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Ectopic expression of CD74 in Ikk?-deleted mouse hepatocytes.

ABSTRACT: CD74, a Type II membrane glycoprotein and MHC class II chaperone involved in antigen processing, is normally expressed by cells associated with the immune system. CD74 also forms heterodimers with CD44 to generate receptors to macrophage migration inhibitory factor (MIF), a proinflammatory cytokine. Following targeted Alb-Cre-mediated deletion of Ikk? in Ikk?(?hep) mice (Ikk?(F/F):Alb-Cre, a strain highly susceptible to chemically induced hepatotoxicity and hepatocarcinogenesis), CD74 is expressed abundantly by adult hepatocytes throughout liver acini, albeit more intensely in midzonal-to-centrilobular regions. By comparison, CD74 expression is not observed in Ikk?(F/F) hepatocytes, nor is it augmented in the livers of Ikk?(+/+):Alb-Cre mice; CD74 is barely detectable in cultured embryonic fibroblasts from Ikk?(-/-) mice. Microarray profiling shows that constitutive CD74 expression in Ikk?(?hep) hepatocytes is accompanied by significantly augmented expression of CD44 and key genes associated with antigen processing and host defense, including MHC class II I-A?, I-A?, and I-E? chains, CIITA and CD86. Taken together, these observations suggest that Ikk?(?hep) hepatocytes might express functional capacities for class II-restricted antigen presentation and heightened responsiveness to MIF-signaling, and also suggest further roles for intrahepatocellular IKK? in the suppression or inactivation of molecules normally associated with the formation and differentiation of cells of the immune system.

SUBMITTER: Koch KS

PROVIDER: S-EPMC2965206 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Ectopic expression of CD74 in Ikkβ-deleted mouse hepatocytes.

Koch Katherine S KS Leffert Hyam L HL

Acta histochemica 20100601 4

CD74, a Type II membrane glycoprotein and MHC class II chaperone involved in antigen processing, is normally expressed by cells associated with the immune system. CD74 also forms heterodimers with CD44 to generate receptors to macrophage migration inhibitory factor (MIF), a proinflammatory cytokine. Following targeted Alb-Cre-mediated deletion of Ikkβ in Ikkβ(Δhep) mice (Ikkβ(F/F):Alb-Cre, a strain highly susceptible to chemically induced hepatotoxicity and hepatocarcinogenesis), CD74 is express ...[more]

PMID: 20569972

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Project description:Primary hepatocyte culture is an important in vitro system for the study of liver functions. In vivo, hepatocytes have high oxidative metabolism. However, oxygen supply by means of diffusion in in vitro static cultures is much less than that by blood circulation in vivo. Therefore, we investigated whether hypoxia contributes to dedifferentiation and deregulated metabolism in cultured hepatocytes. To this end, murine hepatocytes were cultured under static or shaken (60 revolutions per minute) conditions in a collagen sandwich. The effect of hypoxia on hepatocyte cultures was examined by metabolites in media and cells, hypoxia-inducible factors (HIF)-1/2? western blotting, and real-time quantitative polymerase chain reaction for HIF target genes and key genes of glucose and lipid metabolism. Hepatocytes in shaken cultures showed lower glycolytic activity and triglyceride accumulation than static cultures, compatible with improved oxygen delivery and mitochondrial energy metabolism. Consistently, static cultures displayed significant HIF-2? expression, which was undetectable in freshly isolated hepatocytes and shaken cultures. Transcript levels of HIF target genes (glyceraldehyde 3-phosphate dehydrogenase [Gapdh], glucose transporter 1 [Glut1], pyruvate dehydrogenase kinase 1 [Pdk1], and lactate dehydrogenase A [Ldha]) and key genes of lipid metabolism, such as carnitine palmitoyltransferase 1 (Cpt1), apolipoprotein B (Apob), and acetyl-coenzyme A carboxylase 1 (Acc1), were significantly lower in shaken compared to static cultures. Moreover, expression of hepatocyte nuclear factor 4? (Hnf4?) and farnesoid X receptor (Fxr) were better preserved in shaken cultures as a result of improved oxygen delivery. We further revealed that HIF-2 signaling was involved in hypoxia-induced down-regulation of Fxr. Conclusion: Primary murine hepatocytes in static culture suffer from hypoxia. Improving oxygenation by simple shaking prevents major changes in expression of metabolic enzymes and aberrant triglyceride accumulation; in addition, it better maintains the differentiation state of the cells. The shaken culture is, therefore, an advisable strategy for the use of primary hepatocytes as an in vitro model. (Hepatology Communications 2018;2:299-312).

Project description:BackgroundNANOS3 is a gene conserved throughout evolution. Despite the quite low conservation of Nanos sequences between different organisms and even between Nanos paralogs, their role in germ cell development is remarkably universal. Human Nanos3 expression is normally restricted to the gonads and the brain. However, ectopic activation of this gene has been detected in various human cancers. Until now, Nanos3 and other Nanos proteins have been studied almost exclusively in germ cell development.MethodsTransgenic mice were generated by targeted insertion of a human Nanos3 cDNA into the ROSA26 locus. The transgene could be spatiotemporally induced by Cre recombinase activity removing an upstream floxed STOP cassette. A lung tumor model with ectopic Nanos3 expression was based on the lung-specific activation of the reverse tetracycline transactivator gene, in combination with a tetO-CMV promoter controlling Cre expression. When doxycycline was provided to the mice, Cre was activated leading to deletion of TP53 alleles and activation of both oncogenic KRasG12D and Nanos3. Appropriate controls were foreseen. Tumors and tumor-derived cell cultures were analyzed in various ways.ResultsWe describe the successful generation of Nanos3LSL/- and Nanos3LSL/LSL mice in which an exogenous human NANOS3 gene can be activated in vivo upon Cre expression. These mice, in combination with different conditional and doxycycline-inducible Cre lines, allow the study of the role of ectopic Nanos3 expression in several cancer types. The Nanos3LSL mice were crossed with a non-small cell lung cancer (NSCLC) mouse model based on conditional expression of oncogenic KRas and homozygous loss of p53. This experiment demonstrated that ectopic expression of Nanos3 in the lungs has a significant negative effect on survival. Enhanced bronchiolar dysplasia was observed when Nanos3-expressing NSCLC mice were compared with control NSCLC mice. An allograft experiment, performed with cell cultures derived from primary lung tumors of control and Nanos3-expressing NSCLC mice, revealed lymph node metastasis in mice injected with Nanos3-expressing NSCLC cells.ConclusionsA new mouse model was generated allowing examination of Nanos3-associated pathways and investigation of the influence of ectopic Nanos3 expression in various cancer types. This model might identify Nanos3 as an interesting target in cancer therapeutics.

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Ectopic expression of CD74 in Ikk?-deleted mouse hepatocytes.

Publications

Ectopic expression of CD74 in Ikkβ-deleted mouse hepatocytes.

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