MAPK p38 regulates inflammatory gene expression via tristetraprolin: Doing good by stealth.

Tristetraprolin (TTP) is an RNA-destabilizing protein that exerts profound anti-inflammatory effects by inhibiting the expression of tumour necrosis factor and many other inflammatory mediators. The mitogen-activated protein kinase (MAPK) p38 signaling pathway controls the strength and duration of inflammatory responses by regulating both the expression and function of TTP. The kinase MK2 (MAPK activated kinase 2) is activated by MAPK p38, and in turn phosphorylates TTP at two critical serine residues. One consequence of these phosphorylations is the protection of TTP from proteasome-mediated degradation. Another consequence is the loss of mRNA destabilizing activity. The control of TTP expression and function by the MAPK p38 pathway provides an elegant mechanism for coupling the on and off phases of inflammatory responses, and dictating the precise kinetics of expression of individual inflammatory mediators.

Targeting PP2A and proteasome activity ameliorates features of allergic airway disease in mice.

BACKGROUND: Asthma is an allergic airway disease (AAD) caused by aberrant immune responses to allergens. Protein phosphatase-2A (PP2A) is an abundant serine/threonine phosphatase with anti-inflammatory activity. The ubiquitin proteasome system (UPS) controls many cellular processes, including the initiation of inflammatory responses by protein degradation. We assessed whether enhancing PP2A activity with fingolimod (FTY720) or 2-amino-4-(4-(heptyloxy) phenyl)-2-methylbutan-1-ol (AAL(S) ), or inhibiting proteasome activity with bortezomib (BORT), could suppress experimental AAD. METHODS: Acute AAD was induced in C57BL/6 mice by intraperitoneal sensitization with ovalbumin (OVA) in combination with intranasal (i.n) exposure to OVA. Chronic AAD was induced in mice with prolonged i.n exposure to crude house dust mite (HDM) extract. Mice were treated with vehicle, FTY720, AAL(S) , BORT or AAL(S) +BORT and hallmark features of AAD assessed. RESULTS: AAL(S) reduced the severity of acute AAD by suppressing tissue eosinophils and inflammation, mucus-secreting cell (MSC) numbers, type 2-associated cytokines (interleukin (IL)-33, thymic stromal lymphopoietin, IL-5 and IL-13), serum immunoglobulin (Ig)E and airway hyper-responsiveness (AHR). FTY720 only suppressed tissue inflammation and IgE. BORT reduced bronchoalveolar lavage fluid (BALF) and tissue eosinophils and inflammation, IL-5, IL-13 and AHR. Combined treatment with AAL(S) +BORT had complementary effects and suppressed BALF and tissue eosinophils and inflammation, MSC numbers, reduced the production of type 2 cytokines and AHR. AAL(S) , BORT and AAL(S) +BORT also reduced airway remodelling in chronic AAD. CONCLUSION: These findings highlight the potential of combination therapies that enhance PP2A and inhibit proteasome activity as novel therapeutic strategies for asthma.

Cyclooxygenase 2: its regulation, role and impact in airway inflammation.

Cyclooxygenase 2 (COX-2: official gene symbol - PTGS2) has long been regarded as playing a pivotal role in the pathogenesis of airway inflammation in respiratory diseases including asthma. COX-2 can be rapidly and robustly expressed in response to a diverse range of pro-inflammatory cytokines and mediators. Thus, increased levels of COX-2 protein and prostanoid metabolites serve as key contributors to pathobiology in respiratory diseases typified by dysregulated inflammation. But COX-2 products may not be all bad: prostanoids can exert anti-inflammatory/bronchoprotective functions in airways in addition to their pro-inflammatory actions. Herein, we outline COX-2 regulation and review the diverse stimuli known to induce COX-2 in the context of airway inflammation. We discuss some of the positive and negative effects that COX-2/prostanoids can exert in in vitro and in vivo models of airway inflammation, and suggest that inhibiting COX-2 expression to repress airway inflammation may be too blunt an approach; because although it might reduce the unwanted effects of COX-2 activation, it may also negate the positive effects. Evidence suggests that prostanoids produced via COX-2 upregulation show diverse actions (and herein we focus on prostaglandin E2 as a key example); these can be either beneficial or deleterious and their impact on respiratory disease can be dictated by local concentration and specific interaction with individual receptors. We propose that understanding the regulation of COX-2 expression and associated receptor-mediated functional outcomes may reveal number of critical steps amenable to pharmacological intervention. These may prove invaluable in our quest towards future development of novel anti-inflammatory pharmacotherapeutic strategies for the treatment of airway diseases.

IL-17A increases TNF-α-induced COX-2 protein stability and augments PGE2 secretion from airway smooth muscle cells: impact on ß2 -adrenergic receptor desensitization.

BACKGROUND: IL-17A plays an important role in respiratory disease and is a known regulator of pulmonary inflammation and immunity. Recent studies have linked IL-17A with exacerbation in asthma and COPD. We have shown that the enzyme cyclooxygenase-2 (COX-2) and its prostanoid products, prostaglandin E2 (PGE2 ) in particular, are key contributors in in vitro models of infectious exacerbation; however, the impact of IL-17A was not known. METHODS AND RESULTS: We address this herein and show that IL-17A induces a robust and sustained upregulation of COX-2 protein and PGE2 secretion from airway smooth muscle (ASM) cells. COX-2 can be regulated at transcriptional, post-transcriptional and/or post-translational levels. We have elucidated the underlying molecular mechanisms responsible for the sustained upregulation of TNF-α-induced COX-2 by IL-17A in ASM cells and show that is not via increased COX-2 gene expression. Instead, TNF-α-induced COX-2 upregulation is subject to regulation by the proteasome, and IL-17A acts to increase TNF-α-induced COX-2 protein stability as confirmed by cycloheximide chase experiments. In this way, IL-17A acts to amplify the COX-2-mediated effects of TNF-α and greatly enhances PGE2 secretion from ASM cells. CONCLUSION: As PGE2 is a multifunctional prostanoid with diverse roles in respiratory disease, our studies demonstrate a novel function for IL-17A in airway inflammation by showing for the first time that IL-17A impacts on the COX-2/PGE2 pathway, molecules known to contribute to disease exacerbation.

The p38-MK2-HuR pathway potentiates EGFRvIII-IL-1ß-driven IL-6 secretion in glioblastoma cells.

The microenvironment of glioblastoma (GBM) contains high levels of inflammatory cytokine interleukin 6 (IL-6), which contributes to promote tumour progression and invasion. The common epidermal growth factor receptor variant III (EGFRvIII) mutation in GBM is associated with significantly higher levels of IL-6. Furthermore, elevated IL-1ß levels in GBM tumours are also believed to activate GBM cells and enhance IL-6 production. However, the crosstalk between these intrinsic and extrinsic factors within the oncogene-microenvironment of GBM causing overproduction of IL-6 is poorly understood. Here, we show that EGFRvIII potentiates IL-1ß-induced IL-6 secretion from GBM cells. Importantly, exacerbation of IL-6 production is most effectively attenuated in EGFRvIII-expressing GBM cells with inhibitors of p38 mitogen-activated protein kinase (p38 MAPK) and MAPK-activated protein kinase 2 (MK2). Enhanced IL-6 production and increased sensitivity toward pharmacological p38 MAPK and MK2 inhibitors in EGFRvIII-expressing GBM cells is associated with increased MK2-dependent nuclear-cytoplasmic shuttling and accumulation of human antigen R (HuR), an IL-6 mRNA-stabilising protein, in the cytosol. IL-1ß-stimulated activation of the p38 MAPK-MK2-HuR pathway significantly enhances IL-6 mRNA stability in GBM cells carrying EGFRvIII. Further supporting a role for the p38 MAPK-MK2-HuR pathway in the development of inflammatory environment in GBM, activated MK2 is found in more than 50% of investigated GBM tissues and correlates with lower grade and secondary GBMs. Taken together, p38 MAPK-MK2-HuR signalling may enhance the potential of intrinsic (EGFRvIII) and extrinsic (IL-1ß) factors to develop an inflammatory GBM environment. Hence, further improvement of brain-permeable and anti-inflammatory inhibitors targeting p38 MAPK, MK2 and HuR may combat progression of lower grade gliomas into aggressive GBMs.

TLR2 activation causes tachyphylaxis to ß2 -agonists in vitro and ex vivo: modelling bacterial exacerbation.

BACKGROUND: Asthma is a widespread chronic health problem exacerbated by common viral and bacterial infections. Further research is required to understand how infection worsens asthma control in order to advance therapeutic options in the future. Recent research has revealed that ß2 -adrenergic receptor (ß2 -AR) agonists lose bronchodilatory efficacy because the receptor-mediated molecular pathways responsible for their beneficial actions are desensitized by infection. To date, most studies have focussed on viral infection, leaving the impact of bacterial infection on ß2 -AR desensitization relatively under-investigated. We address this in this study. METHODS AND RESULTS: Utilizing an in vitro model of bacterial exacerbation in airway smooth muscle (ASM) cells, we show that activation of toll-like receptor 2 (TLR2; mimicking bacterial infection) in the presence of an inflammatory stimulus leads to ß2 -AR desensitization. This occurs via TLR2-dependent upregulation of cyclooxygenase 2 (COX-2) mRNA expression and increased secretion of PGE2 . Importantly, PGE2 causes heterologous ß2 -AR desensitization and reduces cAMP production in response to short-acting (salbutamol) and long-acting (formoterol) ß2 -agonists. Thus, bacterial infectious stimuli act in a PGE2 -dependent manner to severely curtail the beneficial actions of ß2 -agonists. The impact of ß2 -AR desensitization is demonstrated by reduced gene expression of the critical anti-inflammatory molecule MKP-1 in response to ß2 -agonists, as well as impaired bronchodilation in a mouse lung slices. CONCLUSIONS: Taken together, our results show that, like viruses, bacteria induce prostanoid-dependent ß2 -AR desensitization on ASM cells. Notably, COX-2 inhibition with the specific inhibitor celecoxib represses PGE2 secretion, presenting a feasible pharmacological option for treatment of infectious exacerbation in asthma in the future.

The NLRP3 inflammasome: role in airway inflammation.

Asthma is characterized by airway inflammation, airway hyperresponsiveness and airway remodelling. Uncontrolled airway inflammation or repeated asthma exacerbations can lead to airway remodelling, which cannot be reversed by current pharmacological treatment, and consequently lead to decline in lung function. Thus, it is critical to understand airway inflammation in asthma and infectious exacerbation. The inflammasome has emerged as playing a key role in innate immunity and inflammation. Upon ligand sensing, inflammasome components assemble and self-oligomerize, leading to caspase-1 activation and maturation of pro-IL-1ß and pro-IL-18 into bioactive cytokines. These bioactive cytokines then play a pivotal role in the initiation and amplification of inflammatory processes. In addition to facilitating the proteolytic activation of IL-1ß and IL-18, inflammasomes also participate in cell death through caspase-1-mediated pyroptosis. In this review, we describe the structure and function of the inflammasome and provide an overview of our current understanding of role of the inflammasome in airway inflammation. We focus on nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome as it is the best-characterized subtype shown expressed in airway and considered to play a key role in chronic airway diseases such as asthma.

Corticosteroids and ß2-agonists upregulate mitogen-activated protein kinase phosphatase 1: in vitro mechanisms.

BACKGROUND AND PURPOSE: Airway remodelling is a consequence of long-term inflammation and MAPKs are key signalling molecules that drive pro-inflammatory pathways. The endogenous MAPK deactivator--MAPK phosphatase 1 (MKP-1)--is a critical negative regulator of the myriad pro-inflammatory pathways activated by MAPKs in the airway. EXPERIMENTAL APPROACH: Herein we investigated the molecular mechanisms responsible for the upregulation of MKP-1 in airway smooth muscle (ASM) by the corticosteroid dexamethasone and the ß2-agonist formoterol, added alone and in combination. KEY RESULTS: MKP-1 is a corticosteroid-inducible gene whose expression is enhanced by long-acting ß2-agonists in an additive manner. Formoterol induced MKP-1 expression via the ß2-adrenoceptor and we provide the first direct evidence (utilizing overexpression of PKIα, a highly selective PKA inhibitor) to show that PKA mediates ß2-agonist-induced MKP-1 upregulation. Dexamethasone activated MKP-1 transcription in ASM cells via a cis-acting corticosteroid-responsive region located between -1380 and -1266 bp of the MKP-1 promoter. While the 3'-untranslated region of MKP-1 contains adenylate + uridylate elements responsible for regulation at the post-transcriptional level, actinomycin D chase experiments revealed that there was no increase in MKP-1 mRNA stability in the presence of dexamethasone, formoterol, alone or in combination. Rather, there was an additive effect of the asthma therapeutics on MKP-1 transcription. CONCLUSIONS AND IMPLICATIONS: Taken together, these studies allow us a greater understanding of the molecular basis of MKP-1 regulation by corticosteroids and ß2-agonists and this new knowledge may lead to elucidation of optimized corticosteroid-sparing therapies in the future.

Targeting mitogen-activated protein kinase phosphatase-1 (MKP-1): structure-based design of MKP-1 inhibitors and upregulators.

Mitogen-activated protein kinase phosphatases (MKPs) are dual specificity protein phosphatases (DUSPs) that dephosphorylate both phospho-tyrosine and phospho-threonine residues on mitogen-activated protein kinases (MAPKs). Because the MAPK family of signalling molecules (phospho-p38 MAPK, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)) play essential roles in cell signalling pathways that regulate cell growth and inflammation, controlling MAPK-mediated pathways is a therapeutically attractive strategy. While small molecule MAPK inhibitors have utility, in this review we will focus on exploring the potential of targeting the endogenous MAPK deactivator--MKP-1. Importantly, there is a strong justification for developing both inhibitors and upregulators of MKP-1 because of the diverse roles played by MAPKs in disease: for example, in cancer, MKP-1 inhibitors may prove beneficial, as MKP-1 is overexpressed and is considered responsible for the failure of JNK-driven apoptotic pathways induced by chemotherapeutics; conversely, in inflammatory diseases such as asthma and arthritis, MKP-1 reduces MAPK-mediated signalling and developing novel ligands to upregulate MKP-1 levels would be a therapeutically attractive anti-inflammatory strategy. Thus, in this review we utilise MKP-1 homology modeling to highlight the structural features of MKP-1 inhibitors that permit potent and selective inhibition, and to provide insights into the structural requirements for selective MKP-1 upregulators.

IκBα glutathionylation and reduced histone H3 phosphorylation inhibit eotaxin and RANTES.

Airway smooth muscle cells (ASMCs) secrete eotaxin and RANTES (regulated on activation, normal T-cell expressed and secreted) in response to tumour necrosis factor (TNF)-α, which is inhibited by the nuclear factor (NF)-κB inhibitor dimethylfumarate (DMF). NF-κB/IκB (inhibitor of NF-κB) glutathionylation and changes in chromatin remodelling can inhibit NF-κB activity. In this study, we determined whether NF-κB/IκB glutathionylation and reduced histone H3 phosphorylation might underlie the inhibitory effect of DMF on NF-κB activity, and eotaxin and RANTES secretion. Primary human ASMCs were treated with DMF, diamide and/or glutathione (GSH) ethylester (OEt) prior to TNF-α stimulation and were subsequently analysed by ELISA, electrophoretic mobility shift assay, immunofluorescence, co-immunoprecipitation or immunoblotting. DMF reduced intracellular GSH and induced IκBα glutathionylation (IκBα-SSG), which inhibited IκBα degradation, NF-κB p65 nuclear entry and NF-κB/DNA binding. In addition, DMF inhibited the phosphorylation of histone H3, which was possibly mediated by the inhibitory effect of DMF on mitogen- and stress-activated protein kinase (MSK)-1. However, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase MAPK and MAPK phosphatase-1, upstream of MSK-1, were not inhibited by DMF. Importantly, DMF-mediated effects on NF-κB, histone H3, eotaxin and RANTES were reversed by addition of GSH-OEt. Our data suggest that DMF inhibits NF-κB-dependent eotaxin and RANTES secretion by reduction of GSH with subsequent induction of IκBα-SSG and inhibition of histone H3 phosphorylation. Our findings offer new potential drug targets to reduce airway inflammation in asthma.

Development of inhalable formulations of anti-inflammatory drugs to potentially treat smoke inhalation injury in burn victims.

Injury arising from smoke inhalation is a significant mortality risk in severe burned patients. Inflammatory processes are major contributors to the development of respiratory insufficiency owing to pulmonary oedema, formation of airway fibrin clots and hypoxaemia. Anti-inflammatory and anti-coagulant drugs such as heparin and pentoxifylline are currently systemically administered for the treatment of smoke inhalation. Delivery of these drugs in the form of inhalable particles could be an effective manner to achieve rapid targeted action for acceleration of the treatment. The study developed and characterised a series of spray-dried heparin and pentoxifylline dry powder formulations suitable for inhalation administration. Drug particles were co-spray-dried with leucine in varying ratios. Particle size analysis confirmed all powders (except 2%, w/w, pentoxifylline with 1%, w/w, leucine in spray-drying feed solution) had particle size in the optimal range (< or =5 microm) for deep lung drug deposition. Leucine supplementation dramatically altered heparin surface topography while pentoxifylline formulations were a mixture of elongated needles interspersed with wrinkly particles. Addition of leucine improved fine particle fraction of heparin and pentoxifylline. The study indicated manufacture of inhalable heparin and pentoxifylline was feasible and can potentially be an attractive delivery alternative to the more conventional systemic delivery route.

Inflammatory mediator release in normal bronchial smooth muscle cells is altered by pregnant maternal and fetal plasma independent of asthma.

Studies have shown that pregnancy can alter the pathophysiology of a pre-existing maternal disease such as asthma. However, the mechanisms that alter maternal asthma during pregnancy are presently unknown. Previous work has demonstrated that human bronchial smooth muscle (BSM) cells produce inflammatory factors in response to nonpregnant, atopic plasma. The aim of this study was to determine whether circulating pregnancy-derived factors in maternal and fetal plasma can stimulate inflammatory mediator release in BSM cells in the presence and absence of maternal asthma. Cultured human BSM cells were exposed to maternal and fetal plasma from normal pregnancies and pregnancies complicated by asthma. Inflammatory mediator release was determined by enzyme-linked immunosorbent assay (ELISA). Both maternal and fetal plasma from asthmatic and nonasthmatic individuals significantly increased production of interleukin (IL)-6 (ANOVA, P<0.001), regulated upon activation, normal T-cell expressed and secreted (RANTES) (ANOVA, P<0.01), and soluble intercellular cell-adhesion molecule-1 (sICAM-1) (ANOVA, P<0.01). There was no difference in inflammatory mediator release in response to asthma and nonasthmatic plasma. Eotaxin release was increased by pregnant asthmatic plasma (ANOVA, P<0.05). The results of this study suggest that circulating pregnancy-related factors can activate asthma-associated mediators in BSM cells. This change in BSM function may be one mechanism that contributes to increased asthma severity during pregnancy.

Mechanisms of serum potentiation of GM-CSF production by human airway smooth muscle cells.

Inflammation and vascular leakage are prevalent in asthma. This study aimed to elucidate the mechanisms involved in serum potentiation of cytokine-induced granulocyte macrophage colony stimulating factor (GM-CSF) production by human airway smooth muscle cells and to identify possible factors responsible. Serum-deprived cells at low density were stimulated with TNF-alpha and IL-1beta for 24 h. Human AB serum (10%), inhibitors of RNA and protein synthesis or specific signaling molecules, or known smooth muscle mitogens were then added for 24 h. Culture supernatants were analyzed for GM-CSF levels, and cells were harvested to assess viability, cell cycle progression, GM-CSF-specific mRNA content, and p38 phosphorylation. Serum potentiated GM-CSF release when added before, together with (maximal), or after the cytokines. The potentiation involved both new GM-CSF-specific mRNA production and protein synthesis. The mitogens IGF, PDGF, and thrombin all potentiated GM-CSF release, and neutralizing antibodies for EGF, IGF, and PDGF reduced the serum potentiation. Inhibitor studies ruled as unlikely the involvement of p70(S6kinase) and the MAPK p42/p44, two signaling pathways implicated in proliferation, and the involvement of the MAPK JNK, while establishing roles for p38 MAPK and NF-kappaB in the potentiation of GM-CSF release. Detection of significant p38 phosphorylation in response to serum stimulation, through Western blotting, further demonstrated the involvement of p38. These studies have provided evidence to support p38 being targeted to interrupt the cycle of inflammation, vascular leakage and cytokine production in asthma.

Low lung volume alters contractile properties of airway smooth muscle in sheep.

Breathing at volumes lower than functional residual capacity (FRC) can induce changes in nonasthmatic airways consistent with the behaviour of asthmatic airways. This study investigated the chronic effect of breathing at volumes lower than FRC on the contractility of airway smooth muscle and myosin light chain kinase (MLCK) content and activity. Sheep of three age groups (neonate, adolescent and adult) had their FRC reduced by approximately 25%, for 4 weeks using a leather corset. Contractile responses to carbachol were then recorded in isolated tracheal strips and bronchial rings. MLCK content and activity were assessed by immunoblotting. The rate of stress generation increased in the bronchial smooth muscle of both adult and adolescent but not neonatal corseted sheep: adolescent corseted versus control, 65.0 +/- 4.1 versus 103.4 +/- 7.0 s (to reach 50% maximum stress), respectively; and adult corseted versus control, 57.0 +/- 6.4 versus 93.4 +/- 8.2 s, respectively. This was not due to increases in either bronchial or tracheal smooth muscle amount or MLCK content and activity. The present results indicate that chronic breathing at low lung volumes increases the rate of stress generation in airway smooth muscle.

Human mast cell and airway smooth muscle cell interactions: implications for asthma.

Asthma is characterized by inflammation, hyperresponsiveness, and remodeling of the airway. Human mast cells (HMCs) play a central role in all of these changes by releasing mediators that cause exaggerated bronchoconstriction, induce human airway smooth muscle (HASM) cell proliferation, and recruit and activate inflammatory cells. Moreover, the number of HMCs present on asthmatic HASM is increased compared with that on nonasthmatic HASM. HASM cells also have the potential to actively participate in the inflammatory process by synthesizing cytokines and chemokines and expressing surface molecules, which have the capacity to perpetuate the inflammatory mechanisms present in asthma. This review specifically examines how the mediators of HMCs have the capacity to modulate many functions of HASM; how the synthetic function of HASM, particularly through the release and expression of stem cell factor, has the potential to influence HMC number and activation in an extraordinarily potent and proinflammatory manner; and how these interactions between HMCs and HASM have potential consequences for airway structure and inflammation relevant to the disease process of asthma.

Gingerols and related analogues inhibit arachidonic acid-induced human platelet serotonin release and aggregation.

Gingerols, the active components of ginger (the rhizome of Zingiber officinale, Roscoe), represent a potential new class of platelet activation inhibitors. In this study, we examined the ability of a series of synthetic gingerols and related phenylalkanol analogues (G1-G7) to inhibit human platelet activation, compared to aspirin, by measuring their effects on arachidonic acid (AA)-induced platelet serotonin release and aggregation in vitro. The IC(50) for inhibition of AA-induced (at EC(50)=0.75 mM) serotonin release by aspirin was 23.4+/-3.6 microM. Gingerols and related analogues (G1-G7) inhibited the AA-induced platelet release reaction in a similar dose range as aspirin, with IC(50) values between 45.3 and 82.6 microM. G1-G7 were also effective inhibitors of AA-induced human platelet aggregation. Maximum inhibitory (IC(max)) values of 10.5+/-3.9 and 10.4+/-3.2 microM for G3 and G4, respectively, were approximately 2-fold greater than aspirin (IC(max)=6.0+/-1.0 microM). The remaining gingerols and related analogues maximally inhibited AA-induced platelet aggregation at approximately 20-25 microM. The mechanism underlying inhibition of the AA-induced platelet release reaction and aggregation by G1-G7 may be via an effect on cyclooxygenase (COX) activity in platelets because representative gingerols and related analogues (G3-G6) potently inhibited COX activity in rat basophilic leukemia (RBL-2H3) cells. These results provide a basis for the design of more potent synthetic gingerol analogues, with similar potencies to aspirin, as platelet activation inhibitors with potential value in cardiovascular disease.

Tumor necrosis factor receptor (TNFR) 1, but not TNFR2, mediates tumor necrosis factor-alpha-induced interleukin-6 and RANTES in human airway smooth muscle cells: role of p38 and p42/44 mitogen-activated protein kinases.

Little information is available regarding the mechanisms involved in cytokine-induced synthetic function of human airway smooth muscle (ASM) cells. Here, we report that tumor necrosis factor receptor (TNFR) 1-induced p38 and p42/44 mitogen-activated protein kinase (MAPK) activation modulates tumor necrosis factor-alpha (TNF alpha)-mediated synthetic responses: expression of intercellular adhesion molecule-1 (ICAM-1) and secretion of interleukin (IL)-6 and the regulated-on-activation, normal T-cell expressed and secreted (RANTES) chemokine in human ASM cells. Pretreatment of ASM cells with SB203580, a p38 MAPK inhibitor, slightly enhanced TNF alpha-induced ICAM-1 expression in a dose-dependent manner but partially inhibited secretion of RANTES and IL-6. In contrast, PD98059, a p42/44 inhibitor, reduced ICAM-1 expression by 50% but had no effect on TNF alpha-induced RANTES or IL-6 secretion. SB203580 and PD98059 had little effect on TNF alpha-induced nuclear factor-kappa B (NF-kappa B) activation as determined in cells transfected with a NF-kappa B-luciferase reporter construct. We also found that agonistic antibodies specific for either TNFR1 or TNFR2 stimulated IL-6 and RANTES secretion and activated p38 and p42/44 MAPKs. In addition, both antibodies induced NF-kappa B-mediated gene transcription. Using receptor-specific blocking antibodies, we found that TNFR1 primarily regulates TNF alpha-induced IL-6 and RANTES secretion and activation of p38 and p42/44 MAPK pathways. Interestingly, we found that TNFR1 and TNFR2 are expressed differently on the cell surface of ASM cells. Our data suggest that despite the presence of functional TNFR2, TNFR1 associated with MAPK-dependent and -independent pathways is the primary signaling pathway involved in TNF alpha-induced synthetic functions in ASM cells.

Invited review: the circle of life: cell cycle regulation in airway smooth muscle.

Severe asthma is characterized by increased airway smooth muscle (ASM) mass, due predominantly to ASM hyperplasia. Diverse stimuli, which include growth factors, plasma- or inflammatory cell-derived mediators, contractile agonists, cytokines, and extracellular matrix proteins, induce ASM proliferation. Mitogens act via receptor tyrosine kinase, G protein-coupled receptors, or cytokine receptors, to activate p21ras and stimulate two parallel signaling pathways in ASM cells, namely, the extracellular signal-regulated kinase (ERK) or the phosphatidylinositol 3-kinase (PI3K) pathways. ERK and PI3K regulate cell cycle protein expression and thus modulate cell cycle traversal. ERK activation and downstream effectors of PI3K, such as Rac1 and Cdc42, stimulate expression of cyclin D1, a key regulator of G(1) progression in the mammalian cell cycle. In addition, PI3K activates 70-kDa ribosomal S6 kinase, an enzyme that also regulates the translation of many cell cycle proteins, including the elongation factor E2F. The present review examines the mitogens and critical signal transduction pathways that stimulate ASM cell proliferation. Further study in this area may reveal new therapeutic targets to abrogate ASM hyperplasia in diseases such as asthma and chronic obstructive pulmonary disease.

Activation of class IA PI3K stimulates DNA synthesis in human airway smooth muscle cells.

The precise mechanisms that regulate increases in airway smooth muscle (ASM) mass in asthma are unknown. This study determined whether class IA phosphatidylinositol 3-kinase (PI3K) is sufficient to stimulate DNA synthesis and characterized the PI3K isoforms expressed in human ASM cells. ASM cells express class IA, II, and III PI3K but not class IB. Because thrombin induces ASM cell proliferation, we investigated whether thrombin can stimulate class IA PI3K. Transient transfection of ASM cells with hemagglutinin-tagged p85 PI3K followed by immunostaining revealed that in quiescent cells, p85 was expressed diffusely in the cytoplasm and after stimulation with thrombin p85 translocated to the cell membrane. Microinjection of ASM cells with a dominant negative class IA PI3K inhibited thrombin-induced DNA synthesis by 30% and epidermal growth factor (EGF)- or serum-induced DNA synthesis by 13 and 28%, respectively (P < 0.05 by chi(2) analysis). In parallel experiments, transfection or microinjection of cells with constitutively active PI3K markedly increased DNA synthesis in transfected cells 10.5-fold and in microinjected cells 12.7-fold (P < 0.05 by chi(2) analysis) compared with cells transfected or microinjected with control plasmid. Interestingly, constitutively active PI3K augmented EGF-induced DNA synthesis but had little effect on that induced by serum or thrombin in ASM cells. Collectively, these data suggest that class IA PI3K is activated by thrombin and is sufficient to induce ASM cell growth.