RESUMO
OBJECTIVE: Multidrug resistance protein 2 (MRP2) is a bottleneck in bilirubin excretion. Its loss is sufficient to induce hyperbilirubinaemia, a prevailing characteristic of acute liver failure (ALF) that is closely associated with clinical outcome. This study scrutinises the transcriptional regulation of MRP2 under different pathophysiological conditions. DESIGN: Hepatic MRP2, farnesoid X receptor (FXR) and Forkhead box A2 (FOXA2) expression and clinicopathologic associations were examined by immunohistochemistry in 14 patients with cirrhosis and 22 patients with ALF. MRP2 regulatory mechanisms were investigated in primary hepatocytes, Fxr -/- mice and lipopolysaccharide (LPS)-treated mice. RESULTS: Physiologically, homeostatic MRP2 transcription is mediated by the nuclear receptor FXR/retinoid X receptor complex. Fxr-/- mice lack apical MRP2 expression and rapidly progress into hyperbilirubinaemia. In patients with ALF, hepatic FXR expression is undetectable, however, patients without infection maintain apical MRP2 expression and do not suffer from hyperbilirubinaemia. These patients express FOXA2 in hepatocytes. FOXA2 upregulates MRP2 transcription through binding to its promoter. Physiologically, nuclear FOXA2 translocation is inhibited by insulin. In ALF, high levels of glucagon and tumour necrosis factor α induce FOXA2 expression and nuclear translocation in hepatocytes. Impressively, ALF patients with sepsis express low levels of FOXA2, lose MRP2 expression and develop severe hyperbilirubinaemia. In this case, LPS inhibits FXR expression, induces FOXA2 nuclear exclusion and thus abrogates the compensatory MRP2 upregulation. In both Fxr -/- and LPS-treated mice, ectopic FOXA2 expression restored apical MRP2 expression and normalised serum bilirubin levels. CONCLUSION: FOXA2 replaces FXR to maintain MRP2 expression in ALF without sepsis. Ectopic FOXA2 expression to maintain MRP2 represents a potential strategy to prevent hyperbilirubinaemia in septic ALF.
Assuntos
Fator 3-beta Nuclear de Hepatócito , Falência Hepática Aguda , Proteína 2 Associada à Farmacorresistência Múltipla , Animais , Camundongos , Bilirrubina , Fator 3-beta Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Hiperbilirrubinemia/metabolismo , Hiperbilirrubinemia/patologia , Lipopolissacarídeos/metabolismo , Fígado/metabolismo , Falência Hepática Aguda/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla/metabolismo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
BACKGROUND: When massive necrosis occurs in acute liver failure (ALF), rapid expansion of HSCs called liver progenitor cells (LPCs) in a process called ductular reaction is required for survival. The underlying mechanisms governing this process are not entirely known to date. In ALF, high levels of retinoic acid (RA), a molecule known for its pleiotropic roles in embryonic development, are secreted by activated HSCs. We hypothesized that RA plays a key role in ductular reaction during ALF. METHODS: RNAseq was performed to identify molecular signaling pathways affected by all-trans retinoid acid (atRA) treatment in HepaRG LPCs. Functional assays were performed in HepaRG cells treated with atRA or cocultured with LX-2 cells and in the liver tissue of patients suffering from ALF. RESULTS: Under ALF conditions, activated HSCs secreted RA, inducing RARα nuclear translocation in LPCs. RNAseq data and investigations in HepaRG cells revealed that atRA treatment activated the WNT-ß-Catenin pathway, enhanced stemness genes (SOX9, AFP, and others), increased energy storage, and elevated the expression of ATP-binding cassette transporters in a RARα nuclear translocation-dependent manner. Further, atRA treatment-induced pathways were confirmed in a coculture system of HepaRG with LX-2 cells. Patients suffering from ALF who displayed RARα nuclear translocation in the LPCs had significantly better MELD scores than those without. CONCLUSIONS: During ALF, RA secreted by activated HSCs promotes LPC activation, a prerequisite for subsequent LPC-mediated liver regeneration.
Assuntos
Falência Hepática Aguda , Células-Tronco , Tretinoína , Humanos , Tretinoína/farmacologia , Células-Tronco/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Fígado/efeitos dos fármacos , Receptor alfa de Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico/metabolismo , Técnicas de Cocultura , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismoRESUMO
BACKGROUND & AIMS: Transforming growth factor-ß1 (TGF-ß1) plays important roles in chronic liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD involves various biological processes including dysfunctional cholesterol metabolism and contributes to progression to metabolic dysfunction-associated steatohepatitis and hepatocellular carcinoma. However, the reciprocal regulation of TGF-ß1 signaling and cholesterol metabolism in MASLD is yet unknown. METHODS: Changes in transcription of genes associated with cholesterol metabolism were assessed by RNA sequencing of murine hepatocyte cell line (alpha mouse liver 12/AML12) and mouse primary hepatocytes treated with TGF-ß1. Functional assays were performed on AML12 cells (untreated, TGF-ß1 treated, or subjected to cholesterol enrichment [CE] or cholesterol depletion [CD]), and on mice injected with adenovirus-associated virus 8-control/TGF-ß1. RESULTS: TGF-ß1 inhibited messenger RNA expression of several cholesterol metabolism regulatory genes, including rate-limiting enzymes of cholesterol biosynthesis in AML12 cells, mouse primary hepatocytes, and adenovirus-associated virus-TGF-ß1-treated mice. Total cholesterol levels and lipid droplet accumulation in AML12 cells and liver tissue also were reduced upon TGF-ß1 treatment. Smad2/3 phosphorylation after 2 hours of TGF-ß1 treatment persisted after CE or CD and was mildly increased after CD, whereas TGF-ß1-mediated AKT phosphorylation (30 min) was inhibited by CE. Furthermore, CE protected AML12 cells from several effects mediated by 72 hours of incubation with TGF-ß1, including epithelial-mesenchymal transition, actin polymerization, and apoptosis. CD mimicked the outcome of long-term TGF-ß1 administration, an effect that was blocked by an inhibitor of the type I TGF-ß receptor. In addition, the supernatant of CE- or CD-treated AML12 cells inhibited or promoted, respectively, the activation of LX-2 hepatic stellate cells. CONCLUSIONS: TGF-ß1 inhibits cholesterol metabolism whereas cholesterol attenuates TGF-ß1 downstream effects in hepatocytes.
Assuntos
Fígado Gorduroso , Fator de Crescimento Transformador beta1 , Camundongos , Animais , Fator de Crescimento Transformador beta1/metabolismo , Hepatócitos/metabolismo , Células Estreladas do Fígado/patologia , Linhagem Celular , Fígado Gorduroso/metabolismoRESUMO
One of the most crucial parameters in protein crystallization is pH, as it governs the protein's electrostatic interactions. However, the fundamental role of pH on crystallization still remains unknown. Here, we systematically investigated the crystallization of human insulin (isoelectric point 5.3) at various pHs between 6.0 and 6.7 at different supersaturation ratios, up to 20.9. Our results demonstrate that the pH has an opposing effect on solubility and nucleation rate as a shift in pH toward a more basic milieu increases the solubility by 5-fold while the onset of nucleation was accelerated by a maximum of 8.6-fold. To shed light on this opposing effect, we evaluated the protein-protein interactions as a function of pH by measuring the second virial coefficient and hydrodynamic radius and showed that a change in pH of less than one unit has no significant impact on the protein-protein interactions. As it is widely understood that the increase in protein solubility as a function of pH is due to the increase in the repulsive electrostatic interactions, we have demonstrated that the increase in insulin solubility and decrease in the onset of nucleation are independent of the protein-protein interactions. We hypothesize that it is the electrostatic interactions between both ions and solvent molecules and the protein residues that are governing the crystallization of human insulin. The findings of this study will be of crucial importance for the design of novel crystallization pathways.
RESUMO
Transforming growth factor-ß (TGF-ß) is a potent effector in the liver, which is involved in a plethora of processes initiated upon liver injury. TGF-ß affects parenchymal, non-parenchymal, and inflammatory cells in a highly context-dependent manner. Its bioavailability is critical for a fast response to various insults. In the liver - and probably in other organs - this is made possible by the deposition of a large portion of TGF-ß in the extracellular matrix as an inactivated precursor form termed latent TGF-ß (L-TGF-ß). Several matrisomal proteins participate in matrix deposition, latent complex stabilisation, and activation of L-TGF-ß. Extracellular matrix protein 1 (ECM1) was recently identified as a critical factor in maintaining the latency of deposited L-TGF-ß in the healthy liver. Indeed, its depletion causes spontaneous TGF-ß signalling activation with deleterious effects on liver architecture and function. This review article presents the current knowledge on intracellular L-TGF-ß complex formation, secretion, matrix deposition, and activation and describes the proteins and processes involved. Further, we emphasise the therapeutic potential of toning down L-TGF-ß activation in liver fibrosis and liver cancer.