Metabolic dysfunction-associated steatotic liver disease (MASLD) comprises a spectrum of liver diseases, ranging from liver steatosis to metabolic dysfunction-associated steatohepatitis (MASH), ...increasing the risk of developing cirrhosis and hepatocellular carcinoma (HCC). Fibrosis within MASLD is critical for disease development; therefore, the identification of fibrosis-driving factors is indispensable. We analyzed the expression of interleukin 32 (IL-32) and chemokine CC ligand 20 (CCL20), which are known to be linked with inflammation and fibrosis, and for their expression in MASLD and hepatoma cells. RT-PCR, ELISA and Western blotting analyses were performed in both human liver samples and an in vitro steatosis model. IL-32 and CCL20 mRNA expression was increased in tissues of patients with NASH compared to normal liver tissue. Stratification for patatin-like phospholipase domain-containing protein 3 (PNPLA3) status revealed significance for IL-32 only in patients with I148M (rs738409, CG/GG) carrier status. Furthermore, a positive correlation was observed between IL-32 expression and steatosis grade, and between IL-32 as well as CCL20 expression and fibrosis grade. Treatment with the saturated fatty acid palmitic acid (PA) induced mRNA and protein expression of IL-32 and CCL20 in hepatoma cells. This induction was mitigated by the substitution of PA with monounsaturated oleic acid (OA), suggesting the involvement of oxidative stress. Consequently, analysis of stress-induced signaling pathways showed the activation of Erk1/2 and p38 MAPK, which led to an enhanced expression of IL-32 and CCL20. In conclusion, cellular stress in liver epithelial cells induced by PA enhances the expression of IL-32 and CCL20, both known to trigger inflammation and fibrosis.
Inflammasomes and innate immune cells have been shown to contribute to liver injury, thereby activating Kupffer cells, which release several cytokines, including IL-6, IL-1ß, and TNFα. Augmenter of ...liver regeneration (ALR) is a hepatotropic co-mitogen that was found to have anti-oxidative and anti-apoptotic properties and to attenuate experimental non-alcoholic fatty liver disease (NAFLD) and cholestasis. Additionally, hepatic ALR expression is diminished in patients with NAFLD or cholestasis, but less is known about the mechanisms of its regulation under these conditions. Therefore, we aimed to investigate the role of IL-1ß in ALR expression and to elucidate the molecular mechanism of this regulation in vitro. We found that ALR promoter activity and mRNA and protein expression were reduced upon treatment with IL-1ß. Early growth response protein-1 (Egr-1), an ALR inducer, was induced by IL-1ß but could not activate ALR expression, which may be attributed to reduced Egr-1 binding to the ALR promoter. The expression and nuclear localization of hepatocyte nuclear factor 4 α (HNF4α), another ALR-inducing transcription factor, was reduced by IL-1ß. Interestingly, c-Jun, a potential regulator of ALR and HNF4α, showed increased nuclear phosphorylation levels upon IL-1ß treatment but did not change the expression of ALR or HNF4α. In conclusion, this study offers evidence regarding the regulation of anti-apoptotic and anti-oxidative ALR by IL-1ß through reduced Egr-1 promoter binding and diminished HNF4α expression independent of c-Jun activation. Low ALR tissue levels in NAFLD and cholestatic liver injury may be caused by IL-1ß and contribute to disease progression.
Nonalcoholic fatty liver disease (NAFLD) covers a spectrum from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. Free fatty acids (FFA) induce steatosis and lipo-toxicity and ...correlate with severity of NAFLD. In this study we aimed to investigate the role of exogenous and endogenous ALR (augmenter of liver regeneration) for FFA induced ER (endoplasmatic reticulum) -stress and lipoapoptosis. Primary human hepatocytes or hepatoma cells either treated with recombinant human ALR (rhALR, 15kDa) or expressing short form ALR (sfALR, 15kDa) were incubated with palmitic acid (PA) and analyzed for lipo-toxicity, -apoptosis, activation of ER-stress response pathways, triacylglycerides (TAG), mRNA and protein expression of lipid metabolizing genes. Both, exogenous rhALR and cytosolic sfALR reduced PA induced caspase 3 activity and Bax protein expression and therefore lipotoxicity. Endogenous sfALR but not rhALR treatment lowered TAG levels, diminished activation of ER-stress mediators C-Jun N-terminal kinase (JNK), X-box binding protein-1 (XBP1) and proapoptotic transcription factor C/EBP-homologous protein (CHOP), and reduced death receptor 5 protein expression. Cellular ALR exerts its lipid lowering and anti-apoptotic actions by enhancing FABP1, which binds toxic FFA, increasing mitochondrial β-oxidation by elevating the mitochondrial FFA transporter CPT1α, and decreasing ELOVL6, which delivers toxic FFA metabolites. We found reduced hepatic mRNA levels of ALR in a high fat diet mouse model, and of ALR and FOXA2, a transcription factor inducing ALR expression, in human steatotic as well as NASH liver samples, which may explain increased lipid deposition and reduced β-oxidation in NASH patients. Present study shows that exogenous and endogenous ALR reduce PA induced lipoapoptosis. Furthermore, cytosolic sfALR changes mRNA and protein expression of genes regulating lipid metabolism, reduces ER-stress finally impeding progression of NASH.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Liver regeneration can be impaired by permanent oxidative stress and activation of nuclear factor erythroid 2-related factor 2 (Nrf2), known to regulate the cellular antioxidant response, and has ...been shown to improve the process of liver regeneration. A variety of factors regulate hepatic tissue regeneration, among them augmenter of liver regeneration (ALR), attained great attention as being survival factors for the liver with proproliferative and antiapoptotic properties. Here we determined the Nrf2/antioxidant response element (ARE) regulated expression of ALR and show ALR as a target gene of Nrf2 in vitro and in vivo. The ALR promoter comprises an ARE binding site and, therefore, ALR expression can be induced by ARE-activator tertiary butylhydroquinone (tBHQ) in hepatoma cells and primary human hepatocytes (PHH). Promoter activity and expression of ALR were enhanced after cotransfection of Nrf2 compared with control and dominant negative mutant of Nrf2. Performing partial hepatectomy in livers from Nrf2+/+ mice compared with Nrf2-/- knock-out (KO) mice, we found increased expression of ALR in addition to known antioxidant ARE-regulated genes. Furthermore, we observed increased ALR expression in hepatitis B virus (HBV) compared with hepatitis C virus (HCV) positive hepatoma cells and PHH. Recently, it was demonstrated that HBV infection activates Nrf2 and, now, we add results showing increased ALR expression in liver samples from patients infected with HBV. ALR is regulated by Nrf2, acts as a liver regeneration and antioxidative protein and, therefore, links oxidative stress to hepatic regeneration to ensure survival of damaged cells.
Given the important role of angiogenesis in liver pathology, the current study investigated the role of Runt-related transcription factor 1 (RUNX1), a regulator of developmental angiogenesis, in the ...pathogenesis of non-alcoholic steatohepatitis (NASH). Quantitative RT-PCRs and a transcription factor analysis of angiogenesis-associated differentially expressed genes in liver tissues of healthy controls, patients with steatosis and NASH, indicated a potential role of
in NASH. The gene expression of
was correlated with histopathological attributes of patients. The protein expression of RUNX1 in liver was studied by immunohistochemistry. To explore the underlying mechanisms, in vitro studies using
siRNA and overexpression plasmids were performed in endothelial cells (ECs). RUNX1 expression was significantly correlated with inflammation, fibrosis and NASH activity score in NASH patients. Its expression was conspicuous in liver non-parenchymal cells. In vitro, factors from steatotic hepatocytes and/or VEGF or TGF- significantly induced the expression of
in ECs.
regulated the expression of angiogenic and adhesion molecules in ECs, including CCL2, PECAM1 and VCAM1, which was shown by silencing or over-expression of
. Furthermore,
increased the angiogenic activity of ECs. This study reports that steatosis-induced
augmented the expression of adhesion and angiogenic molecules and properties in ECs and may be involved in enhancing inflammation and disease severity in NASH.
Knowledge about innate antimicrobial defense of the liver is limited. We investigated hepatic expression and regulation of antimicrobial peptides with focus on the human beta defensin-1 (hBD-1).
...Radial diffusion assay was used to analyze antimicrobial activity of liver tissue. Different defensins including hBD-1 and its activator thioredoxin-1 (TXN) were analyzed in healthy and cholestatic liver samples by qPCR and immunostaining. Regulation of hBD-1 expression was studied
and
using bile duct-ligated mice. Regulation of hBD-1
bilirubin and bile acids (BAs) was studied using siRNA.
We found strong antimicrobial activity of liver tissue against
. As a potential mediator of this antimicrobial activity we detected high expression of hBD-1 and TXN in hepatocytes, whereas other defensins were minimally expressed. Using a specific antibody for the reduced, antimicrobially active form of hBD-1 we found hBD-1 in co-localization with TXN within hepatocytes. hBD-1 was upregulated in cholestasis in a graded fashion. In cholestatic mice hepatic AMP expression (Defb-1 and Hamp) was enhanced. Bilirubin and BAs were able to induce hBD-1 in hepatic cell cultures
. Treatment with siRNA and/or agonists demonstrated that the farnesoid X receptor (FXR) mediates basal expression of hBD-1, whereas both constitutive androstane receptor (CAR) and FXR seem to be responsible for the induction of hBD-1 by bilirubin.
hBD-1 is prominently expressed in hepatocytes. It is induced during cholestasis through bilirubin and BAs, mediated by CAR and especially FXR. Reduction by TXN activates hBD-1 to a potential key player in innate antimicrobial defense of the liver.
Hepatic ischemia reperfusion injury (IRI) is a major complication in liver resection and transplantation. Here, we analyzed the impact of recombinant human augmenter of liver regeneration (rALR), an ...anti-oxidative and anti-apoptotic protein, on the deleterious process induced by ischemia reperfusion (IR). Application of rALR reduced tissue damage (necrosis), levels of lipid peroxidation (oxidative stress) and expression of anti-oxidative genes in a mouse IRI model. Damage associated molecule pattern (DAMP) and inflammatory cytokines such as HMGB1 and TNFα, were not affected by rALR. Furthermore, we evaluated infiltration of inflammatory cells into liver tissue after IRI and found no change in CD3 or γδTCR positive cells, or expression of IL17/IFNγ by γδTCR cells. The quantity of Gr-1 positive cells (neutrophils), and therefore, myeloperoxidase activity, was lower in rALR-treated mice. Moreover, we found under hypoxic conditions attenuated ROS levels after ALR treatment in RAW264.7 cells and in primary mouse hepatocytes. Application of rALR also led to reduced expression of chemo-attractants like CXCL1, CXCL2 and CCl2 in hepatocytes. In addition, ALR expression was increased in IR mouse livers after 3 h and in biopsies from human liver transplants with minimal signs of tissue damage. Therefore, ALR attenuates IRI through reduced neutrophil tissue infiltration mediated by lower expression of key hepatic chemokines and reduction of ROS generation.
Augmenter of liver regeneration (ALR), which is critically important in liver regeneration and hepatocyte proliferation, is highly expressed in cirrhotic livers and hepatocellular carcinomas (HCC). ...In the current study, the functional role of ALR in hepatocancerogenesis was analyzed in more detail. HepG2 cells, in which the cytosolic 15 kDa ALR isoform was reexpressed stably, (HepG2-ALR) were used in migration and invasion assays using modified Boyden chambers. Epithelial-mesenchymal transition (EMT) markers were determined in HepG2-ALR cells in vitro and in HepG2-ALR tumors grown in nude mice. ALR protein was quantified in HCC and nontumorous tissues by immunohistochemistry. HepG2-ALR, compared with HepG2 cells, demonstrated reduced cell motility and increased expression of the epithelial cell markers E-cadherin and Zona occludens-1 (ZO-1), whereas SNAIL, a negative regulator of E-cadherin, was diminished. Matrix metalloproteinase MMP1 and MMP3 mRNA expression and activity were reduced. HepG2-ALR cell-derived subcutaneously grown tumors displayed fewer necrotic areas, more epithelial-like cell growth and fewer polymorphisms and atypical mitotic figures than tumors derived from HepG2 cells. Analysis of tumor tissues of 53 patients with HCC demonstrated an inverse correlation of ALR protein with histological angioinvasion and grading. The 15 kDa ALR isoform was found mainly in HCC tissues without histological angioinvasion 0. In summary the present data indicate that cytosolic ALR reduces hepatoma cell migration, augments epithelial growth and, therefore, may act as an antimetastatic and EMT reversing protein.
Background Targeting heat shock protein 90 (HSP90) has gained great interest for cancer therapy. However, in view of novel multimodality therapy approaches for treating hepatic metastases, concerns ...have raised regarding the impact of targeted therapies on liver regeneration and repair. In this study, we investigated the impact of HSP90 inhibition on liver regeneration in murine models. Methods Effects of HSP90 inhibition on the activation of signaling intermediates, expression of vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) were investigated in primary human hepatocytes (PHHs) in vitro. Effects of HSP90 inhibition on liver regeneration and repair were determined in a murine hepatectomy model and in a model with acute carbon tetrachloride (CCl4 )-induced liver damage. Results Inhibition of HSP90 effectively diminished the constitutive phosphorylation of Akt, Erk, and STAT3 in PHHs. Conversely, inhibition of HSP90 significantly increased the expression of both VEGF and HGF mRNA, and induced HSP70 protein in PHH cultures in vitro. In vivo, HSP90 inhibition significantly upregulated constitutive VEGF mRNA and HSP70 in murine livers and did not impair liver re-growth after 70% hepatectomy. Furthermore, BrdUrd-staining and histological quantification of necrotic areas revealed that HSP90 inhibition did not impair liver regeneration following partial hepatectomy, or liver repair that occurs after toxic liver injury with CCl4. Conclusion Targeting HSP90 does not negatively affect the multifactorial process of liver regeneration and repair in vivo. Hence, the use of inhibitors to HSP90 appears to be a valid option for neoadjuvant therapy of liver metastases when subsequent surgery is intended.
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