Darren J. Fernandes

The effect of glucocorticoids on proliferation of human cultured airway smooth muscle

1 Airway smooth muscle proliferation is a significant component of the airway wall remodelling that occurs in asthma. In this study, the effects of glucocorticoids on mitogenic responses of human airway smooth muscle have been examined. 2 Pretreatment of smooth muscle cells with dexamethasone (100 nM, 60 min) inhibited thrombin‐induced increases in [3H]‐thymidine incorporation (DNA synthesis) and cell number. 3 Inhibition of thrombin‐induced [3H]‐thymidine incorporation was also observed with hydrocortisone (0.01‐1 μm) and methylprednisolone (0.001‐0.1 μm) pretreatment. In contrast, pretreatment with either testosterone (0.001‐1 μm), progesterone (0.001‐1 μm), 17β‐oestradiol (0.001‐1 μm), or aldosterone (0.001‐1 μm) had no effect on the response to thrombin. 4 Responses to a range of mitogens including thrombin (0.01–10 u ml−1), epidermal growth factor (EGF, 3–3000 pM), basic fibroblast growth factor (bFGF, 0.3–300 pM) and foetal calf serum (FCS, 0.1–10% v/v) were inhibited by dexamethasone (100 nM) pretreatment. However, the magnitude of the inhibitory effect was dependent on the mitogen, with EGF being the least, and thrombin being the most sensitive to the inhibitory effect. 5 The potency of hydrocortisone as an inhibitor of [3H]‐thymidine incorporation was reduced when FCS (10% v/v, which caused a 40 fold increase in [3H]‐thymidine incorporation) was used as the mitogen in place of thrombin (0.3 u ml−1, which caused a 10 fold increase in [3H]‐thymidine incorporation). 6 The effect of post‐treatment with dexamethasone (100 nM) indicated that addition of the glucocorticoid up to 17–19 h after thrombin (0.3 u ml−1) produced similar degrees of inhibition to those obtained when it was added as a pretreatment. Dexamethasone no longer produced an inhibitory effect if added 21 h or more after the addition of thrombin. 7 These results suggest that glucocorticoids regulate airway smooth muscle proliferation initiated by a range of stimuli. This effect may be of importance in the therapeutic actions of these compounds in asthma, particularly when they are used for prolonged periods of time.

Extracellular Matrix, Integrins, and Mesenchymal Cell Function in the Airways

Subepithelial fibrosis is one of the characteristic features of asthmatic airways. The fibrotic response includes an increase in volume occupied by extracellular matrix (ECM) tissue, and a change in the ECM composition favouring wound type collagens, fibronectin and a number of glycoproteins and proteoglycans normally associated with development. The altered ECM is likely to be deposited by the mesenchymal cells (including (myo) fibroblasts and smooth muscle) that are increased in number in asthmatic airways. In turn, the altered asthmatic ECM is likely to influence the function of the resident airway cells, and may be directly responsible for increasing proliferation, migration, ECM synthesis, inflammatory mediator release, and survival of resident mesenchymal cells. Therefore, the deposited ECM may perpetuate the disease phenotype. The different components of the ECM bi-directionally communicate with cells through a family of transmembrane receptors called integrins. Current research has begun to characterize: 1) the particular ECM components altered in airways disease; 2) the breadth of activity of different ECM components on airway cell function; and 3) the particular integrins responsible for mediating these effects. Further understanding of the role of integrins in transmitting responses of ECM in healthy or diseased airways may lead to novel targets for anti-asthma therapy.

Functional significance of protein kinase A activation by endothelin-1 and ATP: negative regulation of SRF-dependent gene expression by PKA.

Endothelin-1 (ET1) and ATP stimulate contraction and hypertrophy of vascular smooth muscle cells (VSMC) by activating diverse signalling pathways. In this study, we show that in VSMC, ET1 and ATP stimulate transient and sustained activation of protein kinase A (PKA), respectively. Using a dominant negative PKA mutant (PKA-DN), we examined the functional significance of PKA activation in the signalling of ET1 and ATP. Overexpression of PKA-DN did not alter the ET1- or ATP-induced phosphorylation of the extracellular signal-regulated protein kinase, Erk2. ATP stimulated a profound, PKA-dependent activation of cAMP-response element (CRE), whereas the effect of ET1 was negligible. Both ET1 and ATP stimulated serum response factor (SRF)-dependent gene expression. Overexpression of PKA-DN potentiated the effects of ET1 and ATP on SRF activity, whereas stimulation of PKA by isoproterenol, forskolin or by overexpression of the PKA catalytic subunit decreased SRF activity. These data demonstrate that (i) PKA negatively regulates SRF activity and (ii) ET1 and ATP stimulate opposing pathways, whose balance determines the net activity of SRF.

What evidence implicates airway smooth muscle in the cause of BHR

Bronchial hyperresponsiveness (BHR), the occurrence of excessive bronchoconstriction in response to relatively small constrictor stimuli, is a cardinal feature of asthma. Here, we consider the role that airway smooth muscle might play in the generation of BHR. The weight of evidence suggests that smooth muscle isolated from asthmatic tissues exhibits normal sensitivity to constrictor agonists when studied during isometric contraction, but the increased muscle mass within asthmatic airways might generate more total force than the lesser amount of muscle found in normal bronchi. Another salient difference between asthmatic and normal individuals lies in the effect of deep inhalation (DI) on bronchoconstriction. DI often substantially reverses induced bronchoconstriction in normals, while it often has much less effect on spontaneous or induced bronchoconstriction in asthmatics. It has been proposed that abnormal dynamic aspects of airway smooth muscle contraction—velocity of contraction or plasticity-elasticity balance—might underlie the abnormal DI response in asthma. We suggest a speculative model in which abnormally long actin filaments might account for abnormally increased elasticity of contracted airway smooth muscle.

Contribution of the p38MAPK signalling pathway to proliferation in human cultured airway smooth muscle cells is mitogen‐specific

We have investigated the role of p38MAPK in human airway smooth muscle (HASM) proliferation in response to thrombin and bFGF. The regulation of cyclin D1 mRNA, cyclin D1, cyclin E and p21Cip1 protein levels, and the extent of retinoblastoma protein (pRb) phosphorylation in response to activation of p38MAPK have also been examined. Two distinct inhibitors of p38MAPK, SB 203580 (10 μM) and SB 202190 (10 μM), prevented bFGF (0.3–3 nM)‐stimulated cell proliferation, but had no effect on the response to thrombin (0.3–3 U ml−1). In cells incubated with thrombin or bFGF for 20 h, there was an increase in p38MAPK phosphorylation in response to bFGF, but not to thrombin. Thrombin and bFGF‐stimulated increases in ERK phosphorylation and cyclin D1 mRNA and protein levels were not influenced by SB 203580 pre‐treatment. Similarly, cyclin E and p21Cip1 protein levels, measured after 20 h incubation with mitogen, did not appear to be regulated by SB 203580 (10 μM). Although both thrombin and bFGF significantly increased levels of pRb phosphorylation, SB 203580 (10 μM) inhibited only bFGF‐stimulated pRb phosphorylation. In addition, SB 203580 (10 μM) selectively inhibited bFGF‐stimulated DNA synthesis, suggesting that the antimitogenic actions of SB 203580 on pRb phosphorylation cause cell cycle arrest at late G1 phase. In conclusion, these results indicate that p38MAPK is involved in bFGF‐, but not in thrombin‐stimulated HASM proliferation. The activation of the p38MAPK pathway by bFGF, but not by thrombin, regulates the phosphorylation of pRb without influencing cyclin D1 expression.

Inhibition of Transforming Growth Factor β-enhanced Serum Response Factor-dependent Transcription by SMAD7

Transforming growth factor (TGF)-β is present in large amounts in the airways of patients with asthma and with other diseases of the lung. We show here that TGFβ treatment increased transcriptional activation of SM22α, a smooth muscle-specific promoter, in airway smooth muscle cells, and we demonstrate that this effect stems in part from TGFβ-induced enhancement of serum response factor (SRF) DNA binding and transcription promoting activity. Overexpression of Smad7 inhibited TGFβ-induced stimulation of SRF-dependent promoter function, and chromatin immunoprecipitation as well as co-immunoprecipitation assays established that endogenous or recombinant SRF interacts with Smad7 within the nucleus. The SRF binding domain of Smad7 mapped to the C-terminal half of the Smad7 molecule. TGFβ treatment weakened Smad7 association with SRF, and conversely the Smad7-SRF interaction was increased by inhibition of the TGFβ pathway through overexpression of a dominant negative mutant of TGFβ receptor I or of Smad3 phosphorylation-deficient mutant. Our findings thus reveal that SRF-Smad7 interactions in part mediate TGFβ regulation of gene transcription in airway smooth muscle. This offers potential targets for interventions in treating lung inflammation and asthma.

Stimulus-dependent glucocorticoid-resistance of GM-CSF production in human cultured airway smooth muscle.

1 For a subpopulation of asthmatics, symptoms persist even with high doses of glucocorticoids. Glucocorticoids reduce the levels of the proinflammatory and fibrogenic cytokine, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) produced by human cultured airway smooth muscle (ASM). We have contrasted the effects of a synthetic glucocorticoid, dexamethasone, on thrombin‐ and IL‐1α‐stimulated GM‐CSF production in human ASM cells. 2 Although IL‐1α stimulated three‐fold higher levels of GM‐CSF mRNA and protein compared to thrombin, dexamethasone concentration‐dependently reduced IL‐1α‐stimulated GM‐CSF more potently and to a greater extent than the response to thrombin. This pattern of glucocorticoid regulation was also observed at the GM‐CSF mRNA level and was reproduced with other glucocorticoids such as fluticasone propionate. 3 IL‐1α and thrombin stimulated NF‐κB‐dependent luciferase expression equally. Dexamethasone treatment reduced luciferase expression stimulated by both IL‐1α and thrombin. 4 The GM‐CSF mRNA half life was markedly prolonged by IL‐1α compared to thrombin. This IL‐1α‐induced GM‐CSF mRNA stability was prevented by either dexamethasone or the p38MAPK inhibitor, SB203580, neither of which influenced GM‐CSF mRNA stability in thrombin‐treated cells. Dexamethasone inhibited p38MAPK phosphorylation in IL‐1α‐stimulated ASM, whereas thrombin does not stimulate p38MAPK phosphorylation. 5 These data suggest that the mechanism underlying the greater potency and efficacy of glucocorticoids in reducing GM‐CSF synthesis stimulated by IL‐1α depends on inhibition of the involvement of p38MAPK‐induced increases in GM‐CSF message stability.

Differential inhibition of thrombin- and EGF-stimulated human cultured airway smooth muscle proliferation by glucocorticoids

The present study compared the effects of glucocorticoids on thrombin- and EGF-stimulated proliferation in human cultured airway smooth muscle (ASM) to identify pathways that may be differentially regulated by glucocorticoids. Mitogenic responses to thrombin were inhibited by extracellular-regulated kinase (ERK 1/2) and phosphoinositide 3-kinase (PI3K) inhibitors, whereas mitogenic responses to EGF were inhibited by ERK 1/2 and PI3K inhibitors as well as by the p38 mitogen activated protein kinase inhibitor, SB203580 (10 microM). Mitogenic responses to thrombin were more sensitive to inhibition by dexamethasone (Dex) or fluticasone propionate (FP) than were those to EGF. Elevated cyclin D1 protein and mRNA levels induced by thrombin and EGF were attenuated equally by glucocorticoids. The protein or mRNA levels of the cyclin-dependent kinase inhibitors (cdki) p21(Cip1), p27(Kip1) were unaffected by Dex treatment of ASM cells treated with mitogens. The resistance of EGF-induced proliferation to inhibition by glucocorticoids is not associated with a failure to regulate cyclin D1 induction, nor does it appear to be explained by differential regulation of the levels of the cdkis, p21(Cip1) and p27(Kip1).

Resistance of fibrogenic responses to glucocorticoid and 2-methoxyestradiol in bleomycin-induced lung fibrosis in mice.

Bleomycin-induced lung fibrosis in mice reproduces some key features of pulmonary fibrosis in humans including alveolar inflammation, myofibroblast proliferation, and collagen deposition. Glucocorticoids have been used as first-line therapy for the treatment of lung fibrosis, although their clinical efficacy is equivocal. We examined the effect of the glucocorticoid, methylprednisolone (MP), and the estrogen metabolite, 2-methoxyestradiol (2MEO) on bleomycin-induced bronchoalveolar inflammation, fibrosis, and changes in lung function. The characterization of the time-course of the bleomycin-induced fibrosis indicated that lung dry mass and hydroxyproline content showed less variance than histopathological assessment of fibrosis. The bleomycin-induced increases in bronchoalveolar lavage (BAL) fluid cell number and protein levels were not significantly influenced by treatment with either MP (1 mg.(kg body mass)(-1).day(-1), i.p.) or 2MEO (50 mg.(kg body mass)(-1).day(-1), i.p.). Lung fibrosis, measured histopathologically or by hydroxyproline content, was not significantly influenced by either MP or 2MEO treatment, whereas the latter agent did reduce the increment in lung dry mass. The enlargement of alveolar airspaces and the decline in lung compliance were exacerbated by MP treatment. These data suggest that bleomycin-induced pulmonary fibrosis is resistant to inhibition by concurrent treatment with either glucocorticoids or 2MEO.

CAN WE DIFFERENTIATE BETWEEN AIRWAY AND VASCULAR SMOOTH MUSCLE

1. Airway smooth muscle (ASM) has recently been termed the ‘frustrated’ cell of the lung given that contraction of ASM has no proven useful physiological function in adults and yet is indelibly associated with pathological conditions by virtue of its unwanted airflow‐limiting actions in asthma. In contrast, pulmonary vascular smooth muscle contraction plays an essential role in the control of blood flow through the lung.