Myron L. Toews

Inhbition of invasion of murine mammary carcinoma cells by the tyrosine kinase inhibitor genistein

Tyrosine kinases are ubiquitous enzymes that have been shown to be involved in many cellular functions, including growth and differentiation. Recent studies have shown that they are also involved in integrin signal transduction pathways. Since integrins are known to be involved in cellular adhesion and thus in invasion and metastasis, the possible involvement of tyrosine kinases in invasion was tested. Tumor cell invasion was measured using filter inserts coated with Matrigel, a substance that closely resembles the natural basement membrane. A highly metastatic subline of BALB/c mammary carcinoma (410.4) cells was shown to invade nearly three times as much as a low metastatic subline (168.1). Genistein, an inhibitor of tyrosine kinases, was found to inhibit invasion of 410.4 cells with an EC50 of approximately 1 microM. At a concentration of 37 microM, there was almost complete inhibition of invasion by genistein, whereas the structural analog, daidzein, which does not inhibit tyrosine kinases, had only a small effect. At higher concentrations (370 microM), daidzein also caused marked inhibition. Genistein was able to inhibit invasion at concentrations having little effect on cell growth. However, for daidzein, most of the effect on invasion was apparently due to its effect on growth inhibition. The relatively specific effect of genistein to inhibit tumor invasion suggests a role for tyrosine phosphorylation in this process. Genistein or other tyrosine kinase inhibitors may be effective inhibitors of tumor invasion and metastasis.

Lysophosphatidic acid in airway function and disease

Lysophosphatidic acid (LPA) is a bioactive lipid mediator and important component of serum. Studies over the past several years which have documented diverse effects of LPA on multiple types of airway cells and which suggest possible involvement of LPA in lung disease are reviewed here. LPA enhances contractility of airway smooth muscle. It also stimulates proliferation of cultured airway smooth muscle cells and exhibits a striking synergism with epidermal growth factor (EGF) for stimulating mitogenesis. Recent studies of the molecular components and signaling pathways mediating synergism are described, including LPA-induced upregulation of EGF receptors and activation of multiple transcription factors by both LPA and EGF. A model for the effects of LPA and EGF on mitogenesis that includes EGF receptor upregulation and synergism between Ras and Rho for activation of the transcription factor AP-1 is presented. LPA stimulates fibronectin secretion and filopodia extension in airway epithelial cells as well as proliferation and collagen gel contraction by lung fibroblasts. A hypothesis for LPA involvement in the airway repair and remodeling, which contribute to the pathology of asthma and other airway diseases, is presented, and future directions for research into the roles of LPA in airway function and disease are suggested.

Radioligand Binding Methods for Membrane Preparations and Intact Cells

The radioligand binding assay is a relatively simple but powerful tool for studying G protein-coupled receptors. There are three basic types of radioligand binding experiments: (1) saturation experiments from which the affinity of the radioligand for the receptor and the binding site density can be determined; (2) inhibition experiments from which the affinity of a competing, unlabeled compound for the receptor can be determined; and (3) kinetic experiments from which the forward and reverse rate constants for radioligand binding can be determined. Detailed methods for typical radioligand binding assays for G protein-coupled receptors in membranes and intact cells are presented for these types of experiments. Detailed procedures for analysis of the data obtained from these experiments are also given.

Transcription factor activation and mitogenic synergism in airway smooth muscle cells.

Simultaneous treatment of human airway smooth muscle (HASM) cells with lysophosphatidic acid (LPA) and epidermal growth factor (EGF) leads to strikingly synergistic stimulation of mitogenesis. The purpose of this study was to explore potential sites for signal integration mediating synergism, focusing on extracellular signal-regulated kinase (ERK) and transcription factors involved in proliferation and inflammation as likely candidates. Activation of ERK was analysed by immunoblotting. Transcription factor activation was assessed using HASM cells transduced with luciferase reporter gene constructs. LPA and EGF both activated ERK but had no synergistic effect when combined. LPA and EGF both activated activator protein (AP)‐1, cyclic adenosine monophosphate response element-binding protein, nuclear factor of activated T‐cells and the serum response element; however, only AP‐1 activation exhibited synergism. Activation of the inhibitory guanine nucleotide-binding protein and of ERK signalling pathways were required for most transcription factor responses to LPA. In contrast, nuclear factor (NF)‐κB was activated by LPA but not EGF and NF‐κB activation was completely blocked only when Rho was inhibited. Rapid activation of Rho was observed in response to LPA but not to EGF. Importantly, inhibition of Rho selectively blocked synergism in both AP‐1 activation and mitogenesis. In summary, extracellular signal-regulated kinase activation is required for many transcription factor responses to lysophosphatidic acid and epidermal growth factor, however it is not synergistic. Activation of activator protein‐1 is synergistic, and Rho activation by lysophosphatidic acid is required for synergism in both activator protein‐1 activation and mitogenesis.

Pharmacological inhibition of soluble epoxide hydrolase prevents renal interstitial fibrogenesis in obstructive nephropathy

Treating chronic kidney disease (CKD) has been challenging because of its pathogenic complexity. Epoxyeicosatrienoic acids (EETs) are cytochrome P-450-dependent derivatives of arachidonic acid with antihypertensive, anti-inflammatory, and profibrinolytic functions. We recently reported that genetic ablation of soluble epoxide hydrolase (sEH), an enzyme that converts EETs to less active dihydroxyeicosatrienoic acids, prevents renal tubulointerstitial fibrosis and inflammation in experimental mouse models of CKD. Here, we tested the hypothesis that pharmacological inhibition of sEH after unilateral ureteral obstruction (UUO) would attenuate tubulointerstitial fibrosis and inflammation in mouse kidneys and may provide a novel approach to manage the progression of CKD. Inhibition of sEH enhanced levels of EET regioisomers and abolished tubulointerstitial fibrosis, as demonstrated by reduced collagen deposition and myofibroblast formation after UUO. The inflammatory response was also attenuated, as demonstrated by decreased influx of neutrophils and macrophages and decreased expression of inflammatory cytokines keratinocyte chemoattractant, macrophage inflammatory protein-2, monocyte chemotactic protein-1, TNF-α, and ICAM-1 in kidneys after UUO. UUO upregulated transforming growth factor-β1/Smad3 signaling and induced NF-κB activation, oxidative stress, tubular injury, and apoptosis; in contrast, it downregulated antifibrotic factors, including peroxisome proliferator-activated receptor (PPAR) isoforms, especially PPAR-γ. sEH inhibition mitigated the aforementioned malevolent effects in UUO kidneys. These data demonstrate that pharmacological inhibition of sEH promotes anti-inflammatory and fibroprotective effects in UUO kidneys by preventing tubular injury, downregulation of NF-κB, transforming growth factor-β1/Smad3, and inflammatory signaling pathways, and activation of PPAR isoforms. Our data suggest the potential use of sEH inhibitors in treating fibrogenesis in the UUO model of CKD.

Rapid and Reversible Enhancement of Blood–Brain Barrier Permeability Using Lysophosphatidic Acid

The present study characterizes the effects of lysophosphatidic acid (LPA) on blood–brain barrier (BBB) permeability focusing specifically on the time of onset, duration, and magnitude of LPA-induced changes in cerebrovascular permeability in the mouse using both magnetic resonance imaging (MRI) and near infrared fluorescence imaging (NIFR). Furthermore, potential application of LPA for enhanced drug delivery to the brain was also examined by measuring the brain accumulation of radiolabeled methotrexate. Exposure of primary cultured brain microvessel endothelial cells (BMECs) to LPA produced concentration-dependent increases in permeability that were completely abolished by clostridium toxin B. Administration of LPA disrupted BBB integrity and enhanced the permeability of small molecular weight marker gadolinium diethylenetriaminepentaacetate (Gd-DTPA) contrast agent, the large molecular weight permeability marker, IRdye800cwPEG, and the P-glycoprotein efflux transporter probe, Rhodamine 800 (R800). The increase in BBB permeability occurred within 3 minutes after LPA injection and barrier integrity was restored within 20 minutes. A decreased response to LPA on large macromolecule BBB permeability was observed after repeated administration. The administration of LPA also resulted in 20-fold enhancement of radiolabeled methotrexate in the brain. These studies indicate that administration of LPA in combination with therapeutic agents may increase drug delivery to the brain.

Sphingosine 1-Phosphate Potentiates Human Lung Fibroblast Chemotaxis through the S1P2 Receptor

Migration of fibroblasts plays an essential role in tissue repair after injury. Sphingosine 1-phosphate (S1P) is a multifunctional mediator released by many cells that can be released in inflammation and after injury. This study evaluated the effect of S1P on fibroblast chemotaxis toward fibronectin. S1P alone did not affect fibroblast migration, but S1P enhanced fibronectin-directed chemotaxis in a concentration-dependent manner. The effect of S1P was not mimicked by dihydro (dh) S1P or the S1P(1) receptor agonist SEW2871. S1P augmentation of fibroblast chemotaxis, however, was completely blocked by JTE-013, an S1P(2) antagonist, but not by suramin, an S1P(3) antagonist. Suppression of the S1P(2) receptor by small interfering (si)RNA also completely blocked S1P augmentation of fibroblast chemotaxis to fibronectin. S1P stimulated Rho activation and focal adhesion kinase (FAK) phosphorylation, and these were also significantly inhibited by the S1P(2) receptor antagonist (JTE-013) or by S1P(2) siRNA. Further, the potentiation of S1P signaling was blocked by the Rho-kinase inhibitor Y-27632 in a concentration-dependent manner. Inhibition of FAK with siRNA reduced basal chemotaxis toward fibronectin slightly but significantly, and almost completely blocked S1P augmented chemotaxis. These results suggest that S1P-augmented fibroblast chemotaxis toward fibronectin depends on the S1P(2) receptor and requires Rho and Rho-kinase, and FAK phosphorylation. By augmenting fibroblast recruitment, S1P has the potential to modulate tissue repair after injury. The pathways by which S1P mediates this effect, therefore, represent a potential therapeutic target to affect tissue repair and remodeling.

Prostaglandin E2 Inhibits Human Lung Fibroblast Chemotaxis through Disparate Actions on Different E-Prostanoid Receptors

The migration of fibroblasts is believed to play a key role in both normal wound repair and abnormal tissue remodeling. Prostaglandin E (PGE)(2), a mediator that can inhibit many fibroblast functions including chemotaxis, was reported to be mediated by the E-prostanoid (EP) receptor EP2. PGE(2), however, can act on four receptors. This study was designed to determine if EP receptors, in addition to EP2, can modulate fibroblast chemotaxis. Using human fetal lung fibroblasts, the expression of all four EP receptors was demonstrated by Western blotting. EP2-selective and EP4-selective agonists inhibited both chemotaxis toward fibronectin in the blindwell assay and migration in a wound-closure assay. In contrast, EP1-selective and EP3-selective agonists stimulated cell migration in both assay systems. These results were confirmed using EP-selective antagonists. The role of both EP2 and EP4 receptors in mediating the PGE(2) inhibition of chemotaxis was also confirmed by small interfering RNA suppression. Furthermore, the role of EP receptors was confirmed by blocking the expected signaling pathways. Taken together, these results demonstrate that PGE(2) can act on multiple EP receptors in human lung fibroblasts, to exert disparate effects. Alterations in EP receptor expression may have the potential to alter PGE(2) action. Targeting specific EP receptors may offer therapeutic opportunities in conditions characterized by abnormal tissue repair and remodeling.

Replicated, replicable and relevant–target engagement and pharmacological experimentation in the 21st century

A pharmacological experiment is typically conducted to: i) test or expand a hypothesis regarding the potential role of a target in the mechanism(s) underlying a disease state using an existing drug or tool compound in normal and/or diseased tissue or animals; or ii) characterize and optimize a new chemical entity (NCE) targeted to modulate a specific disease-associated target to restore homeostasis as a potential drug candidate. Hypothesis testing necessitates an intellectually rigorous, null hypothesis approach that is distinct from a high throughput fishing expedition in search of a hypothesis. In conducting an experiment, the protocol should be transparently defined along with its powering, design, appropriate statistical analysis and consideration of the anticipated outcome (s) before it is initiated. Compound-target interactions often involve the direct study of phenotype(s) unique to the target at the cell, tissue or animal/human level. However, in vivo studies are often compromised by a lack of sufficient information on the compound pharmacokinetics necessary to ensure target engagement and also by the context-free analysis of ubiquitous cellular signaling pathways downstream from the target. The use of single tool compounds/drugs at one concentration in engineered cell lines frequently results in reductionistic data that have no physiologically relevance. This overview, focused on trends in the peer-reviewed literature, discusses the execution and reporting of experiments and the criteria recommended for the physiologically-relevant assessment of target engagement to identify viable new drug targets and facilitate the advancement of translational studies.

Prostaglandin E2 Stimulates the Production of Vascular Endothelial Growth Factor through the E-Prostanoid–2 Receptor in Cultured Human Lung Fibroblasts

Fibroblasts are the major mesenchymal cells present within the interstitium of the lung and are a major source of vascular endothelial growth factor (VEGF), which modulates the maintenance of pulmonary microvasculature. Prostaglandin E(2) (PGE(2)) acts on a set of E-prostanoid (EP) receptors that activate multiple signal transduction pathways leading to downstream responses. We investigated the modulation by PGE(2) of VEGF release by human lung fibroblasts. Human lung fibroblasts were cultured until reaching 90% confluence in tissue culture plates, after which the culture media were changed to serum-free Dulbeccos modified Eagles medium, with or without PGE(2), and with specific agonists or antagonists for each EP receptor. After 2 days, culture media were assayed for VEGF by ELISA. The results demonstrated that PGE(2) and the EP2 agonist ONO-AE1-259-01 significantly stimulated the release of VEGF in a concentration-dependent manner. Agonists for other EP receptors did not stimulate the release of VEGF. The stimulatory effect of PGE(2) was blocked by the EP2 antagonist AH6809, but was not blocked by antagonists for other EP receptors. The protein kinase-A (PKA) inhibitor KT-5720 also blocked the stimulatory effect of PGE(2). The increased release of VEGF induced by PGE(2) was accompanied by a transient increase in the concentration of VEGF mRNA. These findings demonstrate that PGE(2) can modulate the release of VEGF by human lung fibroblasts through its actions in the EP2 receptor/PKA pathway. This activity may contribute to the maintenance of pulmonary microvasculature in the alveolar wall.