[Show abstract][Hide abstract] ABSTRACT: Following partial hepatectomy, a coordinated series of molecular events occurs to regulate hepatocyte entry into the cell cycle to recover lost mass. In rats during the first 6 h following resection, hepatocytes are primed by a tightly controlled cytokine response to prepare hepatocytes to begin replication. Although it appears to be a critical element driving regeneration, the cytokine response to resection has not yet been fully characterized. Specifically, the role of one of the key response elements to cytokine signaling (NF-κB) remains incompletely characterized. In this study, we present a novel, genome-wide, pattern-based analysis characterizing NF-κB binding during the priming phase of liver regeneration. We interrogated the dynamic regulation of priming by NF-κB through categorizing NF-κB binding in different temporal profiles: immediate sustained response, early transient response, and delayed response to partial hepatectomy. We then identified functional regulation of NF-κB binding by relating the temporal response profile to differential gene expression. We found that NF-κB bound genes govern negative regulation of cell growth and inflammatory response immediately following hepatectomy. NF-κB also transiently regulates genes responsible for lipid biosynthesis and transport as well as induction of apoptosis following hepatectomy. By the end of the priming phase, NF-κB regulation of genes involved in inflammatory response, negative regulation of cell death, and extracellular structure organization became prominent. These results suggest that NF-κB regulates target genes through binding and unbinding in immediate, transient, and delayed patterns. Such dynamic switch-like patterns of NF-κB binding may govern different functional transitions that drive the onset of regeneration.
Frontiers in Physiology 07/2015; 6:189. DOI:10.3389/fphys.2015.00189 · 3.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Effective delivery of oligonucleotides to liver cells in vivo has been of significant interest in the field. Recently developed cationic polymer Polyethylenimine (PEI) with repeating ethylenimine motifs that provide a very high density of positive charges along its backbone have been reported as highly effective in siRNA delivery to target liver cells. This approach has been further optimized for hepatocyte specific delivery through galactose conjugation to target galactose-specific membrane lectins such as the asialoglycoprotein receptor. We compared jetPEI, jetPEI-GAL and jetPEI-Hepatocyte for their efficacy of delivering plasmids and dye labeled siRNA in cultured primary rat hepatocytes as well as in vivo in rat. Our results demonstrate that all three nanoparticle based approaches are effective at in vitro and in vivo delivery. We delivered PGL3 plasmid, FAM green dye and TEX615 red dye to measure fluorescence intensity, indicative of highly significant and efficient translocation of the plasmid or dye labeled siRNA into hepatocytes.
[Show abstract][Hide abstract] ABSTRACT: In this study, we explore effective in vivo plasmid and siRNA delivery vehicle in liver parenchymal cells especially in the hepatocytes. Recently developed biodegradable polymer, PolyJet based DNA transfection reagent that ensures effective and reproducible transfection on a broad ranges of hard-to-transfect mammalian cells, was selected to transfect hepatocytes. We compared PolyJet versus jetPEI and Lipofectamine in HEK293 cells and in primary culture of rat hepatocytes and Kupffer cells. We used TEX615 red dye, pmaxGFP and PGL3 plasmid to measure transfection efficiency. Our results indicated a significant and efficient translocation of the PolyJet conjugated pmaxGFP plasmid or dye labeled siRNA into hepatocytes, Kupffer cells and HEK293 cell lines compared to other transfection reagents. Our results also demonstrated efficient translocation of PGL3 plasmid into hepatocytes as measured by increase in luciferase activity. The transfection of PGL3 was improved through the Polyjet mediated approach as compared to that of Lipofectamine and jetPEI-Hepatocyte.
[Show abstract][Hide abstract] ABSTRACT: Chronic alcohol intake interferes with the onset and progression of liver regeneration after tissue damage; suppression of this repair mechanism is thought to contribute to the development of alcoholic liver disease. Our overall objective is to uncover and characterize the regulatory network affected by chronic alcohol consumption and how alterations in this network lead to an aberrant liver repair and regeneration response. Our strategy is to utilize a combination of experimental analysis of gene expression and transcription factor activity changes combined with computational modeling to evaluate the impact of chronic ethanol consumption on the transcriptional regulatory network. In the present study, we aimed to identify the genes and regulatory patterns associated with the early stage of liver regeneration after partial hepatectomy (PHx) to examine the effects of chronic ethanol intake on this process.
We employed the Lieber-DeCarli alcohol pair-feeding protocol to assess the extent to which the molecular processes underlying liver regeneration in alcohol-fed rats are similar to pair-fed littermate controls in which ethanol calories were replaced by carbohydrates. Our previous studies showed that the alcohol-fed group contained significant changes to transcription factor (TF) localization during the priming phase of liver regeneration (0-6 hours). Genome-wide binding targets of multiple TFs (of NFκB, C/EBPβ, C/EBPα, STAT3) were detected using either Roche NimbleGen ChIP-ChIP microarray platform (ChIP-chip) or ABI SOLiD platform (ChIP-Seq). For each TF, we annotated the localization site data with TF binding site (TFBS) genes and discretized the TFBS data by specifying if a gene was bound by the TF (1) or not bound (0). In order to minimize technical noise, each TFBS was considered to be occupied only if a majority of the biological replicates showed statistically significant binding. We integrated results from multiple TFBS data into dynamic binding activity patterns reflecting the effect of an alcohol diet on combinatorial TF binding during the priming phase of liver regeneration. We examined each pattern for enriched pathways to identify alcohol-affected biological functions.
Our results revealed dynamic changes in binding of several early response TFs at the promoters of key genes differentially co-regulated during alcohol exposure and the onset of liver regeneration. Analysis of binding patterns indicated significant differences in the genome-wide targeting of NFkB activity between chow-fed and isocaloric pair-fed rats. We identified target genes unique to the pair-fed group and unique to the chow-fed group. The former set included genes associated with functions such as positive regulation of caspase activity, protein complex assembly, and apoptosis. The target gene set unique to chow-fed rats included genes associated with functions such as signaling, positive regulation of organelle organization, and positive regulation of cell growth and size. We also identified genes with a delayed increase in NFkB binding activity in the chow-fed group, including genes related to cell adhesion, and genes with an early increase in NFkB binding activity, including genes related to the cell cycle. Given the similarity of physiological response in the two dietary groups, it is likely that these differences in NFkB binding are compensated for elsewhere in the global regulatory network driving liver regeneration.
The binding activity dynamics of the TFs of NFκB, C/EBPβ, C/EBPα, and STAT3 at target genes were altered by chronic alcohol intake. Further analysis of the dynamic patterns led to novel insights about the differences in the transcriptional network subtending liver regeneration between alcohol-fed rats and pair-fed controls. The dominant patterns were either novel in the alcohol group or unique to the control group. Functional association of genes in these key dynamic response patterns revealed pathways such as cellular homeostasis, cell cycle regulation, RNA metabolism, and translation, to be affected by novel and missing activity in the alcohol group. Together, these results indicate that the effects of alcohol on liver regeneration may be mediated by dysregulation of cell cycle related processes and activation of alternative pathways.
Promoter analysis by PAINT revealed that several target genes may be driven by regulatory modules containing co-located binding sites for multiple TFs. We validated a subset of 20 NFκB targets and 5 key target promoters for the other TFs using ChIP-qPCR. Our results reveal that: (1) increased baseline activation of NFκB, C/EBPβ, C/EBPα, STAT3, c-Fos, and c-Jun in regenerating livers is correlated with the promoter binding for a subset of the target genes; (2) similarly, alcohol-fed rats showed a further increase in NFκB binding activity in response to PHx at 6 hours post-PHx occurring at only a subset of target promoters as opposed to pair-fed controls, which showed a consistent increase at all promoters tested during early liver regeneration; and (3) C/EBPβ, C/EBPα, STAT3, c-Fos, and c-Jun binding activity were down regulated at a subset of target promoters in alcohol-fed rats during early liver regeneration.
We conclude that chronic alcohol intake significantly affects the system-wide transcriptional regulatory network, including TF binding to the key genes active during the early stage of liver regeneration. These alterations may be critical in disrupting the normal regulatory network dynamics driving effective liver regeneration.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Adaptation to chronic ethanol (EtOH) treatment of rats results in a changed functional state of the liver and greatly inhibits its regenerative ability, which may contribute to the progression of alcoholic liver disease. METHODS: In this study, we investigated the effect of chronic EtOH intake on hepatic microRNA (miRNA) expression in male Sprague-Dawley rats during the initial 24 hours of liver regeneration following 70% partial hepatectomy (PHx) using miRNA microarrays. miRNA expression during adaptation to EtOH was investigated using RT-qPCR. Nuclear factor kappa B (NFκB) binding at target miRNA promoters was investigated with chromatin immunoprecipitation. RESULTS: Unsupervised clustering of miRNA expression profiles suggested that miRNA expression was more affected by chronic EtOH feeding than by the acute challenge of liver regeneration after PHx. Several miRNAs that were significantly altered by chronic EtOH feeding, including miR-34a, miR-103, miR-107, and miR-122 have been reported to play a role in regulating hepatic metabolism and the onset of these miRNA changes occurred gradually during the time course of EtOH feeding. Chronic EtOH feeding also altered the dynamic miRNA profile during liver regeneration. Promoter analysis predicted a role for NFκB in the immediate-early miRNA response to PHx. NFκB binding at target miRNA promoters in the chronic EtOH-fed group was significantly altered and these changes directly correlated with the observed expression dynamics of the target miRNA. CONCLUSIONS: Chronic EtOH consumption alters the hepatic miRNA expression profile such that the response of the metabolism-associated miRNAs occurs during long-term adaptation to EtOH rather than as an acute transient response to EtOH metabolism. Additionally, the dynamic miRNA program during liver regeneration in response to PHx is altered in the chronically EtOH-fed liver and these differences reflect, in part, differences in miRNA expression between the EtOH-adapted and control livers at the baseline state prior to PHx.
Alcoholism Clinical and Experimental Research 07/2012; 37(Suppl 1). DOI:10.1111/j.1530-0277.2012.01852.x · 3.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The goal of the present study is to characterize the transcriptomic regulatory dynamics operational in the regenerating rat liver affected by chronic alcohol intake. While transcriptomic data on the gene expression changes during normal liver regeneration is increasingly becoming available from multiple studies, the effect of chronic alcohol consumption on these changes is less clear. Such an alcohol-mediated disruption of repair mechanisms presumably contributes to the onset of liver damage in alcoholic liver disease.
We take an integrated experimental and computational approach to investigate the dynamics of the system-wide transcriptional changes, use sensitive bioinformatics methods to infer regulatory modules comprising of transcription factors (TFs) and target genes, and validate these for changes in the activity of TFs and their binding at target gene promoters. In our experimental approach, rats were fed a liquid diet containing 36% of total calories derived from ethanol; corresponding pair-fed calorie-matched controls received liquid diets in which ethanol calories were replaced either by carbohydrate or by fat. After five weeks, rats were subjected to 2/3d PHx by surgical excision of left-lateral and medial (LLM) lobes. Remnant liver tissues were harvested after 1, 6 and 24h to represent the priming phase, early G1, and G1/S transition, respectively. LLM tissues obtained at t=0 served as within-animal controls. We used Affymetrix Rat Gene 1.0 ST arrays with ~25000 probe sets to obtain global gene expression data from each liver sample (n=4 replicate animals, 72 arrays total). Analysis of the gene expression data using a 3-way ANOVA revealed a total of 6893 transcript clusters as responsive to PHx in at least one of the diet groups at either 1, 6 or 24h post PHx. Clustering the sample replicates indicated that the gene expression response to PHx is the primary factor separating the groups.
We developed a novel dynamic response pattern analysis approach in which the average differential gene expression data was discretized to three levels (+1, 0, -1) based on a fold change threshold of 1.5 up or down regulation. Within each diet group, this discretization yielded a dynamic response pattern for each gene, encoded by one of 27 possible ordered sets of the three levels +1, 0, and -1. Pairs of diet groups were compared to count the number of genes that follow each of the possible 27 * 27 (=729) comparative dynamic response patterns. Only a select set of the possible 729 patterns contained more than 25 genes indicating that the ethanol effects are mediated through specific mechanisms. The largest set of genes with different patterns between the diet groups were those with lack of differential regulation in ethanol group. This was followed by another gene set with de novo differential expression in ethanol group but not in that of the control diets. Other patterns involved alterations of dynamic gene expression from a transient response at one of the time points in control groups to a more persistent differential gene expression in ethanol group, and vice versa. Notably, ethanol group showed a marked lack of up regulation of cell cycle related gene expression at 6 and 24h.
In complementary experiments, we detected genome-wide NF-kB binding targets using Roche NimbleGen ChIP-chip microarray platform using Chromatin Immunoprecipitated (ChIP) samples. Our results indicate that PHx induced significant increase in NF-kB genome-wide binding at 1h, more so in chronic ethanol samples (5300 genes) than in controls (4000 genes), with several common targets (~3300 genes). At 6h post PHx, only the control samples showed a further increase in NF-kB promoter binding (~5500 genes). Pathway analysis revealed that NF-kB target genes specific to the chronic ethanol group participate in key processes such as DNA methylation, cell death, proteolysis, histone modification, and regulation of cell cycle. Our dynamic response pattern analysis of NF-kB promoter binding revealed similar alterations seen in gene expression response patterns, notably the conversion of persistent response to a transient response and vice versa in the ethanol group as compared to the control diet groups.
Taken together, our results provide a first system-wide view of the regulatory network dynamics in liver regeneration as it is affected by alcohol, and implicate combinatorial regulatory modules as participating in the adverse liver regeneration response. The above results indicate a phenomenon in which compensatory mechanisms that lead to an adaptive state in alcoholic rats play a role in suppressing the sensitivity of the system to certain perturbations, affecting the ability of the liver to initiate effective repair responses in the face of additional environmental challenges.
Supported by: NIH AA008714, AA014986, AA017261, AA016919, and AA018873.