Discovery of Novel Oleamide Analogues as Brain-Penetrant Positive Allosteric Serotonin 5-HT2C Receptor and Dual 5-HT2C/5-HT2A Receptor Modulators.

The serotonin 5-HT2A receptor (5-HT2AR) and 5-HT2CR localize to the brain and share overlapping signal transduction facets that contribute to their roles in cognition, mood, learning, and memory. Achieving selective targeting of these receptors is challenged by the similarity in their 5-HT orthosteric binding pockets. A fragment-based discovery approach was employed to design and synthesize novel oleamide analogues as selective 5-HT2CR or dual 5-HT2CR/5-HT2AR positive allosteric modulators (PAMs). Compound 13 (JPC0323) exhibited on-target properties, acceptable plasma exposure and brain penetration, as well as negligible displacement to orthosteric sites of ∼50 GPCRs and transporters. Furthermore, compound 13 suppressed novelty-induced locomotor activity in a 5-HT2CR-dependent manner, suggesting 5-HT2CR PAM, but not 5-HT2AR, activity at the level of the whole organism at the employed doses of 13. We discovered new selective 5-HT2CR PAMs and first-in-class 5-HT2CR/5-HT2AR dual PAMs that broaden the pharmacological toolbox to explore the biology of these vital receptors.

Discovery of 4-Phenylpiperidine-2-Carboxamide Analogues as Serotonin 5-HT2C Receptor-Positive Allosteric Modulators with Enhanced Drug-like Properties.

Targeting the serotonin (5-HT) 5-HT2C receptor (5-HT2CR) allosteric site to potentiate endogenous 5-HT tone may provide novel therapeutics to alleviate the impact of costly, chronic diseases such as obesity and substance use disorders. Expanding upon our recently described 5-HT2CR-positive allosteric modulators (PAMs) based on the 4-alkylpiperidine-2-carboxamide scaffold, we optimized the undecyl moiety at the 4-position with variations of cyclohexyl- or phenyl-containing fragments to reduce rotatable bonds and lipophilicity. Compound 12 (CTW0415) was discovered as a 5-HT2CR PAM with improved pharmacokinetics and reduced off-target interactions relative to our previous series of molecules. The in vivo efficacy of compound 12 to potentiate the effects of a selective 5-HT2CR agonist was established in a drug discrimination assay. Thus, 12 is reported as a 5-HT2CR PAM with characteristics suitable for in vivo pharmacological studies to further probe the biological and behavioral mechanisms of allosteric modulation of a receptor important in several chronic diseases.

Activation of Human Peripheral Blood Eosinophils by Cytokines in a Comparative Time-Course Proteomic/Phosphoproteomic Study.

Activated eosinophils contribute to airway dysfunction and tissue remodeling in asthma and thus are considered to be important factors in asthma pathology. We report here comparative proteomic and phosphoproteomic changes upon activation of eosinophils using eight cytokines individually and in selected cytokine combinations in time-course reactions. Differential protein and phosphoprotein expressions were determined by mass spectrometry after 2-dimensional gel electrophoresis (2DGE) and by LC-MS/MS. We found that each cytokine-stimulation produced significantly different changes in the eosinophil proteome and phosphoproteome, with phosphoproteomic changes being more pronounced and having an earlier onset. Furthermore, we observed that IL-5, GM-CSF, and IL-3 showed the greatest change in protein expression and phosphorylation, and this expression differed markedly from those of the other five cytokines evaluated. Comprehensive univariate and multivariate statistical analyses were employed to evaluate the comparative results. We also monitored eosinophil activation using flow cytometry (FC) analysis of CD69. In agreement with our proteomic studies, FC indicated that IL-5, GM-CSF, and IL-3 were more effective than the other five cytokines studied in stimulating a cell surface CD69 increase indicative of eosinophil activation. Moreover, selected combinations of cytokines revealed proteomic patterns with many proteins in common with single cytokine expression patterns but also showed a greater effect of the two cytokines employed, indicating a more complex signaling pathway that was reflective of a more typical inflammatory pathology.

Cytokine-Induced Glucocorticoid Resistance from Eosinophil Activation: Protein Phosphatase 5 Modulation of Glucocorticoid Receptor Phosphorylation and Signaling.

The mechanisms contributing to persistent eosinophil activation and poor eosinopenic response to glucocorticoids in severe asthma are poorly defined. We examined the effect of cytokines typically overexpressed in the asthmatic airways on glucocorticoid signaling in in vitro activated eosinophils. An annexin V assay used to measure eosinophil apoptosis showed that cytokine combinations of IL-2 plus IL-4 as well as TNF-α plus IFN-γ, or IL-3, GM-CSF, and IL-5 alone significantly diminished the proapoptotic response to dexamethasone. We found that IL-2 plus IL-4 resulted in impaired phosphorylation and function of the nuclear glucocorticoid receptor (GCR). Proteomic analysis of steroid sensitive and resistant eosinophils identified several differentially expressed proteins, namely protein phosphatase 5 (PP5), formyl peptide receptor 2, and annexin 1. Furthermore, increased phosphatase activity of PP5 correlated with impaired phosphorylation of the GCR. Importantly, suppression of PP5 expression with small interfering RNA restored proper phosphorylation and the proapoptotic function of the GCR. We also examined the effect of lipoxin A4 on PP5 activation by IL-2 plus IL-4. Similar to PP5 small interfering RNA inhibition, pretreatment of eosinophils with lipoxin A4 restored GCR phosphorylation and the proaptoptotic function of GCs. Taken together, our results showed 1) a critical role for PP5 in cytokine-induced resistance to GC-mediated eosinophil death, 2) supported the dependence of GCR phosphorylation on PP5 activity, and 3) revealed that PP5 is a target of the lipoxin A4-induced pathway countering cytokine-induced resistance to GCs in eosinophils.

Proteomic Analysis in Esophageal Eosinophilia Reveals Differential Galectin-3 Expression and S-Nitrosylation.

BACKGROUND AIMS: Esophageal eosinophilia (EE) can be caused by gastroesophageal reflux disease (GERD), proton-pump inhibitor-responsive EE (PPI-REE) or eosinophilic esophagitis (EoE). This study quantified protein expression and S-nitrosylation (SNO) post-translational modifications in EE to elucidate potential disease biomarkers. METHODS: Proximal and distal esophageal (DE) biopsy proteins in patients with EE and in controls were assayed for protein content and fluorescence-labeled with and without ascorbate treatment. Protein SNO was determined, and selected protein spots were identified by matrix-assisted laser desorption ionization time-of-flight/mass spectrometry. Western blot and ingenuity pathway analysis were performed. RESULTS: Ninety-one of 648 proteins showed differential expression. There were significantly altered levels of abundance for 11 proximal and 14 DE proteins. Hierarchal clustering revealed differential SNO in inflamed tissues, indicating reactive nitrogen/oxygen species involvement. Galectin-3 was upregulated in both proximal (p < 0.04) and distal (p < 0.004) esophageal EE biopsies compared to controls. In distal EE samples, galectin-3 was significantly S-nitrosylated (p < 0.004). Principal component analysis revealed sample group discrimination distally. CONCLUSION: Proteomic analysis in EE esophageal mucosa revealed a distinct abundance and nitrosylation profile, most prominently in distal biopsies. Galectin-3 was upregulated in expression and SNO, which may indicate its potential role in mucosal inflammation. These results call for more studies to be performed to investigate the role of galectin-3 in GERD, PPI-REE and EoE.

Eosinophil resistance to glucocorticoid-induced apoptosis is mediated by the transcription factor NFIL3.

The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the inhibition of inflammatory signaling and induction of eosinophil apoptosis. However, laboratory and clinical observations of GC-resistant asthma suggest that GCs' effects on eosinophil viability may depend on the state of eosinophil activation. In the present study we demonstrate that eosinophils stimulated with IL-5 show impaired pro-apoptotic response to GCs. We sought to determine the contribution of GC-mediated transactivating (TA) and transrepressing (TR) pathways in modulation of activated eosinophils' response to GC by comparing their response to the selective GC receptor (GR) agonist Compound A (CpdA) devoid of TA activity to that upon treatment with Dexamethasone (Dex). IL-5-activated eosinophils showed contrasting responses to CpdA and Dex, as IL-5-treated eosinophils showed no increase in apoptosis compared to cells treated with Dex alone, while CpdA elicited an apoptotic response regardless of IL-5 stimulation. Proteomic analysis revealed that both Nuclear Factor IL-3 (NFIL3) and Map Kinase Phosphatase 1 (MKP1) were inducible by IL-5 and enhanced by Dex; however, CpdA had no effect on NFIL3 and MKP1 expression. We found that inhibiting NFIL3 with specific siRNA or by blocking the IL-5-inducible Pim-1 kinase abrogated the protective effect of IL-5 on Dex-induced apoptosis, indicating crosstalk between IL-5 anti-apoptotic pathways and GR-mediated TA signaling occurring via the NFIL3 molecule. Collectively, these results indicate that (1) GCs' TA pathway may support eosinophil viability in IL-5-stimulated cells through synergistic upregulation of NFIL3; and (2) functional inhibition of IL-5 signaling (anti-Pim1) or the use of selective GR agonists that don't upregulate NFIL3 may be effective strategies for the restoring pro-apoptotic effect of GCs on IL-5-activated eosinophils.

Systems biology approaches to understanding Epithelial Mesenchymal Transition (EMT) in mucosal remodeling and signaling in asthma.

A pathological hallmark of asthma is chronic injury and repair, producing dysfunction of the epithelial barrier function. In this setting, increased oxidative stress, growth factor- and cytokine stimulation, together with extracellular matrix contact produces transcriptional reprogramming of the epithelial cell. This process results in epithelial-mesenchymal transition (EMT), a cellular state associated with loss of epithelial polarity, expression of mesenchymal markers, enhanced mobility and extracellular matrix remodeling. As a result, the cellular biology of the EMT state produces characteristic changes seen in severe, refractory asthma: myofibroblast expansion, epithelial trans-differentiation and subepithelial fibrosis. EMT also induces profound changes in epithelial responsiveness that affects innate immune signaling that may have impact on the adaptive immune response and effectiveness of glucocorticoid therapy in severe asthma. We discuss how this complex phenotype is beginning to be understood using systems biology-level approaches through perturbations coupled with high throughput profiling and computational modeling. Understanding the distinct changes induced by EMT at the systems level may provide translational strategies to reverse the altered signaling and physiology of refractory asthma.

Functional proteomics for the characterization of impaired cellular responses to glucocorticoids in asthma.

In chronic airway inflammatory disorders, such as asthma, glucocorticoid (GC) insensitivity is a challenging clinical problem associated with life-threatening disease progression and the potential development of serious side effects. The mechanism of steroid resistance in asthma remains unclear and may be multifactorial. Excluding noncompliance with GC treatment, abnormal steroid pharmacokinetics, and rare genetic defects in the glucocorticoid receptor (GR), the majority of GC insensitivity in asthma can be attributed to secondary defects related to GR function. Airway inflammatory cells obtained from patients with GC-resistant asthma show a number of abnormalities in cell immune responses to GC, which suggests that there is a causative defect in GR signaling in GC-resistant cells that could be further elucidated by a functional and molecular proteomics approach. Since T cells, eosinophils, and monocytes play a major role in the pathogenesis of airway inflammation, most of the work published to date has focused on these cell types as the primary therapeutic targets in GC-insensitive asthma. We herein review several distinct techniques for the assessment of (1) the cellular response to GCs including the effect of GCs on cell viability, adhesion, and mediator release; (2) the functionality of GC receptors, including phosphorylation of the GR, nuclear translocation, and binding activities; and (3) the characterization of proteins differentially expressed in steroid-resistant cells by comparative 2DE-gel electrophoresis-based techniques and mass spectrometry. These comprehensive approaches are expected to reveal novel candidates for biomarkers of steroid insensitivity, which may lead to the development of effective therapeutic interventions for patients with chronic steroid-resistant asthma.

Altered eosinophil proteome in a patient with hypereosinophilia from acute fascioliasis.

We used comparative proteomics to analyze eosinophils from a patient with hypereosinophilia due to fascioliasis. Using 2-dimensional electrophoresis and mass spectrometry, we demonstrated that the eosinophil proteome was significantly altered compared to those of healthy controls.

Priming of eosinophils by GM-CSF is mediated by protein kinase CbetaII-phosphorylated L-plastin.

The priming of eosinophils by cytokines leading to augmented response to chemoattractants and degranulating stimuli is a characteristic feature of eosinophils in the course of allergic inflammation and asthma. Actin reorganization and integrin activation are implicated in eosinophil priming by GM-CSF, but their molecular mechanism of action is unknown. In this regard, we investigated the role of L-plastin, an eosinophil phosphoprotein that we identified from eosinophil proteome analysis. Phosphoproteomic analysis demonstrated the upregulation of phosphorylated L-plastin after eosinophil stimulation with GM-CSF. Additionally, coimmunoprecipitation studies demonstrated a complex formation of phosphorylated L-plastin with protein kinase CßII (PKCßII), GM-CSF receptor α-chain, and two actin-associated proteins, paxilin and cofilin. Inhibition of PKCßII with 4,5-bis(4-fluoroanilino)phtalimide or PKCßII-specific small interfering RNA blocked GM-CSF-induced phosphorylation of L-plastin. Furthermore, flow cytometric analysis also showed an upregulation of α(M)ß(2) integrin, which was sensitive to PKCßII inhibition. In chemotaxis assay, GM-CSF treatment allowed eosinophils to respond to lower concentrations of eotaxin, which was abrogated by the above-mentioned PKCßII inhibitors. Similarly, inhibition of PKCßII blocked GM-CSF induced priming for degranulation as assessed by release of eosinophil cationic protein and eosinophil peroxidase in response to eotaxin. Importantly, eosinophil stimulation with a synthetic L-plastin peptide (residues 2-19) phosphorylated on Ser(5) upregulated α(M)ß(2) integrin expression and increased eosinophil migration in response to eotaxin independent of GM-CSF stimulation. Our results establish a causative role for PKCßII and L-plastin in linking GM-CSF-induced eosinophil priming for chemotaxis and degranulation to signaling events associated with integrin activation via induction of PKCßII-mediated L-plastin phosphorylation.

Molecular biological effects of selective neuronal nitric oxide synthase inhibition in ovine lung injury.

Neuronal nitric oxide synthase is critically involved in the pathogenesis of acute lung injury resulting from combined burn and smoke inhalation injury. We hypothesized that 7-nitroindazole, a selective neuronal nitric oxide synthase inhibitor, blocks central molecular mechanisms involved in the pathophysiology of this double-hit insult. Twenty-five adult ewes were surgically prepared and randomly allocated to 1) an uninjured, untreated sham group (n = 7), 2) an injured control group with no treatment (n = 7), 3) an injury group treated with 7-nitroindazole from 1-h postinjury to the remainder of the 24-h study period (n = 7), or 4) a sham-operated group subjected only to 7-nitroindazole to judge the effects in health. The combination injury was associated with twofold increased activity of neuronal nitric oxide synthase and oxidative/nitrosative stress, as indicated by significant increases in plasma nitrate/nitrite concentrations, 3-nitrotyrosine (an indicator of peroxynitrite formation), and malondialdehyde lung tissue content. The presence of systemic inflammation was evidenced by twofold, sixfold, and threefold increases in poly(ADP-ribose) polymerase, IL-8, and myeloperoxidase lung tissue concentrations, respectively (each P < 0.05 vs. sham). These molecular changes were linked to tissue damage, airway obstruction, and pulmonary shunting with deteriorated gas exchange. 7-Nitroindazole blocked, or at least attenuated, all these pathological changes. Our findings suggest 1) that nitric oxide formation derived from increased neuronal nitric oxide synthase activity represents a pivotal reactive agent in the patho-physiology of combined burn and smoke inhalation injury and 2) that selective neuronal nitric oxide synthase inhibition represents a goal-directed approach to attenuate the degree of injury.

Toward the Proteome of the Human Peripheral Blood Eosinophil.

Eosinophils are granular leukocytes that have significant roles in many inflammatory and immunoregulatory responses, especially asthma and allergic diseases. We have undertaken a fairly comprehensive proteomic analysis of purified peripheral blood eosinophils from normal human donors primarily employing 2-dimensional gel electrophoresis with protein spot identification by matrix-assisted laser desorption/ionization mass spectrometry. Protein subfractionation methods employed included isoelectric focusing (Zoom(®) Fractionator) and subcellular fractionation using differential protein solubilization. We have identified 3,141 proteins which had Mascot expectation scores of 10(-3) or less. Of these 426 were unique and non-redundant of which 231 were novel proteins not previously reported to occur in eosinophils. Ingenuity Pathway Analysis showed that some 70% of the non-redundant proteins could be subdivided into categories that are clearly related to currently known eosinophil biological activities. Cytoskeletal and associated proteins predominated among the proteins identified. Extensive protein posttranslational modifications were evident, many of which have not been previously reported that reflected the dynamic character of the eosinophil. This dataset of eosinophilic proteins will prove valuable in comparative studies of disease versus normal states and for studies of gender differences and polymorphic variation among individuals.

Lipoxin A4 counterregulates GM-CSF signaling in eosinophilic granulocytes.

Eosinophils are granulated leukocytes that are involved in many inflammation-associated pathologies including airway inflammation in asthma. Resolution of eosinophilic inflammation and return to homeostasis is in part due to endogenous chemical mediators, for example, lipoxins, resolvins, and protectins. Lipoxins are endogenous eicosanoids that demonstrate antiinflammatory activity and are synthesized locally at sites of inflammation. In view of the importance of lipoxins (LXs) in resolving inflammation, we investigated the molecular basis of LXA(4) action on eosinophilic granulocytes stimulated with GM-CSF employing the eosinophilic leukemia cell line EoL-1 as well as peripheral blood eosinophils. We report herein that LXA(4) (1-100 nM) decreased protein tyrosine phosphorylation in EoL-1 cells stimulated with GM-CSF. Additionally, the expression of a number of GM-CSF-induced cytokines was inhibited by LXA(4) in a dose-dependent manner. Furthermore, using a proteomics approach involving mass spectrometry and immunoblot analysis we identified 11 proteins that were tyrosine phosphorylated after GM-CSF stimulation and whose phosphorylation was significantly inhibited by LXA(4) pretreatment. Included among these 11 proteins were alpha-fodrin (nonerythroid spectrin) and actin. Microscopic imaging showed that treatment of EoL-1 cells or blood eosinophils with GM-CSF resulted in the reorganization of actin and the translocation of alpha-fodrin from the cytoplasm to the plasma membrane. Importantly, alpha-fodrin translocation was prevented by LXA(4) but actin reorganization was not. Thus, the mechanism of LXA(4) action likely involves prevention of activation of eosinophilic granulocytes by GM-CSF through inhibition of protein tyrosine phosphorylation and modification of some cytoskeletal components.

Cross-talk between ICAM-1 and granulocyte-macrophage colony-stimulating factor receptor signaling modulates eosinophil survival and activation.

Reversal of eosinophilic inflammation has been an elusive therapeutic goal in the management of asthma pathogenesis. In this regard, GM-CSF is a primary candidate cytokine regulating eosinophil activation and survival in the lung; however, its molecular mechanism of propagation and maintenance of stimulated eosinophil activation is not well understood. In this study, we elucidate those late interactions occurring between the GM-CSF receptor and activated eosinophil signaling molecules. Using coimmunoprecipitation with GM-CSF-stimulated eosinophils, we have identified that the GM-CSF receptor beta-chain (GMRbeta) interacted with ICAM-1 and Shp2 phosphatase, as well as Slp76 and ADAP adaptor proteins. Separate experiments using affinity binding with a tyrosine-phosphorylated peptide containing an ITIM (ICAM-1 residues 480-488) showed binding to Shp2 phosphatase and GMRbeta. However, the interaction of GMRbeta with the phosphorylated ICAM-1-derived peptide was observed only with stimulated eosinophil lysates, suggesting that the interaction of GMRbeta with ICAM-1 required phosphorylated Shp2 and/or phosphorylated GMRbeta. Importantly, we found that inhibition of ICAM-1 in activated eosinophils blocked GM-CSF-induced expression of c-fos, c-myc, IL-8, and TNF-alpha. Moreover, inhibition of ICAM-1 expression with either antisense oligonucleotide or an ICAM-1-blocking Ab effectively inhibited ERK activation and eosinophil survival. We concluded that the interaction between ICAM-1 and the GM-CSF receptor was essential for GM-CSF-induced eosinophil activation and survival. Taken together, these results provide novel mechanistic insights defining the interaction between ICAM-1 and the GM-CSF receptor and highlight the importance of targeting ICAM-1 and GM-CSF/IL-5/IL-3 receptor systems as a therapeutic strategy to counter eosinophilia in asthma.

Negative transcriptional regulation of human colonic smooth muscle Cav1.2 channels by p50 and p65 subunits of nuclear factor-kappaB.

BACKGROUND & AIMS: The expression of Cav1.2 channels in colonic circular smooth muscle cells and the contractility of these cells are suppressed in inflammation. Our aim was to investigate whether the activation of p50 and p65 nuclear factor-kappaB subunits mediates these effects. METHODS: Primary cultures of human colonic circular smooth muscle cells and muscle strips were used. RESULTS: The messenger RNA and protein expression of the pore-forming alpha1C subunit of Cav1.2 channels decreased time dependently in response to tumor necrosis factor alpha. This effect was blocked by prior transient transfection of the cells with antisense oligonucleotides to p50 or p65. The overexpression of p50 and p65 inhibited the constitutive expression of alpha1C. Three putative kappaB binding motifs were identified on the 5' flanking region of exon 1b of the human L-type calcium channel alpha1C gene. Progressive 5' deletions of the promoter and point mutations of the kappaB binding motifs indicated that the two 5' binding sites, but not the third 3' binding site, were essential for the suppression of alpha1C. Transient transfection of human colonic circular muscle strips with antisense oligonucleotides to p50 and p65 decreased expression of the 2 nuclear factor-kappaB units and reversed the suppression of alpha1C, as well as that of the contractile response to acetylcholine, by 24 hours of treatment with tumor necrosis factor alpha. CONCLUSIONS: The activation of p50 and p65 by tumor necrosis factor alpha suppresses the expression of the alpha1C subunit of Cav1.2 channels in human colonic circular smooth muscle cells and their contractile response to acetylcholine. Nuclear factor-kappaB must bind concurrently to the two 5' kappaB motifs on the promoter of alpha1C to produce this effect.

TNFalpha suppresses human colonic circular smooth muscle cell contractility by SP1- and NF-kappaB-mediated induction of ICAM-1.

BACKGROUND & AIMS: Intercellular adhesion molecule 1 (ICAM-1) receptors are expressed at low levels on human colonic circular smooth muscle cells (HCCSMCs) and their expression is increased in patients with Crohn's disease. We investigated the roles of transcription factors Sp1 and nuclear factor kappa B (NF-kappaB) in the regulation of ICAM-1 expression on HCCSMCs and examined whether ICAM-1 expression mediates the suppression of contractility in response to TNFalpha. METHODS: Experiments were performed on primary cultures of HCCSMCs and fresh human colonic circular muscle strips. RESULTS: TNFalpha treatment of HCCSMCs induced rapid and prolonged accumulation of ICAM-1 messenger RNA (mRNA) and protein. NF-kappaB inhibition before, but not after, 1 hour of TNFalpha-stimulation blocked the expression of ICAM-1. TNFalpha significantly enhanced Sp1/DNA binding. Sp1 bound to the 3' flanking region of a variant kappaB site in the -192/-172 region of ICAM-1 promoter. Mutation of this region abolished the response to TNFalpha. The treatment of HCCSMCs with Sp1 antisense oligonucleotides (ODNs) blocked the expression of ICAM-1, but sense ODNs had no effect. Protein kinase C zeta (PKCzeta) inhibition before or 3 hours after stimulation with TNFalpha also blocked the expression of ICAM-1. TNFalpha treatment of circular muscle strips pretreated with ICAM-1 sense ODNs or control medium significantly reduced their response to acetylcholine, whereas pretreatment with antisense ODNs blocked this effect. CONCLUSIONS: The expression of ICAM-1 on HCCSMCs in response to TNFalpha is regulated by transcription factors Sp1 and NF-kappaB binding independently to the -192/-172 region of the ICAM-1 promoter. The expression of ICAM-1 plays a critical role in the suppression of cell contractility in response to TNFalpha.

MAPK activation is involved in posttranscriptional regulation of RSV-induced RANTES gene expression.

Airway epithelial cells represent the primary cell target of respiratory syncytial virus (RSV) infection. They actively participate in the lung immune/inflammatory response that follows RSV infection by expressing chemokines, small chemotactic cytokines that recruit and activate leukocytes. Regulated on activation, normal T cell expressed, and presumably secreted (RANTES) is a member of the CC chemokine subfamily and is strongly chemotactic for T lymphocytes, monocytes, basophils, and eosinophils, cell types that are present or activated in the inflammatory infiltrate that follows RSV infection of the lung. RSV infection of airway epithelial cells induces RANTES expression by increasing gene transcription and stabilizing RNA transcripts. The signaling pathway regulating RANTES gene expression after RSV infection has not been determined. In this study, we examined the role of extracellular signal-regulated kinase (ERK) and p38, members of the mitogen-activated protein (MAP) kinase (MAPK) family, in RSV-induced RANTES production. RSV infection of alveolar epithelial cells induced increased phosphorylation and catalytic activity of ERK and the upstream kinases Raf-1 and MAP ERK kinase. Induction of the MAP signaling cascade required a replication-competent virus. RSV infection of alveolar epithelial cells also induced activation of p38 MAPK. Inhibition of ERK and p38 activation significantly reduced RSV-induced RANTES mRNA and protein secretion without affecting RANTES gene transcription or transcription factor activation. These results indicate that the MAPK signaling cascade regulates RANTES production in alveolar epithelial cells through a posttranscriptional mechanism.