Immature mucosal defenses contribute to increased susceptibility of newborn infants to pathogens. Sparse knowledge of age-dependent changes in mucosal immunity has hampered improvements in neonatal morbidity due to infections. Here, we report that exposure of neonatal mice to commensal bacteria immediately after birth is required for a robust host defense against bacterial pneumonia, the leading cause of death in newborn infants. This crucial window was characterized by an abrupt influx of interleukin (IL)-22 producing group 3 innate lymphoid cells (IL22+ILC3) into the lungs of newborn mice. This influx was dependent on sensing of commensal bacteria by intestinal mucosal dendritic cells. Disruption of postnatal commensal colonization or selective depletion of dendritic cells interrupted the migratory program of lung IL-22+ILC3 and made the newborn mice more susceptible to pneumonia, which was reversed by transfer of commensal bacteria after birth. Thus, the resistance of newborn mice to pneumonia relied on commensal bacteria-directed ILC3-influx into the lungs, which mediated IL-22-dependent host resistance to pneumonia during this developmental window. These data establish that postnatal colonization by intestinal commensal bacteria is pivotal in the development of lung defenses in mice.

Previous studies demonstrated that targeted deletion of the Ron receptor tyrosine kinase (TK) domain in mice leads to marked hepatocyte protection in a well-characterized model of lipopolysaccharide (LPS)-induced acute liver failure in D-galactosamine (GalN)-sensitized mice. Hepatocyte protection in TK2/2 mice was observed despite paradoxically elevated serum levels of tumor necrosis factor alpha (TNF-a). To understand the role of Ron in the liver, purified populations of Kupffer cells and hepatocytes from wildtype (TK1/1) and TK2/2 mice were studied. Utilizing quantitative reverse-transcription polymerase chain reaction (RT-PCR), we demonstrated that Ron is expressed in these cell types. Moreover, we also recapitulated the protected hepatocyte phenotype and exaggerated cytokine production observed in the TK2/2 mice in vivo through the use of purified cultured cells ex vivo. We show that isolated TK2/2 Kupffer cells produce increased levels of TNF-a and select cytokines compared to TK1/1 cells following LPS stimulation. We also show that conditioned media from LPS-treated TK2/2 Kupffer cells was more toxic to hepatocytes than control media, suggesting the exaggerated levels of cytokines produced from the TK2/2 Kupffer cells are detrimental to wildtype hepatocytes. In addition, we observed that TK2/2 hepatocytes were more resistant to cell death compared to TK1/1 hepatocytes, suggesting that Ron functions in both the epithelial and inflammatory cell compartments to regulate acute liver injury. These findings were confirmed in vivo in mice with hepatocyte and macrophage cell-type-specific conditional Ron deletions. Mice with Ron loss selectively in hepatocytes exhibited less liver damage and increased survival compared to mice with Ron loss in macrophages. Conclusion: We dissected cell-type-specific roles for Ron such that this receptor modulates cytokine production from Kupffer cells and inhibits hepatocyte survival in response to injury. (HEPATOLOGY 2011;53:1618-1628

Our previous studies demonstrated that selective overexpression of the Ron receptor tyrosine kinase in the murine mammary epithelium leads to mammary tumor formation. Biochemical analysis of mammary tumor lysates showed that Ron overexpression was associated with increases in β-catenin expression and tyrosine phosphorylation. β-catenin has also been shown to be regulated through tyrosine phosphorylation by the receptor tyrosine kinases Met, Fer, and Fyn. However, the molecular and physiological roles of β-catenin and β-catenin tyrosine phosphorylation downstream of Ron are not known. To investigate this association, we show that Ron and β-catenin are coordinately elevated in human breast cancers. Our data also demonstrate that activation of Ron, through ligand binding by hepatocyte growth factor-like protein (HGFL), induces the tyrosine phosphorylation of β-catenin, primarily on tyrosine residues Tyr 654 and Tyr 670. In addition, HGFL mediated Ron activation induces both β-catenin nuclear localization and transcriptional activity, with Tyr 654 and Tyr 670 residues of β-catenin being critical for these processes. We also demonstrate that a knockdown of Ron in breast cancer cell lines leads to a loss of HGFL-induced β-catenin-dependent transcriptional activation and cell growth which can rescued by activation of canonical Wnt/β-catenin signaling. Moreover, we show that HGFL-dependent Ron activation mediates upregulation of the β-catenin target genes cyclin D1 and c-myc, and that expression of these target genes in breast cancer cells is decreased following inhibition of Ron and/or β-catenin. Finally, we show that genetic ablation of β-catenin in Ron-expressing breast cancer cells decreases cellular proliferation in vitro, as well as mammary tumor growth and metastasis following orthotopic transplantation into the mammary fat pad. Together, our data suggest that β-catenin is a crucial downstream regulator of Ron receptor activation and is an important mediator of mammary tumorigenesis.

The proliferation cell nuclear antigen (PCNA) is a critical protein required for DNA replication in proliferating cells including cancer cells. However, direct inhibition of PCNA in cancer cells has been difficult due to the lack of targetable sites. We previously reported that phosphorylation of tyrosine 211 (Y211) on PCNA is important for the proliferative function of PCNA when this protein is associated with the chromatin in cancer cells. Here, we show that the Y211 phosphorylation of PCNA is a frequent event in advanced prostate cancer. To explore the potential of this signaling event in inhibition of cancer cell growth, we used a synthetic peptide, the Y211F peptide, which when present inhibits phosphorylation of Y211 on endogenous PCNA. Treatment with this peptide, but not a scrambled control peptide, resulted in S-phase arrest, inhibition of DNA synthesis, and enhanced cell death in a panel of human prostate cancer cell lines. In addition, treatment with the Y211F peptide led to decreased tumor growth in PC3-derived xenograft tumors in vivo in nude mice. Our study shows for the first time that PCNA phosphorylation at Y211 is a frequent and biologically important signaling event in prostate cancer. This study also shows a proof of concept that Y211 phosphorylation of PCNA may be used as a therapeutic target in prostate cancer cells, including cells of advanced cancers that are refractory to standard hormonal therapies.