Cellular Microenvironment Stiffness Regulates Eicosanoid Production and Signaling Pathways.

Pathological changes in the biomechanical environment are implicated in the progression of idiopathic pulmonary fibrosis (IPF). Stiffened matrix augments fibroblast proliferation and differentiation and activates TGF-ß1 (transforming growth factor-ß1). Stiffened matrix impairs the synthesis of the antifibrogenic lipid mediator prostaglandin E2 (PGE2) and reduces the expression of the rate-limiting prostanoid biosynthetic enzyme cyclooxygenase-2 (COX-2). We now show that prostaglandin E synthase (PTGES), the final enzyme in the PGE2 biosynthetic pathway, is expressed at lower levels in the lungs of patients with IPF. We also show substantial induction of COX-2, PTGES, prostaglandin E receptor 4 (EP4), and cytosolic phospholipase A2 (cPLA2) expression in human lung fibroblasts cultured in soft collagen hydrogels or in spheroids compared with conventional culture on stiff plastic culture plates. Induction of COX-2, cPLA2, and PTGES expression in spheroid cultures was moderately inhibited by the p38 mitogen-activated protein kinase inhibitor SB203580. The induction of prostanoid biosynthetic enzyme expression was accompanied by an increase in PGE2 levels only in non-IPF-derived fibroblast spheroids. Our study reveals an extensive dysregulation of prostanoid biosynthesis and signaling pathways in IPF-derived fibroblasts, which are only partially abrogated by culture in soft microenvironments.

Annexin A2 contributes to lung injury and fibrosis by augmenting factor Xa fibrogenic activity.

In lung injury and disease, including idiopathic pulmonary fibrosis (IPF), extravascular factor X is converted into factor Xa (FXa), a coagulant protease with fibrogenic actions. Extracellular annexin A2 binds to FXa, augmenting activation of the protease-activated receptor-1 (PAR-1). In this study, the contribution of annexin A2 in lung injury and fibrosis was investigated. Annexin A2 immunoreactivity was observed in regions of fibrosis, including those associated with fibroblasts in lung tissue of IPF patients. Furthermore, annexin A2 was detected in the conditioned media and an EGTA membrane wash of human lung fibroblast (LF) cultures. Incubation with human plasma (5% vol/vol) or purified FXa (15-50 nM) evoked fibrogenic responses in LF cultures, with FXa increasing interleukin-6 (IL-6) production and cell number by 270 and 46%, respectively (P < 0.05, n = 5-8). The fibrogenic actions of plasma or FXa were attenuated by the selective FXa inhibitor apixaban (10 µM, or antibodies raised against annexin A2 or PAR-1 (2 µg/ml). FXa-stimulated LFs from IPF patients (n = 6) produced twice as much IL-6 as controls (n = 10) (P < 0.05), corresponding with increased levels of extracellular annexin A2. Annexin A2 gene deletion in mice reduced bleomycin-induced increases in bronchoalveolar lavage fluid (BALF) IL-6 levels and cell number (*P < 0.05; n = 4-12). Lung fibrogenic gene expression and dry weight were reduced by annexin A2 gene deletion, but lung levels of collagen were not. Our data suggest that annexin A2 contributes to lung injury and fibrotic disease by mediating the fibrogenic actions of FXa. Extracellular annexin A2 is a potential target for the treatment of IPF.

The Coagulant Factor Xa Induces Protease-Activated Receptor-1 and Annexin A2-Dependent Airway Smooth Muscle Cytokine Production and Cell Proliferation.

During asthma exacerbation, plasma circulating coagulant factor X (FX) enters the inflamed airways and is activated (FXa). FXa may have an important role in asthma, being involved in thrombin activation and an agonist of protease-activated receptor-1 (PAR-1). Extracellular annexin A2 and integrins are also implicated in PAR-1 signaling. In this study, the potential role of PAR-1 in mediating the effects of FXa on human airway smooth muscle (ASM) cell cytokine production and proliferation was investigated. FXa (5-50 nM), but not FX, stimulated increases in ASM IL-6 production and cell number after 24- and 48-hour incubation, respectively (P < 0.05; n = 5). FXa (15 nM) also stimulated increases in the levels of mRNA for cytokines (IL-6), cell cycle-related protein (cyclin D1), and proremodeling proteins (FGF-2, PDGF-B, CTGF, SM22, and PAI-1) after 3-hour incubation (P < 0.05; n = 4). The actions of FXa were insensitive to inhibition by hirudin (1 U/ml), a selective thrombin inhibitor, but were attenuated by SCH79797 (100 nM), a PAR-1 antagonist, or Cpd 22 (1 µM), an inhibitor of integrin-linked kinase. The selective targeting of PAR-1, annexin A2, or ß1-integrin by small interfering RNA and/or by functional blocking antibodies also attenuated FXa-evoked responses. In contrast, the targeting of annexin A2 did not inhibit thrombin-stimulated ASM function. In airway biopsies of patients with asthma, FXa and annexin A2 were detected in the ASM bundle by immunohistochemistry. These findings establish FXa as a potentially important asthma mediator, stimulating ASM function through actions requiring PAR-1 and annexin A2 and involving integrin coactivation.

The antimalarial drug artesunate inhibits primary human cultured airway smooth muscle cell proliferation.

Airway smooth muscle (ASM) cell hyperplasia contributes to airway wall remodeling (AWR) in asthma. Glucocorticoids, which are used as first-line therapy for the treatment of inflammation in asthma, have limited impact on AWR, and protracted usage of high doses of glucocorticoids is associated with an increased risk of side effects. Moreover, patients with severe asthma often show reduced sensitivity to glucocorticoids. Artesunate, a semisynthetic artemisinin derivative used to treat malaria with minimal toxicity, attenuates allergic airway inflammation in mice, but its impact on AWR is not known. We examined the effects of artesunate on ASM proliferation in vitro and in vivo. Primary human ASM cells derived from nonasthmatic donors were treated with artesunate before mitogen stimulation. Artesunate reduced mitogen-stimulated increases in cell number and cyclin D1 protein abundance but had no significant effect on ERK1/2 phosphorylation. Artesunate, but not dexamethasone, inhibited phospho-Akt and phospho-p70(S6K) protein abundance. Artesunate, but not dexamethasone, inhibited mitogen-stimulated increases in cell number, cyclin D1, and phospho-Akt protein abundance on ASM cells derived from asthmatic donors. In a murine model of allergic asthma, artesunate reduced the area of α-smooth muscle actin-positive cells and decreased cyclin D1 protein abundance. Our study provides a basis for the future development of artesunate as a novel anti-AWR agent that targets ASM hyperplasia via the PI3K/Akt/p70(S6K) pathway and suggests that artesunate may be used as combination therapy with glucocorticoids.

Bronchial epithelial cells are rendered insensitive to glucocorticoid transactivation by transforming growth factor-ß1.

BACKGROUND: We have previously shown that transforming growth factor-beta (TGF-beta) impairs glucocorticoid (GC) function in pulmonary epithelial cell-lines. However, the signalling cascade leading to this impairment is unknown. In the present study, we provide the first evidence that TGF-beta impairs GC action in differentiated primary air-liquid interface (ALI) human bronchial epithelial cells (HBECs). Using the BEAS-2B bronchial epithelial cell line, we also present a systematic examination of the known pathways activated by TGF-beta, in order to ascertain the molecular mechanism through which TGF-beta impairs epithelial GC action. METHODS: GC transactivation was measured using a Glucocorticoid Response Element (GRE)-Secreted embryonic alkaline phosphatase (SEAP) reporter and measuring GC-inducible gene expression by qRT-PCR. GC transrepression was measured by examining GC regulation of pro-inflammatory mediators. TGF-beta signalling pathways were investigated using siRNA and small molecule kinase inhibitors. GRα level, phosphorylation and sub-cellular localisation were determined by western blotting, immunocytochemistry and localisation of GRα-Yellow Fluorescent Protein (YFP). Data are presented as the mean ± SEM for n independent experiments in cell lines, or for experiments on primary HBEC cells from n individual donors. All data were statistically analysed using GraphPad Prism 5.0 (Graphpad, San Diego, CA). In most cases, two-way analyses of variance (ANOVA) with Bonferroni post-hoc tests were used to analyse the data. In all cases, P <0.05 was considered to be statistically significant. RESULTS: TGF-beta impaired Glucocorticoid Response Element (GRE) activation and the GC induction of several anti-inflammatory genes, but did not broadly impair the regulation of pro-inflammatory gene expression in A549 and BEAS-2B cell lines. TGF-beta-impairment of GC transactivation was also observed in differentiated primary HBECs. The TGF-beta receptor (ALK5) inhibitor SB431541 fully prevented the GC transactivation impairment in the BEAS-2B cell line. However, neither inhibitors of the known downstream non-canonical signalling pathways, nor knocking down Smad4 by siRNA prevented the TGF-beta impairment of GC activity. CONCLUSIONS: Our results indicate that TGF-beta profoundly impairs GC transactivation in bronchial epithelial cells through activating ALK5, but not through known non-canonical pathways, nor through Smad4-dependent signalling, suggesting that TGF-beta may impair GC action through a novel non-canonical signalling mechanism.

Laminin drives survival signals to promote a contractile smooth muscle phenotype and airway hyperreactivity.

Increased airway smooth muscle (ASM) mass is believed to underlie the relatively fixed airway hyperresponsiveness (AHR) in asthma. Developments of therapeutic approaches to reverse airway remodeling are impeded by our lack of insight on the mechanisms behind the increase in mass of contractile ASM cells. Increased expression of laminin, an extracellular matrix protein, is associated with asthma. Our studies investigate the role of laminin-induced ASM survival signals in the development of increased ASM and AHR. Antagonizing laminin integrin binding using the laminin-selective competing peptide, YIGSR, and mimicking laminin with exogenous α2-chain laminin, we show that laminin is both necessary and sufficient to induce ASM cell survival, concomitant with the induction of ASM contractile phenotype. Using siRNA, we show that the laminin-binding integrin α7ß1 mediates this process. Moreover, in laminin-211-deficient mice, allergen-induced AHR was not observed. Notably, ASM cells from asthmatic airways express a higher abundance of intracellular cell survival proteins, consistent with a role for reduced rates of cell apoptosis in development of ASM hyperplasia. Targeting the laminin-integrin α7ß1 signaling pathway may offer new avenues for the development of therapies to reduce the increase in mass of contractile phenotype ASM cells that underlie AHR in asthma.

Transforming growth factor-ß-induced differentiation of airway smooth muscle cells is inhibited by fibroblast growth factor-2.

In asthma, basic fibroblast growth factor (FGF-2) plays an important (patho)physiological role. This study examines the effects of FGF-2 on the transforming growth factor-ß (TGF-ß)-stimulated differentiation of airway smooth muscle (ASM) cells in vitro. The differentiation of human ASM cells after incubation with TGF-ß (100 pM) and/or FGF-2 (300 pM) for 48 hours was assessed by increases in contractile protein expression, actin-cytoskeleton reorganization, enhancements in cell stiffness, and collagen remodeling. FGF-2 inhibited TGF-ß-stimulated increases in transgelin (SM22) and calponin gene expression (n = 15, P < 0.01) in an extracellular signal-regulated kinase 1/2 (ERK1/2) signal transduction-dependent manner. The abundance of ordered α-smooth muscle actin (α-SMA) filaments formed in the presence of TGF-ß were also reduced by FGF-2, as was the ratio of F-actin to G-actin (n = 8, P < 0.01). Furthermore, FGF-2 attenuated TGF-ß-stimulated increases in ASM cell stiffness and the ASM-mediated contraction of lattices, composed of collagen fibrils (n = 5, P < 0.01). However, the TGF-ß-stimulated production of IL-6 was not influenced by FGF-2 (n = 4, P > 0.05), suggesting that FGF-2 antagonism is selective for the regulation of ASM cell contractile protein expression, organization, and function. Another mitogen, thrombin (0.3 U ml(-1)), exerted no effect on TGF-ß-regulated contractile protein expression (n = 8, P > 0.05), α-SMA organization, or the ratio of F-actin to G-actin (n = 4, P > 0.05), suggesting that the inhibitory effect of FGF-2 is dissociated from its mitogenic actions. The addition of FGF-2, 24 hours after TGF-ß treatment, still reduced contractile protein expression, even when the TGF-ß-receptor kinase inhibitor, SB431542 (10 µM), was added 1 hour before FGF-2. We conclude that the ASM cell differentiation promoted by TGF-ß is antagonized by FGF-2. A better understanding of the mechanism of action for FGF-2 is necessary to develop a strategy for therapeutic exploitation in the treatment of asthma.

Plasminogen-stimulated inflammatory cytokine production by airway smooth muscle cells is regulated by annexin A2.

Plasminogen has a role in airway inflammation. Airway smooth muscle (ASM) cells cleave plasminogen into plasmin, a protease with proinflammatory activity. In this study, the effect of plasminogen on cytokine production by human ASM cells was investigated in vitro. Levels of IL-6 and IL-8 in the medium of ASM cells were increased by incubation with plasminogen (5-50 µg/ml) for 24 hours (P < 0.05; n = 6-9), corresponding to changes in the levels of cytokine mRNA at 4 hours. The effects of plasminogen were attenuated by α2-antiplasmin (1 µg/ml), a plasmin inhibitor (P < 0.05; n = 6-12). Exogenous plasmin (5-15 mU/ml) also stimulated cytokine production (P < 0.05; n = 6-8) in a manner sensitive to serine-protease inhibition by aprotinin (10 KIU/ml). Plasminogen-stimulated cytokine production was increased in cells pretreated with basic fibroblast growth factor (300 pM) in a manner associated with increases in urokinase plasminogen activator expression and plasmin formation. The knockdown of annexin A2, a component of the putative plasminogen receptor comprised of annexin A2 and S100A10, attenuated plasminogen conversion into plasmin and plasmin-stimulated cytokine production by ASM cells. Moreover, a role for annexin A2 in airway inflammation was demonstrated in annexin A2-/- mice in which antigen-induced increases in inflammatory cell number and IL-6 levels in the bronchoalveolar lavage fluid were reduced (P < 0.01; n = 10-14). In conclusion, plasminogen stimulates ASM cytokine production in a manner regulated by annexin A2. Our study shows for the first time that targeting annexin A2-mediated signaling may provide a novel therapeutic approach to the treatment of airway inflammation in diseases such as chronic asthma.

Annexin-1 signals mitogen-stimulated breast tumor cell proliferation by activation of the formyl peptide receptors (FPRs) 1 and 2.

The role of the calcium- and phospholipid-binding protein annexin I (ANXA1) in cell cycle regulation has been investigated in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast tumor cell lines. In MCF-7 cells, ANXA1-targeting small interfering RNA (siRNA) reduced ANXA1 mRNA and protein levels and attenuated cell proliferation induced by FCS, estradiol, or epidermal growth factor. Well-characterized agonists for the known ANXA1 receptor, FPR2, including the ANXA1 N-terminal proteolytic product ANXA1(2-26), lipoxin A(4) (LXA(4)), and the synthetic peptide, Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), stimulated proliferation of MCF-7 and MDA-MB-231 cells that was attenuated by incubation with FPR2 antagonists WRW(4) (1 µM) or Boc2 (100 nM) or by siRNA against FPR2. FCS-induced mitogenic responses were attenuated by each of the FPR antagonists and by siRNA against FPR2 and, to a lesser extent, FPR1. LXA(4) increased phosphorylation of Akt, p70(S6K) but not ERK1/2. Increases in cyclin D1 protein induced by FCS or LXA(4) were blocked by the PI3 kinase inhibitor, LY294002, and attenuated by FPR2 antagonism using Boc2. In invasive breast cancer, immunohistochemistry revealed the presence of ANXA1 and its receptor, FPR2, in both tumor epithelium and stromal cells. These observations suggest a novel signaling role for ANXA1 in mitogen-activated proliferation of breast tumor epithelial cells that is mediated via activation of FPR1 and FPR2.

Plasminogen activation by airway smooth muscle is regulated by type I collagen.

Plasmin, the activated protease product of plasminogen, is involved in collagen remodeling, and is strongly implicated in asthma pathophysiology by recent genome-wide association studies. This study examines plasminogen "activation" by airway smooth muscle cells, and its regulation in a fibrotic environment created by culture on type I collagen and incubation with transforming growth factor (TGF)-ß. Urokinase plasminogen activator (uPA) activity was detected in the supernatants of human airway smooth muscle cell cultures maintained in serum-free conditions. Incubation with plasminogen (1.5-50.0 µg/ml, 24 h) increased plasmin activity in a concentration-dependent manner (P < 0.001). uPA activity was higher in cultures maintained on fibrillar type I collagen substrata than in those on plastic, as was plasmin activity after incubation with plasminogen (20 µg/ml). Pretreatment with TGF-ß (100 pM) for 18 hours inhibited plasminogen activation by airway smooth muscle cells maintained on plastic, but not on collagen. TGF-ß stimulated an increase in the level of uPA mRNA in airway smooth muscle cells grown on collagen, but not on plastic. Reducing the levels of ß1-integrin collagen receptor, using interference RNA, attenuated plasmin formation by airway smooth muscle cells grown on collagen, and restored the inhibitory effect of TGF-ß. This study shows that airway smooth muscle activation of plasminogen by uPA is accelerated in a collagen-rich environment in which the inhibitory effect of TGF-ß is attenuated in association with greater uPA expression induced via ß1-integrin signaling. These findings suggest that the plasminogen-activation system involving uPA has the potential to contribute to airway wall remodeling in asthma.

Functional expression of IgG-Fc receptors in human airway smooth muscle cells.

IgE-Fc receptors and IgG-Fc receptors are expressed on hematopoietic cells, but some evidence suggests that these receptors are also found on nonhematopoietic cells, including human airway smooth muscle (hASM) cells. Our study characterizes the expression of IgE-Fc receptors (FcεRI/CD23) and IgG-Fc receptors (FcγRs-I, -II, and -III) in cultured hASM cells by flow cytometry and Western blotting, and the functional activity of receptors was determined through quantification of cell proliferation and released cytokines. Expression of Fc receptor-linked intracellular signaling proteins and phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1/2 and p38(MAPK) in hASM cells was examined by Western blotting. Expression of FcεRI and CD23 was not detectable in hASM cells. However, FcγRI and FcγRII were shown to be expressed on these cells. Specific antibodies, validated using transfected cell lines, revealed that the inhibitory IgG receptor, FcγRIIb, was the most abundant Fc receptor subtype expressed. Although cross-linking FcγR with heat-aggregated γ globulin (HAGG) did not induce detectable cell stimulation, pretreating hASM cells with HAGG significantly inhibited IL-1α-induced increases in cytokine levels and basic fibroblast growth factor-induced cell proliferation. This inhibitory effect of HAGG was abrogated by preincubation of cells with an anti-FcγRIIb antigen-binding fragment (Fab). Expression of proteins involved in the canonical FcγRIIb inhibitory signaling pathway was established in hASM cells. Pretreatment of hASM cells with HAGG significantly inhibited IL-1α- and basic fibroblast growth factor-induced extracellular signal-regulated kinase 1/2 and p38(MAPK) phosphorylation. This study identifies functional expression of FcγRIIb in hASM cells, with the potential to suppress their remodeling and immunomodulatory roles.

Airway smooth muscle remodels pericellular collagen fibrils: implications for proliferation.

Airway wall remodeling comprises a broad range of structural changes including increases in the volume of airway smooth muscle (ASM) and fibrillar collagen. The impact of fibrillar collagen remodeling on ASM proliferation was investigated. Human ASM cultured on type I fibrillar collagen remodeled the collagen substrate by both degradation (collagenolysis) and formation of networks comprised of thicker reticular collagen fibrils (fibrillogenesis). In cultures maintained on fibrillar collagen, the levels of matrix metalloproteases (MMPs) -1 and -14 mRNA and active MMP-2 were higher than in cultures maintained on nonfibrillar type I collagen (gelatin) or plastic. Although there was no apparent increase in cytotoxicity or apoptosis, the number of ASM was lower on fibrillar collagen than on gelatin or plastic for control conditions. Furthermore, maintenance on fibrillar collagen attenuated basic fibroblast growth factor-stimulated increases in cell number and the percentage of cells entering S-phase. In cultures maintained on fibrillar collagen, the MMP inhibitor ilomastat (2.5 microM) 1) attenuated collagenolysis, 2) enhanced fibrillogenesis, and 3) inhibited proliferation. In contrast, knockdown of the beta1-integrin gene in ASM maintained on fibrillar collagen led to an increase in proliferation and reduced MMP-1 and -14 expression. Thus, ASM remodel the pericellular environment by degrading collagen fibrils and spinning them into larger collagen assemblies. Moreover, the collagen fibrils limit proliferation and activate autocrine MMPs in a beta-integrin-dependent manner, suggesting a potential negative feedback on modeling executed through fibrillar collagen activation of beta1-integrins.

Fibrillar collagen clamps lung mesenchymal cells in a nonproliferative and noncontractile phenotype.

Pulmonary fibrosis is characterized by phenotypic changes to mesenchymal cells and an increase in the deposition of fibrillar collagen (fCollagen). This study investigated the effect of type I fCollagen on the phenotypic plasticity of human parenchymal fibroblasts (PFbs) in vitro. Cell numbers were 45% lower when cultured on fCollagen as compared with culture on its degradation product, monomeric collagen (mCollagen). DNA profiles indicated that fCollagen is antiproliferative, rather than proapoptotic. fCollagen suppressed basic fibroblast growth factor-stimulated increases in the levels of cyclin E and CDK2 mRNA. fCollagen also suppressed transforming growth factor-beta (100 pM)-stimulated increases in the mRNA and protein levels of alpha-smooth muscle actin (alpha-SMA), a marker of the myofibroblast phenotype. However, in cells exposed to fCollagen, the levels of matrix metalloproteinase (MMP)-1 and -14 mRNA, as well as active MMP-2 protein, were increased by between two- and fivefold. The MMP inhibitors, ilomastat (10 microM) and doxycycline (30 microM), attenuated the dissolution of collagen fibrils by fibroblasts maintained on fCollagen, with a corresponding decrease in cell number. Ilomastat also reduced alpha-SMA expression and the capacity of PFb to contract three-dimensional fCollagen gels. Thus, exposure of fibroblasts to the fibrillar form of type I collagen in vitro reduces cell proliferation, increases MMP production and activation, and attenuates differentiation of PFb into myofibroblasts. fCollagen appears to apply a phenotypic clamp on lung fibroblasts that may be partially released by autocrine MMP activity.

A Non-canonical Pathway with Potential for Safer Modulation of Transforming Growth Factor-ß1 in Steroid-Resistant Airway Diseases.

Impaired therapeutic responses to anti-inflammatory glucocorticoids (GC) in chronic respiratory diseases are partly attributable to interleukins and transforming growth factor ß1 (TGF-ß1). However, previous efforts to prevent induction of GC insensitivity by targeting established canonical and non-canonical TGF-ß1 pathways have been unsuccessful. Here we elucidate a TGF-ß1 signaling pathway modulating GC activity that involves LIM domain kinase 2-mediated phosphorylation of cofilin1. Severe, steroid-resistant asthmatic airway epithelium showed increased levels of immunoreactive phospho-cofilin1. Phospho-cofilin1 was implicated in the activation of phospholipase D (PLD) to generate the effector(s) (lyso)phosphatidic acid, which mimics the TGF-ß1-induced GC insensitivity. TGF-ß1 induction of the nuclear hormone receptor corepressor, SMRT (NCOR2), was dependent on cofilin1 and PLD activities. Depletion of SMRT prevented GC insensitivity. This pathway for GC insensitivity offers several promising drug targets that potentially enable a safer approach to the modulation of TGF-ß1 in chronic inflammatory diseases than is afforded by global TGF-ß1 inhibition.

Targeted Graphene Oxide Networks: Cytotoxicity and Synergy with Anticancer Agents.

An effective strategy to inhibit endocytosis in cancer cells is presented where modified net-type graphene oxide (GO) sheets, bound with multiple cell surface receptors, are introduced and synthesized as novel anticancer agents. The results suggest that the binding connects GO sheets with neighboring lipid rafts, neutralizes endocytosis, and causes metabolic deprivation. As a result, tumor cell survival and proliferation are reduced. Live cell confocal microscopy imaging reveals that GO-PEGFA (folate-PEGylated GO) (PEG, polyethylene glycol) is internalized by tumor cells, while GO-PEGRGD (tripeptide Arg-Gly-Asp PEGylated GO) associates with the external cell membrane (not internalized). In vitro exposure of tumor cells to GO-PEGFA or GO-PEGRGD reduces the cell viability by 35%, compared to 50% reduction using methotrexate (100 µM). The combination of modified GO sheets with methotrexate or doxorubicin shows a greater toxicity (80% reduction in cell viability) than the individual agents. The proposed setup demonstrates a significant synergy in limiting tumor cell growth.

Casein Kinase 1δ/ε Inhibitor, PF670462 Attenuates the Fibrogenic Effects of Transforming Growth Factor-ß in Pulmonary Fibrosis.

Transforming growth factor-beta (TGF-ß) is a major mediator of fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). However, therapeutic global inhibition of TGF-ß is limited by unwanted immunosuppression and mitral valve defects. We performed an extensive literature search to uncover a little-known connection between TGF-ß signaling and casein kinase (CK) activity. We have examined the abundance of CK1 delta and epsilon (CK1δ/ε) in lung tissue from IPF patients and non-diseased controls, and investigated whether inhibition of CK1δ/ε with PF670462 inhibits pulmonary fibrosis. CK1δ/ε levels in lung tissue from IPF patients and non-diseased controls were assessed by immunohistochemistry. Anti-fibrotic effects of the CK1δ/ε inhibitor PF670462 were assessed in pre-clinical models, including acute and chronic bleomycin mouse models and in vitro experiments on spheroids made from primary human lung fibroblast cells from IPF and control donors, and human A549 alveolar-like adenocarcinoma-derived epithelial cells. Increased expression of CK1δ and ε in IPF lungs compared to non-diseased controls was accompanied by increased levels of the product, phospho-period 2. In vitro, PF670462 prevented TGF-ß-induced epithelial-mesenchymal transition. The stiffness of IPF-derived spheroids was reduced by PF670462 and TGF-ß-induced fibrogenic gene expression was inhibited. The CK1δ/ε inhibitor PF670462 administered systemically or locally by inhalation prevented both acute and chronic bleomycin-induced pulmonary fibrosis in mice. PF670462 administered in a 'therapeutic' regimen (day 7 onward) prevented bleomycin-induced lung collagen accumulation. Elevated expression and activity of CK1 δ and ε in IPF and anti-fibrogenic effects of the dual CK1δ/ε inhibitor, PF670462, support CK1δ/ε as novel therapeutic targets for IPF.

KCa3.1 Ca2+ activated K+ channels regulate human airway smooth muscle proliferation.

Airway smooth muscle cell hyperplasia contributes to airway remodeling and hyperreactivity characteristic of asthma. Changes to potassium channel activity in proliferating human airway smooth muscle (HASM) cells have been described, but no regulatory role in proliferation has been attributed to them. We sought to investigate the expression of the intermediate conductance calcium-activated potassium channel K(Ca)3.1 in HASM cells and investigate its role in proliferation. Smooth muscle cells derived from human airways were grown in vitro and K(Ca)3.1 channel expression was measured using Western blot, RT-PCR, and patch clamp electrophysiology. Pharmacologic inhibitors of the channel were used in assays of cellular proliferation, and flow cytometry was used to identify cell cycle regulation. HASM cells expressed K(Ca)3.1 channel mRNA, protein, and activity with up-regulation evident after transforming growth factor-beta stimulation. Pharmacologic inhibition of K(Ca)3.1 led to growth arrest in cells stimulated to proliferate with mitogens. These inhibitors did not cause cellular toxicity or induce apoptosis. We have demonstrated, for the first time, the expression of K(Ca)3.1 channels in HASM cells. In addition, we have shown that K(Ca)3.1 channels are important in HASM cell proliferation, making these channels a potential therapeutic target in airway remodeling.

The fibrogenic actions of lung fibroblast-derived urokinase: a potential drug target in IPF.

The role of urokinase plasminogen activator (uPA) in idiopathic pulmonary fibrosis (IPF) remains unclear. uPA-generated plasmin has potent fibrogenic actions involving protease activated receptor-1 (PAR-1) and interleukin-6 (IL-6). Here we characterize uPA distribution or levels in lung tissue and sera from IPF patients to establish the mechanism of its fibrogenic actions on lung fibroblasts (LFs). uPA immunoreactivity was detected in regions of fibrosis including fibroblasts of lung tissue from IPF patients (n = 7). Serum uPA levels and activity were also higher in IPF patients (n = 18) than controls (n = 18) (P < 0.05), being negatively correlated with lung function as measured by forced vital capacity (FVC) %predicted (P < 0.05). The culture supernatants of LFs from IPF patients, as compared to controls, showed an increase in plasmin activity after plasminogen incubation (5-15 µg/mL), corresponding with increased levels of uPA and IL-6 (n = 5-6, P < 0.05). Plasminogen-induced increases in plasmin activity and IL-6 levels were attenuated by reducing uPA and/or PAR-1 expression by RNAi. Plasmin(ogen)-induced mitogenesis was also attenuated by targeting uPA, PAR-1 or IL-6. Our data shows uPA is formed in active regions of fibrosis in IPF lung and contributes to LF plasmin generation, IL-6 production and proliferation. Urokinase is a potential target for the treatment of lung fibrosis.

Glucocorticoid resistance of migration and gene expression in a daughter MDA-MB-231 breast tumour cell line selected for high metastatic potential.

Glucocorticoids are commonly used to prevent chemotherapy-induced nausea and vomiting despite a lack of understanding of their direct effect on cancer progression. Recent studies suggest that glucocorticoids inhibit cancer cell migration. However, this action has not been investigated in estrogen receptor (ER)-negative breast tumour cells, although activation of the glucocorticoid receptor (GR) is associated with a worse prognosis in ER-negative breast cancers. In this study we have explored the effect of glucocorticoids on the migration of the ER-negative MDA-MB-231 human breast tumour cell line and the highly metastatic MDA-MB-231-HM.LNm5 cell line that was generated through in vivo cycling. We show for the first time that glucocorticoids inhibit 2- and 3-dimensional migration of MDA-MB-231 cells. Selection of cells for high metastatic potential resulted in a less migratory cell phenotype that was resistant to regulation by glucocorticoids and showed decreased GR receptor expression. The emergence of glucocorticoid resistance during metastatic selection may partly explain the apparent disparity between the clinical and in vitro evidence regarding the actions of glucocorticoids in cancer. These findings highlight the highly plastic nature of tumour cells, and underscore the need to more fully understand the direct effect of glucocorticoid treatment on different stages of metastatic progression.

2-methoxyestradiol is an estrogen receptor agonist that supports tumor growth in murine xenograft models of breast cancer.

PURPOSE: 2-Methoxyestradiol (2MEO) is being developed as a novel antitumor agent based on its antiangiogenic activity, tumor cell cytotoxicity, and apparent lack of toxicity. However, pharmacologic concentrations of 2MEO bind to estrogen receptors (ER). We have therefore examined the ER activity of 2MEO. EXPERIMENTAL DESIGN: Estrogenic actions of 2MEO were evaluated by changes in gene expression of the ER-positive (MCF7) breast tumor cell line and, in vivo, estrogenicity was assessed in breast tumor xenograft models and by measuring endocrine responses in uterus and liver. RESULTS: In the ER-positive breast tumor cell line (MCF7), microarray experiments revealed that 269 of 279 changes in gene expression common to 2MEO and estradiol were prevented by the ER antagonist, ICI 182,780. Changes in the expression of selected genes and their sensitivity to inhibition by ICI 182,780 were confirmed by quantitative reverse transcription-PCR measurement. Activation of ER in MCF7 cells by 2MEO was further confirmed by stimulation of an estrogen response element-dependent reporter gene that was blocked by ICI 182,780 (1 micromol/L). Doses of 2MEO (15-150 mg/kg) that had no antitumor efficacy in either nu/nu BALB/c or severe combined immunodeficient mice bearing ER-negative MDA-MB-435 tumors had uterotropic and hepatic estrogen-like actions. In female nu/nu BALB/c mice inoculated with the estrogen-dependent MCF7 tumor cells, 2MEO (50 mg/kg/d) supported tumor growth. CONCLUSIONS: Tumor growth enhancement by 2MEO at doses generating serum levels (100-500 nmol/L) that have estrogenic activity suggests that a conservative approach to the further clinical evaluation of this agent should be adopted and that its evaluation in breast cancer is inappropriate.