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Altered ECM protein deposition is a feature in asthmatic airways. Fibronectin (Fn), an ECM protein produced by human bronchial epithelial cells (HBECs), is increased in asthmatic airways. This study investigated the regulation of Fn production in asthmatic or nonasthmatic HBECs and whether Fn modulated HBEC proliferation and inflammatory mediator secretion. The signaling pathways underlying transforming growth factor (TGF)-β1-regulated Fn production were examined using specific inhibitors for ERK, JNK, p38 MAPK, phosphatidylinositol 3 kinase, and activin-like kinase 5 (ALK5). Asthmatic HBECs deposited higher levels of Fn in the ECM than nonasthmatic cells under basal conditions, whereas cells from the two groups had similar levels of Fn mRNA and soluble Fn. TGF-β1 increased mRNA levels and ECM and soluble forms of Fn but decreased cell proliferation in both cells. The rate of increase in Fn mRNA was higher in nonasthmatic cells. However, the excessive amounts of ECM Fn deposited by asthmatic cells after TGF-β1 stimulation persisted compared with nonasthmatic cells. Inhibition of ALK5 completely prevented TGF-β1-induced Fn deposition. Importantly, ECM Fn increased HBEC proliferation and IL-6 release, decreased PGE2 secretion, but had no effect on VEGF release. Soluble Fn had no effect on cell proliferation and inflammatory mediator release. Asthmatic HBECs are intrinsically primed to produce more ECM Fn, which when deposited into the ECM, is capable of driving remodeling and inflammation. The increased airway Fn may be one of the key driving factors in the persistence of asthma and represents a novel, therapeutic target. [ABSTRACT FROM AUTHOR]

Cytokines drive many inflammatory diseases, including asthma. Understanding the molecular mechanisms responsible for cytokine secretion will allow us to develop novel strategies to repress inflammation in the future. Harnessing the power of endogenous anti-inflammatory proteins is one such strategy. In this study, we investigate the p38 MAPK-mediated regulatory interaction of two anti-inflammatory proteins, mitogen-activated protein kinase phosphatase 1 (MKP-1) and tristetraprolin (TTP), in the context of asthmatic inflammation. Using primary cultures of airway smooth muscle cells in vitro, we explored the temporal regulation of IL-6 cytokine mRNA expression upon stimulation with TNF-α. Intriguingly, the temporal profile of mRNA expression was biphasic. This was not due to COX-2-derived prostanoid upregulation, increased expression of NLRP3 inflammasome components, or upregulation of the cognate receptor for TNF-α-TNFR1. Rather, the biphasic nature of TNF-α-induced IL-6 mRNA expression was regulated temporally by the RNA-destabilizing molecule, TTP. Importantly, TTP function is controlled by p38 MAPK, and our study reveals that its expression in airway smooth muscle cells is p38 MAPK-dependent and its anti-inflammatory activity is also controlled by p38 MAPK-mediated phosphorylation. MKP-1 is a MAPK deactivator; thus, by controlling p38 MAPK phosphorylation status in a temporally distinct manner, MKP-1 ensures that TTP is expressed and made functional at precisely the correct time to repress cytokine expression. Together, p38 MAPK, MKP-1, and TTP may form a regulatory network that exerts significant control on cytokine secretion in proasthmatic inflammation through precise temporal signaling. [ABSTRACT FROM AUTHOR]

Airway inflammation and respiratory infections are important factors contributing to disease exacerbation in chronic airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle (ASM) cells express Toll-like receptors (TLRs) and may be involved in the amplification of airway inflammatory responses during infectious exacerbations. We determined whether infectious stimuli (mimicked using Pam3CSK4, a synthetic bacterial lipopeptide that binds to TLR2/TLR1) further enhance ASM cell inflammatory responses to TNFα in vitro and the signaling pathways involved. Human ASM cells were pretreated for 1 h with Pam3CSK4 (1 μg/ml) in the absence or presence of TNFα (10 ng/ml), and IL-6 and IL-8 release was measured after 24 h. As expected, stimulation with Pam3CSK4 or TNFα alone induced significant IL-6 and IL-8 release. Furthermore, Pam3CSK4 significantly increased TNFα-induced IL-6 and IL-8 mRNA expression and protein release and neutrophil chemotactic activity. The potentiating effect of Pam3CSK4 on TNFα- induced inflammatory responses was not due to enhanced TLR2 expression nor did it involve augmentation of NF-κB or MAPK signaling pathways. Rather, Pam3CSK4 induced cAMP response element (CRE) binding protein phosphorylation and induced CREmediated transcriptional regulation, suggesting that Pam3CSK4 and TNFα are acting in concert to enhance ASM cytokine secretion via parallel transcriptional pathways. Our findings suggest that ASM cells may be involved in the amplification of airway inflammatory responses during infectious exacerbations in chronic airway disease. [ABSTRACT FROM AUTHOR]

β2-Adrenergic receptor (β2AR) agonists induce airway relaxation via cAMP. Phosphodiesterase (PDE)s degrade and regulate cAMP, and in airway smooth muscle (ASM) cells PDE4D degrades cAMP. Long-acting β2-agonists are now contraindicated as monotherapy for asthma, and increased PDE4D has been speculated to contribute to this phenomenon. In this study we investigated the expression of PDE4D in asthmatic and nonasthmatic ASM cells and its regulation by formoterol and budesonide. Primary ASM cells from people with or without asthma were stimulated with transforming growth factor (TGF)-β1, formoterol, and/or budesonide. PDE4D mRNA was assessed by real-time PCR, or PCR to assess splice variant production. PDE4D protein was assessed by Western blotting, and we investigated the effect of formoterol on cAMP production and PDE activity. Interleukin (IL)-6 was assessed using ELISA. PDE4D mRNA was dose dependently upregulated by formoterol, with a single splice variant, PDE4D5, present. Formoterol did not induce PDE4D protein at time points between 3 to 72 h, whereas it did induce and increase IL-6 secretion. We pretreated cells with actinomycin D and a proteasome inhibitor, MG132, and found no evidence of alterations in mRNA, protein expression, or degradation of PDE4D. Finally PDE activity was not altered by formoterol. This study shows, for the first time, that PDE4D5 is predominantly expressed in human ASM cells from people with and without asthma and that formoterol does not upregulate PDE4D protein production. This leads us to speculate that continual therapy with β2AR agonists is unlikely to cause PDE4-mediated tachyphylaxis. [ABSTRACT FROM AUTHOR]

Increased levels of IL-6 are documented in asthma, but its contribution to the pathology is unknown. Asthma is characterized by airway wall thickening due to increased extracellular matrix deposition, inflammation, angiogenesis, and airway smooth muscle (ASM) mass. IL-6 binds to a specific membrane-bound receptor, IL-6 receptor-α (mIL-6Rα, and subsequently to the signaling protein gp130. Alternatively, IL-6 can bind to soluble IL-6 recpetor-s (sIL-6Rα) to stimulate membrane receptor-deficient cells, a process called trans-signaling. We discovered that primary human ASM cells do not express mIL-6Rα and, therefore, investigated the effect of IL-6 trans-signaling on the pro-remodeling phenotype of ASM. ASM required sIL-6Rα to activate signal transducer and activator 3, with no differences observed between cells from asthmatic subjects compared with controls. Further analysis revealed that IL-6 alone or with sIL-6Rα did not induce release of matrix-stimulating factors (including connective tissue growth factor, fibronectin, or integrins) and had no effect on mast cell adhesion to ASM or ASM proliferation. However, in the presence of sIL-6Rα, IL-6 increased eotaxin and VEGF release and may thereby contribute to local inflammation and vessel expansion in airway walls of asthmatic subjects. As levels of sIL-6Rα are increased in asthma, this demonstration of IL-6 trans-signaling in ASM has relevance to the development of airway remodeling. [ABSTRACT FROM AUTHOR]

Human airway smooth muscle (ASM) plays an immunomodulatory role in asthma. Recently, IL-17A has become of increasing interest in asthma, being found at elevated levels in asthmatic airways and emerging as playing an important role in airway neutrophilia. IL-17A predominantly exerts its neutrophil orchestrating role indirectly via the induction of cytokines by resident airway structural cells. Here, we perform an in vitro study to show that although IL-17A did not induce secretion of the CXC chemokine IL-8 from ASM cells, IL-17A significantly potentiates TNF-α-induced IL-8 protein secretion and gene expression in a concentration- and time-dependent manner (P < 0.05). Levels of IL-8 protein produced after 24 h of incubation with TNF-α were enhanced 2.7-fold in the presence of IL-17A, and conditioned media significantly enhanced neutrophil chemotaxis in vitro. As IL-17A had no effect on the activity of NF-κB, a key transcriptional regulator of IL-8 gene expression, we then examined whether IL-17A acts at the posttranscriptional level. We found that IL-17A significantly augmented TNF-α-induced IL-8 mRNA stability. Interestingly, this enhanced stability occurred via a p38 MAPK-dependent pathway. The decay of IL-8 mRNA transcripts proceeded at a significantly faster rate when cells were pretreated with the p38 MAPK inhibitor SB-203580 (-0.05763 ± 0.01964, t1/2 = 12.0 h), compared with vehicle (-0.0 1030 ± 0.007963, t1/2 = 67.3 h) [results are expressed as decay constant (means ± SE) and half-life (t1/2 in h): P < 0.051. Collectively, these results demonstrate that IL-17A amplifies the synthetic function of ASM cells, acting via a p38 MAPK-dependent posttranscriptional pathway to augment TNF-α-induced secretion of the potent neutrophil chemoattractant IL-8 from ASM cells. [ABSTRACT FROM AUTHOR]

Airway remodeling describes the structural changes that occur in the asthmatic airway that include airway smooth muscle hyperplasia, increases in vascularity due to angiogenesis, and thickening of the basement membrane. Our aim in this study was to examine the effect of transforming growth factor-β on the release of connective tissue growth factor and vascular endothelial growth factor from human airway smooth muscle cells derived from asthmatic and nonasthmatic patients. In addition we studied the immunohistochemical localization of these cytokines in the extracellular matrix after stimulating bronchial rings with transforming growth factor-β. Connective tissue growth factor and vascular endothelial growth factor were released from both cell types and colocalized in the surrounding extracellular matrix. Prostaglandin E2 inhibited the increase in connective tissue growth factor mRNA but augmented the release of vascular endothelial growth factor. Matrix metalloproteinase-2 decreased the amount of connective tissue growth factor and vascular endothelial growth factor, but not fibronectin deposited in the extracellular matrix. This report provides the first evidence that connective tissue growth factor may anchor vascular endothelial growth factor to the extracellular matrix and that this deposition is decreased by matrix metalloproteinase-2 and prostaglandin E2. This relationship has the potential to contribute to the changes that constitute airway remodeling, therefore providing a novel focus for therapeutic intervention in asthma. [ABSTRACT FROM AUTHOR]

The protease-activated receptor-2 (PAR-2) is present on human airway smooth muscle (ASM) cells and can be activated by mast cell tryptase, trypsin, or an activating peptide (AP). Trypsin induced significant increases in PGE[sub 2] release from human ASM cells after 6 and 24 h and also induced cyclooxygenase (COX)-2 mRNA expression and COX-2 protein. Tryptase and the PAR-2 AP did not alter PGE[sub 2] release or COX-2 protein levels, suggesting a lack of PAR-2 involvement. When we compared results in asthmatic and nonasthmatic muscle cells, both trypsin and bradykinin induced less PGE[sub 2] from asthmatic ASM cells, and bradykinin induced significantly less COX-2 mRNA in asthmatic cells. Significantly less PGE[sub 2] was released from proliferating ASM cells from asthmatic patients. In conclusion, trypsin induces PGE[sub 2] release and COX-2 in human ASM cells, which is unlikely to be via PAR-2 activation. In addition, ASM cells from asthmatic patients produce significantly less PGE[sub 2] and COX-2 compared with nonasthmatic cells. These findings may contribute to the increase in muscle mass evident in asthmatic airways. [ABSTRACT FROM AUTHOR]