Transgenic analysis of Smad4

Kotsikoris, Vasilios 2005. Transgenic analysis of Smad4. PhD Thesis, Cardiff University.

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Abstract

Mammary gland involution proceeds through massive, highly controlled epithelial cell apoptosis and tissue remodelling. Thus, mammary gland represents an ideal physiological environment in which to study apoptosis. Recently, the Smad gene family has been identified as mediators of TGF-P superfamily signaling and has been implicated in mediating epithelial cell apoptosis. The Smad family of signal transduction proteins focuses around a central mediator, Smad4, and this report presents a number of analyses which have been undertaken to investigate the potential apoptotic role of Smad4 in the mammary gland. To investigate the role of Smad4 I have utilised an over-expressing Smad4 transgenic mouse. This transgenic mouse has been designed to increase Smad4 protein in the secretory epithelial cells of the mammary gland during pregnancy and lactation by using the biological properties of the BLG promoter. Histological investigation of Smad4 slides indicated accelerated involution and significant increase of apoptosis at day 2 and 3 of involution. Molecular analysis through Western blots for STAT3 and STAT5a levels showed a deregulation of both proteins at the same time points. The mechanism with which transgenic mice regulated apoptosis was prove by Western blots to be via the p27Kipl molecular pathway and independent of p21wa or Box. Microarray analysis for day 3 involuting mice showed an over expression of Vitamin D Receptor. A result which was confirmed by both semi quantitative RT-PCR and Western blot analysis. Raw microarray data showed a down regulation of Methyl Binding Domain 2 (MBD2) in Smad4 transgenic mice indicating that MBD2 could play a role in mammary gland involution. However, analysis of conditional mammary knockout MBD2 mice at day 3 involution did not show any involvement of MBD2 in the regulation mammary gland. Another way of investigating Smad4 was by passing the Smad4 construct to Stat3 mammary knockout animals with the hypothesis that Smad4 could restore STAT3's delayed involution. Based on the deregulation of STAT3 protein levels in the smad4 transgenics the above hypothesis was investigated at day 3 of involution. Phenotypic investigation showed that over expression of smad4 is not able to restore the delayed phenotype of STAT3 knockouts. My final investigation was to identify molecules whose expression is altered by a conditional deletion of STAT3 in the mammary gland. This investigation was approached through microarrays which demonstrated an over expression of the Bone Morphogenic Protein receptor, a result which demonstrates once more the synergistic cooperation of Smads and STATS.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Biosciences
ISBN:	9781303201134
Date of First Compliant Deposit:	30 March 2016
Last Modified:	19 Mar 2016 23:33
URI:	https://orca.cardiff.ac.uk/id/eprint/55961

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