Age-related declines in α-Klotho drive progenitor cell mitochondrial dysfunction and impaired muscle regeneration.

While young muscle is capable of restoring the original architecture of damaged myofibers, aged muscle displays a markedly reduced regeneration. We show that expression of the "anti-aging" protein, α-Klotho, is up-regulated within young injured muscle as a result of transient Klotho promoter demethylation. However, epigenetic control of the Klotho promoter is lost with aging. Genetic inhibition of α-Klotho in vivo disrupted muscle progenitor cell (MPC) lineage progression and impaired myofiber regeneration, revealing a critical role for α-Klotho in the regenerative cascade. Genetic silencing of Klotho in young MPCs drove mitochondrial DNA (mtDNA) damage and decreased cellular bioenergetics. Conversely, supplementation with α-Klotho restored mtDNA integrity and bioenergetics of aged MPCs to youthful levels in vitro and enhanced functional regeneration of aged muscle in vivo in a temporally-dependent manner. These studies identify a role for α-Klotho in the regulation of MPC mitochondrial function and implicate α-Klotho declines as a driver of impaired muscle regeneration with age.

Nickel requires hypoxia-inducible factor-1 alpha, not redox signaling, to induce plasminogen activator inhibitor-1.

Human epidemiological and animal studies have associated inhalation of nickel dusts with an increased incidence of pulmonary fibrosis. At the cellular level, particulate nickel subsulfide inhibits fibrinolysis by transcriptionally inducing expression of plasminogen activator inhibitor (PAI)-1, an inhibitor of the urokinase-type plasminogen activator. Because nickel is known to mimic hypoxia, the present study examined whether nickel transcriptionally activates PAI-1 through the hypoxia-inducible factor (HIF)-1 alpha signaling pathway. The involvement of the NADPH oxidase complex, reactive oxygen species, and kinases in mediating nickel-induced HIF-1 alpha signaling was also investigated. Addition of nickel to BEAS-2B human airway epithelial cells increased HIF-1 alpha protein levels and elevated PAI-1 mRNA levels. Pretreatment of cells with the extracellular signal-regulated kinase inhibitor U-0126 partially blocked HIF-1 alpha protein and PAI-1 mRNA levels induced by nickel, whereas antioxidants and NADPH oxidase inhibitors had no effect. Pretreating cells with antisense, but not sense, oligonucleotides to HIF-1 alpha mRNA abolished nickel-stimulated increases in PAI-1 mRNA. These data indicate that signaling through extracellular signal-regulated kinase and HIF-1 alpha is required for nickel-induced transcriptional activation of PAI-1.

AP-1-dependent induction of plasminogen activator inhibitor-1 by nickel does not require reactive oxygen.

Inhalation of nickel dust has been associated with an increased incidence of pulmonary fibrosis. Nickel may promote fibrosis by transcriptionally activating plasminogen activator inhibitor (PAI)-1 and inhibiting fibrinolysis. The current studies examined whether nickel stimulated the PAI-1 promoter though an oxidant-sensitive activator protein (AP)-1 signaling pathway. Addition of nickel to BEAS-2B human airway epithelial cells stimulated intracellular oxidation, induced c-Jun and c-Fos mRNA levels, increased phospho- and total c-Jun protein levels, and elevated PAI-1 mRNA levels over a 24-h time course. Pretreatment of the cells with antioxidants did not affect increased c-Jun protein or PAI-1 mRNA levels. Expression of the dominant negative inhibitor of AP-1, TAM67, prevented nickel-stimulated AP-1 DNA binding, AP-1-luciferase reporter construct activity, and PAI-1 mRNA levels. Overexpression of c-Jun, however, failed to induce the AP-1 luciferase reporter construct or PAI-1 mRNA levels. These data indicated that nickel activated AP-1 through an oxidant-independent pathway and that basal AP-1 is necessary for nickel-induced expression of PAI-1.

Identification and role of thiols in Toxoplasma gondii egress.

The nucleoside triphosphate hydrolase of Toxoplasma gondii is a potent apyrase that is secreted into the parasitophorous vacuole where it appears to be essentially inactive in an oxidized form. Recent evidence shows that nucleoside triphosphate hydrolase can be activated by dithiothreitol in vivo. On reduction of the enzyme, there is a rapid depletion of host cell ATP. Previous results also demonstrate a dithiothreitol induced egress of parasites from the host cell with a concurrent Ca2+ flux, postulated to be a consequence of the release of ATP-dependent Ca2+ stores within the tubulovesicular network of the parasitophorous vacuole. Reduction of the nucleoside triphosphate hydrolase appears crucial for its activation; however, the exact mechanism of reduction/activation has not been determined. Using a variety of techniques, we show here that glutathione promoters activate a Ca2+ flux and decrease ATP levels in infected human fibroblasts. We further show the in vitro activation of nucleoside triphosphate hydrolase by endogenous reducing agents, one of which we postulate might be secreted into the PV by T. gondii. Our findings suggest that the reduction of the parasite nucleoside triphosphate hydrolase, and ultimately parasite egress, is under the control of the parasites themselves.

Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells.

Low-level arsenite treatment of porcine aortic endothelial cells (PAEC) stimulated superoxide accumulation that was attenuated by inhibitors of NAD(P)H oxidase. To demonstrate whether arsenite stimulated NADPH oxidase, intact PAEC were treated with arsenite for up to 2 h and membrane fractions were prepared to measure NADPH oxidase activity. Arsenite (5 microM) stimulated a twofold increase in activity by 1 h, which was inhibited by the oxidase inhibitor diphenyleneiodonium chloride. Direct treatment of isolated membranes with arsenite had no effect. Analysis of NADPH oxidase components revealed that p67(phox) localized exclusively to membranes of both control and treated cells. In contrast, cytosolic Rac1 translocated to the membrane fractions of cells treated with arsenite or angiotensin II but not with tumor necrosis factor. Immunodepletion of p67(phox) blocked oxidase activity stimulated by all three compounds. However, depleting Rac1 inhibited responses only to arsenite and angiotensin II. These data demonstrate that stimulus-specific activation of NADPH oxidase in endothelial cells was the source of reactive oxygen in endothelial cells after noncytotoxic arsenite exposure.

Integration of the NF-kappaB and mitogen-activated protein kinase/AP-1 pathways at the collagenase-1 promoter: divergence of IL-1 and TNF-dependent signal transduction in rabbit primary synovial fibroblasts.

Collagenase-1 (MMP-1) is a protease that is expressed by stromal cells and that is involved in remodeling of the extracellular matrix. IL-1 and TNF-alpha enhance collagenase secretion by stromal cells, and chronic exposure of cells to these cytokines can contribute to connective tissue disease. In this study, we show that the NF-kappaB pathway is required for activation of collagenase-1 transcription in rabbit primary synovial fibroblasts (RSF). Although both IL-1 and TNF activate NF-kappaB in these cells, only IL-1 induces collagenase-1 transcription. We have reported previously that NF-kappaB and AP-1 cooperate to mediate IL-1-induced MMP-1 transcription. Here, we show that IL-1 is superior to TNF at inducing c-Jun synthesis, phosphorylation and binding activity in RSF. Similarly, IL-1 is more effective at activating the mitogen-activated protein kinases (MAPK), including the extracellular signal-regulated kinases (ERK), which are required for IL-1-induced MMP-1 transcription. Thus stimulation of the ERK and AP-1 pathways is an essential component of MMP-1 transcriptional activation, which is deficient in TNF-treated cells. These studies demonstrate cooperation between the MAPK and NF-kappaB signaling pathways for IL-1-dependent collagenase-1 transcription, and they define a dichotomy of IL-1- and TNF-elicited signaling that is relevant to cytokine-mediated connective tissue disease.

Nickel-induced plasminogen activator inhibitor-1 expression inhibits the fibrinolytic activity of human airway epithelial cells.

One cause of debilitating pulmonary fibrosis is inhalation of insoluble metals. Human epidemiological and animal studies have associated inhalation of nickel dusts with increased incidence of pulmonary fibrosis. However, specific mechanisms for nickel-induced pulmonary fibrosis have yet to be elucidated. The current studies examine the hypothesis that particulate nickel promotes pulmonary fibrosis by inhibiting the fibrinolytic cascade. Since the urokinase-type plasminogen activator (uPA) initiates this cascade, this hypothesis was tested by investigating the effects of noncytotoxic levels of nickel subsulfide on the balance of uPA expression relative to expression of its inhibitor, PAI-1, in cultured human bronchial epithelial cells (BEAS-2B). Exposure to the metal decreased secreted uPA protein levels and activity without affecting uPA mRNA levels. In contrast, these same exposures stimulated transcription of PAI-1, causing prolonged increases in both mRNA and protein levels. Despite partial recovery of uPA protein levels, uPA activity remained depressed for more than 48 h after exposure to nickel due to the continued increase in PAI-1 expression. These data indicate that particulate nickel inhibits the fibrinolytic cascade by increasing the ratio of plasminogen inhibitor to activator. Sustained loss of uPA activity may contribute to nickel-induced pulmonary fibrosis in exposed populations.

Antiplatelet effects of MK-852, a platelet fibrinogen receptor antagonist, in healthy volunteers.

MK-852, a cyclic heptapeptide, is a potent platelet fibrinogen receptor antagonist. When administered to normal healthy male subjects by 1- and 4-hour constant rate intravenous infusions, it provides a generally well-tolerated and reversible means of inhibition of platelet function. At infusion rates of 1 microgram/kg/min for 1 hour and 0.44 microgram/kg/min for 4 hours, respectively, MK-852 extended baseline bleeding time by greater than 2.2-fold and 2.6-fold, inhibited ADP-induced platelet aggregation by 76% and 69%, and inhibited collagen-induced platelet aggregation by 65% and 67%, respectively. The pharmacokinetics of MK-852 include an elimination half-life of approximately 2 hours, total clearance of about 150 ml/min, and volume of distribution of about 18 liters. Examination of the relationship between MK-852 whole-blood concentration in vitro and inhibition of platelet aggregation showed an EC50 of about 55 ng/ml and a Hill coefficient of 1.55. The infusions were generally well tolerated, with no study drug-related changes in blood counts or biochemical profiles.

Arsenic inhibits NF-kappaB-mediated gene transcription by blocking IkappaB kinase activity and IkappaBalpha phosphorylation and degradation.

The inflammatory cytokine, TNF-alpha, induces IL-8 gene transcription via a mechanism involving proteasome-mediated IkappaBalpha degradation and NF-kappaB activation. Here, we investigated whether arsenic, which has been shown to inhibit the ubiquitin-proteasome pathway, could inhibit TNF-alpha-mediated increases in IL-8 expression. Using RT-PCR, we show that the addition of TNF-alpha to human bronchial epithelial (BEAS 2B) or embryonic kidney (HEK293) cells resulted in increased steady-state levels of IL-8 mRNA. This was preceded by a rapid decrease in cellular IkappaBalpha levels, as demonstrated by Western analysis, and an increase in nuclear levels of NF-kappaB, as demonstrated by gel shift analysis. Further demonstrating the activation of NF-kappaB, TNF-alpha induced the transcription of a NF-kappaB-dependent reporter gene. Exposing the cells to 500 microM arsenite, prior to adding TNF-alpha, completely inhibited IkappaBalpha degradation, NF-kappaB translocation, NF-kappaB-dependent gene transcription, and transcription of the endogenous gene for IL-8. In comparison with the proteasome inhibitor MG-132, which does not affect the phosphorylation and ubiquitination of IkappaBalpha, arsenite inhibited the phosphorylation of IkappaBalpha. Furthermore, arsenite directly blocked the activity of IKK, the kinase responsible for IkappaBalpha phosphorylation. These studies demonstrate that high levels of arsenic may inhibit NF-kappaB-mediated gene transcription by specifically blocking IKK activity, thereby limiting the phosphorylation and subsequent degradation of the NF-kappaB inhibitor, IkappaBalpha.

Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor kappaB: differential regulation of collagenase 1 and collagenase 3.

OBJECTIVE: To examine the mechanism of interleukin-1 (IL-1)-induced collagenase 3 (matrix metalloproteinase 13 [MMP-13]) gene expression in cultured chondrocytes for the purpose of better understanding how the gene is induced in these cells, and how it contributes to cartilage degradation in osteoarthritis. METHODS: The transcriptional and posttranscriptional responses of the MMP-13 gene to IL-1 were assessed first. Then, direct inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways and a constitutive repressor of nuclear factor kappaB (NF-kappaB) were used to assess the role of each pathway in IL-1-mediated induction of MMP-13. RESULTS: We found that IL-1 induction of MMP-13 requires p38 activity, c-Jun N-terminal kinase (JNK) activity and NF-kappaB translocation. These results suggest that both NF-kappaB and activator protein 1 transcription factors are necessary for IL-1 induction of MMP-13. We also compared the signaling pathways necessary for IL-1 to stimulate collagenase 1 (MMP-1) in articular chondrocytes and chondrosarcoma cells and found that IL-1 induction of MMP-1 requires different pathways from those required by MMP-13. In chondrosarcoma cells, MMP-1 induction depends on p38 and MEK (an MAPK kinase of the extracellular signal-regulated kinase pathway) and does not require JNK or NF-kappaB. In articular chondrocytes, inhibition of MEK had no effect, while inhibition of p38 gave variable results. CONCLUSION: These studies demonstrate, for the first time, that p38, JNK, and NF-kappaB are required for IL-1 induction of MMP-13. The results also highlight the differential requirements for signaling pathways in the induction of MMP-1 and MMP-13. Additionally, they demonstrate that induction of MMP-1 by IL-1 in chondrocytic cells depends on unique combinations of signaling pathways that are cell type-specific.

Chromium(VI) inhibits the transcriptional activity of nuclear factor-kappaB by decreasing the interaction of p65 with cAMP-responsive element-binding protein-binding protein.

Chromium(VI) regulation of gene expression has been attributed to the generation of reactive chromium and oxygen species, DNA damage, and alterations in mRNA stability. However, the effects of Cr(VI) on signal transduction leading to gene expression are not resolved. Therefore, this study investigated the effects of Cr(VI) on basal and tumor necrosis factor-alpha (TNF-alpha)-induced transcriptional competence of nuclear factor-kappaB (NF-kappaB) in A549 human lung carcinoma cells. Pretreatment of A549 cells with nontoxic levels of Cr(VI) inhibited TNF-alpha-stimulated expression of the endogenous gene for interleukin-8 and of an NF-kappaB-driven luciferase gene construct, but not expression of urokinase, a gene with a more complex promoter. Chromium did not inhibit TNF-alpha-stimulated IkappaBalpha degradation or translocation of NF-kappaB-binding proteins to the nucleus. However, Cr(VI) pretreatments prevented TNF-alpha-stimulated interactions between the p65 subunit of NF-kappaB and the transcriptional cofactor cAMP-responsive element-binding protein-binding protein (CBP). This inhibition was not the result of an effect of chromium on the protein kinase A catalytic activity required for p65/CBP interactions. In contrast, Cr(VI) caused concentration-dependent increases in c-Jun/CBP interactions. These data indicate that nontoxic levels of hexavalent chromium selectively inhibit NF-kappaB transcriptional competence by inhibiting interactions with coactivators of transcription rather than DNA binding.

Inhibition of protein synthesis by chromium(VI) differentially affects expression of urokinase and its receptor in human type II pneumocytes.

Exposure to chromium(VI) increases the incidence of cancer, respiratory distress, and pulmonary fibrosis. The latter is a pathological disorder characterized by decreased urokinase-type plasminogen activator (uPA) activity and fibrinolysis. In this study, treatment of alveolar type II cells (A549) with 1 to 5 microM chromium(VI) for 4 and 12 h decreased both the specific activity and the amount of uPA protein. Chromium reduced uPA protein levels by inhibiting protein synthesis and had no effect on uPA mRNA levels or the rate of uPA protein degradation. In contrast, both mRNA and protein levels for the uPA receptor (uPAR) were increased by treatment with concentrations of chromium(VI) that did not completely inhibit protein synthesis. The chromium-induced increase in uPAR resulted from increased message stability. These data indicate that chromium has differential effects on expression of the proteins in the pulmonary fibrinolytic cascade. The net loss of uPA activity may promote fibrosis following inhalation of chromium(VI).

Low levels of arsenic trioxide stimulate proliferative signals in primary vascular cells without activating stress effector pathways.

Chronic human exposure to low levels of inorganic arsenic increases the incidence of vascular diseases and specific cancers. Exposure of endothelial cells to environmentally relevant concentrations of arsenic trioxide (arsenite) induces oxidant formation, activates the transcription factor NF-kappaB, and increases DNA synthesis (Barchowsky et al., Free Radic. Biol. Med. 21, 783-790, 1996). We show, in the current study, that arsenite induces concentration-dependent cell proliferation or death in primary porcine aortic endothelial cells. Low concentrations caused cell proliferation and were associated with increased superoxide and H(2)O(2) accumulation, cSrc activity, H(2)O(2)-dependent tyrosine phosphorylation, and NF-kappaB-dependent transcription. These concentrations were insufficient to activate MAP kinases. However, the MAP kinases, extracellular signal-regulated kinase and p38, were activated in response to levels of arsenite that caused cell death. These data suggest that arsenite-induced oxidant accumulation and subsequent activation of tyrosine phosphorylation represent a MAPK-independent pathway for phenotypic change and proliferation in vascular cells.

Stimulation of reactive oxygen, but not reactive nitrogen species, in vascular endothelial cells exposed to low levels of arsenite.

Elevated levels of arsenite, the trivalent form of arsenic, in drinking water correlates with increased vascular disease and vessel remodeling. Previous studies from this laboratory demonstrated that environmentally relevant concentrations of arsenite caused oxidant-dependent increases in nuclear transcription factor levels in cultured porcine vascular endothelial cells. The current studies characterized the reactive species generated in these cells exposed to levels of arsenite that initiate cell signaling. These exposures did not deplete 5'-triphosphate, nor did they affect basal or bradykinin-stimulated intracellular free Ca2+ levels, indicating that they were not lethal. Electron paramagnetic resonance (EPR) spectroscopy, including spin trapping with carboxy-PTIO (cPTIO), demonstrated that 5 microM or less of arsenite did not increase *NO levels over a 30-min period relative to *NO release stimulated by bradykinin. However, these same levels of arsenite rapidly increased both oxygen consumption and superoxide formation, as measured by EPR oximetry and spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), respectively. Pretreatment of the cells with DPI, apocynin, or superoxide dismutase abolished arsenite-stimulated DMPO-OH adduct formation. Finally arsenite increased extracellular accumulation of H2O2, measured as oxidation of homovanillic acid, with the same time and dose dependence, as seen for superoxide formation. These data suggest that superoxide and H2O2 are the predominant reactive species produced by endothelial cells after arsenite exposures that stimulate cell signaling and activate transcription factors.

Expression and activity of urokinase and its receptor in endothelial and pulmonary epithelial cells exposed to asbestos.

An elongated endothelial cell phenotype, which demonstrated increased ICAM-1-dependent neutrophil adherence, was induced when these cells were exposed to noncytotoxic concentrations of asbestos (Treadwell et al., Toxicol. Appl. Pharmacol. 139, 62-70, 1996). The present study examined mechanisms underlying this phenotypic change by investigating the effects of asbestos on transcription factor activation and expression of urokinase-type plasminogen activator (uPA) and its receptor uPAR. In situ zymography was used to compare the effects of these fibers on the activity of uPA. Cultures incubated with chrysotile or crocidolite asbestos, but not refractory ceramic fiber 1 (RCF-1), demonstrate localized cleavage of plasminogen, which was inhibited by amiloride. Immunocytochemistry showed that chrysotile-stimulated uPA activity was associated with a time-dependent augmentation of uPAR protein levels. RT-PCR analysis was used to investigate molecular mechanisms for these increases. Chrysotile asbestos, but not RCF-1, increased endothelial cell uPA message, relative to changes in beta-actin mRNA. This response to asbestos was not limited to endothelial cells, since both uPA and uPAR mRNA levels increase in human bronchial epithelial BEAS-2B cells exposed to chrysotile fibers. Finally, both types of asbestos, but not RCF-1, increased nuclear levels of nuclear factor-kappaB (NF-kappa B), a transcription factor common to increased expression of ICAM-1 and uPA. These data demonstrate that asbestos caused fiber-specific activation of endothelial and pulmonary epithelial cells, resulting in phenotypes capable of facilitating tissue remodeling.

Novel triterpenoids suppress inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) in mouse macrophages.

We have synthesized more than 80 novel triterpenoids, all derivatives of oleanolic and ursolic acid, as potential anti-inflammatory and chemopreventive agents. These triterpenoids have been tested for their ability to suppress the de novo formation of two enzymes, inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2), using IFN-gamma-stimulated primary mouse macrophages or lipopolysaccharide (LPS)-activated RAW 264.7 macrophages as assay systems. Two synthetic oleananes, 3,12-dioxoolean-1-en-28-oic acid (TP-69) and 3,11-dioxoolean-1,12-dien-28-oic acid (TP-72), were highly active inhibitors of de novo formation of both iNOS and COX-2. Both TP-69 and TP-72 blocked the increase in iNOS or COX-2 mRNA induced by IFN-gamma or LPS. In addition, TP-72 suppressed NF-KB activation in primary macrophages treated with the combination of IFN-gamma and LPS or IFN-gamma and tumor necrosis factor. The 3-alpha(axial)-epimer of ursolic acid suppressed de novo formation of COX-2, in contrast to naturally occurring 3-beta(equatorial)-ursolic acid. Inhibitory effects of TP-69 or TP-72 on iNOS formation were not blocked by the glucocorticoid receptor antagonist RU-486, indicating that these triterpenoids do not act through the glucocorticoid receptor, nor does TP-72 act as an iNOS or COX-2 enzyme inhibitor when added to RAW cells in which synthesis of these two enzymes in response to LPS has already been induced. It may be possible to develop triterpenoids as useful agents for chemoprevention of cancer or other chronic diseases with an inflammatory component.

Inhibition of NF-kappa B binding to DNA by chromium, cadmium, mercury, zinc, and arsenite in vitro: evidence of a thiol mechanism.

NF-kappa B binding to DNA in the presence of thiol-reactive metals has been explored in vitro. Gel mobility shift assays using total nuclear extracts isolated from tumor necrosis factor alpha-treated A549 cells demonstrated dose-dependent inhibition of NF-kappa B binding by chromium, cadmium, mercury, zinc, and arsenite. Maximum inhibition of binding occurred when these metals were preincubated with the nuclear proteins prior to addition of radiolabeled oligonucleotide. The potency of mercury, cadmium, and zinc for inhibiting binding closely correlated to the affinity of these metals for protein thiols. Further addition of dithiothreitol competitively blocked the effects of all of the metals, except chromium(III), on NF-kappa B binding. This study demonstrates mechanisms for metals to inhibit NF-kappa B-DNA binding through interactions with critical protein sulfhydryls.

Cell signaling pathways elicited by asbestos.

In recent years, it has become apparent that minerals can trigger alterations in gene expression by initiating signaling events upstream of gene transactivation. These cascades may be initiated at the cell surface after interaction of minerals with the plasma membrane either through receptorlike mechanisms or integrins. Alternatively, signaling pathways may be stimulated by active oxygen species generated both during phagocytosis of minerals and by redox reactions on the mineral surface. At least two signaling cascades linked to activation of transcription factors, i.e., DNA-binding proteins involved in modulating gene expression and DNA replication, are stimulated after exposure of lung cells to asbestos fibers in vitro. These include nuclear factor kappa B (NF kappa B) and the mitogen-activated protein kinase (MAPK) cascade important in regulation of the transcription factor, activator protein-1 (AP-1). Both NF kappa B and AP-1 bind to specific DNA sequences within the regulatory or promoter regions of genes that are critical to cell proliferation and inflammation. Unraveling the cell signaling cascades initiated by mineral dusts and pharmacologic inhibition of these events may be important for the control and treatment of mineral-associated occupational diseases.

Increased focal adhesion kinase- and urokinase-type plasminogen activator receptor-associated cell signaling in endothelial cells exposed to asbestos.

Exposure of low-passage endothelial cells in culture to nonlethal amounts of asbestos, but not refractory ceramic fiber-1, increases cell motility and gene expression. These changes may be initiated by the fibers mimicking matrix proteins as ligands for receptors on the cell surface. In the present study, 1- to 3-hr exposures of endothelial cells to 5 mg/cm2 of chrysotile asbestos caused marked cell elongation and motility. However, little morphological change was seen when chrysotile was added to cells pretreated with either mannosamine to prevent assembly of glycophosphatidylinositol (GPI)-anchored receptors or with herbimycin A to inhibit tyrosine kinase activity. Affinity purification of GPI-anchored urokinase-type plasminogen activator receptor (uPAR) from chrysotile-exposed cells demonstrated that asbestos altered the profile of proteins and phosphoproteins complexed with this receptor. Tyrosine kinase activities in the complexes were also increased by asbestos. Immunoprecipitations with selective monoclonal antibodies demonstrated that both chrysotile and crocidolite asbestos increase kinase activities associated with p60 Src or p120 focal adhesion kinase (FAK). Further, chrysotile also changed the profile of proteins and phosphoproteins associated with FAK in intact cells. These data suggest that asbestos initiates endothelial cell phenotypic change through interactions with uPAR-containing complexes and that this change is mediated through tyrosine kinase cascades.

Asbestos causes translocation of p65 protein and increases NF-kappa B DNA binding activity in rat lung epithelial and pleural mesothelial cells.

The mechanisms of cell signaling and altered gene expression by asbestos, a potent inflammatory, fibrogenic, and carcinogenic agent, are unclear. Activation of the transcription factor, nuclear factor (NF)-kappa B, is critical in up-regulating the expression of many genes linked to inflammation and proliferation. Inhalation models of crocidolite- and chrysotile-induced inflammation and asbestosis were used to study the localization of p65, a protein subunit of the NF-kappa B transcription factor, in sham control rats and those exposed to asbestos. In addition, we investigated, using electrophoretic mobility shift analysis, whether in vitro exposure of rat lung epithelial cells and rat pleural mesothelial cells to asbestos increased binding of nuclear proteins, including p65, to the NF-kappa B DNA response element. Furthermore, translocation of p65 into the nucleus was determined by confocal microscopy. In comparison with sham animals, striking increases in p65 immunofluorescence were observed in airway epithelial cells of rats at 5 days after inhalation of asbestos. These increases were diminished by 20 days, the time period necessary for development of fibrotic lesions. In contrast, although inter-animal variability was observed, immunoreactivity for p65 was more dramatic in the interstitial compartment of asbestos-exposed rat lungs at both 5 and 20 days. Changes in p65 expression in pleural mesothelial cells exposed to asbestos in inhalation experiments were unremarkable. Exposure to asbestos also caused significant increases in nuclear protein complexes that bind the NF-kappa B consensus DNA sequence in both rat lung epithelial and rat pleural mesothelial cells. Using confocal microscopy, we observed partial nuclear translocation of p65 in rat pleural mesothelial cells exposed to asbestos. This partial response contrasted with the effects of lipopolysaccharide, which caused rapid and complete translocation of p65 from cytoplasm to nucleus. Our studies are the first to show the presence of the NF-kappa B system in lung tissue and evidence of activation in vitro and in vivo after exposure to a potent inflammatory, fibrinogenic, and carcinogenic environmental agent.