We investigated whether Nuclear Factor-kappa B (NF-κB), which exhibits a regulated pattern of activity during murine mammary gland development, plays an important role during lactation and involution, when milk production ceases and the gland undergoes apoptosis and remodeling. We generated a doxycycline inducible transgenic mouse model to activate NF-κB specifically in the mammary epithelium through expression of a constitutively active form of IKK2, the upstream kinase in the classical NF-κB signaling cascade. We found that activation of NF-κB during involution resulted in a more rapid reduction in milk levels and increased cleavage of caspase-3, an indicator of apoptosis. We also found that activation of NF-κB during lactation with no additional involution signals had a similar effect. The observation that NF-κB is a key regulator of milk production led us to investigate the role of NF-κB during mastitis, an infection of the mammary gland in which milk loss is observed. Mammary gland injection of E. coli LPS resulted in activation of NF-κB and milk loss during lactation. This milk loss was decreased by selective inhibition of NF-κB in mammary epithelium. Together, our data reveal that activation of NF-κB leads to milk clearance in the lactating mammary gland. Therefore, targeting of NF-κB signaling may prove therapeutic during mastitis in humans and could be beneficial for the dairy industry, where such infections have a major economic impact.

A regulated pattern of nuclear factor B (NF-B) activation is essential for normal development of the mammary gland. An increase in NF-B activity has been implicated in breast cancer. We have generated a novel transgenic mouse model to investigate the role of the alternative NF-B pathway in ductal development and identify possible mediators of tumorigenesis downstream of p100/p52. By overexpressing the NF-B p100/p52 subunit in mammary epithelium using the ␤-lactoglobulin milk protein promoter, we found that transgene expression resulted in increased overall NF-B activity during late pregnancy. During pregnancy, p100/p52 expression resulted in delayed ductal development with impaired secondary branching and increased levels of Cyclin D1, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and cyclo-oxygenase-2 (COX-2) in the mammary gland. After multiple pregnancies the p100 transgenics exhibited a ductal thickening accompanied by small hyperplastic foci. In tumors from mice expressing the polyoma middle T oncoprotein (PyVT) in the mammary gland, increased levels of p100/p52 were present at the time of tumor development. These results show that increased p100/p52 disrupts normal ductal development and provides insight into the mechanism by which this may contribute to human breast cancer.The development of the murine mammary gland is a complex process. A simple ductal structure forms during embryogenesis through signaling between the epithelium and surrounding mesenchyme. The cyclic production of hormones promotes further ductal growth and branching during puberty. The majority of mammary gland proliferation is observed in pregnancy, with ductal branching and the formation of secretary lobular alveolar units. Following lactation and the weaning of pups there is a re-modeling of the epithelium termed involution, and the gland returns to the simpler structure observed in virgin animals (1-3).The transcription factor nuclear factor B (NF-B) 2 is involved in the process of mammary development. NF-B is a dimer formed from a multisubunit family consisting of p65 (Rel A), Rel B, c-Rel, p105/p50 (NF-B1), and p100/p52 (NF-B2; Ref. 4). The activation of NF-B is regulated by the binding of inhibitory proteins termed IBs (5). The C-terminals of the p105 and p100 subunits share homology with IB (6, 7). These inhibitory subunits are phosphorylated in response to stimuli by an upstream IB kinase complex (IKK). This contains two kinases, IKK␣ and IKK␤, and a regulatory subunit, IKK␥ (NEMO; Refs. 8 -10). In the canonical NF-B pathway, a dimer composed of the p65 and p50 subunits is held in the cytosol through binding of the inhibitor IB␣. The phosphorylation of IB␣ by IKK␤ acts as a signal for the ubiquitination and degradation of the inhibitor (11). This allows p50/p65 to translocate to the nucleus and bind to the promoter of target genes that are involved in a diverse range of processes including inflammation, cell growth, and apoptosis (4, 12). More recently an alternative pathway of NF-B subunit activation ...

Specific cell and tissue interactions drive the formation and function of the vertebrate cardiovascular systems. Although much attention has been focused on the muscular components of the developing heart, the endocardium plays a key role in the formation of a functioning heart. Endocardial cells exhibit heterogeneity that allows them to participate in events such as the formation of the valves, septation of the outflow tract, and trabeculation. Here we review the contributions of the endocardium to cardiovascular development and outline useful approaches developed in the chick and mouse that have revealed endocardial cell heterogeneity, the signaling molecules that direct endocardial cell behavior, and how these insights have contributed to our understanding of cardiovascular development and disease.

SummaryPseudomonas aeruginosa pneumonia usually results from a deficit of the innate immune system. To investigate whether inflammatory signalling by airway epithelial cells provides a pivotal line of defence against P. aeruginosa infection, we utilized two separate lines of inducible transgenic mice that express a constitutive activator of the nuclear factor kappa-B (NF-kB) pathway (IKTA) or a dominant inhibitor of NF-kB (DNTA) in airway epithelial cells. Compared with control mice, IKTA mice showed an enhanced host response to P. aeruginosa infection with greater neutrophil influx into the lungs, increased expression of Glu-Leu-Arg-positive (ELR + ) CXC chemokines macrophage inflammatory protein-2 and keratinocyte chemoattractant (KC), superior bacterial clearance and improved survival at 24 h after infection. Neutrophil depletion abrogated the improvement in host defence identified in IKTA mice. In contrast, DNTA mice showed impaired responses to P. aeruginosa infection with higher bacterial colony counts in the lungs, decreased neutrophilic lung inflammation and lower levels of KC in lung lavage fluid. DNTA mice given recombinant KC at the time of P. aeruginosa infection demonstrated improved neutrophil recruitment to the lungs and enhanced bacterial clearance. Our data indicate that the NF-kB pathway in airway epithelial cells plays an essential role in defence against P. aeruginosa through generation of CXC chemokines and recruitment of neutrophils.

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