Induction of mammary gland differentiation in transgenic mice by the fatty acid-binding protein MRG

A mammary-derived growth inhibitor-related gene (MRG) was previously identified and characterized. MRG induces differentiation of mammary epithelial cells in vitro and its expression is associated with mammary differentiation. To further define the role of MRG on mammary gland differentiation, a MRG transgenic mice model under the control of mouse mammary tumor virus promoter was established and the effect of MRG on mammary gland differentiation was investigated at histological and molecular levels. Expression of endogenous mouse MRG gene was significantly increased from the non-differentiated gland of control virgin mice to the functionally differentiated gland of pregnant control mice. Whole mount analyses demonstrated that ductal development was not affected by MRG transgene expression. While there was no lobuloalveolar structure in control virgin mice, expression of MRG transgene in the mammary gland resulted in the development of lobuloalveolar-like structure, which mimics the gland from early pregnancy. Consistent with the morphological change, expression of MRG also increased milk protein beta-casein expression in the gland. To study the mechanism of MRG-induced mammary differentiation, we investigated the Stat5 activation in the glands from the transgenic mouse versus virgin control mouse. While activated Stat5 was expressed at the minimal level in the non-differentiated control virgin gland, a significant Stat5 phosphorylation was observed in the virgin transgenic gland. Our data indicate that MRG is a mediator of the differentiating effects of pregnancy on breast epithelium, and overexpression of MRG in young nulliparous mice can induce differentiation.     

Real-time imaging of beta-lactoglobulin-targeted luciferase activity in the mammary glands of transgenic mice.

This study was aimed at establishing a new platform for real-time monitoring of milk-protein gene expression in the mammary glands. A transgenic reporter composed of the beta-lactoglobulin (BLG)/luciferase hybrid gene was targeted to the mammary glands of pregnant and lactating mice and luciferase activity was imaged in vivo with a low-light imaging system. The mammary glands of a 17-day pregnant mouse occupied an area comparable to that of a 6-day lactating mouse. Nevertheless, the intensity of the luciferase signal was much weaker and confined to regions in the inguinal and thoracic glands. A few small and defined locations of higher expression were also detected, indicating diversity in the initiation of this transgenic milk protein expression. In the lactating mice, high inter- and intra-heterogeneity among regions in a particular gland and among glands was demonstrated, and confirmed by ex vivo analysis of luciferase activity in mammary biopsies. The lack of correlation between luciferase activities and levels of beta-casein accumulation in these biopsies resulted, most probably, from the longer half-life of the native milk protein, compared to the activity of the transgenic marker in the tissue. Unilateral sealing of mammary glands for 4 hr resulted in complete abrogation of luciferase activity, establishing the BLG/luciferase transgene as a reliable tool to follow short-term stimuli. Dispersed mammary epithelial cells preserved luciferase activity in culture, and thus could be used for following mammary gland development after re-implantation. The bioluminescence-based methodology presented here eliminates averaging of heterogeneity in gene expression among glands, and misinterpretations resulting from sampling biopsies taken from inactive regions. Imaging luciferase expression in the mammary glands may enable an accurate monitoring of milk-protein gene expression during cyclic periods of development and apoptosis in a limited number of animals, and could be applied for reporting the consequences of selected drugs on milk-protein gene expression.     

Forced activation of Stat5 subjects mammary epithelial cells to DNA damage and preferential induction of the cellular response mechanism during proliferation

Parity‐dependent adenocarcinoma tumors developed in postestropausal transgenic mice expressing a constitutively active Stat5 variant (STAT5ca) in their mammary gland. These tumors maintained elevated expression levels of genes regulating the cellular DNA damage response (DDR) mechanism, compared to the intact gland. No correlation with STAT5ca expression was observed for these genes in the established tumors. However, activated Stat5a in individual cells of the rarely and earlier developed hyperplasia was associated with induced Chk2 activity. Deregulated Stat5 may already cause DNA damage during the fertile period. This hypothesis and the specific vulnerable stage were further studied in mammary epithelial cells that were stably transfected with β‐lactoglobulin (BLG)/STAT5ca and exposed to a reproduced reproductive cycle. During the pregnancy‐like proliferative state, STAT5ca expression was induced by the added lactogenic hormones. Production of reactive oxygen species, rather than proliferation, served as the primary mediator of DNA damage and cellular DDR. Differentiated cells expressed higher levels of STAT5ca and retained the DNA nicks. However, the elevated expression of the genes involved in DDR was downregulated. Higher levels of DNA damage were also detected in the mammary gland of transgenic mice expressing the BLG/STAT5ca during pregnancy and lactation. However, the relative number of damaged cells was much lower than that in the reproduced in vitro stages and the insults were generally associated with apoptosis and DDR. This study implicates pregnancy as the vulnerable stage for deregulated Stat5 activity, and demonstrates that DNA insults in viable differentiated mammary epithelial cells are ignored by the DDR mechanism. J. Cell. Physiol. 226: 616–626, 2011. © 2010 Wiley‐Liss, Inc.