Ron receptor-dependent gene regulation in a mouse model of endotoxin-induced acute liver failure.

BACKGROUND: Prior experimentation has shown that loss of the tyrosine kinase (TK) signaling domain of the Ron receptor leads to marked hepatocyte protection in a model of lipopolysaccharide-induced acute liver failure (ALF) in D-galactosamine (GalN)-sensitized mice. The aim of this study was to identify the role of Ron in the regulation of hepatic gene expression. METHODS: Microarray analyses were performed on liver RNA isolated sequentially from wild-type (WT) and TK-/- mice during the progression of ALF. Gene array data were validated using Western and immunohistochemistry analyses as well as with ex vivo culture systems. RESULTS: At baseline, 101 genes were differentially expressed between WT and TK-/- livers, which regulate processes involved in hypoxia, proliferation, apoptosis and metabolism. One hour after ALF induction, WT livers exhibited increased cytokine expression compared to TK-/- livers, and after 4 hours, an induction of suppressor of cytokine signaling (SOCS) genes as well as JAK-STAT pathway activation were prominent in TK-/- livers compared to controls. CONCLUSION: Our studies suggest a novel hepato-protective mechanism in Ron TK-/- mice wherein increased and sustained SOCS production and JAK-STAT activation in the hepatocyte may inhibit the destructive proinflammatory milieu and promote survival factors which blunt hepatic death and the ensuing development of ALF.

Transcriptional profiles of intestinal tumors in Apc(Min) mice are unique from those of embryonic intestine and identify novel gene targets dysregulated in human colorectal tumors.

The adenomatous polyposis coli (APC) tumor suppressor is a major regulator of the Wnt signaling pathway in normal intestinal epithelium. APC, in conjunction with AXIN and GSK-3beta, forms a complex necessary for the degradation of beta-catenin, thereby preventing beta-catenin/T-cell factor interaction and alteration of growth-controlling genes such as c-MYC and cyclin D1. Inappropriate activation of the Wnt pathway, via Apc/APC mutation, leads to gastrointestinal tumor formation in both the mouse and human. In order to discover novel genes that may contribute to tumor progression in the gastrointestinal tract, we used cDNA microarrays to identify 114 genes with altered levels of expression in Apc(Min) mouse adenomas from the duodenum, jejunum, and colon. Changes in the expression of 24 of these 114 genes were not observed during mouse development at embryonic day 16.5, postnatal day 1, or postnatal day 14 (relative to normal adult intestine). These 24 genes are not previously known Wnt targets. Seven genes were validated by real-time reverse transcription-PCR analysis, whereas four genes were validated by in situ hybridization to mouse adenomas. Real-time reverse transcription-PCR analysis of human colorectal cancer cell lines and adenocarcinomas revealed that altered expression levels were also observed for six of the genes Igfbp5, Lcn2, Ly6d, N4wbp4 (PMEPA1), S100c, and Sox4.

Isoforms of the APC tumor suppressor and their ability to inhibit cell growth and tumorigenicity.

Mutation of the APC tumor suppressor gene is one of the earliest events in the development of most colorectal tumors. The APC gene encodes multiple protein isoforms through a complicated pattern of expression and alternative splicing. The role that each isoform plays in cellular physiology is unknown, although the presence of some of these isoforms in postmitotic cells suggests a role in controlling cell growth or promoting differentiation. Three APC isoforms that differ in their amino-terminal domains were evaluated by gene transfer experiments using a colon cancer cell line that lacks functional APC. All three isoforms alter cellular morphology and affect cell growth by elongating the G1 phase of the cell cycle. The conventional APC and brain-specific APC isoforms suppress the tumorigenic phenotype of cultured cells, while the 0.3 APC isoform does not. In support of these experiments, BrdU incorporation as a marker for S-phase entry occurs at a higher level in transiently transfected cells with 0.3 APC when compared to cells transfected with the other isoforms. All three APC isoforms colocalize with microtubules and dramatically reduce beta-catenin activity to the same extent in transiently transfected cancer cells, suggesting that the different effects of each isoform on tumorigenesis may be nontranscriptional in origin.

The role of TFIIB-RNA polymerase II interaction in start site selection in yeast cells.

Previous studies have established a critical role of both TFIIB and RNA polymerase II (RNAPII) in start site selection in the yeast Saccharomyces cerevisiae. However, it remains unclear how the TFIIB-RNAPII interaction impacts on this process since such an interaction can potentially influence both preinitiation complex (PIC) stability and conformation. In this study, we further investigate the role of TFIIB in start site selection by characterizing our newly generated TFIIB mutants, two of which exhibit a novel upstream shift of start sites in vivo. We took advantage of an artificial recruitment system in which an RNAPII holoenzyme component is covalently linked to a DNA-binding domain for more direct and stable recruitment. We show that TFIIB mutations can exert their effects on start site selection in such an artificial recruitment system even though it has a relaxed requirement for TFIIB. We further show that these TFIIB mutants have normal affinity for RNAPII and do not alter the promoter melting/scanning step. Finally, we show that overexpressing the genetically isolated TFIIB mutant E62K, which has a reduced affinity for RNAPII, can correct its start site selection defect. We discuss a model in which the TFIIB-RNAPII interaction controls the start site selection process by influencing the conformation of PIC prior to or during PIC assembly, as opposed to PIC stability.