Impaired cell proliferation in regenerating liver of 3 β-hydroxysterol Δ14-reductase (TM7SF2) knock-out mice

The liver is the most important organ in cholesterol metabolism, which is instrumental in regulating cell proliferation and differentiation. The gene Tm7sf2 codifies for 3 β-hydroxysterol-Δ¹⁴-reductase (C14-SR), an endoplasmic reticulum resident protein catalyzing the reduction of C14-unsaturated sterols during cholesterol biosynthesis from lanosterol. In this study we analyzed the role of C14-SR in vivo during cell proliferation by evaluating liver regeneration in Tm7sf2 knockout (KO) and wild-type (WT) mice. Tm7sf2 KO mice showed no alteration in cholesterol content. However, accumulation and delayed catabolism of hepatic triglycerides was observed, resulting in persistent steatosis at all times post hepatectomy. Moreover, delayed cell cycle progression to the G1/S phase was observed in Tm7sf2 KO mice, resulting in reduced cell division at the time points examined. This was associated to abnormal ER stress response, leading to alteration in p53 content and, consequently, induction of p21 expression in Tm7sf2 KO mice. In conclusion, our results indicate that Tm7sf2 deficiency during liver regeneration alters lipid metabolism and generates a stress condition, which, in turn, transiently unbalances hepatocytes cell cycle progression.     

Depletion of Tip60/Kat5 affects the hepatic antioxidant system in mice

Tat-interactive protein 60 kDa (TIP60, also known as lysine acetyltransferase 5 [KAT5]) is a member of the MYST protein family with histone acetyltransferase activity. Recent studies have reported that TIP60 has multiple functions in many signal transduction mechanisms, especially p53-mediated apoptosis. Although the activation of apoptosis signaling pathways requires the presence of cellular reactive oxygen species (ROS) at a certain level, an imbalance between the production and consumption of ROS in cells results in oxidative stress (OS). In this study, we investigated for the first time how the absence of the Tip60 gene in the liver affects gene expression, enzyme activity, and protein expression of the hepatic antioxidant members localized in the cytoplasm, including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST). First, we successfully generated liver-specific Tip60 knockout mice (mutants) using Cre/LoxP recombination. The reduced glutathione level and nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) expression, a marker of OS, increased significantly in the Tip60 mutant liver. Gene expression, activity, and protein expression of the enzymatic antioxidant system, including SOD, CAT, GR, GPx, and GST were investigated in mutants and control groups. Despite a significant correlation between the gene, enzyme activity, and protein content for CAT and GR, this was not true for SOD and GPx. The overall results suggest that TIP60 acts on the hepatic antioxidant system both at the gene and protein levels, but the actual effect of the deletion of Tip60 is observed at the protein level, especially for SOD and GPx.     

LTW4 Protein on Mouse Chromosome 1 Is a Member of a Family of Antioxidant Proteins

Based on its map position, polymorphism pattern, and expression in the kidney, the gene encoding liver 20,000–30,000 MW protein 4 (LTW4) can be considered a potential candidate for theJckm2modifying locus, which mediates the severity of polycystic kidney disease in thejuvenile cystic kidneymouse. Using two-dimensional gel electrophoresis, we identified variants of a 26-kDa polypeptide that differed in their isoelectric points between the C57BL/6J and the DBA/2J inbred strains in a pattern similar to that originally described for LTW4 protein. N-terminal amino acid sequence was obtained by microsequencing analysis, and full-length clones were obtained by RT-PCR amplification and characterized. The map position of the cloned gene was determined and corresponded to that previously described forLtw4.The gene has homology to a class of proteins characterized as thiol-specific antioxidants that are protective against damage caused by oxidative stress. The murine MER5 gene is also a member of this gene family and has recently been renamedAntioxidant protein 1 (Aop1),based on its functional characterization. We therefore propose that the gene encoding LTW4 be calledAop2.     

Zonation of Nitrogen and Glucose Metabolism Gene Expression upon Acute Liver Damage in Mouse

Zonation of metabolic activities within specific structures and cell types is a phenomenon of liver organization and ensures complementarity of variant liver functions like protein production, glucose homeostasis and detoxification. To analyze damage and regeneration of liver tissue in response to a toxic agent, expression of liver specific enzymes was analyzed by in situ hybridization in mouse over a 6 days time course following carbon tetrachloride (CCl₄) injection. CCl₄ mixed with mineral oil was administered to BALB/c mice by intraperitoneal injection, and mice were sacrificed at different time points post injection. Changes in the expression of albumin (Alb), arginase (Arg1), glutaminase 2 (Gls2), Glutamine synthetase (Gs), glucose-6-phosphatase (G6pc), glycogen synthase 2 (Gys2), Glycerinaldehyd-3-phosphat-Dehydrogenase (Gapdh), Cytochrom p450 2E1 (Cyp2e1) and glucagon receptor (Gcgr) genes in the liver were studied by in situ hybridization and qPCR. We observed significant changes in gene expression of enzymes involved in nitrogen and glucose metabolism and their local distribution following CCl₄ injury. We also found that Cyp2e1, the primary metabolizing enzyme for CCl₄, was strongly expressed in the pericentral zone during recovery. Furthermore, cells in the damaged area displayed distinct gene expression profiles during the analyzed time course and showed complete recovery with strong albumin production 6 days after CCl₄ injection. Our results indicate that despite severe damage, liver cells in the damaged area do not simply die but instead display locally adjusted gene expression supporting damage response and recovery.     

Ribosome biogenesis gene DEF/UTP25 is essential for liver homeostasis and regeneration

Hepatocytes are responsible for diverse metabolic activities in a liver. Proper ribosome biogenesis is essential to sustain the function of hepatocytes. There are approximately 200 factors involved in ribosome biogenesis; however, few studies have focused on the role of these factors in maintaining liver homeostasis. The digestive organ expansion factor (def) gene encodes a nucleolar protein Def that participates in ribosome biogenesis. In addition, Def forms a complex with cysteine protease Calpain3 (Capn3) and recruits Capn3 to the nucleolus to cleave protein targets. However, the function of Def has not been characterized in the mammalian digestive organs. In this report, we show that conditional knockout of the mouse def gene in hepatocytes causes cell morphology abnormality and constant infiltration of inflammatory cells in the liver. As age increases, the def conditional knockout liver displays multiple tissue damage foci and biliary hyperplasia. Moreover, partial hepatectomy leads to sudden acute death to the def conditional knockout mice and this phenotype is rescued by intragastric injection of the anti-inflammation drug dexamethasone one day before hepatectomy. Our results demonstrate that Def is essential for maintaining the liver homeostasis and liver regeneration capacity in mammals.

     

The chemokine SDF-1 regulates blastema formation during zebrafish fin regeneration

The work presented in this study focuses on blastema formation in epimorphic regeneration. We describe the expression pattern of Sdf1a and Sdf1b (the chemokines stromal-cell-derived factor-1a and 1b) and their two receptors Cxcr4a and Cxcr4b during zebrafish fin regeneration. We demonstrate that Sdf1a/Cxcr4a plays a critical role in fin regeneration and more precisely in epidermal cell proliferation, an important process for blastema formation. In mammals, a single cxcr4 gene is involved both in chemotaxis and cell proliferation and survival; we discuss in this study a possible functional division of the two cxcr4 zebrafish genes.     

Rice <Emphasis Type="Italic">SERK1</Emphasis> gene positively regulates somatic embryogenesis of cultured cell and host defense response against fungal infection

Here we report on the isolation and characterization of a somatic embryogenesis receptor-like kinase (OsSERK1) gene in rice (Oryza sativa). The OsSERK1 gene belongs to a small subfamily of receptor-like kinase genes in rice and shares a highly conserved gene structure and extensive sequence homology with previously reported plant SERK genes. Though it has a basal level of expression in various rice organs/tissues, as high expression level was detected in rice callus during somatic embryogenesis. Suppression of OsSERK1 expression in transgenic calli by RNA interference resulted in a significant reduction of shoot regeneration rate (from 72% to 14% in the japonica rice Zhonghua11). Overexpression of OsSERK1, however, increased the shoot regeneration rate (from 72% to 86%). Interestingly, OsSERK1 is significantly activated by the rice blast fungus, particularly during the incompatible interaction, and is associated with host cell death in Sekigushi lesion mimic mutants. This gene is also inducible by defense signaling molecules such as salicylic acid, jasmonic acid, and abscisic acid. Furthermore, constitutive overexpression of OsSERK1 in two rice cultivars led to an increase in host resistance to the blast fungus. Our data suggest that OsSERK1 may partially mediate defense signal transduction in addition to its basic role in somatic embryogenesis.     

Functional annotation of mouse mutations in embryonic stem cells by use of expression profiling

Expression profiling offers a potential high-throughput phenotype screen for mutant mouse embryonic stem (ES) cells. We have assessed the ability of expression arrays to distinguish among heterozygous mutant ES cell lines and to accurately reflect the normal function of the mutated genes. Two ES cell lines hemizygous for overlapping regions of mouse Chromosome (Chr) 5 differed substantially from the wildtype parental line and from each other. Expression differences included frequent downregulation of hemizygous genes and downstream effects on genes mapping to other chromosomes. Some genes were affected similarly in each deletion line, consistent with the overlap of the deletions. To determine whether such downstream effects reveal pathways impacted by a mutation, we examined ES cell lines heterozygous for mutations in either of two well-characterized genes. A heterozygous mutation in the gene encoding the cell cycle regulator, cyclin D kinase 4 (Cdk4), affected expression of many genes involved in cell growth and proliferation. A heterozygous mutation in the ATP binding cassette transporter family A, member 1 (Abca1) gene, altered genes associated with lipid homeostasis, the cytoskeleton, and vesicle trafficking. Heterozygous Abca1 mutation had similar effects in liver, indicating that ES cell expression profile reflects changes in fundamental processes relevant to mutant gene function in multiple cell types.     

Rap1‐mediated nucleosome displacement can regulate gene expression in senescent cells without impacting the pace of senescence

Cell senescence is accompanied, and in part mediated, by changes in chromatin, including histone losses, but underlying mechanisms are not well understood. We reported previously that during yeast cell senescence driven by telomere shortening, the telomeric protein Rap1 plays a major role in reprogramming gene expression by relocalizing hundreds of new target genes (called NRTS, for n ew R ap1 t argets at s enescence) to the promoters. This leads to two types of histone loss: Rap1 lowers histone level globally by repressing histone gene expression, and it also causes local nucleosome displacement at the promoters of upregulated NRTS. Here, we present evidence of direct binding between Rap1 and histone H3/H4 heterotetramers, and map amino acids involved in the interaction within the Rap1 SANT domain to amino acids 392–394 (SHY). Introduction of a point mutation within the native RAP1 locus that converts these residues to alanines (RAP1^SHY), and thus disrupts Rap1‐H3/H4 interaction, does not interfere with Rap1 relocalization to NRTS at senescence, but prevents full nucleosome displacement and gene upregulation, indicating direct Rap1‐H3/H4 contacts are involved in nucleosome displacement. Consistent with this, the histone H3/H4 chaperone Asf1 is similarly unnecessary for Rap1 localization to NRTS but is required for full Rap1‐mediated nucleosome displacement and gene activation. Remarkably, RAP1^SHY does not affect the pace of senescence‐related cell cycle arrest, indicating that some changes in gene expression at senescence are not coupled to this arrest.