糖调节蛋白78抗四氯化碳诱导的人HepG2细胞脂肪合成

为研究糖调节蛋白78（glucose regulated protein 78, GRP78）对肝细胞脂肪变性的影响,采用四氯化碳（carbon tetrachloride, CCl₄）刺激人肝癌HepG₂细胞,油红O染色证实,CCl₄作用HepG₂细胞后,细胞浆中脂肪颗粒明显增加,同时固醇调节元件结合蛋白1(sterol regulatory element binding protein 1, SREBP-1)蛋白水平和3羟3甲基戊二酸单酰CoA还原酶（HMGCoA还原酶）mRNA水平分别为对照组的1.55倍和1.70倍.构建人GRP78启动子荧光素酶报告基因载体pGL3/hGRP78P转染人肝癌HepG₂细胞后,结果发现,CCl₄促进GRP78基因转录,转录活性为诱导前的1.92倍. 构建人GRP78 RNAi沉默质粒pSuper/GRP78转染人肝癌HepG₂细胞后,该质粒能特异性沉默内源性GRP78;内源性GRP78沉默后的人肝癌HepG₂细胞经CCl₄诱导, HMGCoA还原酶mRNA和SREBP-1蛋白的表达较对照组进一步升高,分别为对照组的1.48倍和2.38倍;人肝癌HepG₂细胞GRP78的体外过表达能降低CCl₄介导的HMGCoA还原酶mRNA和SREBP-1蛋白诱导表达,分别为对照组的78.5％和51.5％;油红O染色进一步证实,GRP78过表达可明显减少脂肪颗粒在HepG2细胞浆中的集聚.综上表明,GRP78可抑制CCl₄的SREBP-1和HMGCoA还原酶的诱导表达以及HepG₂细胞脂肪变性,提示GRP78的表达增加在肝细胞脂肪变性损伤过程中具有潜在的保护作用.     

TNFR1-mediated signaling is important to induce the improvement of liver fibrosis by bone marrow cell infusion

The importance of TNF-α signals mediated by tumor necrosis factor receptor type 1 (TNFR1) in inflammation and fibrosis induced by carbon tetrachloride (CCl₄), and in post-injury liver regeneration including a GFP/CCl₄ model developed as a liver repair model by bone marrow cell (BMC) infusion, was investigated. In mice in which TNFR1 was suppressed by antagonist administration or by knockout, liver fibrosis induced by CCl₄ was significantly decreased. In these mice, intrahepatic macrophage infiltration and TGF-β1 expression were reduced and stellate cell activity was decreased; however, expression of MMP-9 was also decreased. With GFP-positive BMC (TNFR1 wild-type, WT) infusion in these mice, fibrosis proliferation, including host endogenous intrahepatic macrophage infiltration, TGF-β1 expression and stellate cell activity, increased significantly. There was no significant increase of MMP-9 expression. In this study, TNFR1 in hosts had a promoting effect on CCl₄-induced hepatotoxicity and fibrosis, whereas BMC infusion in TNFR1 knockout mice enhanced host-derived intrahepatic inflammation and fibrosis proliferation. These findings differed from those in WT recipient mice, in which improvement in inflammation and fibrosis with BMC infusion had previously been reported. TNFR1-mediated signaling might be important to induce the improvement of liver fibrosis by bone marrow cell infusion.     

MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl₄-treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation.     

Inhibition of hepatic damage and liver fibrosis by brain natriuretic peptide

Anti-fibrotic and organ protective effects of brain natriuretic peptide (BNP) have been reported. In this study, effects of BNP on liver fibrosis were examined in the carbon tetrachloride (CCl₄)-induced liver fibrosis model using BNP-transgenic (Tg) and wild-type (WT) mice. Twice-a-week intraperitoneal injections of CCl₄ for 8 weeks resulted in massive liver fibrosis, augmented transforming growth factor (TGF)-β₁ and type I procollagen α₁ chain (Col1a1) mRNA expression, and the hepatic stellate cell (HSC) activation in WT mice, all of which were significantly suppressed in Tg mice. These observations indicate that BNP inhibits liver fibrosis by attenuating the activation of HSCs.     

Attenuation of unfolded protein response and apoptosis by mReg2 induced GRP78 in mouse insulinoma cells

Murine regenerating (mReg) genes have been implicated in preserving islet cell biology. Expanding on our previous work showing that overexpression of mReg2 protects MIN6 insulinoma cells against streptozotocin-induced apoptosis, we now demonstrate that mReg2 induces glucose-regulated peptide 78 (GRP78) expression via the Akt–mTORC1 axis and protects MIN6 cells against ER stress induced by thapsigargin and glucolipotoxicity. Activation of mTORC1 activity results from both mReg2-induced increased mTOR phosphorylation as well as increased expression of Raptor and GβL. Inhibition of Akt and mTORC1 blunted the ability of mReg2 to induce GRP78 and attenuate unfolded protein response (UPR). Knockdown of GRP78 sensitized the cells overexpressing mReg2 to UPR without affecting its ability to activate Akt–mTORC1 signaling. Induced expression of mReg2 may protect insulin producing cells from ER stress in diabetes.     

T cell receptor-mediated signaling induces GRP78 expression in T cells: the implications in maintaining T cell viability

The 78-kDa glucose-regulated protein (GRP78) is an important molecular chaperone in the endoplasmic reticulum (ER) induced by various stresses. This study showed that stimulation with anti-CD3 mAb, PMA plus ionomycin, or an antigen increased the levels of GRP78 mRNA in primary T cells, which was inhibited by Ca²⁺ chelators EGTA and BAPTA-AM and by an inhibitor of calcineurin FK506. In addition, the specific knockdown of GRP78 protein expression induced apoptosis in mouse EL-4 T cell line associated with CHOP induction and caspase-3 activation. Furthermore, overexpression of GRP78 inhibited PMA/ionomycin-induced cell death in EL-4 cells. Collectively, GRP78 expression is induced by TCR activation via a Ca²⁺-dependent pathway and may play a critical role in maintaining T cell viability in the steady and TCR-activated states. These results suggest a novel regulatory mechanism and an essential function of GRP78 in T cells.     

Fibroblast growth factor 2 facilitates the differentiation of transplanted bone marrow cells into hepatocytes

We have developed an in vivo mouse model, the green fluorescent protein (GFP)/carbon tetrachloride (CCl₄) model, and have previously reported that transplanted GFP-positive bone marrow cells (BMCs) differentiate into hepatocytes via hepatoblast intermediates. Here, we have investigated the growth factors that are closely related to the differentiation of transplanted BMCs into hepatocytes, and the way that a specific growth factor affects the differentiation process in the GFP/CCl₄ model. We performed immunohistochemical analysis to identify an important growth factor in our model, viz., fibroblast growth factor (FGF). In liver samples, the expression of FGF1 and FGF2 and of FGF receptors (FGFRs; FGFR1, FGFR2) was significantly elevated with time after bone marrow transplantation (BMT) compared with other factors, and co-expression of GFP and FGFs or FGFRs could be detected. We then analyzed the effect and molecular mechanism of FGF signaling on the enhancement of BMC differentiation into hepatocytes by immunohistochemistry, immunoblotting, and microarray analysis. Treatment with recombinant FGF (rFGF), especially rFGF2, elevated the repopulation rate of GFP-positive cells in the liver and significantly increased the expression of both Liv2 (hepatoblast marker) and albumin (hepatocyte marker). Administration of rFGF2 at BMT also raised serum albumin levels and improved the survival rate. Transplantation of BMCs with rFGF2 specifically activated tumor necrosis factor-alpha (TNF-α) signaling. Thus, FGF2 facilitates the differentiation of transplanted BMCs into albumin-producing hepatocytes via Liv2-positive hepatoblast intermediates through the activation of TNF-α signaling. Administration of FGF2 in combination with BMT improves the liver function and prognosis of mice with CCl₄-induced liver damage. This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (nos. 13470121, 13770262, 15790348, 16390211, and 16590597) and for translational research from the Ministry of Health, Labor and Welfare (H-trans-5).     

Expression of the 78 kD glucose-regulated protein is induced by endoplasmic reticulum stress in the development of hepatopulmonary syndrome

Objective

This study is to explore the role of 78 kD glucose-regulated protein (GRP78) in the development of hepatopulmonary syndrome (HPS) in rats.

Methods

The rat model of liver cirrhosis and HPS were induced with multiple pathogenic factors. Hematoxylin and eosin (H & E) staining was performed to detect the pathological changes of the lung and liver tissues. The levels of alanine transferase (ALT), endotoxin, and tumor necrosis factor-α (TNF-α) in plasma and TNF-α and malondialdehyde (MDA) in lung tissues were detected. RT-PCR and Western blotting were conducted to detect the mRNA and protein expression levels of GRP78 in lungs.

Results

The plasma endotoxin level was gradually increased as HPS developed, and the mRNA and protein expression levels of GRP78 in lungs were also increased as the disease progressed. The levels of ALT and TNF-α in plasma and the contents of TNF-α and MDA in lung tissues were gradually increased along with the disease progression, with a strong positive correlation. Compared with controls, the plasma TNF-α level and the mRNA and protein expression levels of GRP78 in lung tissues were significantly higher in rats with HPS. The levels of endotoxin and ALT in plasma and the level of MDA in lungs were significantly higher in rats with HPS than controls.

Conclusions

The increased GRP78 expression is indicative of endoplasmic reticulum stress response during HPS, which may play an important role in the disease pathogenesis.     

PI3‐kinase/Akt pathway‐regulated membrane transportation of acid‐sensing ion channel 1a/Calcium ion influx/endoplasmic reticulum stress activation on PDGF‐induced HSC Activation

Acid‐sensing ion channel 1a (ASIC1a) allows Na⁺ and Ca²⁺ flow into cells. It is expressed during inflammation, in tumour and ischaemic tissue, in the central nervous system and non‐neuronal injury environments. Endoplasmic reticulum stress (ERS) is caused by the accumulation of misfolded proteins that interferes with intracellular calcium homoeostasis. Our recent reports showed ASIC1a and ERS are involved in liver fibrosis progression, particularly in hepatic stellate cell (HSC) activation. In this study, we investigated the roles of ASIC1a and ERS in activated HSC. We found that ASIC1a and ERS‐related proteins were up‐regulated in carbon tetrachloride (CCl₄)‐induced fibrotic mouse liver tissues, and in patient liver tissues with hepatocellular carcinoma with severe liver fibrosis. The results show silencing ASIC1a reduced the expression of ERS‐related biomarkers GRP78, Caspase12 and IREI‐XBP1. And, ERS inhibition by 4‐PBA down‐regulated the high expression of ASIC1a induced by PDGF, suggesting an interactive relationship. In PDGF‐induced HSCs, ASIC1a was activated and migrated to the cell membrane, leading to extracellular calcium influx and ERS, which was mediated by PI3K/AKT pathway. Our work shows PDGF‐activated ASIC1a via the PI3K/AKT pathway, induced ERS and promoted liver fibrosis progression.     

Toll like receptor 2 knock-out attenuates carbon tetrachloride (CCl4)-induced liver fibrosis by downregulating MAPK and NF-κB signaling pathways

Innate immune signaling associated with Toll-like receptors (TLRs) is a key pathway involved in the progression of liver fibrosis. In this study, we reported that TLR2 is required for hepatic fibrogenesis induced by carbon tetrachloride (CCl₄). After CCl₄ treatment, TLR2^−/− mice had reduced liver enzyme levels, diminished collagen deposition, decreased inflammatory infiltration and impaired activation of hepatic stellate cells (HSCs) than wild type (WT) mice. Furthermore, after CCl₄ treatment, TLR2^−/− mice demonstrated downregulated expression of profibrotic and proinflammatory genes and impaired mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NF-κB) activation than WT mice. Collectively, our data indicate that TLR2 deficiency protects against CCl₄-induced liver fibrosis.     

Effect of hypoxic treatment on bone marrow cells that are able to migrate to the injured liver

Restricted numbers and poor regenerative properties limit the use of adult stem cells. We tested the effect of hypoxic treatment as a method by which to increase cell migration. Bone marrow cells (BMCs) were cultured under oxygen saturations of 0.1, 3, and 20% for 24 h. After hypoxic treatment, BMCs of apoptotic fraction were decreased. The expression of CXCR4 was noticeably increased in the hypoxia-treated BMCs and their migration in response to SDF-1α was enhanced compared with cells cultured under normoxic condition. Hypoxic BMCs had a higher degree of engraftment to the CCl₄-injured liver than the normoxic cells. Hypoxic treatment of BMCs may have merits in decreasing apoptosis of those cells as well as in enhancing cellular migration to SDF-1α, the chemokine which binds to BMCs expressed CXCR4 and to the injured tissue, such as CCl₄ damaged liver.     

Mesenchymal stem cells restore CCl4‐induced liver injury by an antioxidative process

We have investigated BM (bone marrow)‐derived MSCs (mesenchymal stem cells) for the treatment of liver injury. It was hypothesized that MSC‐mediated resolution of liver injury could occur through an antioxidative process. After being injected with CCl₄ (carbon tetrachloride), mice were injected with syngenic BM‐derived MSCs or normal saline. Oxidative stress activity of the MSCs was determined by the analysis of ROS (reactive oxygen species) and SOD (superoxide dismutase) activity. In addition, cytoprotective genes of the liver tissue were assessed by real‐time PCR and ARE (antioxidant‐response element) reporter assay. Up‐regulated ROS of CCl₄‐treated liver cells was attenuated by co‐culturing with MSCs. Suppression of SOD by adding an SOD inhibitor decreased the effect of MSCs on injured liver cells. MSCs significantly increased SOD activity and inhibited ROS production in the injured liver. The gene expression levels of Hmox‐1 (haem oxygenase‐1), BI‐1 (Bax inhibitor‐1), HGF (hepatocyte growth factor), GST (glutathione transferase) and Nrf2 (nuclear factor‐erythoid 2 p45 subunit‐related factor 20), attenuated by CCl₄, were increased up to basal levels after MSC transplantation. In addition, MSCs induced an ARE, shown by luciferase activity, which represented a cytoprotective response in the injured liver. Evidence of a new cytoprotective effect is shown in which MSCs promote an antioxidant response and supports the potential of using MSC transplantation as an effective treatment modality for liver disease.     

Induction of expression and co-localization of heat shock polypeptides with the polyalanine expansion mutant of poly(A)-binding protein N1 after chemical stress

Formation of nuclear inclusions consisting of aggregates of a polyalanine expansion mutant of nuclear poly(A)-binding protein (PABPN1) is the hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a late onset autosomal dominant disease. Patients with this disorder exhibit progressive swallowing difficulty and drooping of their eye lids, which starts around the age of 50. Previously we have shown that treatment of cells expressing the mutant PABPN1 with a number of chemicals such as ibuprofen, indomethacin, ZnSO₄, and 8-hydroxy-quinoline induces HSP70 expression and reduces PABPN1 aggregation. In these studies we have shown that expression of additional HSPs including HSP27, HSP40, and HSP105 were induced in mutant PABPN1 expressing cells following exposure to the chemicals mentioned above. Furthermore, all three additional HSPs were translocated to the nucleus and probably helped to properly fold the mutant PABPN1 by co-localizing with this protein.     

Glucose-regulated protein 78 may play a crucial role in promoting the pulmonary microvascular remodeling in a rat model of hepatopulmonary syndrome

Objective

This study is to investigate the role of glucose-regulated protein 78 (GRP78) in the pulmonary microvascular remodeling during hepatopulmonary syndrome (HPS) development.

Methods

The rat models with liver cirrhosis and HPS were induced by multiple pathogenic factors for 4 to 8 wk. The concentrations of alanine transferase (ALT) and endotoxin in plasma were detected in the models, followed by the detection of GRP78 expression. RT-PCR, quantitative real-time PCR and Western blotting were employed to assess the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), respectively. Immunohistochemistry staining was used to examine the expression of a specific vascular marker, factor VIII-related antigen (FVIII-RAg), and several cell proliferation- and apoptosis-related proteins, including CHOP/GADD153, caspase-12, Bcl-2 and nuclear factor (NF)-κB.

Results

The levels of endotoxin and ALT in plasma were gradually increased as the disease progressed, so did GRP78, which were in a positive correlation. The expression levels of VEGF (both mRNA and protein) and FVIII-RAg were significantly elevated in the HPS models, indicating active angiogenesis, which was also positively correlated with GRP78 expression. Furthermore, the expression levels of the pro-apoptotic proteins of CHOP/GADD153 and caspase-12 were dramatically decreased, while the anti-apoptotic proteins of Bcl-2 and NF-κB were significantly elevated, in the HPS models. There were also close correlation between these proteins and GRP78.

Conclusions

Over-expression of GRP78 in lungs may be the critical pathogenic factor for HPS. Through promoting cell proliferation and survival and inhibiting apoptosis, GRP78 may promote the pulmonary microvascular remodeling in HPS pathogenesis. Our results provide a potential therapeutic target for clinical prevention and treatment for HPS and related complications.     

Role of the unfolded protein response in cell death

Unfolded protein response (UPR) is an important genomic response to endoplasmic reticulum (ER) stress. The ER chaperones, GRP78 and Gadd153, play critical roles in cell survival or cell death as part of the UPR, which is regulated by three signaling pathways: PERK/ATF4, IRE1/XBP1 and ATF6. During the UPR, accumulated unfolded protein is either correctly refolded, or unsuccessfully refolded and degraded by the ubiquitin-proteasome pathway. When the unfolded protein exceeds a threshold, damaged cells are committed to cell death, which is mediated by ATF4 and ATF6, as well as activation of the JNK/AP-1/Gadd153-signaling pathway. Gadd153 suppresses activation of Bcl-2 and NF-κB. UPR-mediated cell survival or cell death is regulated by the balance of GRP78 and Gadd153 expression, which is coregulated by NF-κB in accordance with the magnitude of ER stress. Less susceptibility to cell death upon activation of the UPR may contribute to tumor progression and drug resistance of solid tumors.