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ABSTRACT: Hepatocyte nuclear factor (HNF)-4α is a key member of the transcription factor network regulating hepatocyte differentiation and function. Genetic and molecular evidence suggests that expression of HNF-4α is mainly regulated at the transcriptional level. Activation of HNF-4A gene involves the interaction of distinct sets of transcription factors and co-transcription factors within enhancer and promoter regions. Here we study the inhibitory effect of microRNAs (miRNA) on the 3'-untranslated region (3'-UTR) of HNF-4A mRNA. The potential recognition elements of a set of miRNAs were identified utilizing bioinformatics analysis. The family members of miR-34 and miR-449, including miR-34a, miR-34c-5p and miR-449a, share the same target elements located at two distinct locations within the 3'-UTR of HNF-4A. The over-expression of miR-34a, miR-34c-5p or miR-449a in HepG2 cells led to a significant decrease in the activity of luciferase reporter carrying 3'-UTR of HNF-4A. The repressive effect on reporter activity was partially or fully eliminated when one or two of the binding site(s) for miR-34a/miR-34c-5p/miR-449a were deleted within the 3'-UTR. The protein level of HNF-4α was dramatically reduced by over-expression of miR-34a, miR-34c-5p and miR-449a, which correlates with a decrease in the binding activity of HNF-4α and transactivation of HNF-4α target genes. These results suggest that the recognition sites of miR-34a, miR-34c-5p and miR-449a within 3'-UTR of HNF-4A are functional. The mechanism of down-regulation of the binding activity and transactivation of HNF-4α by the miRNAs involves the decrease in HNF-4α protein level via miRNAs selectively targeting HNF-4A 3'-UTR, leading to the translational repression of HNF-4α expression.
Biochimica et Biophysica Acta 01/2013; · 4.66 Impact Factor
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ABSTRACT: Hepatocyte nuclear factor-4α (HNF-4α), a liver-enriched transcription factor, is essential for liver development and function. HNF-4α regulates a large number of liver-specific genes, many of which are modulated by injury. While HNF-4α function is regulated by phosphorylation, only a limited number of phosphorylation sites in HNF-4α have been identified, and the roles of HNF-4α phosphorylation after injury are unexplored. To address these issues, we have carried out an extensive quantitative mass spectrometry (MS)-based analysis of HNF-4α serine and threonine phosphorylation in response to cytokine stimulation. Studies were performed in HNF-4α-enriched HepG2 cells treated with cytokines for 3 h or left untreated, followed by chemical derivatization of the phosphoserine and phosphothreonine residues using stable isotopic variants of dithiothreitol (DTT) and MS analysis. This has allowed the identification and relative quantification of 12 serine/threonine phosphorylation sites in HNF-4α. Eight of these phosphorylation sites and their sensitivity to cytokine stimulation have not been previously reported. We found that cytokine treatment leads to an increase of HNF-4α phosphorylation in several phosphopeptides. The phosphorylation of HNF-4α mediated by protein kinase A (PKA) significantly reduces HNF-4α binding activity, which mimics the repressive effect of cytokines on HNF-4α binding, and the inhibition of PKA activity by PKA inhibitor can partially recover the reduced HNF-4α binding activity induced by cytokines. These results suggest that the mechanism that alters HNF-4α binding after cytokine stimulation involves modulation of specific HNF-4α phosphorylation dependent, in part, on a PKA signaling pathway.
Biochemistry 06/2011; 50(23):5292-300. · 3.42 Impact Factor
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ABSTRACT: The hepatic acute phase response(APR) is an organ-specific response to a diverse array of insults and is largely under transcriptional control. Liver-specific transcription factors, hepatic nuclear factors (HNFs)-1α and 4α play important roles in maintenance of liver phenotype and function and their binding activity changes early after injury. However, their roles in modulation of the liver's response over time are not defined.
C57/BL6 mice were anesthetized and exposed to 95°C water for 10 s to create a 15% body surface area full-thickness burn. At specific time points, the mice were sacrificed. An ELISA for IL-6 was performed on serum and hepatic mRNA levels for fibrinogen-γ and serum amyloid A(SAA)-3 were obtained through polymerase chain reaction (PCR). Transcriptional factor binding activity was assessed with electrophoretic mobility shift assays.
Serum IL-6 levels peaked at 3 h and fibrinogen-γ and SAA mRNA levels increased more than 6-fold at 12 h before returning to control levels at 48 h. The binding activity of HNF-4α and HNF-1α rapidly declined after injury (1.5 h) but recovered to near control level at 24 and 6 h, respectively.
Changes in HNF-4α and HNF-1α binding occurred before changes in acute phase protein mRNA levels and were preceded by the peak in IL-6 levels. The rapid suppression and reconstitution of liver-specific transcription factor binding after injury may represent a mechanism that allows the normal liver phenotype to change and an injury-response phenotype to prevail. This mechanism in the liver's adaptive response to injury suggests a central role for both HNF-4α and HNF-1α in transcriptional regulation of the hepatic APR.
Journal of Surgical Research 05/2011; 175(2):298-304. · 2.25 Impact Factor
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ABSTRACT: The acute-phase response (APR) is critical to the body's ability to successfully respond to injury. A murine model of closed unilateral femur fractures and bilateral femur fracture were used to study the effect of injury magnitude on this response.
Standardized unilateral femur fracture and bilateral femur fracture in mice were performed. The femur fracture sites, livers, and serum were harvested over time after injury. Changes in mRNA expression of cytokines, hepatic acute-phase proteins, and serum cytokines overtime were measured.
There was a rapid and short-lived hepatic APR to fracture injuries. The overall pattern in both models was similar. Both acute-phase proteins' mRNA (fibrinogen-γ and serum amyloid A-3) showed increased mRNA expression over baseline within the first 48 hours and their levels positively correlated with the extent of injury. However, increased severity of injury resulted in a delayed induction of the APR. A similar effect on the gene expression of cytokines (interleukin [IL]-1β, IL-6, and tumor necrosis factor-α) at the fracture site was seen. Serum IL-6 levels increased with increased injury and showed no delay between injury models.
Greater severity of injury resulted in a delayed induction of the liver's APR and a diminished expression of cytokines at the fracture site. Serum IL-6 levels were calibrated to the extent of the injury, and changes may represent mechanisms by which the local organ responses to injury are regulated by the injury magnitude.
The Journal of trauma 04/2011; 70(4):948-53. · 2.48 Impact Factor
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ABSTRACT: Hepatocyte nuclear factor 4α (HNF4α), a liver-specific transcription factor, plays a significant role in liver-specific functions. However, its functions are poorly understood in the regulation of the inflammatory response. In order to obtain a genomic view of HNF4α in this context, microarray analysis was used to probe the expression profile of an inflammatory response induced by cytokine stimulation in a model of HNF4α knock-down in HepG2 cells.
The expression of over five thousand genes in HepG2 cells is significantly changed with the dramatic reduction of HNF4α concentration compared to the cells with native levels of HNF4α. Over two thirds (71%) of genes that exhibit differential expression in response to cytokine treatment also reveal differential expression in response to HNF4α knock-down. In addition, we found that a number of HNF4α target genes may be indirectly mediated by an ETS-domain transcription factor ELK1, a nuclear target of mitogen-activated protein kinase (MAPK).
The results indicate that HNF4α has an extensive impact on the regulation of a large number of the liver-specific genes. HNF4α may play a role in regulating the cytokine-induced inflammatory response. This study presents a novel function for HNF4α, acting not only as a global player in many cellular processes, but also as one of the components of inflammatory response in the liver.
BMC Genomics 02/2011; 12:128. · 4.07 Impact Factor
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ABSTRACT: Transthyretin is a negative acute phase protein whose serum level decreases during the acute phase response. Transthyretin gene expression in the liver is regulated at the transcriptional level, and is controlled by hepatocyte nuclear factor (HNF)-4α and other HNFs. The site-directed mutagenesis of HNF-4, HNF-1, HNF-3 and HNF-6 binding sites in the transthyretin proximal promoter dramatically decreases transthyretin promoter activity. Interestingly, the mutation of the HNF-4 binding site not only abolishes the response to HNF-4α, but also reduces significantly the response to other HNFs. However, mutation of the HNF-4 binding site merely affects the specific binding of HNF-4α, but not other HNFs, suggesting that an intact HNF-4 binding site not only provides a platform for specific interaction with HNF-4α, but also facilitates the interaction of HNF-4α with other HNFs. In a cytokine-induced acute phase response cell culture model, we observed a significant reduction in the binding of HNF-4α, HNF-1α, HNF-3β and HNF-6α to the transthyretin promoter, which correlates with a decrease in transthyretin expression after injury. These findings provide new insights into the mechanism of the negative transcriptional regulation of the transthyretin gene after injury caused by a decrease in the binding of HNFs and a modulation in their coordinated interactions.
FEBS Journal 10/2010; 277(19):4066-75. · 3.79 Impact Factor
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ABSTRACT: HNF-4alpha (hepatocyte nuclear factor-4alpha) is a key regulator of liver-specific gene expression. To understand the mechanisms governing the regulation of HNF-4alpha function during the APR (acute-phase response), the effects of transcription co-activators, including p300, PGC-1alpha (peroxisome-proliferator-activated receptor-gamma co-activator-1alpha) and SRC (steroid receptor co-activator)-1alpha were investigated in an injury cell model. We have shown previously that the HNF-4alpha-sensitive APR genes ApoB (apolipoprotein B), TTR (transthyretin) and alpha1-AT (alpha1-antitrypsin) were regulated at the DNA binding and transcriptional levels after cytokine stimulation. We now show that co-activators have a differential impact on the transactivation of HNF-4alpha-sensitive genes via HNF-4alpha-binding sites in ApoB, TTR or alpha1-AT promoters. PGC-1alpha strongly enhances the transactivation of ApoB and alpha1-AT and, to a lesser extent, of TTR, whereas SRC-1alpha and p300 only have a weak or no effect on these three genes. More importantly, it was found that PGC-1alpha has a novel role in the modulation of the binding ability of HNF-4alpha in response to cytokine treatment. Using in vitro and in vivo approaches, electrophoretic mobility-shift and chromatin immunoprecipitation assays, we demonstrate that the reduced HNF-4alpha-DNA binding ability induced by cytokines is eliminated by overexpression of PGC-1alpha. Cytokine treatment does not significantly alter the protein levels of HNF-4alpha and PGC-1alpha, but it does reduce the recruitment of PGC-1alpha to HNF-4alpha-binding sites and thereby decreases transcriptional activity. These results establish the importance of PGC-1alpha for HNF-4alpha function and describe a new HNF-4alpha-dependent regulatory mechanism that is involved in the response to injury.
Biochemical Journal 06/2008; 415(2):289-96. · 4.90 Impact Factor
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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 4alpha (HNF-4alpha) 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-4alpha-binding sites in their promoter regions and are transcriptionally regulated by HNF-4alpha. Utilizing a cytokine induced acute phase response in HepG2 cells, we investigated the role of HNF-4alpha in regulating the transcription of three HNF-4alpha sensitive genes, alpha1-antitrypsin (alpha1-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-4alpha. However, the unique mRNA expression patterns of alpha1-AT, TTR, and ApoB in response to cytokine treatment were abrogated in HepG2 cells with dramatically reduced HNF-4alpha protein concentrations. The mechanism by which HNF-4alpha mediates this injury response is through site-specific alterations in HNF-4alpha-binding abilities and transactivation potentials. Cytokine treatment phosphorylates HNF-4alpha, which directly affects HNF-4alpha activity. Our results demonstrate that HNF-4alpha is a crucial mediator in the regulation of alpha1-AT, TTR, and ApoB gene expression before and after injury, providing evidence of a novel role for HNF-4alpha in the control of the liver's acute phase response.
Journal of Molecular Biology 09/2007; 371(2):323-35. · 4.00 Impact Factor
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ABSTRACT: The effects of salicylate on the phosphorylation and nuclear translocation of signal transducers and activators of transcription (STATs) induced by interferon-gamma (IFN-gamma) were studied in rat cardiac fibroblasts as a possible model for the anti-inflammatory effects of salicylate on this signaling pathway. Salicylate inhibited the tyrosine phosphorylation of both STAT1 and STAT3, but had a more pronounced effect on STAT3 activation. Salicylate pretreatment prevented both the nuclear translocation and the DNA-binding activity of STAT1 and STAT3, assessed by immunoblotting and gel shift assays, respectively. In addition to causing phosphorylation at tyrosine residues, IFN-gamma also phosphorylated STAT3 and STAT1 at serine 727. Salicylate attenuated both tyrosine and serine phosphorylations of STAT3, and also suppressed extracellular signal-regulated kinase (ERK) activation, implicating the effect of salicylate on ERK as a possible mechanism for attenuating STAT3 activation. The possibility that salicylate might affect signaling cascades by altering the redox state of the cells was examined, and its effects differed from those of other reducing agents. Salicylate did attenuate the effects of hydrogen peroxide on STAT phosphorylation, consistent with a mechanism involving an interaction between salicylate and reactive oxygen species within the cell.
Biochemical Pharmacology 05/2002; 63(7):1197-207. · 4.70 Impact Factor
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ABSTRACT: -The anti-inflammatory effects of salicylate are well known, but the intracellular mechanisms underlying those effects remain to be clarified and are not explained solely by an influence on cyclooxygenase activity. In the present study, we have used cardiac fibroblasts stimulated by either angiotensin II (Ang II) or platelet-derived growth factor (PDGF) to demonstrate an inhibitory effect of salicylate on the phosphorylation of the nonreceptor tyrosine kinases, proline-rich tyrosine kinase 2 (PYK2) and c-Src, by immunoprecipitation and immunoblotting methods. This inhibition was dose dependent, with a clear effect observed at concentrations between 5 and 20 mmol/L salicylate. Intracellular Ca(2+) chelation and protein kinase C (PKC) inhibition reduced Ang II and PDGF-induced PYK2 and c-Src phosphorylation. Salicylate significantly inhibited the phosphorylation of both of the tyrosine kinases activated by either ionophore A23187 or thapsigargin treatment, which led to an elevation of cytosolic Ca(2+). Activation of PKC by phorbol ester phosphorylated both PYK2 and Src, and this effect also was attenuated by salicylate. In contrast, salicylate had no effect on either the transactivation of the epidermal growth factor receptor by Ang II or the phosphorylation of phospholipase C-gamma by PDGF. These studies indicate a novel site of action for salicylate on PYK2 and c-Src phosphorylation and suggest that this inhibitory effect on these important signaling intermediates may be through a Ca(2+)- and PKC-dependent mechanism.
Hypertension 02/2001; 37(1):148-153. · 6.21 Impact Factor
