Search

Understanding mechanisms in lineage differentiation is critical for organ development, pathophysiology and oncogenesis. To determine whether microRNAs (miRNA) may serve as drivers or adjuncts in hepatic differentiation, we studied human embryonic stem cell-derived hepatocytes and primary hepatocytes representing fetal or adult stages. Model systems were used for hepatic lineage advancement or regression under culture conditions with molecular assays. Profiles of miRNA in primary fetal and adult hepatocytes shared similarities and distinctions from pluripotent stem cells or stem cell-derived early fetal-like hepatocytes. During phenotypic regression in fetal or adult hepatocytes, miRNA profiles oscillated to regain stemness-associated features that had not been extinguished in stem cell-derived fetal-like hepatocytes. These oscillations in stemness-associated features were not altered in fetal-like hepatocytes by inhibitory mimics for dominantly-expressed miRNA, such as hsa-miR-99b, -100, -214 and -221/222. The stem cell-derived fetal-like hepatocytes were permissive for miRNA characterizing mature hepatocytes, including mimics for hsa-miR-122, -126, -192, -194 and -26b, although transfections of the latter did not advance hepatic differentiation. Examination of genome-wide mRNA expression profiles in stem cell-derived or primary fetal hepatocytes indicated targets of highly abundant miRNA regulated general processes, e.g., cell survival, growth and proliferation, functional maintenance, etc., without directing cell differentiation. Among upstream regulators of gene networks in stem cell-derived hepatocytes included HNF4A, SNAI1, and others, which affect transcriptional circuits directing lineage development or maintenance. Therefore, miRNA expression oscillated in response to microenvironmental conditions, whereas lineage-specific transcriptional regulators, such as HNF4A, were necessary for directing hepatic differentiation. This knowledge will be helpful for understanding the contribution of stem cells in pathophysiological states and oncogenesis, as well as for applications of stem cell-derived hepatocytes. [ABSTRACT FROM AUTHOR]

Abstract: Following injury, a large number of hepatic acute phase genes are rapidly modulated at the transcriptional level to restore metabolic homeostasis and limit tissue damage. Hepatocyte nuclear factor 4α (HNF-4α) is a liver-enriched transcription factor that controls embryonic liver development and regulates tissue-specific gene expression in adult liver cells. Many genes encoding acute phase proteins contain HNF-4α-binding sites in their promoter regions and are transcriptionally regulated by HNF-4α. Utilizing a cytokine induced acute phase response in HepG2 cells, we investigated the role of HNF-4α in regulating the transcription of three HNF-4α sensitive genes, α1-antitrypsin (α1-AT), transthyretin (TTR), and apolipoprotein B (ApoB) after injury. The transcriptional behavior of all three genes depends, in part, on the intracellular concentrations of HNF-4α. However, the unique mRNA expression patterns of α1-AT, TTR, and ApoB in response to cytokine treatment were abrogated in HepG2 cells with dramatically reduced HNF-4α protein concentrations. The mechanism by which HNF-4α mediates this injury response is through site-specific alterations in HNF-4α-binding abilities and transactivation potentials. Cytokine treatment phosphorylates HNF-4α, which directly affects HNF-4α activity. Our results demonstrate that HNF-4α is a crucial mediator in the regulation of α1-AT, TTR, and ApoB gene expression before and after injury, providing evidence of a novel role for HNF-4α in the control of the liver''s acute phase response. [Copyright &y& Elsevier]

Background Recent data highlighted the role of nuclear receptors in the transcriptional regulation of the limiting enzyme of bile acid synthesis, cholesterol 7α-hydroxylase, in cellular and animal models. This study was designed to analyze the effects of age on cholesterol 7α-hydroxylase and related nuclear receptor expression in human livers. Design Surgical liver biopsies were obtained in 23 patients requiring operation on the gastrointestinal tract. mRNA levels of cholesterol 7α-hydroxylase and related nuclear receptors and co-activators were assayed by quantitative real-time RT-PCR. Serum levels of 7α-hydroxy-4-cholesten-3-one, a marker of bile acid synthesis, were assayed by gas-liquid chromatography:mass spectrometry. Results Ageing was inversely correlated with serum 7α-hydroxy-4-cholesten-3-one and with cholesterol 7α-hydroxylase mRNA levels ( r = –0·44 and r = −0·45 on a semi-log scale, respectively, P < 0·05). Among different nuclear factors, cholesterol 7α-hydroxylase mRNA best correlated with hepatocyte nuclear factor-4 ( r = 0·55 on a log scale, P < 0·05); hepatocyte nuclear factor-4 levels were also inversely correlated with age ( r = –0·64 on a semi-log scale, P < 0·05). Age was inversely correlated with serum insulin-like growth factor-I levels, which were directly correlated with hepatocyte nuclear factor-4 and cholesterol 7α-hydroxylase expression. No suppressive effect of short heterodimer partner expression on cholesterol 7α-hydroxylase was observed. Conclusions Ageing associates with reduced bile acid synthesis, possibly related to decreased hepatic expression of hepatocyte nuclear factor-4 and consequently of cholesterol 7α-hydroxylase. Age-related modifications of the growth hormone/insulin-like growth factor axis might play a role. These findings may help to elucidate the pathophysiology of age-related modifications of cholesterol metabolism. [ABSTRACT FROM AUTHOR]

The multiprotein Mediator coactivator complex is universally required for transcription of metazoan genes. It has been proposed to function by interfacing between transcriptional activators and the RNA polymerase II machinery. However, in vitro transcription systems reconstituted from homogeneous preparations of RNA polymerase II, the general transcription initiation factors, and the cofactor PC4 display relatively robust activator (HNF-4)-dependent activity, which, nonetheless, can be further stimulated by Mediator. By contrast, an unfractionated nuclear extract-based system in which Mediator has been immunodepleted displays a near-absolute dependence on ectopic Mediator. Here, we identified and purified an activity, MSA-2, that confers extract-like Mediator responsiveness to our reconstituted system. Mass spectrometric analyses identified its two constituent polypeptides as hSpt5 and hSpt4, which also comprise the elongation factor DSIF. Mechanistically, MSA-2/DSIF acts by restricting overall transcription in the pure system, thereby imposing a strong Mediator dependence. Our data thus point to potential mechanisms for Mediator function beyond its presently believed role in promoting the initial formation of the RNA polymerase II-containing preinitiation complex. [ABSTRACT FROM AUTHOR]

The suppression of hypoxia-induced erythropoietin (EPO) expression by inflammatory cytokines like interleukin-1 (IL-1) contributes to the development of the anemia of chronic disease (ACD). However, the precise mechanism of this suppression is unclear. The 3’-EPO enhancer mediates the transcriptional response to hypoxia by binding several transcription factors, including hypoxia-inducible factor, hepatocyte nuclear factor-4α (HNF-4α) and chicken ovalbumin upstream promoter transcription factor. We investigated whether IL-1β inhibits the activity of the 3’-EPO enhancer via HNF-4α. IL-1β inhibited HNF-4α mRNA expression and caused proteasome-dependent degradation of HNF-4α protein, which resulted in a strongly reduced DNA-binding activity of HNF-4α. Reporter gene assays revealed that IL-1β caused a complete suppression of the hypoxic inducibility of the 3′ enhancer via inhibition of HNF-4α. We conclude that IL-1β, at least partially, reduces hypoxia-induced EPO expression by down-regulation of HNF-4α. [ABSTRACT FROM AUTHOR]

Direct repeat motifs composed of two hexamer half-sites spaced by a single nucleotide (DR-1) are recognized by several members of the nuclear hormone receptor superfamily. We examined, by means of gene transfection assays, the interplay between the DR-1-binding nuclear receptors commonly expressed in liver, peroxisome proliferator-activated receptor a (PPARα), hepatocyte nuclear factor-4 (HNF-4), and chicken ovalbumin upstream transcription factor I (COUP-TFI). Both PPARα and HNF-4 efficiently bound to the acyl-CoA oxidase gene enhancer element, but PPARα exhibited much stronger transactivation than HNF-4. As a result, HNF-4 suppressed the gene-activating function of PPARα, when they were expressed together, due to competition for a common binding site. On the other hand, HNF-4, but not PPARα, effectively bound to the apolipoprotein CIII gene element, and activated gene transcription. PPARα had no effect even when co-expressed with HNF-4. COUP-TFI bound to both elements, and suppressed the gene activation by PPARα and HNF-4. Thus, these nuclear receptors have individual functions in gene regulation, and exhibit complex compound effects when they co-exist. [ABSTRACT FROM AUTHOR]