Two distinct Notch signals, Delta-like 4/Notch1 and Jagged-1/Notch2, antagonistically regulate chemical hepatocarcinogenesis in mice.

Notch signaling is one of the most common drivers of carcinogenesis in many types of cancers, including hepatocellular carcinoma (HCC); however, it occasionally suppresses tumor progression. Moreover, it is virtually unknown how different sets of Notch ligands and receptors regulate the HCC development. In this study, we demonstrate that the expression of the Notch ligands, Delta-like 4 (Dll4) and Jagged-1 (Jag1), is upregulated during diethylnitrosamine-induced hepatocarcinogenesis. Dll4 is detected in the preneoplastic hepatocytes and HCC cells, but not in the normal hepatocytes, while Jag1 is expressed in the desmin-positive mesenchymal cells. Hepatocyte-specific Dll4 knockout abolishes the Notch1 signaling and suppresses the tumor progression. In contrast, Jag1 deletion induces the ectopic expression of Dll4 in hepatocytes along with the loss of Notch2 signaling, leading to the tumor progression. These results indicate that the two distinct Notch signals, Dll4/Notch1 and Jag1/Notch2, are antagonistic to each other, exerting opposite effects on HCC progression.

Visualization and isolation of zone-specific murine hepatocytes that maintain distinct cytochrome P450 oxidase expression in primary culture.

Parenchymal hepatocytes are responsible for most of the metabolic functions of the liver, but exhibit distinct functional properties depending on their localization within the hepatic lobule. Cytochrome P450 oxidases represent a family of drug-metabolizing enzymes, which are expressed predominantly in hepatocytes localized in the centrilobular area (zone 3). The present study describes a unique transgenic mouse strain that distinguishes zone 3 hepatocytes from periportal zone 1 hepatocytes by the intensity of EGFP fluorescence. Both zone 1 and zone 3 hepatocytes isolated from these mice showed the same zone-specific gene expression patterns as in liver tissue in vivo. Experiments using primary cultures of hepatocytes indicated that a combination of low oxygen concentration and activation of Wnt/ß-catenin signaling maintained the expression of zone 3-specific P450 drug-metabolizing enzymes, which was characterized by their susceptibility to acetaminophen-induced mitochondrial dysfunction. These zone-specific hepatocytes provide a useful system in the research area of liver pathophysiology and drug development.

Identification of a novel alpha-fetoprotein-expressing cell population induced by the Jagged1/Notch2 signal in murine fibrotic liver.

The liver is well known to possess high regenerative capacity in response to partial resection or tissue injury. However, liver regeneration is often impaired in the case of advanced liver fibrosis/cirrhosis when mature hepatocytes can hardly self-proliferate. Hepatic progenitor cells have been implicated as a source of hepatocytes in regeneration of the fibrotic liver. Although alpha-fetoprotein (AFP) is known as a clinical marker of progenitor cell induction in injured/fibrotic adult liver, the origin and features of such AFP-producing cells are not fully understood. Here, we demonstrate a unique and distinct AFP-expressing cell population that is induced by the Jagged1/Notch2 signal in murine fibrotic liver. Following repeated carbon tetrachloride injections, a significant number of AFP-positive cells with high proliferative ability were observed along the fibrous septa depending on the extent of liver fibrosis. These AFP-positive cells exhibited features of immature hepatocytes that were stained positively for hepatocyte-lineage markers, such as albumin and hepatocyte nuclear factor 4 alpha, and a stem/progenitor cell marker Sox9. A combination of immunohistological examination of fibrotic liver tissues and coculture experiments with primary hepatocytes and hepatic stellate cells indicated that increased Jagged1 expression in activated hepatic stellate cells stimulated Notch2 signaling and up-regulated AFP expression in adjacent hepatocytes. The mobilization and proliferation of AFP-positive cells in fibrotic liver were further enhanced after partial hepatectomy, which was significantly suppressed in Jagged1-conditional knockout mice. Finally, forced expression of the intracellular domain of Notch2 in normal liver induced a small number of AFP-expressing hepatocytes in vivo. Conclusion: Insight is provided into a novel pathophysiological role of Jagged1/Notch2 signaling in the induction of AFP-positive cells in fibrotic liver through the interaction between hepatocytes and activated hepatic stellate cells. (Hepatology Communications 2017;1:215-229).

Natural OX40L expressed on human T cell leukemia virus type-I-immortalized T cell lines interferes with infection of activated peripheral blood mononuclear cells by CCR5-utilizing human immunodeficiency virus.

BACKGROUND: OX40 ligand (OX40L) co-stimulates and differentiates T cells via ligation of OX40 that is transiently induced on T cells upon activation, resulting in prolonged T cell survival and enhanced cytokine production by T cells. This view has led to the targeting of OX40 as a strategy to boost antigen specific T cells in the context of vaccination. In addition, the ligation of OX40 has also been shown to inhibit infection by CCR5-utilizing (R5) but not CXCR4-utilizing (X4) human immunodeficiency virus type-1 (HIV-1) via enhancement of production of CCR5-binding ß-chemokines. It was reasoned that human T cell leukemia virus type-I (HTLV-1) immortalized T cell lines that express high levels of OX40L could serve as an unique source of physiologically functional OX40L. The fact that HTLV-1+ T cell lines simultaneously also express high levels of OX40 suggested a potential limitation. RESULTS: Results of our studies showed that HTLV-1+ T cell lines bound exogenous OX40 but not OX40L, indicating that HTLV-1+ T cell lines express an active form of OX40L but an inactive form of OX40. Anti-OX40 non-blocking monoclonal antibody (mAb), but not blocking mAb, stained HTLV-1+ T cell lines, suggesting that the OX40 might be saturated with endogenous OX40L. Functionality of the OX40L was confirmed by the fact that a paraformaldehyde (PFA)-fixed HTLV-1+ T cell lines inhibited the infection of autologous activated peripheral blood mononuclear cells (PBMCs) with R5 HIV-1 which was reversed by either anti-OX40L blocking mAb or a mixture of neutralizing mAbs against CCR5-binding ß-chemokines. CONCLUSIONS: Altogether, these results demonstrated that autologous T cell lines immortalized by HTLV-1 can be utilized as a conventional source of physiologically functional OX40L.