A transgenic analysis of the role of Smad4 in the mammary gland

• Main Author: Duff, Eleanor K.
• Published: University of Edinburgh 2001
• Subjects: 571.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.649757

To investigate the effects of Smad proteins in normal euploid cells, various Smads were over-expressed in embryonic stem cells (ES cells). Analysis of ligand-specific Smads 1, 2, 3 and the central mediator Smad4, showed that only Smad4 could induce both a G1 arrest and apoptosis. This confirmed a role for Smad4 in engaging apoptosis, and these studies were then extended to an <i>in vivo </i>model of apoptosis namely involution of the mammary gland. This was undertaken using two transgenic approaches. First, a mammary-specific Smad4 transgenic strain was created which utilised the ovine beta-lactoglobulin gene (BLG) promoter to drive Smad4 expression to the mammary epithelium during lactation. Remarkably, mice transgenic for Smad4 and characterised as over expressing Smad4 throughout lactation and involution showed no overt phenotype. This negative result may arise because levels of Smad4 are already limiting in the normal mammary gland, because functional activity of Smad4 is being repressed, or even because Smad4 does not mediate a central role in the apoptotic response in the mammary gland. The second transgenic approach was to create mice with a conditional Smad4 allele using the Cre-Lox system. A floxed targeting construct was successfully used to target ES cells and these were then subsequently used to create chimeras. Germline transmission of the floxed Smad4 allele and subsequent crossing to a BLG-Cre transgenic will generate a powerful tool to investigate the requirement for Smad4 specifically within the mammary epithelium, Smad-STAT interactions were analysed during involution of the mammary gland. Other workers had previously demonstrated these pathways interact <i>in vitro</i> and this was confirmed <i>in vivo</i> with data suggesting a novel role for STAT3 as an inhibitor of Smad activity during involution. In conclusion this thesis has addressed the role of the Smad proteins, and especially the role of the central mediator Smad4, in mediating apoptosis. I have shown that Smad4 can mediate apoptosis in normal euploid cells in culture, but that over expression of Smad4 in the mammary epithelium apparently fails to alter the programme of involution.