Gene Expression Profiles Of Hepatocytes Research Articles

8236 Hepatocyte Vitamin D Receptor (VDR) Regulates Liver Regeneration

Abstract Disclosure: J.E. Gunton: None. Harendran Elangovan [1], Rebecca A. Stokes [1], Jeremy Keane [1], Sarinder Chahal [1], Caroline Samer [1], Miguel Agoncillo [1], Josephine Yu [1], Jennifer Chen [1], Michael Downes 2, Ronald M. Evans 2, Christopher Liddle [1],2, Jenny E. Gunton [1] [1]Centre for Diabetes, Obesity and Endocrinology, The Westmead Institute for Medical Research, University of Sydney, Westmead, NSW, Australia 2 Salk Institute for Biological Studies, La Jolla, CA. USA Background: Vitamin D signals through the vitamin D receptor (VDR) to induce its end-organ effects. Hepatic stellate cells control development of liver fibrosis in response to stressors and vitamin D signaling decreases fibrogenesis. VDR expression in hepatocytes, however, is low in healthy liver, and the role of VDR in hepatocyte proliferation is unclear. Methods: In these studies, hepatocyte-VDR null mice (hVDR) were used to assess the role of VDR and vitamin D signaling in hepatic regeneration. Mice underwent 2/3 partial hepatectomy and liver regeneration and function were studied. Results: hVDR mice had impaired liver regeneration with impaired hepatocyte proliferation. This was associated with significant differential changes in bile salts. Notably, mice lacking hepatocyte VDR had significant increases in expression of conjugated bile acids after partial hepatectomy, consistent with failure to normalize hepatic function by the 14-day time point tested. CDCA (chenodeoxycholate) is an FXR ligand, which was increased after surgery in controls but not in hVDR mice. FXR signaling is important for hepatocyte proliferation. Real-time PCR of hVDR and control livers showed significant changes in expression of cell cycle genes including cyclins D1 and E1 and cyclin-dependent kinase 2. Gene expression profiling of hepatocytes treated with vitamin D or control showed regulation of groups of genes involved in liver proliferation, hepatitis, liver hyperplasia / hyperproliferation and liver necrosis / cell death. Conclusions: Together these studies demonstrate an important functional role for VDR in hepatocytes during liver regeneration. Combined with the known profibrotic effects of impaired VDR signaling in stellate cells, these studies provide a mechanism whereby vitamin D deficiency would both reduce hepatocyte proliferation and permit fibrosis, leading to significant liver compromise. Presentation: 6/2/2024

Far-infrared radiation alleviates steatohepatitis and fibrosis in metabolic dysfunction-associated fatty liver disease

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a disease that causes an abnormal accumulation of fat in the liver, triggering inflammation and fibrosis, the mechanism of which is not fully understood and for which there is a lack of specific drug therapy. Far-infrared radiation (FIR) has demonstrated evident therapeutic efficacy across various diseases, and novel nanomaterial graphene patches can emit it through electric heating. This study aimed to investigate the potential protective effects of FIR against MAFLD. Mice were fed with a MCD diet to mimic MAFLD progression, and histopathology analysis, biochemical analysis, RT-qPCR, and Western blotting analysis were performed to assess the effect of FIR on MAFLD in vivo. The effect of FIR treatment on MAFLD in vitro was investigated by biochemical analysis and gene expression profiling of hepatocytes. Mice subjected to the MCD diet and treated with FIR exhibited reduced hepatic lipid deposition, inflammation, fibrosis and liver damage. The therapeutic effect exerted by FIR in mice may be caused by the enhancement of AMPK phosphorylation and inhibition of the TGFβ1-SMAD2/3 pathway. Besides, FIR intervention alleviated MAFLD in hepatocytes in vitro and the results were verified by gene expression profiling. Our results revealed a promising potential of FIR as a novel therapeutic approach for MAFLD.

Vitamin D Receptor Regulates Liver Regeneration After Partial Hepatectomy in Male Mice.

Vitamin D signals through the vitamin D receptor (VDR) to induce its end-organ effects. Hepatic stellate cells control development of liver fibrosis in response to stressors and vitamin D signaling decreases fibrogenesis. VDR expression in hepatocytes is low in healthy liver, and the role of VDR in hepatocyte proliferation is unclear. Hepatocyte-VDR null mice (hVDR) were used to assess the role of VDR and vitamin D signaling in hepatic regeneration. hVDR mice have impaired liver regeneration and impaired hepatocyte proliferation associated with significant differential changes in bile salts. Notably, mice lacking hepatocyte VDR had significant increases in expression of conjugated bile acids after partial hepatectomy, consistent with failure to normalize hepatic function by the 14-day time point tested. Real-time PCR of hVDR and control livers showed significant changes in expression of cell-cycle genes including cyclins D1 and E1 and cyclin-dependent kinase 2. Gene expression profiling of hepatocytes treated with vitamin D or control showed regulation of groups of genes involved in liver proliferation, hepatitis, liver hyperplasia/hyperproliferation, and liver necrosis/cell death. Together, these studies demonstrate an important functional role for VDR in hepatocytes during liver regeneration. Combined with the known profibrotic effects of impaired VDR signaling in stellate cells, the studies provide a mechanism whereby vitamin D deficiency would both reduce hepatocyte proliferation and permit fibrosis, leading to significant liver compromise.

G-quadruplexes sense natural porphyrin metabolites for regulation of gene transcription and chromatin landscapes

BackgroundG-quadruplexes (G4s) are unique noncanonical nucleic acid secondary structures, which have been proposed to physically interact with transcription factors and chromatin remodelers to regulate cell type-specific transcriptome and shape chromatin landscapes.ResultsBased on the direct interaction between G4 and natural porphyrins, we establish genome-wide approaches to profile where the iron-liganded porphyrin hemin can bind in the chromatin. Hemin promotes genome-wide G4 formation, impairs transcription initiation, and alters chromatin landscapes, including decreased H3K27ac and H3K4me3 modifications at promoters. Interestingly, G4 status is not involved in the canonical hemin-BACH1-NRF2-mediated enhancer activation process, highlighting an unprecedented G4-dependent mechanism for metabolic regulation of transcription. Furthermore, hemin treatment induces specific gene expression profiles in hepatocytes, underscoring the in vivo potential for metabolic control of gene transcription by porphyrins.ConclusionsThese studies demonstrate that G4 functions as a sensor for natural porphyrin metabolites in cells, revealing a G4-dependent mechanism for metabolic regulation of gene transcription and chromatin landscapes, which will deepen our knowledge of G4 biology and the contribution of cellular metabolites to gene regulation.

Chemical conversion of aged hepatocytes into bipotent liver progenitor cells.

In the aging society, understanding the influence of hepatocyte age on hepatocyte donation may inform efforts to expand alternative cell sources to mitigate liver donor shortage. A combination of the molecules Y27632, A-83-01, and CHIR99021 has been used to reprogram rodent young hepatocytes into chemically induced liver progenitor (CLiP) cells; however, whether it could also reprogram aged hepatocytes has not yet been elucidated. Primary hepatocytes were isolated from aged and young donor rats, respectively. Hepatic histological changes were evaluated. Differences in gene expression in hepatocytes were identified. The in vitro reprogramming plasticity of hepatocytes as evidenced by CLiP conversion and the hepatocyte and cholangiocyte maturation capacity of reprogrammed CLIPs were analyzed. The effect of hepatocyte growth factor (HGF) on cell propagation was also investigated. The histological findings revealed ongoing liver damage with inflammation, fibrosis, senescence, and ductular reaction in aged livers. Microarray analysis showed altered gene expression profiles in hepatocytes from aged donors, especially with regard to metabolic pathways. Aged hepatocytes could be converted into CLiPs (Aged-CLiPs) expressing progenitor cell markers, but with a relatively low proliferative rate compared with young hepatocytes. Aged-CLiPs possessed both hepatocyte and cholangiocyte maturation capacity. HGF facilitated CLiP conversion in aged hepatocytes, which was partly related to the activation of Erk1 and Akt1 signaling. Aged rat hepatocytes have retained reprogramming plasticity as evidenced by CLiP conversion in culture. HGF promoted proliferation and CLiP conversion in aged hepatocytes. Hepatocytes from aged donors may be used as an alternative cell source to mitigate donor shortage.

Deciphering hepatocellular responses to metabolic and oncogenic stress.

Each cell type responds uniquely to stress and fractionally contributes to global and tissue-specific stress responses. Hepatocytes, liver macrophages (MΦ), and sinusoidal endothelial cells (SEC) play functionally important and interdependent roles in adaptive processes such as obesity and tumor growth. Although these cell types demonstrate significant phenotypic and functional heterogeneity, their distinctions enabling disease-specific responses remain understudied. We developed a strategy for the simultaneous isolation and quantification of these liver cell types based on antigenic cell surface marker expression. To demonstrate the utility and applicability of this technique, we quantified liver cell-specific responses to high-fat diet (HFD) or diethylnitrosamine (DEN), a liver-specific carcinogen, and found that while there was only a marginal increase in hepatocyte number, MΦ and SEC populations were quantitatively increased. Global gene expression profiling of hepatocytes, MΦ and SEC identified characteristic gene signatures that define each cell type in their distinct physiological or pathological states. Integration of hepatic gene signatures with available human obesity and liver cancer microarray data provides further insight into the cell-specific responses to metabolic or oncogenic stress. Our data reveal unique gene expression patterns that serve as molecular "fingerprints" for the cell-centric responses to pathologic stimuli in the distinct microenvironment of the liver. The technical advance highlighted in this study provides an essential resource for assessing hepatic cell-specific contributions to metabolic and oncogenic stress, information that could unveil previously unappreciated molecular mechanisms for the cellular crosstalk that underlies the continuum from metabolic disruption to obesity and ultimately hepatic cancer.

Dose-response analysis of the effects of persistent organic pollutants (POPs) on gene expression in human hepatocytes

Determination of gene expression profile alterations following dose-dependent exposure to toxicants will greatly improve the utility of arrays in identifying biomarkers and elucidating modes of toxicity. In the present study, the effects of increasing doses of persistent organic pollutants (POPs) on the expression of toxicity-related genes were evaluated. Human hepatocellular carcinoma (HepG2) cells were exposed to nontoxic (NT) and 20% inhibitory concentrations (IC20) of the well-known toxic PoP compounds, toxaphene (TOX) and chlordane (CD), for 48 h. Transcriptomic profiling showed differential gene expression patterns and a profound effect on gene expression changes in the IC20 exposure group. Through clustering analysis, 98 NT- and 336 IC20-specific genes and 73 genes expressed dose-dependently were identified. Gene Ontology (GO) analysis identified several key pathways related to the adverse health effects of POPs. In conclusion, this report describes changes in gene expression profiles in hepatocytes in response to different doses of POPs and relates these changes to hepatotoxicity-related pathways. Moreover, novel genes and pathways that may potentially play a role in the prevention of liver disease due to POP exposure were identified.

Rat hepatocytes weighted gene co-expression network analysis identifies specific modules and hub genes related to liver regeneration after partial hepatectomy.

The recovery of liver mass is mainly mediated by proliferation of hepatocytes after 2/3 partial hepatectomy (PH) in rats. Studying the gene expression profiles of hepatocytes after 2/3 PH will be helpful to investigate the molecular mechanisms of liver regeneration (LR). We report here the first application of weighted gene co-expression network analysis (WGCNA) to analyze the biological implications of gene expression changes associated with LR. WGCNA identifies 12 specific gene modules and some hub genes from hepatocytes genome-scale microarray data in rat LR. The results suggest that upregulated MCM5 may promote hepatocytes proliferation during LR; BCL3 may play an important role by activating or inhibiting NF-kB pathway; MAPK9 may play a permissible role in DNA replication by p38 MAPK inactivation in hepatocytes proliferation stage. Thus, WGCNA can provide novel insight into understanding the molecular mechanisms of LR.

Interleukin 18 accelerates the hepatic cell proliferation in rat liver regeneration after partial hepatectomy

Interleukin 18 (IL-18) is a proinflammatory cytokine with an ability to accelerate cell proliferation through activating other factors. However, little is yet understood of the role of IL-18 in the regulation of liver regeneration (LR). To study the effect of IL-18 on LR, the gene expression profiles of hepatocytes isolated from rat regenerative liver were determined by Rat Genome 230 2.0 microarray. Next, the synergistic effects of genes associated to IL-18 pathway were analyzed by expression profile function Et. Then, the expression level of IL-18 was examined by RT-PCR and ELISA. Finally, the effect of IL-18 on hepatocyte proliferation was detected by injecting recombinant rat IL-18 (rrIL-18) into rats immediately after partial hepatectomy (PH) and the rate of hepatocyte proliferation was detected by BrdU labeling. The microarray result showed that the expressions of 13 genes of IL-18 pathway and 49 cell proliferation genes regulated by the pathway were significantly altered at transcriptional level. The Et values of three branches of IL-18 pathway, NF-κB, p38 and JNK, were markedly enhanced during the priming and progressing phases of rat LR. The mRNA level of IL-18 was significantly elevated at 2 and 36h, and its level in plasma was also significantly increased at 2h, and reached the peaks at 12h and 48h after PH (p<0.05). The number of BrdU positive cells was dramatically increased in rats treated with IL-18 compared to PBS control group (p<0.01). In conclusion, IL-18 promotes rat hepatocyte proliferation in the LR priming and progressing phases after PH.

Functional Based Analysis and Visualization of Gene Expression Data from Hepatocytes Grown on Different Substrates

The ability to grow a fully functional Hepatocyte in vitro would be a prodigious breakthrough for modern day drug testing and regenerative medicine. However there are many factors affecting Hepatocyte functionality in vitro that need to be studied before this is possible, like the effect of the underlying substrate that the Hepatocyte is grown on. To better understand this interaction, gene expression profiles of Hepatocytes were measured on four different substrates: Corning(r) CellBIND(r), Collagen, Corning Matrigel(r) and Locust Bean Gum (S906). Here, we present data analysis methods that enable data reduction, identification of differentially expressed genes, and grouping of genes with similar intensities by implementing data clustering. The optimum number of clusters in a given data set is estimated using the gap statistic method. We further employ Cytoscape to help visualize the data and incorporate GeneMania to help identify potentially interesting patterns across substrates. One of the interesting findings from our analysis suggests up-regulation of genes involved in vasculature development on all four substrates. Such findings can be used to design new experiments that can provide further insight on the interactions between hepatocytes and their substrates and help understand the mechanism behind responses of hepatocytes on these substrates.

Molecular perturbations restrict potential for liver repopulation of hepatocytes isolated from non–heart-beating donor rats

Organs from non-heart-beating donors are attractive for use in cell therapy. Understanding the nature of molecular perturbations following reperfusion/reoxygenation will be highly significant for non-heart-beating donor cells. We studied non-heart-beating donor rats for global gene expression with Affymetrix microarrays, hepatic tissue integrity, viability of isolated hepatocytes, and engraftment and proliferation of transplanted cells in dipeptidyl peptidase IV-deficient rats. In non-heart-beating donors, liver tissue was morphologically intact for >24 hours with differential expression of 1, 95, or 372 genes, 4, 16, or 34 hours after death, respectively, compared with heart-beating donors. These differentially expressed genes constituted prominent groupings in ontological pathways of oxidative phosphorylation, adherence junctions, glycolysis/gluconeogenesis, and other discrete pathways. We successfully isolated viable hepatocytes from non-heart-beating donors, especially up to 4 hours after death, although the hepatocyte yield and viability were inferior to those of hepatocytes from heart-beating donors (P < 0.05). Similarly, although hepatocytes from non-heart-beating donors engrafted and proliferated after transplantation in recipient animals, this was inferior to hepatocytes from heart-beating donors (P < 0.05). Gene expression profiling in hepatocytes isolated from non-heart-beating donors showed far greater perturbations compared with corresponding liver tissue, including representation of pathways in focal adhesion, actin cytoskeleton, extracellular matrix-receptor interactions, multiple ligand-receptor interactions, and signaling in insulin, calcium, wnt, Jak-Stat, or other cascades. Liver tissue remained intact over prolonged periods after death in non-heart-beating donors, but extensive molecular perturbations following reperfusion/reoxygenation impaired the viability of isolated hepatocytes from these donors. Insights into molecular changes in hepatocytes from non-heart-beating donors offer opportunities for improving donor cell viability, which will advance the utility of non-heart-beating donor organs for cell therapy or other applications.

Dose-response functional gene analysis by exposure to 3 different polycyclic aromatic hydrocarbons in human hepatocytes

Polycyclic aromatic hydrocarbons (PAHs) are known as carcinogen and have been studied to show modulation of gene expression by exposure to various PAHs. However, few studies have been reported on microarray analysis of dose-response relationships of gene expression patterns. For comprehensive examination of dose-response effects of PAHs on gene expression, we elicited the genes which were changed more than 1.5-fold by analysis of gene expression profiles in human hepatocellular carcinoma (HepG2) cells, exposed for 48 h to nontoxic (NT) and IC20 doses of 3 different PAHs (benzo[a]anthracene (B[a]A), benzo[k]fluoranthene (B[k]F) and indeno[1,2,3-c,d]pyrene (IND)) by using human oligonucleotide chip. Transcriptomic profiling shows different gene expression patterns in NT and IC20 exposure groups and shows higher sensitivity to gene alteration in IC20 exposure group than NT group. Through the clustering analysis of gene expression profiles, we identified 7 up- and 3 down-regulated NT dose specific genes and 401 up- and 562 downregulated IC20 dose specific genes. After Gene Ontology (GO) analysis on IC20 dose specific genes, we determined several key pathways which are known as related to increase hepatotoxicity such as metabolism of xenobiotics by cytochrome P450, Jak-STAT signaling pathway, cytokine-cytokine receptor interaction and complement and coagulation cascade. But we did not find hepatotoxicity-related pathways through GO analysis on NT dose-specific genes. Genes that are expressed in only IC20 exposure group were regarded as biomarker of PAHs-induced hepatotoxicity. In conclusion, this study describes changes in gene expression profiles in hepatocytes in response to exposure to 3 PAHs with different doses and relates these gene expression changes to hepatotoxicity related pathways. Moreover, potential new leads to genes and pathways that could play a role in liver disease prevention by PAHs were identified.

Gene expression profiles of hepatocytes treated with La(NO3)3 of rare earth in rats.

To compare the gene expression between La(NO(3))(3)-exposed and control rats in vivo. Rats were fed La(NO(3))(3) once daily at a dose of 20 mg/kg for one month by gavage. Gene expression of hepatocytes was detected using mRNA differential display (DD) technique and cDNA microarray and compared between treated and control groups. Six differentially expressed sequence tags were cloned by DD, of which five were up regulated and one was down regulated in treated rats. Two sequences were determined. One band was novel. The other shared 100% sequence homology with AU080263 Sugano mouse brain mncb Mus musculus cDNA clone MNCb-5435 5'. With DNA microarray, 136 differentially expressed genes were identified including 131 over-expressed genes and 5 under-expressed genes. Most of these differentially expressed genes were cell signal and transmission genes, genes associated with metabolism, protein translation and synthesis. La(NO(3))(3) could change the expression levels of some kinds of genes. Further analysis of the differentially expressed genes would be helpful for understanding the wide biological effect spectrum of rare earth elements.

Isolated human hepatocytes in culture display markedly different gene expression patterns depending on attachment status

In vitro human hepatocyte cultures are a key tool in the investigation of xenobiotic toxicity and metabolism. In most in vitro hepatocyte studies, the cells are allowed to adhere to an extracellular matrix, such as collagen. Unfortunately, the ability of freshly isolated hepatocytes to adhere to collagen varies from donor to donor. We used microarray analysis to determine what gene expression differences exist between hepatocytes in suspension and hepatocytes attached to collagen. Results from different donors showed a considerable difference in gene expression patterns between the two hepatocyte populations. In addition, we also compared the gene expression profiles of hepatocytes in culture with liver tissue. The results showed that both hepatocytes in suspension and hepatocytes attached to collagen display significant gene expression differences compared with liver tissue. Finally, we show that both populations of hepatocytes are responsive to dexamethasone and regulate some of the same genes. Overall, our results suggest that either significant gene expression changes occur in isolated hepatocytes or that suspended and attached cells represent different populations of hepatocytes found in intact livers.