The fibrinolytic system: A new target for treatment of depression with psychedelics.

Current understanding of the neurobiology of depression has grown over the past few years beyond the traditional monoamine theory of depression to include chronic stress, inflammation and disrupted synaptic plasticity. Tissue plasminogen activator (tPA) is a key factor that not only promotes fibrinolysis via the activation of plasminogen, but also contributes to regulation of synaptic plasticity and neurogenesis through plasmin-mediated activation of a probrain derived neurotrophic factor (BDNF) to mature BDNF. ProBDNF activation could potentially be supressed by competition with fibrin for plasmin and tPA. High affinity binding of plasmin and tPA to fibrin could result in a decrease of proBDNF activation during brain inflammation leading to fibrosis further perpetuating depressed mood. There is a paucity of data explaining the possible role of the fibrinolytic system or aberrant extravascular fibrin deposition in depression. We propose that within the brain, an imbalance between tPA and urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) and neuroserpin favors the inhibitors, resulting in changes in neurogenesis, synaptic plasticity, and neuroinflammation that result in depressive behavior. Our hypothesis is that peripheral inflammation mediates neuroinflammation, and that cytokines such as tumor necrosis factor alpha (TNF-α) can inhibit the fibrinolytic system by up- regulating PAI-1 and potentially neuroserpin. We propose that the decrement of the activity of tPA and uPA occurs with downregulation of uPA in part involving the binding and clearance from the surface of neural cells of uPA/PAI-1 complexes by the urokinase receptor uPAR. We infer that current antidepressants and ketamine mitigate depressive symptoms by restoring the balance of the fibrinolytic system with increased activity of tPA and uPA with down-regulated intracerebral expression of their inhibitors. We lastly hypothesize that psychedelic 5-ht2a receptor agonists, such as psilocybin, can improve mood through anti- inflammatory and pro-fibrinolytic effects that include blockade of TNF-α activity leading to decreased PAI-1 activity and increased clearance. The process involves disinhibition of tPA and uPA with subsequent increased cleavage of proBDNF which promotes neurogenesis, decreased neuroinflammation, decreased fibrin deposition, normalized glial-neuronal cross-talk, and optimally functioning neuro-circuits involved in mood. We propose that psilocybin can alleviate deleterious changes in the brain caused by chronic stress leading to restoration of homeostatic brain fibrinolytic capacity leading to euthymia.

Plasminogen activator inhibitor-1 inhibits factor VIIa bound to tissue factor.

BACKGROUND AND OBJECTIVE: A growing body of experimental evidence supports broad inhibitory and regulatory activity of plasminogen activator inhibitor 1 (PAI-1). The present study was designed to investigate whether PAI-1 inhibits factor (F) VIIa complexed with tissue factor (TF), a well-known procoagulant risk factor. METHODS AND RESULTS: The ability of PAI-1 to inhibit FVIIa-TF activity was evaluated in both clotting and factor X (FX) activation assays. PAI-1 and its complex with vitronectin inhibit: (i) clotting activity of FVIIa-TF (PAI-1(IC50) , 817 and 125 nm, respectively); (ii) FVIIa-TF-mediated FX activation (PAI-1(IC50) , 260 and 50 nm, respectively); and (iii) FVIIa bound to TF expressed on the surface of stimulated endothelial cells (PAI-1(IC50) , 260 and 120 nm, respectively). The association rate constant (k(a)) for PAI-1 inhibition of FVIIa-TF was determined using a chromogenic assay. K(a) for PAI-1 inhibition of FVIIa bound to relipidated TF is 3.3-fold higher than that for FVIIa bound to soluble TF (k(a) = 0.09 ± 0.01 and 0.027 ± 0.03 µm(-1) min(-1), respectively). Vitronectin increases k(a) for both soluble and relipidated TF by 3.5- and 30-fold, respectively (to 0.094 ± 0.020 and 2.7 ± 0.2 µm(-1) min(-1)). However, only a 3.5- to 5.0-fold increase in the acylated FVIIa was observed on SDS PAGE in the presence of vitronectin for both relipidated and soluble TF, indicating fast formation of PAI-1/vitronectin/FVIIa/relipidated TF non-covalent complex. CONCLUSIONS: Our results demonstrate potential anticoagulant activity of PAI-1 in the presence of vitronectin, which could contribute to regulation of hemostasis under pathological conditions such as severe sepsis, acute lung injury and pleural injury, where PAI-1 and TF are overexpressed.

Plasmin enhances cell surface tissue factor activity in mesothelial and endothelial cells.

BACKGROUND: Mesothelial cells that line the thoracic cavity play an important role in maintaining the local balance between procoagulant and fibrinolytic activity, a role akin to the endothelial cells in blood vessels. The mechanism(s) responsible for increased tissue factor (TF) expression in mesothelial cells in response to injury are at present unclear. OBJECTIVE: To investigate whether plasmin or thrombin, two major proteases that may be generated on the pleural surface upon injury, induce TF expression in human pleural mesothelial cells (HMC) and elucidate the underlying mechanism(s). METHODS: Confluent monolayers of HMC and human umbilical vein endothelial cells (HUVEC) were exposed to plasmin or thrombin for varying time periods and TF expression was analyzed by measuring its activity in a factor Xa generation assay, TF antigen levels by immunoblot analysis and TF mRNA by Northern blot analysis. RESULTS: Both plasmin and thrombin treatments increased cell surface TF activity in HMC by 3- to 4-fold. In contrast to thrombin, plasmin-induced TF activity is not dependent on the de novo synthesis of TF. In HUVEC, plasmin had a minimal effect on unperturbed HUVEC whereas it markedly increased TF activity of activated HUVEC. Plasmin treatment neither affected anionic phospholipid levels at the cell surface nor released protein disulfide isomerase, an oxidoreductase protein that was newly described to play a role in TF activation. Plasmin cleaved cell-associated TFPI. CONCLUSION: Thrombin up-regulates TF activity in HMC through the transcriptional activation of TF whereas plasmin increases TF activity by inactivating the cell-associated TFPI by a limited proteolysis.

Thrombin-thrombomodulin inhibits prourokinase-mediated pleural mesothelial cell-dependent fibrinolysis.

Fibrin deposition is a hallmark of pleural inflammation and loculation but understanding of mechanisms by which mesothelial cells regulate intrapleural fibrinolysins remains incomplete. We speculated that pleural mesothelial cells regulate local fibrinolytic capacity via processing of single chain urokinase type plasminogen activator (scuPA). Pretreatment of human pleural mesothelial (MeT-5A) cells with TGF-beta or thrombin, either alone or in combination, inhibited urokinase (uPA)-mediated fibrinolysis by MeT-5A. Thrombin, unlike TGF-beta, inhibited fibrinolysis without induction of PAI-1, suggesting that thrombin-mediated cleavage of scuPA inhibits the fibrinolytic capacity of MeT-5A cells. Thrombin cleaves both purified scuPA as well as that secreted by MeT-5A cells and cell surface thrombomodulin accelerates thrombin-mediated cleavage of scuPA to inhibit cellular fibrinolytic activity. Molecular dynamics analyses demonstrated that thrombin-cleaved scuPA (uPAt) do not acquire a catalytically active conformation and that secondary plasminogen binding sites of uPA implicated in plasminogen activation are distorted in uPAt, explaining, at least in part, why uPAt is a poor enzyme. uPAt was detectable in transudative and exudative pleural effusions from patients. Intrapleural administration of scuPA generated increased levels of uPAt in PF of rabbits with pleural injury and loculation induced by tetracycline in vivo. This pathway is operative in diverse forms of pleural injury, restricts the urokinase-dependent fibrinolytic capacity of pleural mesothelial cells and contributes to local control of fibrinolytic activity via processing of endogenous or exogenous scuPA within the pleural compartment.

Tissue factor in experimental acute lung injury.

Acute lung injury (ALI) is characterized by fibrin deposition in the tissue and vascular spaces. Coagulation is activated after exposure to endotoxin or bacteria, and a procoagulant environment rapidly develops in the vascular, interstitial, and alveolar spaces of the lung. These changes are tissue factor (TF)-dependent and associated with increases in inflammatory cytokines. Procoagulant changes also occur in the lungs of patients with the acute respiratory distress syndrome (ARDS), suggesting that epithelial inflammation activates the extrinsic pathway. Many inflammatory mediators have specific effects on coagulation; however, the role of TF in regulation of pulmonary inflammatory responses is less clear. Here we report initial data on blockade of TF-initiated coagulation in baboons with Escherichia coli sepsis-induced ALI, using active site-inactivated FVIIa (FVIIai ASIS). Treatment with FVIIai prevented plasma fibrinogen depletion and attenuated fibrin deposition in the tissues. The drug also decreased systemic cytokine responses and inflammatory changes in the lung, including neutrophil infiltration, and decreased edema. Coagulation blockade with FVIIai improved lung function by preserving gas exchange and compliance, decreased pulmonary hypertension, and enhanced renal function. These results show that TF-FVIIa complex is an important regulatory site for the pathologic response of the lung to sepsis.

Coagulation blockade prevents sepsis-induced respiratory and renal failure in baboons.

Sepsis-induced tissue factor (TF) expression activates coagulation in the lung and leads to a procoagulant environment, which results in fibrin deposition and potentiates inflammation. We hypothesized that preventing initiation of coagulation at TF-Factor VIIa (FVIIa) complex would block fibrin deposition and control inflammation in sepsis, thereby limiting acute lung injury (ALI) and other organ damage in baboons. A model of ALI was used in which adult baboons were primed with killed Escherichia coli (1 x 10(9) CFU/kg), and bacteremic sepsis was induced 12 h later by infusion of live E. coli at 1 x 10(10) CFU/kg. Animals in the treatment group were given a competitive inhibitor of TF, site-inactivated FVIIa (FVIIai), intravenously at the time of the infusion of live bacteria and monitored physiologically for another 36 h. FVIIai dramatically protected gas exchange and lung compliance, prevented lung edema and pulmonary hypertension, and preserved renal function relative to vehicle (all p < 0.05). Treatment attenuated sepsis-induced fibrinogen depletion (p < 0.01) and decreased systemic proinflammatory cytokine responses, for example, interleukin 6 (p < 0.01). The protective effects of TF blockade in sepsis-induced ALI were confirmed by using tissue factor pathway inhibitor. The results show that TF-FVIIa complex contributes to organ injury in septic primates in part through selective stimulation of proinflammatory cytokine release and fibrin deposition.

Urokinase induces expression of its own receptor in Beas2B lung epithelial cells.

Interaction between the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) localizes cellular proteolysis and promotes cellular proliferation and migration. The interaction between uPA and uPAR at the surface of epithelial cells thereby contributes to the pathogenesis of lung inflammation and neoplasia. In this study, we sought to determine if uPA itself alters uPAR expression by lung epithelial cells. uPA enhanced uPAR expression as well as (125)I-uPA binding in Beas2B lung epithelial cells in a time- and concentration-dependent manner. The uPA-mediated induction of uPAR is not accomplished through its receptor and requires enzymatic activity. The low molecular weight fragment of uPA, lacking the receptor binding domain, was as potent as intact two-chain uPA in inducing expression of uPAR at the cell surface. Plasmin, the end product of plasminogen activation, did not alter uPA-mediated uPAR expression. Induction of uPAR by uPA represents a novel pathway by which epithelial cells can regulate uPAR-dependent cellular responses that may contribute to stromal remodeling in lung injury or neoplasia.

Vitronectin adsorption to chrysotile asbestos increases fiber phagocytosis and toxicity for mesothelial cells.

Biological modification of asbestos fibers can alter their interaction with target cells. We have shown that vitronectin (VN), a major adhesive protein in serum, adsorbs to crocidolite asbestos and increases fiber phagocytosis by mesothelial cells via integrins. Because chrysotile asbestos differs significantly from crocidolite in charge and shape, we asked whether VN would also adsorb to chrysotile asbestos and increase its toxicity for mesothelial cells. We found that VN, either from purified solutions or from serum, adsorbed to chrysotile but at a lower amount per surface area than to crocidolite. Nevertheless, VN coating increased the phagocytosis of chrysotile as well as of crocidolite asbestos. VN coating of both chrysotile and crocidolite, but not of glass beads, increased intracellular oxidation and apoptosis of mesothelial cells. The additional apoptosis could be blocked by integrin-ligand blockade with arginine-glycine-aspartic acid peptides, confirming a role for integrins in the fiber-induced toxicity. We conclude that VN increases the phagocytosis of chrysotile as well as of crocidolite asbestos and that phagocytosis is important in fiber-induced toxicity for mesothelial cells.

Tissue factor pathway inhibitor expression by human pleural mesothelial and mesothelioma cells.

The mesothelial lining of the pleura and malignant mesothelioma promote fibrin deposition in pleural injury or neoplasia via expression of tissue factor (TF). It was hypothesized that these cells might also regulate intrapleural coagulation by elaborating TF pathway inhibitor (TFPI). TFPI activity and antigen in pleural fluids were assayed from patients with congestive heart failure (CHF), pneumonia, empyema, metastatic pleural cancer and malignant mesothelioma. The authors also assessed expression of TF and TFPI messenger ribonucleic acid (mRNA) as well as TFPI activity and antigen by human pleural mesothelial cells, malignant mesothelioma cells (MS-1 cell line) and human lung fibroblasts. Immunohistochemical analyses of normal, fibrotic, and neoplastic pleura were performed to determine whether TFPI antigen was expressed in vivo. The study revealed that TFPI was present in transudates from patients with CHF and exudative pleural effusions from patients with pneumonia, empyema or pleural carcinoma. TFPI mRNA, activity and antigen were expressed by pleural mesothelial cells, MS-1 cells and lung fibroblasts. Cytokines and serum stimulated a significant early increase in TF mRNA levels with minimal enhancement of TFPI mRNA, activity and antigen levels. TFPI antigen was found in normal, fibrotic and neoplastic pleural tissues. The current observations indicate that tissue factor pathway inhibitor is locally expressed in pleural disease, but that it does not prevent the development of a prothrombotic environment favouring local fibrin deposition in pleural inflammation or cancer.

Post-transcriptional regulation of urokinase mRNA. Identification of a novel urokinase mRNA-binding protein in human lung epithelial cells in vitro.

We sought to determine if urokinase expression is regulated at the post-transcriptional level in cultured lung epithelial cells. We also sought to determine if differences in urokinase expression by cultured human lung carcinoma and non-malignant lung epithelial subtypes were attributable to post-transcriptional regulatory mechanisms. Urokinase was expressed by phenotypically diverse lung carcinoma cell lines as well as non-malignant small airway epithelial cells and bronchial epithelial cells. Using gel mobility shift and UV cross-linking assays, we identified a 30-kDa urokinase mRNA-binding protein that selectively bound to a 66-nucleotide protein-binding fragment of urokinase mRNA. The urokinase mRNA-binding protein is found in the cytosolic but not nuclear extracts of non-malignant lung epithelial cells; whereas, it is found in the nuclear but not cytosolic extracts of selected malignant carcinoma-derived cells that express relatively large amounts of urokinase. Chimeric beta-globin/urokinase cDNA containing the urokinase mRNA-binding protein binding sequence destabilized otherwise stable beta-globin mRNA. Our results demonstrate that urokinase gene expression in lung epithelial and lung carcinoma-derived cells is regulated at the post-transcriptional level. The mechanism involves an interaction between a 66-nucleotide sequence of the urokinase mRNA 3'-untranslated region with a newly recognized urokinase mRNA-binding protein to regulate urokinase mRNA stability.

Posttranscriptional regulation of plasminogen activator inhibitor-1 in human lung carcinoma cells in vitro.

Plasminogen activator inhibitor-1 (PAI-1), the major circulating inhibitor of urokinase [urokinase-type plasminogen activator (uPA)], has been linked to the pathogenesis of lung cancer. PAI-1 belongs to the serpin family of inhibitors and inhibits both free urokinase (uPA) and receptor-bound urokinase (uPA receptor). Although PAI-1 has been related to a poor prognosis in lung carcinoma, mechanisms that regulate its expression in human lung cancer cells are not well understood. We used cultured human small cell and non-small cell lung carcinoma cell lines as model systems to elucidate the regulatory mechanisms that control expression of PAI-1. Levels of PAI-1 protein were significantly increased in selected lung carcinoma cells compared with those in normal small-airway epithelial cells. Corresponding steady-state levels of PAI-1 mRNA were similarly increased in these cells. The half-life of PAI-1 mRNA was prolonged in these lung carcinoma cell lines after transcriptional or translational blockade. We identified a 60-kDa protein that binds the 3'-untranslated region of PAI-1, and complex formation of this binding protein with PAI-1 mRNA reciprocally correlates with mRNA stability. The findings demonstrate that expression of PAI-1 is regulated at the posttranscriptional level in small cell- and non-small cell-derived human lung carcinoma cell lines. Altered regulation of PAI-1 at the posttranscriptional level may contribute to relative overexpression by malignant lung epithelial cells. A newly identified regulatory protein that binds to the 3'-untranslated region of PAI-1 mRNA appears to be involved in the posttranscriptional regulation of PAI-1 gene expression by human lung carcinoma cells in vitro.

Posttranscriptional regulation of urokinase receptor gene expression in human lung carcinoma and mesothelioma cells in vitro.

The urokinase-type plasminogen activator (uPA) interacts with its receptor (uPAR) to promote proteolysis as well as cell proliferation and migration. These functions contribute to the pathogenesis of neoplastic growth and invasiveness. Expression of uPAR in tumor extracts also inversely correlates with prognosis in many forms of cancer. In this study, we sought to determine if differences in uPAR expression were distinguishable between cultured human lung carcinoma and malignant mesothelioma subtypes. We also sought to determine if, as in malignant mesothelioma cells, uPAR expression is regulated at the posttranscriptional level in cultured malignant lung carcinoma cells. Using 125I-uPA binding and ligand blotting techniques, uPAR was expressed by phenotypically diverse lung carcinoma cell lines, including the H460, H157 and H1395 non-small cell lines and the H146 small cell lung carcinoma line. Increased uPAR expression was also detected in spindle-shaped (M33K) and epithelioid (M9K and MS-1) malignant mesothelioma cells. Selected mediators, including TGF-beta, TNF-alpha, LPS and PMA, uniformly enhanced uPAR expression in each of the tumor cell lines. Steady state uPAR mRNA expression was determined by RNase protection assay and correlated directly with the changes in cell surface uPAR expression. By gel mobility shift and UV-cross linking assays, a uPAR mRNA binding protein (uPAR mRNABp) implicated in the posttranscriptional control of message stability, was identified in each of the cell lines. Expression of uPAR and its message in cultured lung carcinoma and malignant mesothelioma cells is similarly influenced by effectors present in the tumor microenvironment. Regulation of the uPAR message occurs at the posttranscriptional level in cultured small and non-small cell lung carcinoma cells as well as spindle-shaped and fibrous malignant mesothelioma cell lines. Posttranscriptional regulation of uPAR in all these cells involves the interaction of the uPAR mRNABp with uPAR mRNA, which promotes uPAR mRNA destabilization.

Asbestos upregulates expression of the urokinase-type plasminogen activator receptor on mesothelial cells.

Inhalation of asbestos is associated with pathologic changes in the pleural space, including pleural thickening, pleural plaques, and mesothelioma. These processes are characterized by altered local proteolysis, cellular proliferation, and cell migration, suggesting that the urokinase-type plasminogen activator receptor (uPAR) could be involved in the pathogenesis of asbestos-induced pleural disease. We hypothesized that mesothelial cell uPAR expression is induced by exposure to asbestos. To test this hypothesis, we used complementary techniques in rabbit and human mesothelial cells to determine whether uPAR expression is altered by exposure to asbestos. uPAR expression was induced by chrysotile and crocidolite asbestos, but not by wollastonite, as indicated by binding of radiolabeled urokinase-type plasminogen activator (uPA) to rabbit or human mesothelial cells. uPA was not induced by fiber exposure. Exposure to exogenous uPA increased uPA activity of cells exposed to wollastonite but not asbestos-treated MeT5A cells. uPAR expression increased further when asbestos was preincubated with vitronectin (VN) or serum. Increases in uPAR expression were confirmed by binding of uPA to uPAR in cell membrane preparations and immunofluorescent staining of uPAR at the cell surface, and were associated with increases in steady-state uPAR messenger RNA. Mesothelial cell uPAR expression was also induced by media from monocytes cultured with asbestos incubated with VN and serum. By antibody neutralization, the latter effect appeared to be in part mediated by transforming growth factor-beta. We found that asbestos increases uPAR at the surface of rabbit and human mesothelial cells, suggesting that altered expression of this receptor could be involved in asbestos-induced remodeling of the pleural mesothelium.

Urokinase-type plasminogen activator induces tyrosine phosphorylation of a 78-kDa protein in H-157 cells.

Studies from our laboratory have shown that exposure of human lung epithelial cells to urokinase plasminogen activator (uPA) induces their proliferation. This effect of uPA is likely to occur via activation of signal transduction pathways. To elucidate uPA-induced signal transduction mechanisms, we exposed H-157 cells to uPA and determined the induced tyrosine phosphorylation profile of proteins. We demonstrate that, in these cells, uPA prominently induced tyrosine phosphorylation of a 78-kDa protein. This effect was observed as early as 30 min and was sustained for at least 24 h. Treatment of cells with agents that abrogate uPA receptor (uPAR) function, including neutralizing anti-uPAR antibody, phosphatidylinositol-specific phospholipase C, or a selective antagonist that blocks the association of uPA with uPAR (A5 compound), all failed to prevent uPA-induced tyrosine phosphorylation. B-428, an active site inhibitor of uPA activity, prevented the uPA effect. Treatment of cells with hepatocyte growth factor, vascular endothelial growth factor, or transforming growth factor-beta, all of which are known to be activated by a uPA-dependent pathway, did not stimulate tyrosine phosphorylation of the 78-kDa protein. uPA induced an increase in [(3)H]thymidine incorporation into DNA, and cell numbers were unaffected in the presence of A5. These results demonstrate that, in H-157 cells, uPA induces tyrosine phosphorylation of a 78-kDa protein via a proteolysis-dependent but uPAR-independent mechanism. This novel signaling pathway represents a putative mechanism by which uPA could influence epithelial cell proliferation.

Regulation of tissue factor pathway inhibitor expression in smooth muscle cells.

Tissue factor pathway inhibitor (TFPI) is the primary physiological inhibitor that regulates tissue factor-induced blood coagulation. TFPI is thought to be synthesized, in vivo, primarily by microvascular endothelial cells. Little is known about how TFPI is regulated under pathophysiological conditions. In this study, we determined mechanisms by which TFPI expression is regulated by human pulmonary artery smooth muscle cells (PASMC), because these cells contribute to remodeling of the pulmonary vasculature in disease. PASMC in culture constitutively synthesize and secrete TFPI. Exposure of PASMC to phorbol myristate acetate, lipopolysaccharide, tumor necrosis factor alpha, thrombin, interleukin-1, and transforming growth factor-beta had no significant effect on expression of TFPI by PASMC. By contrast, treatment of PASMC with serum and basic fibroblast growth factor (bFGF)/heparin markedly upregulated the expression of TFPI activity and antigen. On Western blot analysis, a protein consistent with full-length TFPI (42 kD) was identified in the conditioned media of PASMC, and the levels of the protein were much higher in the conditioned media of serum and bFGF/heparin-treated cells. Northern blot analysis showed that PASMC constitutively express TFPI mRNA, and treatment of cells with serum and bFGF/heparin had a minimal effect on the steady-state levels of TFPI mRNA. Nuclear run-on analysis did not show a significant increase in the transcriptional rate of TFPI gene in PASMC treated with serum or bFGF/heparin. Cycloheximide, but not actinomycin-D, treatment inhibited the serum and bFGF/heparin-induced increase in TFPI activity in PASMC. In conclusion, our data demonstrate that PASMC constitutively synthesize and secrete TFPI and serum or bFGF upregulate its expression, suggesting that growth factors that can stimulate the vessel wall in vivo might locally regulate TFPI expression. Our study also suggests that control of TFPI expression by serum or bFGF occurs via translational rather than transcriptional regulation.

alpha-thrombin inhibits interleukin-6-induced Stat3 signaling and gp130 gene expression in primary cultures of human lung fibroblasts.

Exposure of primary human lung fibroblasts (HLF) to interleukin-6 (IL-6) rapidly induced Stat3 (signal transducers and activators of transcription 3) tyrosine phosphorylation. In these cells, alpha-thrombin did not induce tyrosine phosphorylation of Stat3; however, it potently induced its serine phosphorylation. Interestingly, a short pretreatment of cells with alpha-thrombin significantly inhibited IL-6-induced tyrosine phosphorylation of Stat3. The inhibition by alpha-thrombin was attenuated if cells were pretreated with U0126, a specific inhibitor of the mitogen-activated protein (MAP) kinase kinase 1 (MAPKK1). Exposure of HLF cells to IL-6 induced a twofold increase in gp130 mRNA levels; however, alpha-thrombin inhibited this IL-6-induced response almost to control levels. These results demonstrate, for the first time, that in HLF cells alpha-thrombin inhibits IL-6-induced Stat3 signaling via activation of MAPKK1 and that this cross-talk regulates IL-6-induced gp130 gene expression.

Chemokine involvement in tetracycline-induced pleuritis.

Sclerosants such as tetracycline (TCN) have often been used in the control of malignant pleural effusions. Although the resultant inflammatory response is probably important in the ensuing pleural fibrosis, the signals responsible for the cellular influx into the pleural space following TCN instillation are not well understood. This study, therefore, sought to determine whether the chemokines interleukin-8 (IL-8), growth-related protein (Gro), and monocyte chemotactic protein-1 (MCP-1) were locally elaborated within the first 72 h following intrapleural TCN administration. TCN induced an exudative effusion with high lactate dehydrogenase activity. Although there was no significant change in the pleural fluid total leukocyte content, the median polymorphonuclear neutrophil concentration decreased from 1.067x10(6) to 2.03x10(5) cells x mL(-1) between 24 and 72 h, whereas the median macrophage concentration increased from 1.44x10(5) to 5.98x10(5) cells x mL(-1) over the same period. Furthermore, IL-8, Gro and MCP-1 concentrations decreased between 24 and 72 h. Immunocytochemistry indicated expression of IL-8 by pleural mesothelial cells 24 h, but not 72 h, following TCN administration. The data suggest that local elaboration of interleukin-8 and growth-related protein, in part of mesothelial origin, may influence neutrophil recruitment in tetracycline-induced pleuritis.

A urokinase receptor mRNA binding protein-mRNA interaction regulates receptor expression and function in human pleural mesothelioma cells.

Human pleural malignant mesothelioma (MS-1) or mesothelial (MeT5A) cells express the multifunctional urokinase receptor (uPAR) which influences neoplastic propagation via contributions to cellular proteolysis, migration, and mitogenesis. Recently, we reported that a 51-nucleotide fragment of the uPAR mRNA coding region contains regulatory information for uPAR message stability and that a cytoplasmic uPAR mRNA binding protein (uPAR mRNABp) specifically bound to this sequence in temporal association with uPAR mRNA destabilization in MS-1 cells. To determine if the uPAR mRNA-uPAR mRNABp interaction is a determinant of uPAR message stability as well as uPAR expression, we further characterized this cis-trans interaction and created stable transfected cell lines designed to exploit the interaction and to increase uPAR at the cell surface. The uPAR mRNABp was purified from MS-1 cells, has an apparent molecular mass of 50 kDa, selectively binds to the 51-nt fragment of the uPAR coding region, and does not degrade uPAR mRNA. To determine the role of the uPAR mRNABp on receptor expression, we overexpressed a chimeric beta-globin/uPAR/beta-globin mRNA containing the 51-nt binding fragment of uPAR mRNA in MS-1 cells and found that uPAR at the cell surface increased by twofold as measured by [125I]uPA binding or ligand blotting. Cellular proliferation of uPA-treated cells and invasiveness was similarly increased. The increase in cell surface uPAR was due to commensurately increased uPAR mRNA. The results suggest that competition between the overexpressed 51-nt fragment of the uPAR coding region and the wild-type uPAR mRNA transcript for uPAR mRNABp binding enables the cells to translate and express more uPAR at the cell surface. The interaction between the uPAR mRNABp and uPAR mRNA regulates message stability as well as uPAR expression by MS-1 cells.