17b-Estradiol (E 2 ) was shown to exert neuroprotective effects both in in vitro and in vivo models of stroke. Although these effects of E 2 are known to require estrogen receptor-a (ERa), the cellular target of estrogen-mediated neuroprotection remains unknown. Using cell type-specific ER mutant mice in an in vivo model of stroke, we specifically investigated the role of ERa in neuronal cells versus its role in the microglia in the mediation of neuroprotection by estrogens. We generated and analyzed two different tissue-specific knockout mouse lines lacking ERa either in cells of myeloid lineage, including microglia, or in the neurons of the forebrain. Both E 2 -treated and E 2 -untreated mutant and control mice were subjected to a permanent middle cerebral artery occlusion for 48 h, and the infarct volume was quantified. Although the infarct volume of E 2 -treated female myeloid-specific ERa knockout mice was similar to that of E 2 -treated control mice, both male and female neuron-specific ERa mutant mice had larger infarcts than did control mice after E 2 treatment. We conclude that neuronal ERa in female and male mice mediates neuroprotective estrogen effects in an in vivo mouse model of stroke, whereas microglial ERa is dispensable.

About 5% to 10% of human gastric tumors harbor oncogenic mutations in the KRAS pathway, but their presence alone is often insufficient for inducing gastric tumorigenesis, suggesting a requirement for additional mutagenic events or microenvironmental stimuli, including inflammation. Assessing the contribution of such events in preclinical mouse models requires Cre recombinase-mediated conditional gene expression in stem or progenitor cells of normal and transformed gastric epithelium. We therefore constructed a bacterial artificial chromosome containing transgene (Tg), comprising the regulatory elements of the trefoil factor 1 (Tff1) gene and the tamoxifen-inducible Cre recombinase (CreERT2)-coding sequence. The resulting Tg (Tff1-CreERT2) mice were crossed with mice harboring conditional oncogenic mutations in Kras or Braf. The administration of tamoxifen to the resulting adult Tg(Tff1-CreERT2);Kras LSL-G12D/þ and Tg(Tff1-CreERT2);Braf LSL-V600E/þ mice resulted in gastric metaplasia, inflammation, and adenoma development, characterized by excessive STAT3 activity. To assess the contribution of STAT3 to the spontaneously developing gastric adenomas in gp130

Aberrant activation of the latent transcription factor STAT3 and its downstream targets is a common feature of epithelial-derived human cancers, including those of the gastrointestinal tract. Mouse models of gastrointestinal malignancy implicate Stat3 as a key mediator of inflammatory-driven tumorigenesis, in which its cytokine/gp130/Janus kinase (Jak)-dependent activation provides a functional link through which the microenvironment sustains tumor promotion. Although therapeutic targeting of STAT3 is highly desirable, such molecules are not available for immediate clinical assessment. Here, we investigated whether the smallmolecule Jak1/2 inhibitor AZD1480 confers therapeutic benefits in two mouse models of inflammationassociated gastrointestinal cancer, which are strictly dependent of excessive Stat3 activation. We confirm genetically that Cre-mediated, tumor cell-specific reduction of Stat3 expression arrests the growth of intestinaltype gastric tumors in gp130 F/F mice. We find that systemic administration of AZD1480 readily replicates this effect, which is associated with reduced Stat3 activation and correlates with diminished tumor cell proliferation and increased apoptosis. Likewise, AZD1480 therapy also conferred a cytostatic effect on established tumors in a colitis-associated colon cancer model in wild-type mice. As predicted from our genetic observations in gp130 F/F mice, the therapeutic effect of AZD1480 remains fully reversible upon cessation of compound administration. Collectively, our results provide the first evidence that pharmacologic targeting of excessively activated wild-type Jak kinases affords therapeutic suppression of inflammation-associated gastrointestinal cancers progression in vivo. Mol Cancer Ther; 13(2); 468-74. Ó2014 AACR.

No abstract

Temporal and spatial regulation of genes mediated by tissue-specific promoters and conditional gene expression systems provide a powerful tool to study gene function in health, disease, and during development. Although transgenic mice expressing the Cre recombinase in the gastric epithelium have been reported, there is a lack of models that allow inducible and reversible gene modification in the stomach. Here, we exploited the gastrointestinal epithelium-specific expression pattern of the three trefoil factor (Tff) genes and bacterial artificial chromosome transgenesis to generate a novel mouse strain that expresses the CreERT2 recombinase and the reverse tetracycline transactivator (rtTA). The Tg(Tff1-CreERT2;Tff2-rtTA;Tff3-Luc) strain confers tamoxifen-inducible irreversible somatic recombination and allows simultaneous doxycycline-dependent reversible gene activation in the gastric epithelium of developing and adult mice. This strain also confers luciferase activity to the intestinal epithelium to enable in vivo bioluminescence imaging. Using fluorescent reporters as conditional alleles, we show Tff1-CreERT2 and Tff2-rtTA transgene activity in a partially overlapping subset of long-term regenerating gastric stem/progenitor cells. Therefore, the Tg(Tff1-CreERT2;Tff2-rtTA;Tff3-Luc) strain can confer intermittent transgene expression to gastric epithelial cells that have undergone previous gene modification, and may be suitable to genetically model therapeutic intervention during development, tumorigenesis, and other genetically tractable diseases. Birth Defects Research (Part A) 106:626-635, 2016. © 2016 Wiley Periodicals, Inc.

<p>Extended methods and materials are provided as a supplement to the methods section in the main manuscript and to describe additional methods from the supplementary data.</p>

<p>Supplementary Figure Legends</p>

<p>PDF file - 141K, Table S1. Primers used for quantitative PCR analysis. Table S2. Antibodies (all diluted 1:1000) used for immunohistochemistry and Western Blot analysis.</p>