Conditional deletion of the bcl-x gene from mouse mammary epithelium results in accelerated apoptosis during involution but does not compromise cell function during lactation.

In the mammary gland Bcl-x is the most abundant cell survival factor from the Bcl-2 family. Since Bcl-x null mice die around day 12 of embryogenesis, the relevance of this protein in organ development and function is poorly understood. In erythroid cells bcl-x gene expression is controlled by cytokines and the transcription factor Stat5 (signal transducer and activator of transcription). However, we identified that bcl-x RNA levels in mammary tissue from prolactin receptor- and Stat5-null mice were indistinguishable from wild type mice. We have proposed that Bcl-x might control the survival of mammary epithelial cells throughout pregnancy, lactation, and the early stages of involution, and we have now tested this hypothesis through the conditional deletion of the bcl-x gene from mouse mammary epithelium. Conditional (floxed) bcl-x alleles were excised from alveolar cells during pregnancy using a Cre transgene under the control of the whey acidic protein gene promoter. Deletion of the bcl-x gene from the entire epithelial compartment (ducts and alveoli) was achieved by expressing Cre-recombinase under control of the mouse mammary tumor virus long terminal repeat. The absence of Bcl-x did not compromise proliferation and differentiation of mammary ductal and alveolar epithelial cells in virgin mice and during pregnancy and lactation. However, epithelial cell death and tissue remodeling were accelerated in the bcl-x conditional knockout mice during the first stage of involution. Concomitant deletion of the bax gene did not significantly modify the Bcl-x phenotype. Our results suggest that Bcl-x is not essential during mammopoiesis, but is critical for controlled apoptosis during the first phase of involution.

Signal transducer and activator of transcription (Stat) 5 controls the proliferation and differentiation of mammary alveolar epithelium.

Functional development of mammary epithelium during pregnancy depends on prolactin signaling. However, the underlying molecular and cellular events are not fully understood. We examined the specific contributions of the prolactin receptor (PrlR) and the signal transducers and activators of transcription 5a and 5b (referred to as Stat5) in the formation and differentiation of mammary alveolar epithelium. PrlR- and Stat5-null mammary epithelia were transplanted into wild-type hosts, and pregnancy-mediated development was investigated at a histological and molecular level. Stat5-null mammary epithelium developed ducts but failed to form alveoli, and no milk protein gene expression was observed. In contrast, PrlR-null epithelium formed alveoli-like structures with small open lumina. Electron microscopy revealed undifferentiated features of organelles and a perturbation of cell-cell contacts in PrlR- and Stat5-null epithelia. Expression of NKCC1, an Na-K-Cl cotransporter characteristic for ductal epithelia, and ZO-1, a protein associated with tight junction, were maintained in the alveoli-like structures of PrlR- and Stat5-null epithelia. In contrast, the Na-Pi cotransporter Npt2b, and the gap junction component connexin 32, usually expressed in secretory epithelia, were undetectable in PrlR- and Stat5-null mice. These data demonstrate that signaling via the PrlR and Stat5 is critical for the proliferation and differentiation of mammary alveoli during pregnancy.

Experimental mouse genetics-- answering fundamental questions about mammary gland biology.

Contemporary gene-targeting techniques now make it possible to alter specific genes in the genome. As a result, a plethora of mouse models have been generated that allow researchers to dissect cell-signaling pathways involved in mammary gland development and in breast cancer. But what have we learned so far? What do these models teach us? This review presents a global picture of how the analyses and comparison of individual knockout mouse models provide important insights into basic mammary gland biology. Particular emphasis is placed upon what is currently known about the signaling pathways involved in the establishment of the mammary ductal tree, and its subsequent proliferation at pregnancy and lactation. In addition to these well-established pathways, we address recent data that describe the role of lesser-known genes in the development of the mammary epithelium.

Phosphate transport via Na+ -Pi cotransport and anion exchange in lactating rat mammary tissue.

Inorganic orthophosphate (Pi) transport, using 32P-labelled orthophosphate as tracer, by lactating rat mammary tissue has been examined using both tissue explants and the intact perfused gland. Pi uptake was predominantly via a Na+ -dependent pathway. Li+, however, unlike choline, was able to partially substitute for Na+. In addition, Pi release from tissue explants preloaded with 32Pi was stimulated by reversing the Na+ gradient. Thus transferring mammary explants from a buffer containing Na+ to one which was Na+ free (choline replacement) doubled the Pi efflux rate constant. The uptake of Pi by tissue explants was saturable with respect to external Pi, having apparent K(m) and V(max) values of 1.13 mM and 3.36 mmol (kg cell water)-1 (15 min)-1, respectively. The stimulation of Pi uptake by tissue explants by external Na+ was also saturable; the K(m) for Na+ was 9.7 mM. These results, taken together, suggest that the Na+ -dependent pathway is a Na+ -Pi cotransport mechanism. The transport of Pi by the perfused lactating rat mammary gland was examined using a rapid, paired-tracer dilution technique. Pi uptake by the perfused gland was found to be Na+ dependent and displayed saturable kinetics. The results suggest that the Na+ -Pi cotransporter is situated at the basolateral aspect of the secretory cells. The release of Pi from preloaded tissue explants was trans-accelerated by external Pi but not by Cl- or SO4(2-). However, external Pi stimulated Pi efflux with low affinity.