FasL Protein Levels Research Articles

Estrogen preserves Fas ligand levels by inhibiting microRNA-181a in bone marrow-derived mesenchymal stem cells to maintain bone remodeling balance

Estrogen protects bone loss by promoting Fas ligand (FasL) transcription in osteoclasts and osteoblasts to induce apoptosis of osteoclasts. Bone marrow-derived mesenchymal stem cells (BMMSCs) express FasL protein, which is necessary for BMMSCs to induce T-cell apoptosis in cell therapy. However, the physiologic function of FasL in BMMSCs is unknown. In this study, using an in vitro coculture system and an in vivo BMMSC transplantation assay, we found that BMMSCs potently induced apoptosis of osteoclasts through the FasL/Fas pathway. Estrogen was necessary for this process as a promoter of FasL protein accumulation in BMMSCs. Furthermore, estrogen elevated FasL protein accumulation, not by increasing FasL gene transcription, but through microRNA-mediated posttranscriptional regulation. In brief, estrogen down-regulated expression of miR-181a, a negative modulator of FasL targeting the 3'-UTR of FasL mRNA. Estrogen deficiency resulted in excessive miR-181a, which decreased FasL protein levels to suppress BMMSC-induced osteoclast apoptosis. Furthermore, knockdown of miR-181a recovered the BMMSC defect to induce osteoclast apoptosis during estrogen deficiency. Taken together, our results showed that estrogen preserves FasL protein accumulation by inhibiting miR-181a expression in BMMSCs to maintain bone remodeling balance, suggesting a novel mechanism by which estrogen preserves bone mass.

Interleukin 17 regulates Mer tyrosine kinase-positive cells in Pseudomonas aeruginosa keratitis.

To determine if IL-17 regulates Mer tyrosine kinase-positive (MerTK+) cells in Pseudomonas aeruginosa keratitis. Interleukin 17 was tested in normal and infected cornea of susceptible C57BL/6 and resistant BALB/c mice. The latter were treated with recombinant mouse (rm) IL-17; both groups were treated with IL-17 neutralizing antibody. Mice were infected, and clinical score, PCR, ELISA, and myeloperoxidase (MPO) assays tested expression of proinflammatory and anti-inflammatory mediators and polymorphonuclear neutrophilic leukocyte (PMN) infiltrate. Fas and Fas ligand (FasL) protein levels were assessed in both mouse strains, while MerTK+ cells were examined by immunostaining and cell sorting before and after IL-17 neutralization. The IL-17 mRNA and protein were higher in C57BL/6 versus BALB/c cornea after infection. The rmIL-17 treatment of BALB/c mice modified proinflammatory and anti-inflammatory mediators, but clinical score and MPO assay revealed no differences. However, only BALB/c mice treated with IL-17 neutralizing antibody showed increased disease, macrophage inflammatory protein (MIP) 2, and MPO levels. Fas and FasL protein levels, elevated earlier in BALB/c versus C57BL/6 mice, correlated with significantly more MerTK+ cells in BALB/c cornea at 3 days after infection. Neutralization of IL-17 in C57BL/6 mice elevated MerTK+ cells, while similar treatment of BALB/c mice significantly decreased them. These data provide evidence that IL-17 expression is higher in C57BL/6 versus BALB/c cornea after infection and that the latter group has more MerTK+ cells. Exogenous rmIL-17 failed to shift the disease response in resistant mice, but its neutralization resulted in worsened disease and reduced MerTK+ cells. Neutralization of IL-17 in C57BL/6 mice increased MerTK+ cells but did not dramatically shift the disease response.

Inhibition of eIF5A Results in Aberrant Uterine Natural Killer Cell Function and Embryo Loss in Mice

The role of eukaryotic initiation factor 5A (eIF5A) in feto-maternal immunotolerance is poorly understood. The effects of N1-guanyl-1,7-diaminoheptane (GC7), an inhibitor of eIF5A, on the proportion and function of natural killer (NK) cell subsets were investigated using flow cytometry, immunofluorescence, CCK8 assay, TUNEL assay, DNA fragmentation analysis, mitochondrial membrane potential assay, and Western blotting. Inhibition of eIF5A by GC7 increased embryo loss and reduced the percentage of NK cells in the uterus and spleen. GC7 treatment caused inhibition of NK cell proliferation in a time- and dose-dependent manner. GC7 also induced apoptosis of NK cells. GC7 treatment increased the protein levels of FasL, bax, p53, and cleaved caspase-3. Moreover, GC7 caused loss of mitochondrial membrane potential in NK cells. Inhibition of eIF5A results in aberrant NK cell function and increased embryo loss.

Impact of MiR-21 on the Expression of FasL in the Presence of TGF–β1

Micro-ribonucleic acids (miR) are small, noncoding RNA molecules 19 to 25 nucleotides in length that typically function as negative regulators of expression for many target genes involved in cell proliferation, differentiation, and apoptosis. However, the effects of miR-21 on keloid fibroblasts are currently unknown. The authors investigate whether miR-21, a specific miR implicated in multiple aspects of keloid fibroblasts, affects the expression of Fas ligand (FasL) in the presence of transforming growth factor (TGF)-β1. The relationship between TGF-β1 and miR-21 expression was investigated by TaqMan quantitative real-time polymerase chain reaction (Life Technologies, Grand Island, New York). FasL protein was determined by Western blotting, and regulation of cell proliferation/migration/apoptosis ability by TGF-β1 inhibitor or plasmid was evaluated respectively by EdU incorporation, Transwell assay, and flow cytometry analysis. Fibroblasts from keloid tissue were confirmed to express high levels of TGF-β1 and miR-21 compared with normal skin fibroblasts. Expression of TGF-β1 and miR-21 was positively correlated in fibroblasts. In addition, cells transfected with TGF-β1 inhibitor or miR-21 inhibitor showed significant increases in FasL protein levels and number of apoptotic cells compared with control cells, whereas cell growth and migration significantly decreased. The opposite results could also be confirmed when TGF-β1 was upregulated in normal skin fibroblasts. TGF-β1 could effectively influence cell proliferation, apoptosis, and migration via its control of miR-21. These findings also identify a novel mechanism of interaction between TGF-β1 and miR-21 in the regulation of FasL protein, which is involved in keloid formation.

Up-regulation of Fas Ligand Expression by Sirtuin 1 in both Flow-restricted Vessels and Serum-stimulated Vascular Smooth Muscle Cells

To study the role of sirtuin 1 (SIRT1) in Fas ligand (FasL) expression regulation during vascular lesion formation and to elucidate the potential mechanisms. SIRT1 and FasL protein levels were detected by Western blotting in either mouse arteries extract or the whole rat aortic vascular smooth muscle cell (VSMC) lysate. Smooth muscle cell (SMC)-specific human SIRT1 transgenic (Tg) C57BL/6 mice and their littermate wild-type (WT) controls underwent complete carotid artery ligation (ligation groups) or the ligation-excluded operation (sham groups). The carotid arteries were collected 1 day after operation. Reverse transcription-polymerase chain reaction was performed to detect the mRNA levels of SIRT1 and FasL. Luciferase reporter assays were performed to detect the effect of WT-SIRT1, a dominant-negative form of SIRT1 (SIRT1H363Y), and GATA-6 on the promoter activity of FasL. Flow cytometry assay was applied to measure the hypodiploid DNA content of VSMC so as to monitor cellular apoptosis. SIRT1 was expressed in both rat aortic VSMCs and mouse arteries. Forced SIRT1 expression increased FasL expression both in injured mouse carotid arteries 1 day after ligation (P<0.001) and VSMCs treated with serum (P<0.05 at the transcriptional level, P<0.001 at the protein level). No notable apoptosis was observed. Furthermore, transcription factor GATA-6 increased the promoter activity of FasL (P<0.001). The induction of FasL promoter activity by GATA-6 was enhanced by WT-SIRT1 (P<0.001), while SIRT1H363Y significantly relieved the enhancing effect of WT-SIRT1 on GATA-6 (P<0.001). Overexpression of SIRT1 up-regulates FasL expression in both flow-restricted mouse carotid arteries and serum-stimulated VSMCs. The transcription factor GATA-6 participates in the transcriptional regulation of FasL expression by SIRT1.

Dysregulation of the Fas/FasL system in mononuclear cells recovered from peritoneal fluid of women with endometriosis

In endometriosis, regurgitating endometrial cells fail to undergo apoptosis and implant themselves outside the uterus, particularly in the peritoneum. We studied Fas and FasL behaviour by evaluating the percentages of mFas and mFasL-bearing mononuclear cells from peritoneal fluid, the level of Fas and FasL gene expression at both mRNA and protein levels in the same cells, and the sFas and sFasL values in peritoneal fluid of 80 endometriotic women, at four stages of disease severity. We found no variation in percentage of mFas-bearing mononuclear cells; high and unchanging levels of Fas mRNA and protein, and high and invariable sFas values. Overproduction of sFas antagonises mFas function and plays a role as a decoy in the peritoneal fluid. The mFasL-bearing mononuclear cells and protein levels decreased from the minimal to the severe stage of disease. In contrast to FasL protein, FasL mRNA was overexpressed throughout the course of the disease. sFasL values were high and increased as the disease worsened. Our results showed a non-linear ratio between FasL mRNA and FasL protein levels. Abnormally elevated FasL mRNA may be due to dysregulation in several mechanisms controlling mRNA turnover. The high level of sFasL would be expected to down-regulate FasL activity and compete with the membrane form for mFas binding. As a consequence, mFas-bearing mononuclear cells may be unable to kill and in turn, may themselves become targets for killing by FasL-expressing endometriotic cells.

Hexachlorobenzene induces deregulation of cellular growth in rat liver

Hexachlorobenzene (HCB) is an organochlorine pesticide widely distributed in the biosphere. The aim of the present study was to investigate the effect of HCB on the homeostasis of liver cell growth, analyzing parameters of cell proliferation and apoptosis, in HCB (0.1, 1, 10 and 100mg/kg body weight)-treated rats, during 4 weeks. Cell proliferation and ERK1/2 phosphorylation, associated with survival mechanisms, were increased at HCB 100mg/kg. The pesticide increased the number of apoptotic cells, and the activation of caspase-3, -9 and -8, in a dose-dependent manner, suggesting that HCB-induced apoptosis is mediated by caspases. Increased Fas and FasL protein levels indicate that the death receptor pathway is also involved. This process is associated with decreased Bid, and increased cytosolic cytochrome c protein levels. Transforming growth factor-beta1 (TGF-β1) intervenes in apoptotic and/or proliferative processes in hepatocytes. TGF-β1 cDNA and protein levels are dose-dependently increased, suggesting that this cytokine might be involved in HCB-induced dysregulation of cell proliferation and apoptosis. In conclusion, this study reports for the first time that HCB induces loss of the homeostatic balance between cell growth and cell death in rat liver. Induced apoptosis occurs by mechanisms involving signals emanating from death receptors, and the mitochondrial pathway.

Cyclic-stretch induces the apoptosis of myoblast by activation of Caspase-3 protease in a magnitude-dependent manner

Although many studies have been performed investigating the effects of mechanical stress on the generation and differentiation of myoblasts, little is known about the effects of different magnitudes of mechanical stretch on apoptosis in these cells. The aim of this study was to investigate the effects of different magnitudes of cyclic stretch on apoptosis levels in myoblasts and to investigate the possible mechanisms involved. Myoblasts were cultured on flexible membranes and subjected to cyclic strain stress in a magnitude-dependent manner (6%, 12% or 20% surface elongation). Apoptosis rates were evaluated using flow cytometry, transmission electron microscopy, and caspase assays. Fas/FasL expression was determined by Western blot. The application of different magnitudes of cyclic-stretch-induced a magnitude-dependent increase in apoptosis and caspase-3 activity in cultured myoblasts. Furthermore, inhibition of caspase-3 prevented stretch-induced apoptosis in myoblasts but did not change Fas and FasL protein levels. These data indicate that cyclic stretch induces a magnitude- and caspase-3-dependent increase of apoptosis in cultured myoblasts in vitro. Mechanical forces induced activation of caspase-3 through signalling pathways independent of the Fas/FasL system. These results suggest the existence of a novel mechanism for the regulation of myoblast apoptosis by cyclic stretch.

Immune evasion of human lung carcinoma cell A549 suppressed by human lymphoma cell Jurkat via Fas/FasL pathway

背景与目的当机体发生肿瘤时，肿瘤细胞可以多种方式逃避免疫系统的监控而分裂生长，这就是肿瘤的免疫逃逸。研究表明，肿瘤细胞自身凋亡减少以及机体免疫细胞凋亡增加是肿瘤免疫逃逸的重要机制。Fas/FasL系统是介导凋亡的重要分子体系，本研究旨在通过观察人淋巴瘤细胞株Jurkat诱导人肺癌细胞株A549凋亡过程中凋亡信号分子Fas、FasL及Caspase-8表达的改变，从而探讨Fas/FasL途径在肺癌细胞免疫逃逸中的作用。方法人肺癌细胞株A549与人淋巴瘤细胞株Jurkat以不同比例分别进行共同培养，采用台盼蓝拒染法检测两种细胞存活率；流式细胞术（flow cytometry, FCM）检测两种细胞的凋亡率；Western blot技术检测A549细胞中Fas、FasL及Caspase-8蛋白表达水平。结果随着Jurkat/A549细胞比例逐渐增大，A549细胞的凋亡率明显增加，Jurkat细胞凋亡率显著减少；同时A549细胞中Fas及Caspase-8蛋白的表达水平明显上调，而对FasL蛋白的表达水平无明显影响。结论Jurkat细胞可能通过Fas/FasL途径介导了人肺癌细胞株A549的凋亡，从而抑制了A549细胞的免疫逃逸。

Localization of apoptosis proteins and lymphocyte subsets in chronic rejection of human liver allograft

AbstractIn chronic liver rejection lymphocyte mediated processes lead to chronic inflammation, necrosis and repair mechanisms. The aim of the present study was to investigate the expression of apoptosis related proteins (FAS/APO-1, FAS-L, Bcl-2, Bax, TNF-α, and INF-γ). ApopTag reaction and immunohistochemistry were performed on liver samples of chronically rejected allografts and compared with normal donor livers. In chronic rejection, apoptosis was detected in pericentral hepatocytes and in the biliary epithelium. Bcl-2 was strongly expressed on lymphocytes around the bile ducts, but not on the biliary epithelium itself. Bax, FAS, TNF-αand INF-γwere present in pericentral areas. T-cells showed up around bile ducts, whereas macrophages around pericentral areas. In pericentral areas apoptosis seems to be fostered through TNF-αand INF-γand by the lack of Bcl-2. Based on these results both downregulation and upregulation of apoptotic proteins can be observed in chronic liver allograft rejection: FAS is upregulated in biliary epithelium and zone 2, protein levels of FASL remain unchanged, BAX is upregulated in zone 3, BCL2 is downregulated in both biliary epithelium and zone 1 and both TNFa and IFN are upregulated in zone 3. Our results suggest that the balance between pro- and antiapoptotic patterns was shifted to the proapoptotic side, mainly in the centrilobular area of the hepatic lobule, and in the bile ducts. According to these findings in chronic rejection the predictive sites of apoptosis are the biliary epithelium and the pericentral areas.

Inhibition of NF-kappa B can enhance Fas-mediated apoptosis in leukemia cell line HL-60

This study explored the effects of nuclear factor-kappa B (NF-κB) inhibitor Bay 11-7082 on Fas/FasL system and Fas-mediated apoptosis in cell line HL-60 cells. The mRNA and protein levels of Fas, FasL, and X-linked inhibitor of apoptosis protein (XIAP) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FCM); the level of sFasL was evaluated by enzyme-linked immunosorbent assay (ELISA); and apoptosis was determined by FCM. After treatment with Bay 11-7082, the mRNA and protein levels of FasL and XIAP in HL-60 cells were significantly lower than in the controls (P<0.05), but the mRNA and protein levels of Fas and sFasL did not change significantly (P>0.05). Apoptotic rate of HL-60 cells treated with Bay 11-7082 was significantly higher than in the controls (P<0.05). Therefore, we conclude that Bay 11-7082 can enhance Fas-mediated apoptosis in HL-60 cells by downregulating FasL and XIAP levels.

Gallic Acid Induces Apoptosis of Lung Fibroblasts via a Reactive Oxygen Species-Dependent Ataxia Telangiectasia Mutated-p53 Activation Pathway

Idiopathic pulmonary fibrosis (IPF) is a progressive chronic disorder characterized by the activation of fibroblasts and the overproduction of extracellular matrix. Fibroblast resistance to apoptosis leads to increased fibrosis. Targeting fibroblasts with apoptotic agents represents a major therapeutic intervention for debilitating IPF. Gallic acid (3,4,5-trihydroxybenzoic acid), a naturally occurring plant phenol, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. However, the effects of gallic acid on lung fibroblasts have not been investigated. The aim of the present study is to determine the effects of gallic acid on primary cultured mouse fibroblasts. Our results showed that gallic acid induces the apoptotic death of fibroblasts via both intrinsic and extrinsic apoptotic pathways by the elevation of PUMA, Fas, and FasL protein levels. Moreover, intracellular reactive oxygen species (ROS) generation and 8-hydroxy-2'-deoxyguanosine production were observed in gallic acid-stimulated fibroblasts. Mechanistic studies showed that gallic acid induces early phosphorylation of p53(Ser18) and histone 2AX(Ser139) (H2AX) via ataxia telangiectasia mutated (ATM) activation in response to ROS-provoked DNA damage. When mouse lung fibroblasts were treated with caffeine, an ATM kinase inhibitor, the levels of p53, phosphorylated p53(Ser18), and cell death induced by gallic acid were significantly attenuated. Additionally, pretreatment with antioxidants drastically inhibited the gallic acid-induced 8-hydroxy-2'-deoxyguanosine (8-OH-dG) formation and phosphorylation of p53(Ser18) and ATM(Ser1981), as well as apoptosis. Our results provide the first evidence of the activation of ROS-dependent ATM/p53 signaling as a critical mechanism of gallic acid-induced cell death in primary cultured mouse lung fibroblasts.

Recombinant tissue metalloproteinase inhibitor-3 protein induces apoptosis of murine osteoblast MC3T3-E1

Tissue inhibitor of metalloproteinases (TIMPs) plays an essential role in the regulation of bone metabolism. Here we report that recombinant tissue metalloproteinase inhibitor-3 (TIMP-3) protein induces the apoptosis of MC3T3-E1 osteoblasts. Cell apoptosis was detected by sandwich-enzyme-immunoassay. Fas and Fasl protein levels were determined by Western blot analysis. The enzyme substrate was used to assess the activation of caspase-3 and caspase-8. The phosphorylation of JNK, p38 and ERK1/2 was examined by Western blot analysis. The ELISA suggested that TIMP-3 promoted MC3T3-E1 cell apoptosis. TIMP-3 treatment induced the expression of Fas and Fasl proteins, and the activation of caspase-8 and caspase-3. TIMP-3 treatment induced p38 and ERK phosphorylation. SB203580 and PD98059, the inhibitor of p38 and ERK, respectively, abolished the TIMP-3 effect on osteoblast apoptosis. In conclusion, the signal pathway through which TIMP-3 induces MC3T3-E1 cell apoptosis, mediated by Fas and involves the p38 and ERK signal transduction pathways.

Fas/FasL-mediated apoptosis in the arcuate nucleus and medial preoptic area of male ArKO mice is ameliorated by selective estrogen receptor alpha and estrogen receptor beta agonist treatment, respectively

The aromatase (ArKO) knockout mouse is estrogen deficient. Our previous analysis revealed apoptosis of dopaminergic neurons in the arcuate nucleus (Arc) and medial preoptic area (MPO) of 1-year-old male ArKO mice. We sought to determine which estrogen receptor (ER) is involved in the anti-apoptotic action of estrogen. Male ArKO (9.5-month-old) mice were treated with 16α-LE 2 (ERα-specific agonist) or 8β-VE 2 (ERβ-specific agonist). Daily injections (6 weeks) with 16α-LE 2 prevented dopaminergic cell death in the Arc of male ArKO mice, with no significant effect of 8β-VE 2 treatment. In contrast, 8β-VE 2 prevented dopaminergic cell death in the MPO, while 16α-LE 2 had no significant effect. Concomitant decreases in Fas and FasL protein levels were found upon 16α-LE 2 and 8β-VE 2 treatment in the Arc and MPO, respectively. Our results indicate that anti-apoptotic effects of estrogen are ER mediated, and the specific ER subtype involved in regulating apoptosis depends on the particular brain nucleus in question.

Hyperoxia-induced apoptosis and Fas/FasL expression in lung epithelial cells

Alveolar epithelial apoptosis is an important feature of hyperoxia-induced lung injury in vivo and has been described in the early stages of bronchopulmonary dysplasia (chronic lung disease of preterm newborn). Molecular regulation of hyperoxia-induced alveolar epithelial cell death remains incompletely understood. In view of functional involvement of Fas/FasL system in physiological postcanalicular type II cell apoptosis, we speculated this system may also be a critical regulator of hyperoxia-induced apoptosis. The aim of this study was to investigate the effects of hyperoxia on apoptosis and apoptotic gene expression in alveolar epithelial cells. Apoptosis was studied by TUNEL, electron microscopy, DNA size analysis, and caspase assays. Fas/FasL expression was determined by Western blot analysis and RPA. We determined that in MLE-12 cells exposed to hyperoxia, caspase-mediated apoptosis was the first morphologically and biochemically recognizable mode of cell death, followed by necrosis of residual adherent cells. The apoptotic stage was associated with a threefold upregulation of Fas mRNA and protein expression and increased susceptibility to direct Fas receptor activation, concomitant with a threefold increase of FasL protein levels. Fas gene silencing by siRNAs significantly reduced hyperoxia-induced apoptosis. In murine fetal type II cells, hyperoxia similarly induced markedly increased Fas/FasL protein expression, confirming validity of results obtained in transformed MLE-12 cells. Our findings implicate the Fas/FasL system as an important regulator of hyperoxia-induced type II cell apoptosis. Elucidation of regulation of hyperoxia-induced lung apoptosis may lead to alternative therapeutic strategies for perinatal or adult pulmonary diseases characterized by dysregulated type II cell apoptosis.

Fas/FasL-mediated apoptosis in perinatal murine lungs.

Postcanalicular lung development is characterized by a time-specific increase in alveolar epithelial type II cell apoptosis. We have previously demonstrated that, in fetal rabbits, developmental type II cell apoptosis coincides with transient upregulation of the cell death regulator Fas ligand (FasL). The aims of this study were 1) to determine the spatiotemporal patterns of pulmonary apoptosis and Fas/FasL gene expression in the murine model [embryonic day 17 (E17) through postnatal day 5 (P5)], and 2) to investigate the functional involvement of the Fas/FasL system by determining the effect of Fas activation and inhibition on perinatal pulmonary apoptosis. The apoptotic activity of alveolar epithelial type II cells, determined by combined TUNEL labeling and anti-surfactant protein B immunohistochemistry, showed a dramatic increase during the perinatal transition (type II cell apoptotic index <0.1% at E17, 1.5% at P1-P3, and 0.3% at P5). This timing of enhanced type II cell apoptosis coincided with a robust 14-fold increase in Fas mRNA and protein levels and a threefold increase in FasL protein levels; both Fas and FasL immunolocalized to type II and bronchial epithelial cells. In vitro and in vivo exposure of fetal and postnatal murine type II cells to anti-Fas antibody induced a fourfold increase in apoptotic activity that was prevented by administration of a broad-spectrum caspase inhibitor; the pulmonary apoptotic activity of Fas-deficient lpr mice remained unchanged. Conversely, administration of a caspase inhibitor to newborn mice (P1) resulted in marked diminution of pulmonary apoptotic activity. These combined findings strongly implicate the Fas/FasL system as a critical regulator of perinatal type II cell apoptosis. The developmental time dependence of apoptosis-related events in the murine model should facilitate investigations of the regulation of perinatal pulmonary apoptotic gene expression.

Mesangial cell Fas ligand: upregulation in human lupus nephritis and NF-kappaB-mediated expression in cultured human mesangial cells.

Fas ligand (FasL) is a well-known death factor; however, the role of FasL in the regulation of human glomerulonephritis remains unclear. We investigated the renal expression and localization of FasL in various forms of human glomerulonephritis by immunohistochemistry, utilizing confocal laser scanning microscopy. We further evaluated cytokine-induced FasL expression via nuclear factor (NF)kappaB in cultured human mesangial cells (HMC). The level of soluble FasL was measured by a specific enzyme-linked immunosorbent assay (ELISA). The frequency of glomerular FasL-positive cases was higher in lupus nephritis (37.9%) as compared with other forms of glomerulonephritis (8.7%). The glomerular FasL score in proliferative lupus nephritis was significantly higher than that in nonproliferative forms. Patients with a high apoptosis score, severe microhematuria, proteinuria, or decreased renal function had a high FasL score. Double immunolabelling demonstrated that the most prevalent phenotypes of FasL-positive cells were mesangial cells. In cultured HMC, interleukin (IL)1beta, lipopolysaccharide (LPS), or gamma interferon (IFN) upregulated membrane-bound FasL. IL1beta significantly, and LPS or gammaIFN weakly activated NFkappaB, but none of these agents activated NFkappaB/Rel-related nuclear factor of activated T cells (NFATc) or IFN regulatory factor-1. IL1beta-mediated NFkappaB was completely inhibited in the presence of lactacystin, a potent inhibitor of NFkappaB. Lactacystin-mediated inhibition of NFkappaB reduced FasL protein levels. Matrix metalloproteinase (MMP)-7, but not other MMPs (1, 2, 3, 8, or 9), significantly sensitized HMC to release soluble FasL after IL1beta stimulation. The results suggest that: (1) upregulation of mesangial FasL may contribute to the glomerular inflammation in proliferative lupus nephritis in vivo; (2) proinflammatory cytokines, in particular IL1beta, produced in nephritis can upregulate FasL via the transcription factor NFkappaB in HMC; and (3) MMP-7-mediated release of soluble FasL could control the mesangial inflammation.

Nitric oxide and cyclic guanosine monophosphate stimulate apoptosis via activation of the Fas-FasL pathway.

Inappropriately exaggerated response of pulmonary vascular cells to inflammatory mediators may be one mechanism that leads to acute (or adult) respiratory distress syndrome. Nitric oxide (NO) is induced following such exaggerated responses and may have a variety of biological effects, including induction of apoptosis. The mechanism by which NO causes apoptosis is unknown; however, Fas (CD95) and Fas ligand (FasL) (CD95L) have been implicated. We hypothesized that NO-induced apoptosis in pulmonary vascular smooth muscle cells is mediated through a Fas-FasL pathway. Cultured human and rat pulmonary artery smooth muscle cells (PASMCs) were exposed to soluble FasL (0-5 ng/ml), the NO donor S(G)-nitroso-N-acetyl pencillamine (SNAP) (0-50 microg/ml), and/or anti-FasL (0-100 microg/ml) for 12 h. Apoptosis was measured using in situ DNA nick end labeling and flow cytometry. Changes in Fas and FasL protein levels were assessed via Western blot analysis. Messenger RNA (mRNA) abundance of apoptosis-related genes was determined using a ribonuclease protection assay. Rat PASMCs exposed to FasL show a dose-dependent increase in apoptosis. Human PASMCs are less responsive to FasL. Addition of anti-FasL to rat PASMCs treated with 10(-5) M SNAP decreases apoptosis levels compared to SNAP treated alone. FasL and Fas receptor proteins are increased in response to 10(-3) to 10(-4) M SNAP or 10(-6) M 8-bromo-cyclic guanosine monophosphate (cGMP). The mRNA abundance of Fas, FasL, and other apoptosis-related genes is increased in response to 10(-6) M 8-bromo-cGMP but not 8-bromo-cyclic adenosine monophosphate. Nitric oxide-induced apoptosis in rat and human PASMCs is mediated, at least in part, through the Fas-FasL pathway, with cGMP increasing the expression of Fas and FasL.

Fas ligand expression coincides with alveolar cell apoptosis in late-gestation fetal lung development.

Apoptosis plays a central role in the cellular remodeling of the developing lung. We determined the spatiotemporal patterns of the cell death regulators Fas and Fas ligand (FasL) during rabbit lung development and correlated their expression with pulmonary and type II cell apoptosis. Fetal rabbit lungs (25-31 days gestation) were assayed for apoptotic activity by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and DNA size analysis. Fas and FasL expression were analyzed by RT-PCR, immunoblot, and immunohistochemistry. Type II cell apoptosis increased significantly on gestational day 28; the type II cell apoptotic index increased from 0.54 +/- 0.34% on gestational day 27 to 3.34 +/- 1.24% on day 28, P < 0.01 (ANOVA). This corresponded with the transition from the canalicular to the terminal sac stage of development. The day 28 rise in epithelial apoptosis was synchronous with a robust if transient 20-fold increase in FasL mRNA and a threefold increase in FasL protein levels. In contrast, Fas mRNA levels remained constant, suggestive of constitutive expression. Fas and FasL proteins were immunolocalized to alveolar type II cells and bronchiolar Clara cells. The correlation of this highly specific pattern of FasL expression with alveolar epithelial apoptosis and remodeling implicates the Fas/FasL system as a potentially important regulatory pathway in the control of postcanalicular alveolar cytodifferentiation.