Sublytic C5b-9 triggers glomerular mesangial cell apoptosis via XAF1 gene activation mediated by p300-dependent IRF-1 acetylation

The apoptosis of glomerular mesangial cells (GMCs) in rat Thy-1 nephritis (Thy-1N), a model of human mesangioproliferative glomerulonephritis (MsPGN), is accompanied by sublytic C5b-9 deposition. However, the mechanism by which sublytic C5b-9 induces GMC apoptosis is unclear. In the present studies, the effect of X-linked inhibitor of apoptosis-associated factor 1 (XAF1) expression on GMC apoptosis and the role of p300 and interferon regulatory factor-1 (IRF-1) in mediating XAF1 gene activation were determined, both in the GMCs induced by sublytic C5b-9 (in vitro) and in the renal tissues of rats with Thy-1N (in vivo). The in vitro studies demonstrated that IRF-1-enhanced XAF1 gene activation and its regulation by p300-mediated IRF-1 acetylation were involved in GMC apoptosis induced by sublytic C5b-9. The element of IRF-1 binding to XAF1 promoter and two acetylated sites of IRF-1 protein were also revealed. In vivo, silence of p300, IRF-1 or XAF1 genes in the renal tissues diminished GMC apoptosis and secondary GMC proliferation as well as urinary protein secretion in Thy-1N rats. Together, these data implicate that sublytic C5b-9 induces the expression of both p300 and IRF-1, as well as p300-dependent IRF-1 acetylation that may contribute to XAF1 gene activation and subsequent GMC apoptosis in Thy-1N rats.     

The complement C5b-9 complexes induced injury of glomerular mesangial cells in rats with Thy-1 nephritis by increasing nitric oxide synthesis

Thy-1 nephritis (Thy-1 N), namely, anti-Thy-1 or anti-thymocyte serum (ATS) induced nephritis (ATSN), is a typical model of human mesangioproliferative glomerulonephritis. The pathologic changes of glomerular mesangial cells (GMCs) in Thy-1 N are complement-dependent, especially C5b-9 complexes, but the role of C5b-9 in the mechanism of Thy-1 N has not been defined. Because previous studies have demonstrated that sublytic C5b-9 can increase production of several inflammatory mediators from resident glomerular cells, we utilized the isolated human membrane-bound C5b-9 complexes to stimulate the cultured rat GMCs and examined whether the GMCs can also induce the synthesis of nitric oxide (NO) in vitro. Simultaneously, the effects of antiserum against rat C5b-9 and NG-monomethyl-L-arginine (L-NMMA, NO inhibitor), including interfering with the formation of C5b-9, reducing NO production and GMCs injury were observed. The results showed that sublytic C5b-9 can increase synthesis of inducible NO from the stimulated GMCs, and that the anti-C5b-9 antiserum can obviously inhibit the pathologic changes in Thy-1 N, while L-NMMA can decrease the GMCs damage although the effect is not so significant as that of the anti-C5b-9 antiserum. These findings indicate that the synthesis of NO by GMCs can be promoted by sublytic C5b-9, and that lesions of GMCs in rats with Thy-1 N are prevented by either inhibiting C5b-9 formation or NO elevation in advance. The pathologic changes of GMCs in Thy-1 N are indeed complement C5b-9-dependent, and the glomerular injury can be mediated in part through elevation of NO from the GMCs after the sublytic C5b-9 stimulation.     

Gene expression profile and overexpression of apoptosis-related genes (NGFI-B and Gadd 45 γ) in early phase of Thy-1 nephritis model

Mesangioproliferative glomerulonephritis (MPGN) is a disease of high incidence in humans. Rat Thy-1 nephritis (Thy-1 N), namely, anti-thymocyte serum (ATS)-induced nephritis, is considered to be an animal model for studying MPGN. Although previous studies have demonstrated that glomerular mesangial cell (GMCs) injury might be a feature of Thy-1 N, the mechanism of the disease (i.e., GMC apoptosis) remains unclear. We have examined the pathologic changes of GMCs and the gene expression profile of renal tissues in Thy-1 N. The pathologic changes of Thy-1 N include three phages: GMC apoptosis (40 min), necrosis (2 h), and proliferation (5 days). Many TUNEL-positive cells are found 40 min after administration of ATS. Concomitantly, 341 genes are up-regulated, whereas 392 genes are down-regulated, as shown by microarrays analysis. The mRNA and protein of two of the up-regulated genes (nerve growth factor induced protein I-B, NGFI-B; growth arrest- and DNA-damage-inducible protein 45 gamma, Gadd 45 γ) in the GMC apoptotic phase of Thy-1 N are markedly elevated, as observed by real-time polymerase chain reaction and immunohistochemistry. Our data indicate that pathologic changes of Thy-1 N are involved in the abnormal gene profile. The overexpression of the NGFI-B and Gadd 45 γ genes may be associated with GMC apoptosis of Thy-1 N.This work was supported by grants (no. 30571728, 30471615, and no. 03KJA310074) from the National Natural Science Foundations of China and Jiangsu Province.     

Effects of PI3-k/Akt short hairpin RNA on proliferation, fibronectin production and synthesis of thrombospondin-1 and transforming growth factor-β1 in glomerular mesangial cells induced by sublytic C5b-9 complexes

Objectives: To explore proliferation of glomerular mesangial cells (GMC) and secretion of extracelluar matrix (fibronectin induced by sublytic C5b-9 complexes), and then ascertain the role of phosphatidylinositol 3-kinase (PI3-k)/Akt signal pathway in these processes, by using small hairpin RNAs.
Material and methods: The expression of cyclin D₂, ³H-thymidine into DNA and production of fibronectin including thrombospondin-1 and transforming growth factor-β₁ in the GMCs stimulated by sublytic C5b-9 or transfected with expression vectors of PI3-k and Akt short hairpin RNA or LY294002 (PI3-k inhibitor) were measured by Real-time quantitative polymerase chain reaction (PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and ³H-thymidine incorporation (³H-TdR), respectively.
Results: The expression of cyclin D₂, ³H-thymidine into DNA and fibronectin in the GMCs stimulated by sublytic C5b-9 could all be increased, and the elevations of these parameters mentioned above were also markedly reduced in the GMCs transfected with vectors of PI3-k and Akt short hairpin RNA or LY294002, respectively.
Conclusions: These data indicate that sublytic C5b-9 can promote proliferation of GMCs and secretion of fibronectin as well as synthesis of thrombospondin-1 and transforming growth factor-β₁. The PI3-k/Akt signal pathway in these reactions, mediated by sublytic C5b-9 complexes, may play at least a partial role.     

Sublytic C5b‐9 induces proliferation of glomerular mesangial cells via ERK5/MZF1/RGC‐32 axis activated by FBXO28‐TRAF6 complex

Mesangioproliferative glomerulonephritis (MsPGN) is characterized by the proliferation of glomerular mesangial cells (GMCs) and accumulation of extracellular matrix (ECM), followed by glomerulosclerosis and renal failure of patients. Although our previous studies have demonstrated that sublytic C5b‐9 complex formed on the GMC membrane could trigger GMC proliferation and ECM expansion of rat Thy‐1 nephritis (Thy‐1N) as an animal model of MsPGN, their mechanisms are still not fully elucidated. In the present studies, we found that the levels of response gene to complement 32 (RGC‐32), myeloid zinc finger 1 (MZF1), phosphorylated extracellular signal‐regulated kinase 5 (phosphorylated ERK5, p‐ERK5), F‐box only protein 28 (FBXO28) and TNF receptor‐associated factor 6 (TRAF6) were all markedly up‐regulated both in the renal tissues of rats with Thy‐1N (in vivo) and in the GMCs upon sublytic C5b‐9 stimulation (in vitro). Further in vitro experiments revealed that up‐regulated FBXO28 and TRAF6 could form protein complex binding to ERK5 and enhance ERK5 K63‐ubiquitination and subsequent phosphorylation. Subsequently, ERK5 activation contributed to MZF1 expression and MZF1‐dependent RGC‐32 up‐regulation, finally resulting in GMC proliferative response. Furthermore, the MZF1‐binding element within RGC‐32 promoter and the functions of FBXO28 domains were identified. Additionally, knockdown of renal FBXO28, TRAF6, ERK5, MZF1 and RGC‐32 genes respectively markedly reduced GMC proliferation and ECM production in Thy‐1N rats. Together, these findings indicate that sublytic C5b‐9 induces GMC proliferative changes in rat Thy‐1N through ERK5/MZF1/RGC‐32 axis activated by the FBXO28‐TRAF6 complex, which might provide a new insight into MsPGN pathogenesis.     

Inhibition of oligodendrocyte apoptosis by sublytic C5b-9 is associated with enhanced synthesis of bcl-2 and mediated by inhibition of caspase-3 activation

We have previously shown that generation of sublytic C5b-9, the membrane attack complex of complement, induces oligodendrocytes to enter cell cycle and reduces apoptotic cell death in vitro. In the present study, the cellular factors involved in apoptosis of oligodendrocyte progenitor cells and oligodendrocytes, and the inhibitory effect of C5b-9 on apoptotic process were investigated. Oligodendrocyte progenitor cells identified by mAb A2B5 that were isolated from neonatal rat brains were differentiated into oligodendrocytes in serum-free defined medium. The differentiation, which occurs simultaneously with apoptotic cell death, was associated with a rapid loss of bcl-2 mRNA and increased expression of caspase-3 mRNA. Activation of caspase-3 in differentiating cells was demonstrated by the generation of 17- and 12-kDa fragments of caspase-3 proenzyme and by cleavage of poly(ADP-ribose) polymerase, a specific caspase-3 substrate. Cell death associated with differentiation was inhibited by the caspase-3 inhibitor DEVD-CHO in a dose-dependent manner. Assembly of sublytic C5b-9 resulted in inhibition of caspase-3 activation. In addition, synthesis of BCL-2 protein in oligodendrocytes was significantly increased by C5b-9. The TNF-alpha-induced apoptosis of oligodendrocytes was also inhibited by C5b-9. These results indicate that up-regulation of BCL-2 protein and inhibition of caspase-3 activation are potential mechanisms by which C5b-9 increases survival of oligodendrocyte in vitro and possibly in vivo during inflammation and immune-mediated demyelination affecting the CNS.     

C5b-9 terminal complex protects oligodendrocytes from apoptotic cell death by inhibiting caspase-8 processing and up-regulating FLIP

Activation of the terminal complement cascade involving C5 to C9 proteins has a beneficial role for oligodendrocytes (OLG) in experimental allergic encephalomyelitis, an animal model of multiple sclerosis, by protecting them from apoptotic cell death. We have previously shown that sublytic C5b-9 complexes, through posttranslational regulation of Bad, inhibit the mitochondrial pathway of apoptosis induced by serum deprivation. In the present study, we examined the possible involvement of the caspase-8 and Fas pathway in OLG apoptosis and the role of C5b-9 in this process. In a serum-free defined medium, OLG undergo apoptosis and differentiation concomitantly. Under this condition, we found that caspase-8 processing was increased in association with Bid cleavage and markedly reduced expression of cellular FLIP long isoform protein. The caspase-8 inhibitor Z-IETD-FMK inhibited cell death associated with differentiation in a dose-dependent manner. Exposure to C5b-9 induced an inhibition of caspase-8 activation, Bid cleavage, and a significant increase in expression of cellular FLIP long isoform. These C5b-9 effects were reversed by PI3K inhibitor LY294002. C5b-9 also down-regulated the expression of FasL and the Fas-induced apoptosis. These data suggest that C5b-9 through PI3K signaling can rescue OLG from Fas-mediated apoptosis by regulating caspase-8 processing.     

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

Background

Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

Methods

CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

Results

Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2^′-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O₂.

Conclusion

These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.     

HSP70 inhibits stress-induced cardiomyocyte apoptosis by competitively binding to FAF1

Stress-induced cardiomyocyte apoptosis plays an important role in the pathogenesis of a variety of cardiovascular diseases. Our early studies showed that HSP70 effectively inhibited apoptosis, but the underlying mechanism remained unclear. Fas-associated factor 1 (FAF1) is a member of the Fas death-inducing signaling complex (Fas-DISC) that acts upstream of caspase-8. We investigated the interactions among FAF1, HSP70, and FAS in stressed cardiomyocytes to elucidate the protective mechanism of HSP70. FAS and caspase-3/8 activity was higher in cardiomyocytes undergoing stress-induced apoptosis in restraint-stressed rats compared with cardiomyocytes in non-stressed rats, which indicated that the Fas signaling pathway was activated after restraint stress. Geranylgeranylacetone (GGA) induced an increase in HSP70 expression, which reduced stress-induced apoptosis. Additionally, overexpression of HSP70 via transfection with the pEGFP-rHSP70 plasmid attenuated norepinephrine (NE)-induced apoptosis. FAF1 expression increased during stress-induced apoptosis, and overexpression of FAF1 exacerbated NE-induced apoptosis. We also found that HSP70 interacted with FAF1. Overexpression of HSP70 inhibited the binding of FAF1 to FAS in H9C2 cells, which indicated that HSP70 suppressed NE-induced apoptosis by competitively binding to FAF1. An N-terminal deletion mutant of HSP70 (HSP70-△N) was unable to interact with FAF1. After HSP70-△N was transfected into H9C2 cells, the cells were unable to attenuate the NE-induced increases in caspase-8 and apoptosis. These results indicate that the 1–120 sequence of HSP70 binds to FAF1, which alters the interactions between FAS and FAF1 and inhibits the activation of the Fas signaling pathway and apoptosis.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0589-9) contains supplementary material, which is available to authorized users.     

Arachidonic acid mobilization and phosphoinositide turnover by the terminal complement complex, C5b-9, in rat oligodendrocyte x C6 glioma cell hybrids

Previously, we have shown that rat oligodendrocytes release phospholipid and generate arachidonic acid (AA) and leukotriene B4 in response to sublytic C5b-9 formation. In the present study, we investigated the biochemical pathways by which C5b-9 generates AA from clone ROC-1, a fusion product of rat oligodendrocytes and C6 glioma. Cells were incubated for 24 h in the presence of [3H]AA or [3H]myoinositol. They were then sensitized with antibody against hybrid cell stroma and treated for 1 h with C9-depleted human serum (C9D-HS) or C9D-HS reconstituted with C9. Alternatively, cells were treated with C8,C9D-HS or C8,C9D-HS reconstituted with C8 or C8 plus C9 for 1 h. Qualitative and quantitative analysis of the released [3H]AA and [3H]myoinositol radiolabeled products were performed by thin layer chromatography/autoradiography and anion exchange chromatography, respectively. The major [3H]AA radiolabeled products after C5b-9 stimulation comigrated with intact phospholipid and AA standards, and the major [3H]myoinositol radiolabeled product was inositol-1-phosphate. Treatment of cells with phospholipase A2 inhibitors, mepacrine and bromophenacyl bromide, abolished AA release by C5b-9. In the absence of extracellular Ca2+, C5b-9 also failed to induce the release of AA. Interestingly, 1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinases, inhibited AA release by C5b-9, whereas AA release stimulated by the calcium ionophore A23187 was not blocked by H-7. The results suggest that AA generation by C5b-9 from the ROC-1 clone involves activation of Ca2+-dependent phospholipase A2 which is regulated by protein kinase-dependent mechanisms.     

Protease Activity of Procaspase-8 Is Essential for Cell Survival by Inhibiting Both Apoptotic and Nonapoptotic Cell Death Dependent on Receptor-interacting Protein Kinase 1 (RIP1) and RIP3

Caspase-8 has an important role as an initiator caspase during death receptor-mediated apoptosis. Moreover, it has been reported to contribute to the regulation of cell fate in various types of cells including T-cells. In this report, we show that caspase-8 has an essential role in cell survival in mouse T-lymphoma-derived L5178Y cells. The knockdown of caspase-8 expression decreased the growth rate and increased cell death, both of which were induced by the absence of protease activity of procaspase-8. The cell death was associated with reactive oxygen species (ROS) accumulation, caspase activation, and autophagosome formation. The cell death was inhibited completely by treatment with ROS scavengers, but only partly by treatment with caspase inhibitors, expression of Bcl-xL, and knockdown of caspase-3 or Atg-7 which completely inhibits apoptosis or autophagosome formation, respectively, indicating that apoptosis and autophagy-associated cell death are induced simultaneously by the knockdown of caspase-8 expression. Further analysis indicated that RIP1 and RIP3 regulate this multiple cell death, because the cell death as well as ROS production was completely inhibited by not only treatment with the RIP1 inhibitor necrostatin-1, but also by knockdown of RIP3. Thus, in the absence of protease activity of procaspase-8, RIP1 and RIP3 simultaneously induce not only nonapoptotic cell death conceivably including autophagic cell death and necroptosis but also apoptosis through ROS production in mouse T-lymphoma cells.     

Changes of subcellular localization of Thy-1 antigen during thymic myoid cell differentiation

Using a quantitative enzyme immunoassay, Thy-1 antigen expressed by a rat myoid cell line R615B2 was detected mainly on the cell surface at a single cell stage, whereas at the stage of forming myotubes, Thy-1 was found predominantly in the cytoplasm. The muscle specific creatine kinase activity also increased in association with the shift of Thy-1 from the cell surface to the cytoplasm, suggesting biological significance of Thy-1 redistribution in muscle differentiation from single cells to multinucleated cells.     

Inhibition of aquaporin-1 expression by RNAi protects against aristolochic acid I-induced apoptosis in human proximal tubular epithelial (HK-2) cells

The aim of this study was to investigate the protective effect of inhibition of aquaporin-1 (AQP1) expression against aristolochic acid I (AA-I)-induced apoptosis. HK-2 cells impaired by AA-I were used in this study as the cell model of aristolochic acid nephropathy. Apoptosis was studied by different methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assays, flow cytometry, and caspase 3 activity assays. We compared AA-I-mediated apoptosis in HK-2 cells with or without knockdown of AQP1 expression by RNA interference. MTT assays showed that AA-I inhibited the viability of HK-2 cells in a time- and concentration-dependent manner. Apoptosis was evidenced by the results of the Annexin V/propidium iodide assay and the occurrence of a sub-G1 peak in cell-cycle analysis. The activity of caspase 3 was found to have been increased by AA-I in a concentration-dependent manner. However, AQP1 RNA interference provided protection against injury in cells treated with AA-I (40 μM) for 24 h and attenuated the number of apoptotic cells. These results suggested that AQP1 plays an important role in AA-I-induced apoptosis and that inhibition of AQP1 expression may protect HK-2 cells from AA-I-induced apoptotic damage.     

长链非编码RNAIFNG-AS1靶向miR-19b-1-5p调控oxLDL诱导的血管内皮细胞增殖、凋亡的机制研究

为了探讨长链非编码RNA干扰素活化基因的反义核糖核酸(lncRNA IFNG-AS1)对氧化型低密度脂蛋白(oxLDL)诱导的人脐静脉血管内皮细胞EVC-304增殖、凋亡的影响和调控机制,该研究采用100 μg/mL的oxLDL分别处理转染si-IFNG-AS1、miR-19b-1-5p mimics或共转染si-IF...     

Long non-coding RNA ZFAS1 alleviates sepsis-induced myocardial injury via target miR-34b-5p/SIRT1

Long non-coding RNA ZFAS1 is down-regulated in sepsis. However, whether ZFAS1 participates in sepsis-induced cardiomyopathy (SIC) remains largely unknown. LPS injection to rats was used to establish an in vivo sepsis model, while LPS stimulation with H9C2 cell was used to mimic an in vitro sepsis-induced myocardial injury model. Western blots and quantitative RT-PCR were performed to evaluate protein and mRNA levels, respectively. ELISA was conducted to determine cytokine levels in supernatant. Flow cytometry was used to test apoptosis. Dual-luciferase assay was performed to validate binding between ZFAS1 and miR-34b-5p, miR-34b-5p and SIRT1. Our data revealed that ZFAS1 and SIRT1 were down-regulated, while miR-34b-5p was up-regulated in LPS-induced H9C2 cells. Inhibition of miR-34b-5p or overexpression of ZFAS1 alleviated inflammatory response and cell apoptosis in LPS-stimulated H9C2 cells. A mechanism study revealed that ZFAS1 sponged miR-34b-5p and thus elevated expression of SIRT1, which was prohibited by miR-34b-5p. ZFAS1 alleviated inflammatory response and cell apoptosis in LPS-stimulated H9C2 cells via the miR-34b-5p/SIRT1 axis, providing novel potential therapeutic targets for SIC.     

MFN1介导的线粒体融合在心肌细胞凋亡中的作用研究

目的:探讨线粒体融合关键蛋白MFN1介导的线粒体融合在调控心肌细胞凋亡中的作用。方法:通过si RNA降低体外培养H9C2心肌细胞中MFN1的表达后,采用Western blot检测线粒体细胞色素c(Cyto c)释放及其下游凋亡效应分子Caspase9与Caspase3活性,流式细胞术检测细胞内活性氧(ROS)的产生情况,流式细胞术检测细胞凋亡的情况。结果:干扰MFN1可显著促进H9C2心肌细胞内细胞色素c由线粒体释放至胞浆,促进Caspase9与Caspase3的激活,增加细胞内活性氧ROS产生并提高细胞凋亡率(均P0.05)。结论:MFN1介导的线粒体融合可保护心肌细胞凋亡,其机制可能与抑制ROS产生与细胞色素C释放有关。     

Ghrelin inhibits apoptosis signal-regulating kinase 1 activity via upregulating heat-shock protein 70

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), which has been originally isolated from rat stomach. It has been reported that ghrelin inhibited apoptosis in several cells, such as cardiomyocytes, endothelial cells, adipocyte, adrenal zona glomerulosa cells, pancreatic beta-cells, osteoblastic MC3T3-E1 cells, intestinal epithelial cells and hypothalamic neurons. However, it is unknown whether heat-shock protein 70 (HSP70) or apoptosis signal-regulating kinase 1 (ASK1) is the important target molecule which mediates the anti-apoptotic effects of ghrelin. We show that ghrelin inhibited ASK1 activity induced by sodium nitroprusside (SNP), inhibited ASK1-mediated caspase 3 activation and apoptosis in PC12 cells. Ghrelin promoted expression of HSP70. Quercetin, an inhibitor of HSP70, blocked the effects of ghrelin on ASK1 activity. Thus, ghrelin inhibits ASK1-mediated apoptosis and ASK1 activation by a mechanism involving induction of HSP70 expression. The results of the present study suggest the therapeutic potential of ghrelin for some pathological processes or disorders.