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Lipopolysaccharide Reverses Hepatic Stellate Cell Activation Through Modulation of cMyb, Small Mothers Against Decapentaplegic, and CCAAT/Enhancer-Binding Protein C/EBP Transcription Factors.

ABSTRACT: BACKGROUND AND AIMS:During liver injury, quiescent hepatic stellate cells (qHSCs) transdifferentiate into proliferative and fibrogenic activated myofibroblastic phenotype (activated hepatic stellate cell; aHSCs) expressing smooth muscle α-actin (αSMA) and platelet-derived growth factor beta receptor (PDGFβR). Their interactions with gut-derived bacterial lipopolysaccharide (LPS) are implicated in hepatic fibrogenesis. However, LPS can also attenuate fibrogenic characteristics of aHSCs. APPROACH AND RESULTS:We examined molecular mechanisms of antifibrogenic effects of LPS on aHSCs in vitro and in vivo. Culture-activated rat HSCs were exposed to 0-100 ng/mL of LPS or its active component, diphosphoryl-lipid A (DPLA), and parameters of fibrosis and inflammatory cytokines/chemokines were determined by qRT-PCR, western, and immunohistochemical analyses. In vivo, HSCs were activated by repeated CCl4 administration to rats every 3 days for 3 or 8 weeks, then challenged with LPS (5 mg/kg; IP). HSCs were isolated 24 hours later, and fibrogenic/inflammatory parameters were analyzed. LPS induced phenotypic changes in aHSCs (rounding, size reduction) and loss of proliferation. LPS down-regulated expression of αSMA, PDGFβR, transforming growth factor beta receptor 1 (TGFβR1), collagen 1α1 (Col1α1), and fibronectin while up-regulating tumor necrosis factor alpha, interleukin-6, and C-X-C motif chemokine ligand 1 expression. LPS did not increase peroxisome proliferation-activated receptor gamma expression or lipid accumulation typical of qHSCs. DPLA elicited the same effects as LPS on aHSCs, indicating specificity, and monophosphoryl lipid A down-regulated fibrogenic markers, but elicited very weak inflammatory response. LPS down-regulated the expression of cMyb, a transcription factor for αSMA, and up-regulated small mother against decapentaplegic (SMAD)7 and CCAAT/enhancer-binding protein (C/EBP)δ, the transcriptional inhibitors of Col1α1 expression. In vivo LPS treatment of aHSCs inhibited their proliferation, down-regulated PDGFβR, αSMA, TGFβR1, Col1α1, and cMyb expression, and increased expression of SMAD7, C/EBPα, and C/EBPδ. CONCLUSIONS:In conclusion, LPS induces a unique phenotype in aHSCs associated with down-regulation of key fibrogenic mechanisms and thus may have an important role in limiting fibrosis.

SUBMITTER: Sharma A

PROVIDER: S-EPMC8009050 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionThe c-Jun coactivator, Jun activation-domain binding protein 1 (Jab1) also known as the fifth component of the COP9 signalosome complex (CSN5), is a novel candidate oncogene whose aberrant expression contributes to the progression of breast carcinoma and other human cancers. The mechanism of Jab1 gene expression and its deregulation in cancer cells remains to be identified. We therefore investigated the transcriptional regulatory mechanisms of Jab1 expression in human breast carcinoma cells.MethodsTo identify potential regulators of Jab1 transcription, we cloned the 5' upstream region of the human Jab1 gene and mapped its transcriptional start site. We identified binding sequences for the CCAAT/enhancer binding protein (C/EBP) and GATA, as well as a signal transducer and activator of transcription-3 (Stat3) consensus sequence overlapping the C/EBP site, using 5'- deletion analysis and a gene reporter assay. Mutational analysis of these binding sites was performed to confirm their roles in promoting Jab1 transcription in breast cancer cells. We further confirmed these binding sites using electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays. We also analyzed whether the siRNA-mediated inactivation of Stat3 and Src could reduce Jab1-promoter activity and whether interleukine-6 (IL-6) could mediate increased Jab1 expression through Stat3 signaling.ResultsWe identified binding sequences for C/EBP, GATA, as well as a Stat3 consensus sequence overlapping the C/EBP site in the promoter region of Jab1. C/EBP-beta2 is a potential transcriptional activator of Jab1 and mutation of the C/EBP/Stat3 binding site significantly reduced Jab1-promoter activity. In addition, inhibiting Stat3 significantly reduced Jab1-promoter activation. EMSA and ChIP assays confirmed that C/EBP, GATA1 and Stat3 bind to Jab1 promoter in breast carcinoma cells. We also found that Src, an activator of Stat3, is involved in Jab1-promoter activation. siRNA knockdown of Src reduced the Jab1-promoter activity, similar to the results seen when Stat3 was inhibited in breast carcinoma cells. Interestingly, reactivation of Stat3 in normal mammary epithelial cells (MCF-10A, MCF-10F) is sufficient to reactivate Jab1 expression. Treatment with the cytokine IL-6 resulted in increased Jab1 expression that was blocked by inhibition of Stat3.ConclusionsThese findings reveal a novel mechanism of Jab1 gene regulation and provide functional and mechanistic links between the Src/Stat3 and IL-6/Stat3 signaling axes that are involved in the activation of Jab1 transcription and regulation of this novel oncogenic protein.

Project description:BACKGROUND: Even though many functions of protein-x from the Hepatitis B virus (HBV) have been revealed, the nature of protein-x is yet unknown. This protein is well-known for its transactivation activity through interaction with several cellular transcription factors, it is also known as an oncogene. In this work, we have presented computational approaches to design a model to show the structure of protein-x and its respective binding sites associated with the CCAAT/enhancer-binding protein ? (C/EBP?). C/EBP? belongs to the bZip family of transcription factors, which activates transcription of several genes through its binding sites in liver and fat cells. The C/EBP? has been shown to bind and modulate enhancer I and the enhancer II/core promoter of HBV. In this study using the bioinformatics tools we tried to present a reliable model for the protein-x interaction with C/EBP?. RESULTS: The amino acid sequence of protein-x was extracted from UniProt [UniProt:Q80IU5] and the x-ray crystal structure of the partial CCAAT-enhancer ? [PDB:1NWQ] was retrieved from the Protein Data Bank (PDB). Similarity search for protein-x was carried out by psi-blast and bl2seq using NCBI [GenBank: BAC65106.1] and Local Meta-Threading-Server (LOMETS) was used as a threading server for determining the maximum tertiary structure similarities. Advanced MODELLER was implemented to design a comparative model, however, due to the lack of a suitable template, Quark was used for ab initio tertiary structure prediction.The PDB-blast search indicated a maximum of 23% sequence identity and 33% similarity with crystal structure of the porcine reproductive and respiratory syndrome virus leader protease Nsp1? [PDB:3IFU]. This meant that protein-x does not have a suitable template to predict its tertiary structure using comparative modeling tools, therefore we used QUARK as an ab initio 3D prediction approach. Docking results from the ab initio tertiary structure of protein-x and crystal structure of the C/EBP?- DNA region [PDB:1NWQ] illustrated the protein-binding site interactions. Indeed, the N-terminal part of 1NWQ has a high affinity for certain regions in protein-x (e.g. from Ala76 to Ser101 and Thr105 to Glu125). CONCLUSION: In this study, we predicted the structure of protein-x of HBV in interaction with C/EBP?. The docking results showed that protein-x has an interaction synergy with C/EBP?. However, despite previous experimental data, protein-x was found to interact with DNA. This can lead to a better understanding of the function of protein-x and may provide an opportunity to use it as a therapeutic target.

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Lipopolysaccharide Reverses Hepatic Stellate Cell Activation Through Modulation of cMyb, Small Mothers Against Decapentaplegic, and CCAAT/Enhancer-Binding Protein C/EBP Transcription Factors.

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