during development; however, various factors modulate their activity. Among these factors are the Secreted Frizzled-Related Proteins (SFRP). We previously isolated the SFRP-4 gene from an involuting rat mammary gland and later showed that transgenic mice inappropriately expressing SFRP-4 during lactation exhibited a high level of apoptosis with reduced survival of progeny.ResultsIn order to address the questions related to the mechanism of Wnt signalling and its inhibition by SFRP-4 which we report here, we employed partially-purified Wnt-3a in a co-culture model system. Ectopic expression of SFRP-4 was accomplished by infection with a pBabepuro construct. The co-cultures comprised Line 31E mouse mammary secretory epithelial cells and Line 30F, undifferentiated, fibroblast-like mouse mammary cells. In vitro differentiation of such co-cultures can be demonstrated by induction of the β-casein gene in response to lactogenic hormones.We show here that treatment of cells with partially-purified Wnt-3a initiates Dvl-3, Akt/PKB and GSK-3β hyperphosphorylation and β-catenin activation. Furthermore, while up-regulating the cyclin D1 and connexin-43 genes and elevating transepithelial resistance of Line 31E cell monolayers, Wnt-3a treatment abrogates differentiation of co-cultures in response to the lactogenic hormones prolactin, insulin and glucocorticoid. Cells which express SFRP-4, however, are largely unaffected by Wnt-3a stimulation. Since a physical association between Wnt-3a and SFRP-4 could be demonstrated with immunoprecipitation/Western blotting experiments, this interaction, presumably owing to the Frizzled homology region typical of all SFRPs, explains the refractory response to Wnt-3a which was observed.ConclusionThis study demonstrates that Wnt-3a treatment activates the Wnt signalling pathway and interferes with in vitro differentiation of mammary co-cultures to β-casein production in response to lactogenic hormones. Similarly, in another measure of differentiation, following Wnt-3a treatment mammary epithelial cells could be shown to up-regulate the cyclin D1 and connexin-43 genes while phenotypically they show increased transepithelial resistance across the cell monolayer. All these behavioural changes can be blocked in mammary epithelial cells expressing SFRP-4. Thus, our data illustrate in an in vitro model a mechanism by which SFRP-4 can modulate a differentiation response to Wnt-3a.
Highlights
Conserved Wnt ligands are critical for signalling during development; various factors modulate their activity
Because Secreted Frizzled-Related Proteins (SFRP)-4 is strongly upregulated during involution of the mouse mammary gland and is otherwise virtually absent from this tissue, we wanted to develop a simplified mammary model allowing the study of its effects on Wnt signalling in vitro
In order to be able to study the effects of ectopically-expressed SFRP-4, we used partially-purified Wnt-3a prepared by a convenient protocol based on that described by Willert et al [8]
Summary
Conserved Wnt ligands are critical for signalling during development; various factors modulate their activity. Signalling by the Wnt family of secreted glycoprotein ligands is essential both in embryonic [6,7] as well as adult development, where they are implicated in the maintenance of stem cell pools [8,9,10,11,12], in tissue differentiation programs [11,12,13] and in cell survival [13,14]. The insoluble nature of Wnts was explained by the observation that these proteins are palmitoylated and, more hydrophobic than was initially predicted from the primary amino acid sequence [8]
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