Hepatocyte Nuclear Factor 3β Research Articles

Genotoxic carcinogen 7,12-dimethylbenz[a]anthracene inhibits gap junction intercellular communication through post-transcriptional and post-translational processing involved in connexin 43 stability.

Gap junctional intercellular communication (GJIC) is composed of connexin (Cx) and plays an important role in maintaining intracellular homeostasis. Loss of GJIC is involved in the early stages of cancer pathways of non-genotoxic carcinogens; however, the effect of genotoxic carcinogens, including polycyclic aromatic hydrocarbons (PAHs), on GJIC function remains unclear. Therefore, we determined whether and how a representative PAH 7,12-dimethylbenz[a]anthracene (DMBA) suppresses GJIC in WB-F344cells. First, DMBA significantly inhibited GJIC and dose-dependently reduced Cx43 protein and mRNA expression. In contrast, Cx43 promoter activity was upregulated after DMBA treatment via the induction of specificity protein 1 and hepatocyte nuclear factor 3β, indicating that the promoter-independent loss of Cx43 mRNA can be associated with the inhibition of mRNA stability, which was verified by actinomycin D assay. In addition to a decrease in mRNA stability involved in human antigen R, we also observed DMBA-induced acceleration of Cx43 protein degradation, which was closely related to the loss of GJIC through Cx43 phosphorylation via MAPK activation. In conclusion, the genotoxic carcinogen DMBA suppresses GJIC by inhibiting post-transcriptional and post-translational processing of Cx43. Our findings suggest that the GJIC assay is an efficient short-term screening test for predicting the carcinogenic potential of genotoxic carcinogens.

Subacute ruminal acidosis downregulates FOXA2, changes oxidative status, and induces autophagy in the livers of dairy cows fed a high-concentrate diet

The purpose of this experiment was to investigate high-concentrate feeding-induced changed status of oxidative and autophagy in the livers of dairy cows. Hepatocyte nuclear factor 3β (FOXA2) was reported in cases of liver fibrosis, glucolipid metabolism, and hepatocyte differentiation, but not in cases liver damage in cows fed a high-concentrate diet. Therefore, we also aimed to explore the potential role of FOXA2 in SARA-induced liver damage. We divided 12 mid-lactating Holstein cows into 2 groups and fed them a high-concentrate (HC group, forage:concentrate = 4:6) and a low-concentrate (forage:concentrate = 6:4) diet. After a 2-wk adaptation period and a 3-wk experimental period, peripheral blood was collected for determination of antioxidant enzyme activity, and liver tissue was collected to examine genes and proteins. On d 20 and 21 of the experiment, rumen fluid was collected, and the pH was measured. A significant difference in rumen fluid pH was found between the 2 groups (low-concentrate: 6.10 ± 0.07 vs. HC: 5.59 ± 0.09). The rumen fluid pH in the HC group was less than 5.6 at 2 time points, indicating that SARA was successfully induced. Lipopolysaccharide (0.24 ± 0.10 vs. 0.42 ± 0.12) and malondialdehyde (1.46 ± 0.25 vs. 2.94 ± 0.65) increased, whereas superoxide dismutase (14.06 ± 0.63 vs. 11.71 ± 0.64), reduced glutathione (14.48 ± 2.25 vs. 6.82 ± 0.67), and the total antioxidant capacity (0.43 ± 0.03 vs. 0.30 ± 0.03) decreased in the peripheral blood of the HC group. Moreover, in liver tissue from the HC group, catalase (0.71 ± 0.03 vs. 0.49 ± 0.03) and superoxide dismutase (27.46 ± 1.90 vs. 20.32 ± 1.54) were decreased, whereas malondialdehyde (0.21 ± 0.03 vs. 0.28 ± 0.03) was elevated. Meanwhile, we observed lower gene expression of CAT (1.00 ± 0.15 vs. 0.64 ± 0.17), NAD(P)H quinone dehydrogenase 1 (NQO1; 1.00 ± 0.09 vs. 0.47 ± 0.14), glutathione peroxidase 1 (GPX1; 1.03 ± 0.27 vs. 0.55 ± 0.09), SOD1 (1.01 ± 0.17 vs. 0.76 ± 0.17), and SOD3 (1.02 ± 0.21 vs. 0.55 ± 0.16) in the liver tissue of the HC group. Furthermore, western blot analysis showed that high-concentrate feeding led to decreased sirtuin-1 (SIRT1) (1.00 ± 0.10 vs. 0.62 ± 0.15) and FOXA2 (1.02 ± 0.19 vs. 0.68 ± 0.18), elevated autophagy-related protein microtubule associated protein 1 light chain 3 II (MAP1LC3-II; 1.00 ± 0.32 vs. 1.98 ± 0.83) and autophagy related 5 (ATG5; 1.00 ± 0.30 vs. 1.80 ± 0.27), and suppressed antioxidant signaling pathway-related protein nuclear factor erythroid 2-like 2 (NFE2L2; 1.00 ± 0.18 vs. 0.61 ± 0.30) and heme oxygenase 1 (HMOX1; 1.00 ± 0.48 vs. 0.38 ± 0.25) in liver tissue. Overall, these data indicated that SARA elevated systematic oxidative status and enhanced autophagy in the liver, and suppressed SIRT1 and FOXA2 may mediate enhanced oxidative damage and autophagy in the livers of dairy cows fed a high-concentrate diet.

Hepatocyte Nuclear Factor 3β Plays a Suppressive Role in Colorectal Cancer Progression

Background and Objective: Hepatocyte nuclear factor 3β (HNF3β) is a key transcription factor in the development of the gastrointestinal tract. However, only few studies have examined its' expression, function and potential clinical significance in colorectal cancer tumorigenesis and progression.Methods: HNF3β expression in colorectal cancer tissue samples of 174 patients was assessed by immunohistochemistry. The results were analyzed with respect to patients' clinicopathological characteristics and survival. Following the in vitro cell transfection, MTT, wound healing, and Transwell assays were used to test cell proliferation, migration, and invasion, respectively. Western blot was used to examine IL6, JAK1, and STAT3 protein expression. The potential for tumor formation was evaluated using a mouse xenograft model.Results: HNF3β expression was lower in colon cancer tissue compared to normal tissue and correlated with UICC clinical stage (P = 0.001), depth of invasion (P = 0.004), regional lymph node metastasis (P = 0.007), distant metastasis (P = 0.048), and poor survival (P < 0.001) in patients with colorectal cancer. Furthermore, HNF3β overexpression impeded proliferation, migration and invasion of SW480 cells via JAK-STAT3 signaling in vitro. Moreso, HNF3β overexpression showed a significant growth inhibition of subcutaneous xenograft tumors in vivo.Conclusions: The results show that HNF3β acts as a suppressor of colorectal cancer progression and decreased HNF3 β expression is closely related to the poor prognosis. Thus, HNF3β may be a potential molecular target for inhibition of colorectal cancer cells and development of new anti-tumor therapies.

Simulation of Embryonic Environments Produced by the Effect of Estradiol on Induced Pluripotent Stem Cells

Estradiol (E2) is one of the sex steroid hormones, whose various well-known functions include control of the female reproductive system, causing breast cancers, and inhibiting arterial sclerosis. In this study to investigate how E2 affected cell differentiation of induced pluripotent stem cells (iPSCs), we used different methods to culture hiPSCs, the undifferentiated state, longterm culture without passages (natural differentiation), embryoid bodies (EBs), and accelerated differentiation by activin, focusing on marker gene expression, including Brachyury, hepatocyte nuclear factor (HNF) 3β, estrogen receptor α, and other markers. When iPSCs began to differentiate by responding to E2, Brachyury expression decreased regardless of the amount of E2, cell line, or protocol. Other markers were up or down-regulated, but did not show any tendency except in the early stage of EBs. The definite timing that E2 had the strongest effect on the aspect of differentiation was the EB stage analogous to gastrulation, in which E2 repressed Brachyury expression and induced HNF3β at its restricted amount. These results suggest that the first target of E2 is the gastrulation stage.

Expression of hepatocyte nuclear factor 3β and its correlation with clinical parameters in prostate cancer

Objective To detect the expression of hepatocyte nuclear factor 3β (Foxa2) in prostate cancer and benign prostatic hyperplasia (BPH), and to investigate its clinical relationship with prostate cancer. Methods A retrospective review was performed on 48 cases of prostate cancer and 10 cases of BPH undergoing operation. The expression of Foxa2 protein was detected by immunohistochemistry. Integrated clinical stage, Gleason score, distant metastases, and preoperative prostate specific antigen (PSA) level were analyzed. Results The expression of Foxa2 in prostate cancer was significantly higher than in BPH [weakly positive expression: 18.7% (9/48) vs. 90.0% (9/10); moderate positive expression: 56.3% (27/48) vs. 10.0% (1/10); strong positive expression: 25.0% (12/48) vs. 0.0% (0/10); P=0.000). The expression of Foxa2 (weakly positive expression, moderate positive expression and strong positive expression) in each group was as follows: Gleason score group [2 - 4 score group: 60.0% (3/5), 40.0% (2/5) and 0.0% (0/5); 5-7 score group: 14.3% (4/28), 67.8% (19/28) and 17.9% (5/28); 8-10 score group: 13.3% (2/15), 40.0% (6/15) and 46.7% (7/15). Metastasis group (negative group: 22.6% (7/31), 64.5% (20/31) and 12.9% (4/31); positive group 11.8% (2/17), 41.2% (7/17) and 47.0% (8/17). Clinical stage group (T1-T2 group: 22.7% (5/22), 68.2% (15/22), 9.1% (2/22); T3-T4 group: 15.4% (4/26), 46.1% (12/26), 38.5% (10/26). Preoperative PSA level group (PSA ≤ 10 ng/ml group: 12.5% (1/8), 87.5% (7/8) and 0.0% (0/8); 10-20 ng/ml group: 28.6% (4/14), 57.1% (8/14) and 14.3% (2/14); ≥ 20 ng/ml group: 15.4% (4/26), 46.2% (12/26) and 38.4% (10/26). 2-year biochemical recurrence group (positive group: 25.0% (1/4), 50.0% (2/4) and 25.0% (1/4); negative group: 28.6% (2/7), 57.1% (4/7) and 14.3% (1/7). Foxa2 was associated with Gleason score (P=0.023), invasion and metastasis (P= 0.032). The expression of Foxa2 was positively correlated with the clinical stage (P=0.064) and preoperative PSA level (P=0.115), but didn’t reach statistical significance. No statistically significant differences were found in the 2-year biochemical recurrence rate (P=0.906). Conclusion Our findings unveil the relationship between Foxa2 and prostate cancer development. Key words: Hepatocyte nuclear factor 3β; Prostate cancer; Invasion; Metastasis

Transcriptional Regulation of CYP2B6 Expression by Hepatocyte Nuclear Factor 3β in Human Liver Cells.

CYP2B6 plays an increasingly important role in xenobiotic metabolism and detoxification. The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) have been established as predominant regulators for the inductive expression of CYP2B6 gene in human liver. However, there are dramatic interindividual variabilities in CYP2B6 expression that cannot be fully explained by the CAR/PXR-based modulation alone. Here, we show that expression level of CYP2B6 was correlated with that of hepatocyte nuclear factor 3β (HNF3β) in human primary hepatocytes prepared from 35 liver donors. Utilizing recombinant virus-mediated overexpression or knockdown of HNF3β in HepG2 cells, as well as constructs containing serial deletion and site-directed mutation of HNF3β binding motifs in CYP2B6 luciferase reporter assays, we demonstrated that the presence or lack of HNF3β expression markedly correlated with CYP2B6 gene expression and its promoter activity. Novel enhancer modules of HNF3β located upstream of the CYP2B6 gene transcription start site were identified and functionally validated as key elements governing HNF3β-mediated CYP2B6 expression. Chromatin immunoprecipitation assays in human primary hepatocytes and surface plasmon resonance binding affinity experiments confirmed the essential role of these enhancers in the recruitment of HNF3β to the promoter of CYP2B6 gene. Overall, these findings indicate that HNF3β represents a new liver enriched transcription factor that is involved in the transcription of CYP2B6 gene and contributes to the large interindividual variations of CYP2B6 expression in human population.

Forkhead box protein A2 and T helper type 2-mediated pulmonary inflammation.

The transcription factor forkhead box protein A2 (FOXA2, also known as hepatocyte nuclear factor 3β or transcription factor 3β), has been found to play pivotal roles in multiple phases of mammalian life, from the early development to the organofaction, and subsequently in homeostasis and metabolism in the adult. In the embryonic development period, FOXA2 is require d for the formation of the primitive node and notochord, and its absence results in embryonic lethality. Moreover, FOXA2 plays an important role not only in lung development, but also in T helper type 2 (Th2)-mediated pulmonary inflammation and goblet cell hyperplasia. In this article, the role of FOXA2 in lung development and Th2-mediated pulmonary inflammation, as well as in goblet cell hyperplasia, is reviewed. FOXA2 deletion in airway epithelium results into Th2-mediated pulmonary inflammation and goblet cell hyperplasia in developing lung. Leukotriene pathway and signal transducers and activators of transcription 6 pathway may mediate this inflammation through recruitment and activation of denditric cell during lung developments. FOXA2 is a potential treatment target for lung diseases with Th2 inflammation and goblet cell hyperplasia, such as asthma and chronic obstructive pulmonary disease.

Production of hepatocyte-like cells from human umbilical vein mesenchymal stem cells.

The human umbilical vein, as a readily available stem cell source, is a good alternative to harvest mesenchymal stem cells. Human umbilical cord vein mesenchymal stem cells have recently been isolated and have demonstrated the ability to differentiate into various cell types such as fat, bone, cartilage and neuronal cells. In this study, we have investigated whether human umbilical cord vein mesenchymal stem cells are also able to differentiate into hepatocyte-like cells. Hepatic differentiation was performed with a 2-step protocol and the use of hepatocyte growth factor and oncostatin M for cell culture. During four weeks of induction, most cells displayed a cuboidal morphology. Immunological analysis indicated that umbilical cord vein mesenchymal stem cells-derived hepatocyte-like cells expressed liver-specific protein markers such as albumin and cytokeratin-18. The hepatocyte-like cells also displayed several characteristics of hepatocytes, including expression of transthyretin, glucose 6-phosphatase, cytokeratin-8,18, alpha-fetoprotein, hepatocyte nuclear factor-3β and albumin. The result of indocyanine green cell uptake, as a test substance to evaluate hepatocyte-like cell function, was positive for differentiated cells. Glycogen storage was examined by periodic acid-Schiff staining. Accumulation of intracellular glycogen was detected in the hepatocyte-like cells. Based on these observations, we have concluded that umbilical cord vein mesenchymal stem cells are endowed with hepatogenic potential and may provide a stem cell source to be used as cell therapy for liver diseases.

MicroRNA-141 inhibits cell proliferation and invasion and promotes apoptosis by targeting hepatocyte nuclear factor-3β in hepatocellular carcinoma cells.

BackgroundHepatocyte nuclear factor-3β (HNF-3β) plays a critical role in hepatocyte differentiation and controls liver-specific gene expression during the development of hepatocellular carcinoma (HCC), but the molecular basis of this process has not been fully elucidated. microRNAs (miRNAs) are powerful, post-transcriptional regulators of gene expression. Whether miRNAs can impact the effects of HNF-3β in HCC is still unknown.MethodsHNF-3β and miR-141 expression levels were detected in HepG2 cells, using real-time quantitative RT-PCR (qRT-PCR). Luciferase reporter assays and Western blots were used to validate HNF-3β as a direct target gene of miR-141. Cell proliferation, invasion, and apoptosis were also examined to confirm whether miR-141 could impact on HNF-3β in HCC.ResultsIn this study, we found that HNF-3β protein levels were consistently upregulated in HCC clinical tissues compared with matched normal adjacent tissues. However, the mRNA levels of HNF-3β varied in random tissues, suggesting that a post-transcriptional mechanism was involved in its regulation. We used bioinformatic analyses to search for miRNAs that could potentially target HNF-3β, and identified specific targeting sites for miR-141 in the 3′-untranslated region (3′-UTR) of the HNF-3β gene. By overexpressing miR-141 in HepG2 cells, we experimentally validated that miR-141 directly regulated HNF-3β expression. Furthermore, the biological consequences of targeting HNF-3β by miR-141 were examined using cell proliferation, invasion and apoptosis assays in vitro. We demonstrated that the repression of HNF-3β by miR-141 suppressed the proliferation and invasion and promoted the apoptosis of HepG2 cells.ConclusionsmiR-141 functions as a tumor suppressor in HCC cells through the inhibition of HNF-3β translation.

Generation of insulin-producing cells from rat mesenchymal stem cells using an aminopyrrole derivative XW4.4

Type 1 diabetes mellitus (T1DM), a multisystem disease with both biochemical and anatomical/structural consequences, is a major health concern worldwide. Pancreatic islet transplantation provides a promising treatment for T1DM. However, the limited availability of islet tissue or new sources of insulin producing cells (IPCs) that are responsive to glucose hinder this promising approach. Though slow, the development of pancreatic beta-cell lines from rodent or human origin has been steadily progressing. Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent, culture-expanded, non-hematopoietic cells that are currently being investigated as a novel cellular therapy. The in vitro differentiation potential of IPCs has raised hopes for a treatment of clinical diseases associated with autoimmunity.We screened for small molecules that induce pancreatic differentiation of IPCs. There are some compounds which showed positive effects on the DTZ staining. The aminopyrrole derivative compound XW4.4 which shows the best activity among them was found to induce pancreatic differentiation of rat MSCs (rMSCs). The in vitro studies indicated that treatment of rMSCs with compound XW4.4 resulted in differentiated cells with characteristics of IPCs including islet-like clusters, spherical, grape-like morphology, insulin secretion, positive for dithizone, glucose stimulation and expression of pancreatic endocrine cell marker genes. The data has also suggested that hepatocyte nuclear factor 3β (HNF 3β) may be involved in pancreatic differentiation of rMSCs when treated with XW4.4. Results indicate that XW4.4 induced rMSCs support the efforts to derive functional IPCs and serve as a means to alleviate limitations surrounding islet cell transplantation in the treatment of T1DM.

Estrogen receptor α or β loss in the colon of Min/+ mice promotes crypt expansion and impairs TGFβ and HNF3β signaling

Adenomatous polyposis coli (APC)-regulated Wnt and transforming growth factor-β (TGFβ) signaling cooperate in the intestine to maintain normal enterocyte functions. Human clinical trials showed that estrogen [17β-estradiol (E2)], the ligand of nuclear receptors estrogen receptor α (ERα) and ERβ, inhibited colorectal cancer (CRC) in women. Consistent with this finding, we reported that E2, ERα and ERβ suppressed intestinal tumorigenesis in the C57BL/6J-Min/+ (Min/+) mouse, a CRC model. Here, we extended our results with further comparisons of colon and small intestine from intact female Apc (+/+) (WT), Min/+ and ER-deficient Min/+ mice. In the colon of ER-deficient Min/+ mice, ER loss reduced TGFβ signaling in crypt base cells as evidenced by minimal expression of the effectors Smad 2, 3 and 4 in these strains. We also found reduced expression of Indian hedgehog (Ihh), bone morphogenetic protein 4 and hepatocyte nuclear factor 3β or FoxA2, factors needed for paracrine signaling between enterocytes and mesenchyme. In proximal colon, ER loss produced a >10-fold increased incidence of crypt fission, a marker for wound healing and tumor promotion. These data, combined with our previous work detailing the specific roles of E2, ERα and ERβ in the colon, suggest that ER activity helps to maintain the intestinal stem cell (ISC) microenvironment by modulating epithelial-stromal crosstalk in ways that regulate cytokine, Wnt and Ihh availability in the extracellular matrix (ECM).

Nonviral Cell Labeling and Differentiation Agent for Induced Pluripotent Stem Cells Based on Mesoporous Silica Nanoparticles

The generation of induced pluripotent stem cells (iPSCs) is an innovative personalized-regenerative technology, which can transform own-self somatic cells into embryonic stem (ES)-like cells, which have the potential to differentiate into all cell types of three dermal lineages. However, how to quickly, efficiently, and safely produce specific-lineage differentiation from pluripotent-state cells and iPSCs is still an open question. The objective of the present study was to develop a platform of a nonviral gene delivery system of mesoporous silica nanoparticles (MSNs) to rapidly generate iPSC-derived definitive-lineage cells, including endodermal-differentiated cells. We also evaluated the feasibility and efficiency of FITC-conjugated MSNs (FMSNs) for labeling of iPSCs and utilized the multifunctional properties of FMSNs for a suitable carrier for biomolecule delivery. We showed that FMSNs of various surface charges could be efficiently internalized by iPSCs without causing cytotoxicity. The levels of reactive oxygen species and pluripotent status, including in vitro stemness signatures and in vivo teratoma formation, remained unaltered. Notably, positive-charged FMSN enhanced cellular uptake efficiency and retention time. Moreover, when using positive-charged FMSN to deliver hepatocyte nuclear factor 3β (HNF3β) plasmid DNA (pDNA), the treated iPSCs exhibited significantly improved definitive endoderm formation and further quickly differentiated into hepatocyte-like cells with mature functions (low-density lipoprotein uptake and glycogen storage) within 2 weeks in vitro. Double delivery of pHNF3β further improved mRNA expression levels of liver-specific genes. These findings reveal the multiple advantages of FMSNs to serve as ideal vectors not only for stem cell labeling but also for safe gene delivery to promote the production of hepatocyte-like cells from iPSCs.

Human liver fatty acid binding protein (hFABP1) gene is regulated by liver-enriched transcription factors HNF3β and C/EBPα

The human liver fatty acid binding protein (hFABP1) participates in cellular long-chain fatty acid trafficking and regulation of lipid metabolism and changes in hFABP1 are associated with an increased risk for type 2 diabetes, cardiovascular disease (CVD), and metabolic syndromes. Gene regulation of hFABP1 is not fully understood. Therefore, in the present study, the full length hFABP1 promoter (nucleotides −2125 to +51) and a series of truncated promoter regions were cloned. A luciferase reporter assay revealed that nucleotides −255 to +50 in the promoter region contained full of maximum hFABP1 promoter activity compared with the full length promoter. Furthermore high activity was shown when the plasmid was transfected into liver-derived cells such as the human hepatoblastoma cell line HepG2 and the hepatoma cell line Huh7. TFSEARCH and TESS programs were used to predict potential transcription factor binding sites. Two putative binding sites for the liver-enriched transcription factors hepatocyte nuclear factor 3β (HNF3β) and CCAAT/enhancer binding protein α (C/EBPα) were identified in the −255 nt to −155 nt hFABP1 promoter region. Site-directed mutagenesis of these two sites reduced dramatically hFABP1 promoter activity. In addition, the electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) revealed that these binding sites were recognized by HNF3β and C/EBPα respectively. Overexpression of HNF3β and C/EBPα enhanced the transcription of hFABP1 and consequently improved the protein level of hFABP1 in HepG2 cells, while knockdown of HNF3β and C/EBPα showed the inverse effects. Taken together, the hFABP1 gene is highly transcribed in liver-derived cells, and regulated predominantly by liver-enriched transcription factors HNF3β and C/EBPα.

Docosahexaenoic acid suppresses apolipoprotein A-I gene expression through hepatocyte nuclear factor-3β

Dietary fish-oil supplementation has been shown in human kinetic studies to lower the production rate of apolipoprotein (apo) A-I, the major protein component of HDL. The underlying mechanism responsible for this effect is not fully understood. We investigated the effect and the mechanism of action of the very-long-chain n-3 (omega-3) polyunsaturated fatty acid docosahexaenoic acid (DHA), relative to the saturated fatty acid palmitic acid (PA), on the hepatic expression of apo A-I in HepG2 cells. HepG2 cells were treated with different doses of DHA and PA (0-200 μmol/L). mRNA expression levels of apo A-I were assessed by real-time polymerase chain reaction, and apo A-I protein concentrations were measured by immunoassay. DHA dose-dependently suppressed apo A-I mRNA levels and also lowered apo A-I protein concentrations in the media, with maximum effects at 200 μmol/L. This concentration of fatty acids was used in all subsequent experiments. To elucidate the mechanism mediating the reduction in apo A-I expression by DHA, transfection experiments were conducted with plasmid constructs containing serial deletions of the apo A-I promoter. The DHA-responsive region was mapped to the -185 to -148 nucleotide region of the apo A-I promoter, which binds the hepatocyte nuclear factor (HNF)-3β. Nuclear extracts from cells treated with DHA or PA had a similar nuclear abundance of HNF-3β. However, electrophoresis mobility shift assays showed less binding of HNF-3β to the -180 to -140 sequence of the apo A-I promoter than did PA-treated cells. As shown by chromatin immunoprecipitation analysis, less HNF-3β was recruited to the apo A-I promoter in DHA-treated cells than in PA-treated cells, which supports the concept of an interference of DHA with the binding of HNF-3β to the apo A-I promoter. These findings suggest that, in human hepatoma HepG2 cells, DHA inhibits the binding of HNF-3β to the apo A-I promoter, resulting in the repression of apo A-I promoter transactivity and thus a reduction in apo A-I expression.

Investigation of Hepatoprotective Activity of Induced Pluripotent Stem Cells in the Mouse Model of Liver Injury

To date liver transplantation is the only effective treatment for end-stage liver diseases. Considering the potential of pluripotency and differentiation into tridermal lineages, induced pluripotent stem cells (iPSCs) may serve as an alternative of cell-based therapy. Herein, we investigated the effect of iPSC transplantation on thioacetamide- (TAA-) induced acute/fulminant hepatic failure (AHF) in mice. Firstly, we demonstrated that iPSCs had the capacity to differentiate into hepatocyte-like cells (iPSC-Heps) that expressed various hepatic markers, including albumin, α-fetoprotein, and hepatocyte nuclear factor-3β, and exhibited biological functions. Intravenous transplantation of iPSCs effectively reduced the hepatic necrotic area, improved liver functions and motor activity, and rescued TAA-treated mice from lethal AHF. 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate cell labeling revealed that iPSCs potentially mobilized to the damaged liver area. Taken together, iPSCs can effectively rescue experimental AHF and represent a potentially favorable cell source of cell-based therapy.

137P In vitro differentiation of human umbilical vein mesenchymal stem cells into hepatocyte-like cells

Human umbilical vein, as a readily available source of stem cells, is one of the good alternative source for mesenchymal stem cells. Human umbilical cord vein mesenchymal stem cells (UVMSCs) have been recently isolated and demonstrated the ability to differentiate into various cell types such as fat, bone, cartilage and neuronal cells. In this study, we have investigated whether human UVMSC are also able to differentiate into hepatocyte-like cells. Hepatic differentiation was done with a 2-step protocol and with the use of hepatocyte growth factor and oncostatin M. The cells were cultured with hepatocyte growth factor (HGF) and oncostatin M (OSM). During 4 weeks of induction most of the cells displayed a cuboidal morphology. Immunological analysis indicated that UVMSCs-derived hepatocyte-like cells expressed liver-specific protein markers such as albumin (ALB) and cytokeratin-18 (CK-18). The hepatocyte-like cells also displayed several characteristics of hepatocytes, including expression of transthyretin, glucose-6-phosphatase (G6P), cytokeratin-8,18 (CK-8,18), alpha-fetoprotein (AFP), hepatocyte nuclear factor-3β (AFP) and ALB. The result of indocyanine green (ICG) cell uptake as a test substance to evaluate hepatocyte-like cells function was positive for differentiated cells. Glycogen storage was examined by periodic acid–Schiff (PAS) staining and accumulation of intracellular glycogen were detected in hepatocyte-like cells. Based on these observations it can be concluded that UVMSCs encompass suitable hepatogenic potential which may provide a source of stem cells for cell therapy in liver diseases.

Activation of interleukin-6-induced glycoprotein 130/signal transducer and activator of transcription 3 pathway in mesenchymal stem cells enhances hepatic differentiation, proliferation, and liver regeneration

Adult bone marrow-derived mesenchymal stem cells (MSCs) exist in all living species and are capable of differentiating into different types of specific cells. In this study, we demonstrate the therapeutic effectiveness of rat MSC transplantation in D-galactosamine (GalN)-induced acute liver injury and identified the novel pathways which are involved in hepatic differentiation of MSCs. In vivo, intraportal transplantation with 5 × 10(6) MSCs at 24 hours after GalN administration resulted in significant reduction in serum levels of alanine aminotransferase, aspartate aminotransferase, and total bilirubin compared to the control group. Engrafted MSCs actively proliferated, differentiated, and further enhanced hepatocyte proliferation activity. In vitro, coculture of MSCs with GalN-induced injured hepatocytes showed efficient differentiation and was evidenced by progressive increase in messenger RNA levels of hepatic markers, including albumin, α-fetoprotein, CCAAT-enhancer binding protein α, α-1-antitryspin, and hepatocyte nuclear factor-3β. Immunofluorescent staining revealed that these cells were positive for albumin, α-fetoprotein, and cytokeratin 18, but not clusters of differentiation 34, cytokeratin 19, or OV6. During hepatic differentiation, signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling were constantly activated, and a gradual down-regulation of β-catenin expression in messenger RNA and protein levels was detected. Hyper-interleukin-6 fusion protein but not interleukin-6 (IL-6) alone caused reduction in β-catenin expression associated with the up-regulation of Wnt-5a in MSCs via activating the glycoprotein 130 (gp130)-mediated STAT3 signaling pathway, which indicates the operation of the trans-signaling mechanism. Activation of IL-6/gp130-mediated STAT3 signaling pathway in MSCs triggered wound healing, cell migration, and proliferation. In conclusion, transplantation of MSCs promotes cell proliferation and organ repair, and activation of IL-6/gp130-mediated STAT3 signaling pathway via soluble IL-6 receptor is crucial in hepatic differentiation of MSCs.

15-Hydroxyprostaglandin Dehydrogenase is a Target of Hepatocyte Nuclear Factor 3β and a Tumor Suppressor in Lung Cancer

The forkhead transcription factor hepatocyte nuclear factor 3beta (HNF3beta) is essential in foregut development and the regulation of lung-specific genes. HNF3beta expression leads to growth arrest and apoptosis in lung cancer cells and HNF3beta is a candidate tumor suppressor in lung cancer. In a transcriptional profiling study using a conditional cell line system, we now identify 15-PGDH as one of the major genes induced by HNF3beta expression. 15-PGDH is a critical metabolic enzyme of proliferative prostaglandins, an antagonist to cyclooxygenase-2 and a tumor suppressor in colon cancer. We confirmed the regulation of 15-PGDH expression by HNF3beta in a number of systems and showed direct binding of HNF3beta to 15-PGDH promoter elements. Western blotting of lung cancer cell lines and immunohistochemical examination of human lung cancer tissues found loss of 15-PGDH expression in approximately 65% of lung cancers. Further studies using in vitro cell-based assays and in vivo xenograft tumorigenesis assays showed a lack of in vitro but significant in vivo tumor suppressor activity of 15-PGDH via an antiangiogenic mechanism analogous to its role in colon cancer. In summary, we identify 15-PGDH as a direct downstream effector of HNF3beta and show that 15-PGDH acts as a tumor suppressor in lung cancer.

Hepatic differentiation of human bone marrow-derived mesenchymal stem cells by tetracycline-regulated hepatocyte nuclear factor 3β

Human bone marrow-derived mesenchymal stem cells (BM-MSCs) are expected to be a potential source of cells for transplantation. Although recent reports have shown that isolated MSCs can differentiate into hepatocytes, the efficiency of differentiation is insufficient for therapeutic application. To circumvent this problem, it is necessary to understand the mechanisms of hepatic differentiation of human BM-MSCs. Hepatocyte nuclear factor 3beta (HNF3beta), a forkhead/winged helix transcription factor, is essential for liver development. In the present study, we established a tetracycline (Tet)-regulated expression system for HNF3beta in UE7T-13 BM-MSCs. HNF3beta expression significantly enhanced expression of albumin, alpha-fetoprotein (AFP), tyrosine amino transferase (TAT) and epithelial cell adhesion molecule (EpCAM) genes. The differentiated cells showed hepatocyte-specific functions including glycogen production and urea secretion. During treatment with the Tet-on system for 8 days, over 80% of UE7T-13 cells turned out to express albumin. Furthermore, the combination of Tet with basic fibroblast growth factor (bFGF) efficiently induced the genes such as albumin and TAT, which are associated with maturity of hepatocytes; however, it suppressed genes such as AFP and EpCAM, which are associated with immaturity of hepatocytes, suggesting that Tet-induced HNF3beta expression sensitizes BM-MSCs to bFGF signals. Finally, the results of the present study suggest that down-regulation of Wnt/beta-catenin signals caused by translocation of beta-catenin to cytoplasmic membrane is associated with hepatic differentiation of human BM-MSCs. HNF3beta expression induced efficient differentiation of UE7T-13 human BM-MSCs.

The Locus Control Region Activates Serpin Gene Expression through Recruitment of Liver-Specific Transcription Factors and RNA Polymerase II

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 comprises 11 serpin genes, many of which are expressed specifically in hepatic cells. Previous studies identified a locus control region (LCR) upstream of the human alpha1-antitrypsin (alpha1AT) gene that is required for gene activation, chromatin remodeling, and histone acetylation throughout the proximal serpin subcluster. Here we show that the LCR interacts with multiple liver-specific transcription factors, including hepatocyte nuclear factor 3beta (HNF-3beta), HNF-6alpha, CCAAT/enhancer binding protein alpha (C/EBPalpha), and C/EBPbeta. RNA polymerase II is also recruited to the locus through the LCR. Nongenic transcription at both the LCR and an upstream regulatory region was detected, but the deletion of the LCR abolished transcription at both sites. The deletion of HNF-3 and HNF-6 binding sites within the LCR reduced histone acetylation at both the LCR and the upstream regulatory region and decreased the transcription of the alpha1AT, corticosteroid binding globulin, and protein Z-dependent protease inhibitor genes. These results suggest that the LCR activates genes in the proximal serpin subcluster by recruiting liver-specific transcription factors and components of the general transcription machinery to regulatory regions upstream of the alpha1AT gene.