Characterization of regulatory transcriptional mechanisms in hepatocyte lipotoxicity.
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ABSTRACT:
SUBMITTER: Perez-Schindler J
PROVIDER: S-EPMC9262951 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Hepatocyte nuclear factor 4α (HNF4α) is a master regulator of development and function of digestive tissues. The HNF4A gene uses two separate promoters P1 and P2, with P1 products predominant in adult liver, whereas P2 products prevalent in fetal liver, pancreas, and liver/colon cancer. To date, the mechanisms for the regulation of HNF4A and the dynamic switch of P1-HNF4α and P2-HNF4α during ontogenesis and carcinogenesis are still obscure. Our study validated the previously reported self-stimulation of P1-HNF4α but invalidated the reported synergism between HNF4α and HNF1α. HNF4A-AS1, a long noncoding RNA, is localized between the P2 and P1 promoters of HNF4A. We identified critical roles of P1-HNF4α in regulating the expression of HNF4A-AS1 and its mouse ortholog Hnf4a-os. Paired box 6 (PAX6), a master regulator of pancreas development overexpressed in colon cancer, cooperated with HNF1α to induce P2-HNF4α but antagonized HNF4α in HNF4A-AS1 expression. Thus, PAX6 may be important in determining ontogenic and carcinogenic changes of P2-HNF4α and HNF4A-AS1 in the pancreas and intestine. We also interrogated transactivation activities on multiple gene targets by multiple known and novel HNF4α mutants identified in patients with maturity onset diabetes of the young 1 (MODY1) and liver cancer. Particularly, HNF4α-D78A and HNF4α-G79S, two mutants found in liver cancer with mutations in DNA-binding domain, displayed highly gene-specific transactivation activities. Interestingly, HNF4α-Q277X, a MODY1 truncation mutant, antagonized the transactivation activities of HNF1α and farnesoid X receptor, key regulators of insulin secretion. Taken together, our study provides novel mechanistic insights regarding the transcriptional regulation and transactivation activity of HNF4α in digestive tissues.
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Project description:Lipotoxicity induced by saturated fatty acids (SFAs) plays a central role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); however, the exact mechanisms remain to be fully elucidated. Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide-dependent deacetylase located primarily inside mitochondria. In this study, we demonstrated that an SFA-rich high-fat diet (HFD) was more detrimental to the liver than an isocaloric unsaturated HFD rich in fatty acids. Unexpectedly, SIRT3 expression and activity were significantly elevated in the livers of mice exposed to the SFA-rich HFD. Using cultured HepG2 and AML-12 hepatocytes, we demonstrated that unlike monounsaturated fatty acids, SFAs up-regulate SIRT3 expression and activity. SIRT3 overexpression renders both the liver and hepatocytes susceptible to palmitate-induced cell death, which can be alleviated by SIRT3 small interfering RNA (siRNA) transfection. In contrast, SIRT3 suppression protects hepatocytes from palmitate cytotoxicity. Further studies revealed that SIRT3 acts as a negative regulator of autophagy, thereby enhancing the susceptibility of hepatocytes to SFA-induced cytotoxicity. Mechanistic investigations revealed that SIRT3 overexpression causes manganese superoxide dismutase deacetylation and activation, which depleted intracellular superoxide contents, leading to adenosine monophosphate-activated protein kinase (AMPK) inhibition and mammalian target of rapamycin C1 activation, resulting in autophagy suppression. In contrast, SIRT3 siRNA gene silencing enhanced autophagy flux. A similar result was observed in the liver tissue of SIRT3 knockout mice.Our data indicate that SIRT3 is a negative regulator of autophagy whose activation by SFAs contributes to lipotoxicity in hepatocytes and suggest that restraining SIRT3 overactivation can be a potential therapeutic choice for the treatment of NAFLD as well as other metabolic disorders, with lipotoxicity being the principal pathomechanism. (Hepatology 2017;66:936-952).
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Project description:Hemicellulose, the second most abundant plant biomass fraction after cellulose, is widely viewed as a potential substrate for the production of liquid fuels and other value-added materials. Degradation of hemicellulose by filamentous fungi requires production of many different enzymes, which are induced by biopolymers or its derivatives and regulated mainly at the transcriptional level through transcription factors (TFs). Neurospora crassa, a model filamentous fungus, expresses and secretes enzymes required for plant cell wall deconstruction. To better understand genes specifically associated with degradation of hemicellulose, we applied secretome and transcriptome analysis to N. crassa grown on beechwood xylan. We identified 34 secreted proteins and 353 genes with elevated transcription on xylan. The xylanolytic phenotype of strains with deletions in genes identified from the secretome and transcriptome analysis of the wild type was assessed, revealing functions for known and unknown proteins associated with hemicellulose degradation. By evaluating phenotypes of strains containing deletions of predicted TF genes in N. crassa, we identified a TF (XLR-1; xylan degradation regulator 1) essential for hemicellulose degradation that is an ortholog to XlnR/XYR1 in Aspergillus and Trichoderma species, respectively, a major transcriptional regulator of genes encoding both cellulases and hemicellulases. Deletion of xlr-1 in N. crassa abolished growth on xylan and xylose, but growth on cellulose and cellulolytic activity were only slightly affected. To determine the regulatory mechanisms for hemicellulose degradation, we explored the transcriptional regulon of XLR-1 under xylose, xylanolytic, and cellulolytic conditions. XLR-1 regulated only some predicted hemicellulase genes in N. crassa and was required for a full induction of several cellulase genes. Hemicellulase gene expression was induced by a combination of release from carbon catabolite repression (CCR) and induction. This systematic analysis illustrates the similarities and differences in regulation of hemicellulose degradation among filamentous fungi.
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