Human Bronchial Smooth Muscle Cells Research Articles

Cigarette smoke and its component acrolein augment IL-8/CXCL8 mRNA stability via p38 MAPK/MK2 signaling in human pulmonary cells

Interleukin-8 (IL-8/CXCL8) is an important neutrophil chemoattractant known to be elevated in the airways of cigarette smokers and in patients with chronic obstructive pulmonary disease (COPD). We examined the acute effect of aqueous cigarette smoke extract (CSE) on IL-8 expression in primary human pulmonary cells, in particular in normal human bronchial smooth muscle cells (HBSMCs). IL-8 mRNA levels increased upon CSE exposure in a concentration- and time-dependent manner, and such an effect was accompanied by IL-8 secretion. CSE-evoked elevation of IL-8 mRNA was mimicked by its component acrolein. Both CSE and acrolein induced p38 mitogen-activated protein kinase (MAPK) phosphorylation, accompanied by the phosphorylation of MAPK-activated kinase 2 (MK2), a known downstream substrate of the p38 MAPK, both in HBSMCs and in human airway epithelial cells. Furthermore, pharmacological inhibition of p38 MAPK or MK2 strongly accelerated the decay of IL-8 mRNA levels upon stimulation with CSE or acrolein and subsequent blockade of mRNA neosynthesis with actinomycin D in pulmonary structural cells (HBSMCs and airways epithelial cells) as well as in human alveolar macrophages. Conversely, pharmacological inhibition of ERK1/2 signaling inhibited CSE-induced steady-state levels of IL-8 mRNA without affecting mRNA stability, thus suggesting inhibition at the transcriptional level. In sum, p38 MAPK/MK2 signaling is an important posttranscriptional mechanism underlying upregulation of IL-8 mRNA levels elicited by CSE and acrolein. Given the pivotal role of IL-8 in neutrophil chemotaxis and activation, our results shed light on the mechanisms through which cigarette smoke can initiate inflammation in the lung.

Synthesis and evaluation of indole-containing 3,5-diarylisoxazoles as potential pro-apoptotic antitumour agents

A series of novel indole-containing diarylisoxazoles has been synthesised, based on our previous work on the synthesis and pro-apoptotic antitumour activity of indole-based diaryl 1,2,4-oxadiazoles. Concise synthetic routes to both 3-(indol-2-yl)-5-phenylisoxazoles and 5-(indol-2-yl)-3-phenylisoxazoles have been developed with full regiochemical control, bearing substituents on the indole ring, indole nitrogen, and/or phenyl group. Additionally a series of the related 5-(1H-indol-5-yl)-3-phenylisoxazoles has been prepared. In vitro evaluation in human cancer cell lines Colo320 (colon) and Calu-3 (lung) revealed preferential antiproliferative activity within the 5-(indol-5-yl)-3-phenylisoxazole series (low micromolar IC50). Further analysis revealed the ability of the indol-5-yl series to induce expression of effector caspases-3 and -7, and retention of viability of the human bronchial smooth muscle cell (BSMC) control cell population (particularly for compounds 18c and 18e).

Murine T Cell-Derived Contractile Activity for Bronchial Smooth Muscle Cells

Background: Adoptive transfer of helper T (Th) cells conferred a late asthmatic response upon antigen challenge. A possible production of a contractile activity for bronchial smooth muscle (BSM) by activated Th cells was examined. Method: Murine Th clones were stimulated with immobilized anti-CD3 antibody in the presence or absence of anti-CD28 antibody. Culture supernatants were dialyzed and then applied to the collagen gels containing cultured human BSM cells. Results: Culture supernatants of activated but not resting murine Th clones that conferred a late asthmatic response, induced the contraction of human BSM cell-containing collagen gels. Conclusion: Activated Th cells produce a contractile activity for BSM in vitro.

Calcitriol Decreases Expression of Importin α3 and Attenuates RelA Translocation in Human Bronchial Smooth Muscle Cells

A potent immunomodulatory role of Vitamin D in both innate and adaptive immunity has recently been appreciated. In allergic asthma, activation of NF-кB induces transcription of various cytokines and chemokines involved in allergic airway inflammation. The nuclear import of activated NF-кB p50/RelA subunit is dependent on importin α3 (KPNA4) and importin α4 (KPNA3). In this study, we examined the role of importin α3 in immunomodulatory effect of calcitriol in human bronchial smooth muscle cells (HBSMCs). Cultured HBSMCs were stimulated with calcitriol in the presence and absence of cytokines, TNF-α, IL-1β, and IL-10. The mRNA transcripts of importin α3 and α4 were analyzed using qPCR while protein expression of importin α3, α4 and nuclear RelA was analyzed by immunoblotting. Calcitriol significantly decreased mRNA and protein expression of importin α3 as well as nuclear protein expression of NF-кB p65 (RelA). The decreased activation of RelA by calcitriol was confirmed by decreased release of RelA-inducible molecules, including IL-5, IL-6 and IL-8, by HBSMCs upon calcitriol treatment. Calcitriol attenuated the effect of TNF-α and IL-1β to upregulate mRNA and protein expression of importin α3. IL-10 significantly decreased the TNF-α induced expression of importin α3 and this effect was further potentiated by calcitriol. These data suggest that under inflammatory conditions, calcitriol decreases the expression of importin α3 resulting in decreased nuclear import of activated RelA. This could be a novel mechanism by which calcitriol could exert its immunomodulatory effects to reduce allergic airway inflammation and thus may alleviate the symptoms in allergic asthma.

Adenylyl cyclase 6 defines a distinct cAMP compartment that increases somatostatin expression by airway smooth muscle cells

All adenylyl cyclases (AC) catalyze synthesis of cAMP, but the 9 isoforms differ in localization, regulation and interactions with other signaling molecules. Human bronchial smooth muscle cells (hBSMC) express AC isoforms 2, 4, and 6. Distinct pools of cAMP maintained by AKAPs/PDEs allow cells to respond differently to activation of various GPCR/ACs. Our goal was to examine AC isoform‐specific effects on gene expression. We overexpressed AC2 (non‐raft localized isoform) or AC6 (lipid raft localized isoform) in hBSMC. A PCR array showed a number of genes that were differentially regulated when AC2 or AC6 were overexpressed and activated by forskolin (Fsk). AC6 increased Fsk‐stimulated expression of somatostatin (SST), while overexpression of AC2 led to Fsk‐induced SST mRNA levels below control cells. Activation of Gs coupled GPCR induced SST expression to levels greater than Fsk, suggesting that GPCR might also invoke a non‐cAMP pathway. βAR‐induced SST expression was similar in control cells and those overexpressing AC2 or AC6, while EP2R‐induced SST expression was enhanced by overexpression of AC6 but not AC2. Overexpression of either AC increased Fsk‐stimulated cAMP, but receptor‐stimulated cAMP levels differed between AC2 and AC6. cAMP driven SST expression appears to be coupled to AC6, but not AC2, illustrating that cAMP compartments differentially regulate gene expression. Supported by HL079166.

Interleukin-13-induced activation of signal transducer and activator of transcription 6 is mediated by an activation of Janus kinase 1 in cultured human bronchial smooth muscle cells

BackgroundThe current study was carried out to identify the JAK molecule(s) that is involved in the IL-13-induced activation of STAT6 in cultured human bronchial smooth muscle cells (hBSMCs). MethodsCultured hBSMCs were stimulated with IL-13 in the absence and presence of JAK inhibitor-I (a nonspecific JAKs inhibitor), tyrphostin-AG490 (a specific JAK2 inhibitor), WHI-P131 (a specific JAK3 inhibitor), or tyrphostin-AG9 (a specific Tyk2 inhibitor), and levels of phosphorylated STAT6 were measured by immunoblot analyses. ResultsThe IL-13-induced phosphorylation of STAT6 was abolished by JAK inhibitor-I, whereas the other inhibitors had no significant effect. ConclusionThese findings indicate that the STAT6 phosphorylation/activation induced by IL-13 is mediated by an activation of JAK1 in cultured hBSMCs.

Increased Expression of Importin α3 (KPNA4) and Decreased VDR in the Lung of OVA-Sensitized and Challenged Mice

The nuclear import of p65 and p50 subunits of activated NF-kappaB is dependent on importin α3 (KPNA4) and importin α4 (KPNA3). We previously reported that pro-inflammatory cytokines increase the expression of importin α3 and decrease the expression of VDR in Human bronchial smooth muscle cells (HBSMCs). Our results also demonstrate that co- stimulation with calcitriol attenuated the increased expression of importin α3 thereby leading to decrease in the activated NF-κB in the nucleus through a mechanism mediated by VDR.

Albuterol Isomers Modulate Platelet-Activating Factor Synthesis and Receptor Signaling in Human Bronchial Smooth Muscle Cells

Background: Racemic albuterol is a 50:50 mixture of the (R)- and (S)-enantiomers of albuterol. Its clinical efficacy resides in the (R)-enantiomer (levalbuterol). Studies have shown that (S)-albuterol induces human bronchial smooth muscle cell (HBSMC) proliferation via a pathway linked to platelet-activating factor (PAF), but the underlying mechanism by which (S)-albuterol augments PAF effects is not clear. In this study, we compared effect of levalbuterol and (S)-albuterol on PAF receptor (PAFr)-mediated signaling and PAF metabolism by HBSMCs after incubation with the albuterol isomers. Methods: PAF binding and inositol phosphate (IP₃) release were studied on adherent cultured cells. PAFr protein expression was measured by Western blotting, PAF synthesis and catabolism were measured in membrane and cytosolic proteins of cells incubated with albuterol isomers. Results: Compared to control conditions, (S)-albuterol increased PAF binding by 70% after 30 min of preincubation and by 150% after 24 h of preincubation. Levalbuterol had no effect on PAF binding under both conditions. (S)-albuterol also augmented PAF stimulation of IP₃ release, while levalbuterol and the racemic mixture had no effect. WEB 2170, a PAFr antagonist, inhibited the ability of (S)-albuterol to increase PAF binding or stimulate IP₃ release. (S)-albuterol stimulated PAFr protein expression. With PAF metabolism, (S)-albuterol treatment augmented PAF synthesis, but significantly inhibited PAF catabolism. Conclusions: Our data suggest that one mechanism by which (S)-albuterol stimulates HBSMC proliferation involves upregulation of PAFr-mediated effects including increased PAF synthesis and decreased PAF catabolism.

Nogo-B regulates migration and contraction of airway smooth muscle cells by decreasing ARPC 2/3 and increasing MYL-9 expression

BackgroundAbnormal proliferation, apoptosis, migration and contraction of airway smooth muscle (ASM) cells in airway remodeling in asthma are basically excessive repair responses to a network of inflammatory mediators such as PDGF, but the mechanisms of such responses remain unclear. Nogo-B, a member of the reticulum family 4(RTN4), is known to play a key role in arteriogenesis and tissue repair. Further studies are needed to elucidate the role of Nogo-B in airway smooth muscle abnormalities.MethodsA mouse model of chronic asthma was established by repeated OVA inhalation and subjected to Nogo-B expression analysis using immunohistochemistry and Western Blotting. Then, primary human bronchial smooth muscle cells (HBSMCs) were cultured in vitro and a siRNA interference was performed to knockdown the expression of Nogo-B in the cells. The effects of Nogo-B inhibition on PDGF-induced HBSMCs proliferation, migration and contraction were evaluated. Finally, a proteomic analysis was conducted to unveil the underlying mechanisms responsible for the function of Nogo-B.ResultsTotal Nogo-B expression was approximately 3.08-fold lower in chronic asthmatic mice compared to naïve mice, which was obvious in the smooth muscle layer of the airways. Interference of Nogo-B expression by siRNA resulted nearly 96% reduction in mRNA in cultured HBSMCs. In addition, knockdown of Nogo-B using specific siRNA significantly decreased PDGF-induced migration of HBSMCs by 2.3-fold, and increased the cellular contraction by 16% compared to negative controls, but had limited effects on PDGF-induced proliferation. Furthermore, using proteomic analysis, we demonstrate that the expression of actin related protein 2/3 complex subunit 5 (ARPC 2/3) decreased and, myosin regulatory light chain 9 isoform a (MYL-9) increased after Nogo-B knockdown.ConclusionsThese data define a novel role for Nogo-B in airway remodeling in chronic asthma. Endogenous Nogo-B, which may exert its effects through ARPC 2/3 and MYL-9, is necessary for the migration and contraction of airway smooth muscle cells.

Pterostilbene Suppresses Benzo[a]pyrene-Induced Airway Remodeling

This study has two novel findings: it is not only the first to demonstrate inflammatory cytokines, which are produced by the bronchial epithelium after exposure to benzo[a]pyrene (BaP) and contribute to airway remodeling by increasing human bronchial smooth muscle cells (BSMC) proliferation and migration, but also the first to reveal that pterostilbene, a constituent of grapes and berries, reverses BaP-mediated airway remodeling. Human bronchial epithelial cell lines BEAS-2B and HBE135-E6E7 (HBE) were treated with BaP, and then the condition medium (CM) was harvested, which was then added to BSMC. Cultures of BSMC with BaP-BEAS-2B-CM and -HBE-CM increased BSMC proliferation and migration, which are major features in asthma remodeling. Exposure of BEAS-2B and HBE to BaP caused epithelial cells to produce inflammatory cytokines IL-8, which subsequently induced BSMC proliferation and migration. Moreover, pterostilbene is more potent than resveratrol in suppressing BaP-mediated airway remodeling. This study suggests that pterostilbene is capable of preventing BaP-associated asthma.

Aged Red Garlic Extract Reduces Cigarette Smoke Extract‐induced Cell Death in Human Bronchial Smooth Muscle Cells by Increasing Intracellular Glutathione Levels

Increasing antioxidant capacity has been proposed as a promising strategy to prevent cigarette smoke-induced lung diseases. This study tested whether garlic extracts prevented cigarette smoke extract (CSE)-induced cell death in human bronchial smooth muscle cells (HBSMCs). Garlic extracts were prepared from fresh raw garlic (FRG), aged black garlic (ABG) and aged red garlic (ARG). Treatment of HBSMCs with 10% CSE induced cell death accompanied by activation of caspase. Of the garlic extracts, treatment with ARG extract reduced CSE-induced cell death. The combination of ARG extract with CSE attenuated the CSE-induced reduction in glutathione (GSH) content, generation of reactive oxygen species (ROS) and induction of heme oxygenase-1 expression compared with CSE treatment without ARG extract. Furthermore, the combination of L-BSO, a GSH synthesis inhibitor, with ARG and CSE extracts failed to increase the intracellular GSH content and cell viability. Taken together, these results demonstrate that ARG extract reduces CSE-induced cell death by increasing GSH content and reducing ROS generation in HBSMCs.

IL‐13 mediates PDGF‐induced bronchial smooth muscle cell proliferation: Involvement of oxidant signaling

Smooth muscle cell (SMC) hyperplasia contributes to airway hyperresponsiveness and remodeling in asthma. IL‐13, a Th2 cytokine, is a critical mediator of asthma pathogenesis through its inflammatory and growth promoting functions. The present study investigated the role of IL‐13 in PDGF‐induced human bronchial SMC (HBSMC) growth. Cultured HBSMCs showed basal level of IL‐13 mRNA as observed by nested PCR that was upregulated by PDGF. Further, PDGF‐induced HBSMC growth was significantly inhibited by pretreatment with IL‐13 neutralizing antibody, implicating IL‐13 in PDGF‐induced HBSMC growth. Since oxidant signaling plays an important part in PDGF‐induced cell growth, we tested the involvement of metal‐catalyzed oxidation (MCO). Inhibitors of MCO, deferoxamine and HBED, inhibited PDGF‐induced increase in IL‐13 mRNA levels as well as PDGF‐induced HBSMC growth, suggesting a role for MCO in PDGF‐mediated effects on HBSMCs. Our data shows that PDGF, a potent growth factor involved in airway remodeling, causes human bronchial SMC proliferation by upregulating IL‐13 levels through oxidant signaling.This work was supported by NIH.

β‐adrenergic receptors stimulate IL‐6 production in human bronchial smooth muscle cells: enhancement by non‐raft AC2 but not lipid raft AC6

In human bronchial smooth muscle cells (hBSMC) various isoforms of adenylyl cyclase (AC) are localized to different compartments: AC6 in lipid rafts and caveolae, AC2 outside of rafts. This localization of AC isoforms associates them with different receptors, G proteins and downstream signaling proteins. We hypothesized that specific AC isoforms overexpressed in hBSMC might have differing effects on gene expression. We overexpressed AC2 and AC6 and measured a variety of genes whose expression is influenced by cAMP. Cells were treated with vehicle or the AC activator, forskolin (Fsk). Interleukin 6 (IL‐6) mRNA levels were enhanced by Fsk, particularly when AC2 was overexpressed. Fsk‐stimulated IL‐6 mRNA levels (qRT‐PCR) peaked in 1 hr while protein levels (ELISA) peaked at 24 hr. Agonists selective for Gs‐coupled GPCR were tested for their ability to stimulate IL‐6 in AC2 or AC6 overexpressing cells. Increases in cAMP and IL‐6 were observed in response to isoproterenol, PGE2, butaprost and beraprost. AC2, but not AC6, overexpression increased IL‐6 production by these agonists. Increasing cAMP levels via Gs/Fsk synergy led to no increase in IL‐6 as compared to agonists alone. These data imply that AC2 signaling can lead to IL‐6 production by hBSMC but that little correlation exists between cellular cAMP levels and the level of this pro‐inflammatory cytokine produced. Supported by NIH (HL079166).

Proteomic identification of protein targets for 15-deoxy-Δ(12,14)-prostaglandin J2 in neuronal plasma membrane.

15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is one of factors contributed to the neurotoxicity of amyloid β (Aβ), a causative protein of Alzheimer's disease. Type 2 receptor for prostaglandin D2 (DP2) and peroxysome-proliferator activated receptorγ (PPARγ) are identified as the membrane receptor and the nuclear receptor for 15d-PGJ2, respectively. Previously, we reported that the cytotoxicity of 15d-PGJ2 was independent of DP2 and PPARγ, and suggested that 15d-PGJ2 induced apoptosis through the novel specific binding sites of 15d-PGJ2 different from DP2 and PPARγ. To relate the cytotoxicity of 15d-PGJ2 to amyloidoses, we performed binding assay [3H]15d-PGJ2 and specified targets for 15d-PGJ2 associated with cytotoxicity. In the various cell lines, there was a close correlation between the susceptibilities to 15d-PGJ2 and fibrillar Aβ. Specific binding sites of [3H]15d-PGJ2 were detected in rat cortical neurons and human bronchial smooth muscle cells. When the binding assay was performed in subcellular fractions of neurons, the specific binding sites of [3H]15d-PGJ2 were detected in plasma membrane, nuclear and cytosol, but not in microsome. A proteomic approach was used to identify protein targets for 15d-PGJ2 in the plasma membrane. By using biotinylated 15d-PGJ2, eleven proteins were identified as biotin-positive spots and classified into three different functional proteins: glycolytic enzymes (Enolase2, pyruvate kinase M1 (PKM1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), molecular chaperones (heat shock protein 8 and T-complex protein 1 subunit α), cytoskeletal proteins (Actin β, F-actin-capping protein, Tubulin β and Internexin α). GAPDH, PKM1 and Tubulin β are Aβ-interacting proteins. Thus, the present study suggested that 15d-PGJ2 plays an important role in amyloidoses not only in the central nervous system but also in the peripheral tissues.

Ginger suppresses phthalate ester-induced airway remodeling.

This study has two novel findings: it is not only the first to demonstrate inflammatory cytokines, which are produced by the bronchial epithelium after exposure to phthalate esters and contribute to airway remodeling by increasing human bronchial smooth muscle cells (BSMC) migration and proliferation, but it is also the first to reveal that ginger reverses phthalate ester-mediated airway remodeling. Human bronchial epithelial cell lines BEAS-2B and HBE135-E6E7 (HBE) were treated with butylbenzyl phthalate (BBP), bis(2-ethylhexyl) phthalate (BEHP), dibutyl phthalate (DBP), and diethyl phthalate (DEP), and the conditioned medium (CM) was harvested and then added to BSMC. Cultures of BSMC with BBP-, BEHP-, DBP-, and DEP-BEAS-2B-CM and DEP-HBE-CM increased BSMC proliferation and migration, which are major features in asthma remodeling. Exposure of BEAS-2B and HBE to DBP caused epithelial cells to produce inflammatory cytokines IL-8 and RANTES, which subsequently induced BSMC proliferation and migration. Depleting both IL-8 and RANTES completely reversed the effect of DBP-BEAS-2B-CM and DBP-HBE-CM-mediated BSMC proliferation and migration, suggesting this effect is a synergistic influence of IL-8 and RANTES. Moreover, [6]-shogaol, [6]-gingerol, [8]-gingerol, and [10]-gingerol, which are major bioactive compounds present in Zingiber officinale , suppress phthalate ester-mediated airway remodeling. This study suggests that ginger is capable of preventing phthalate ester-associated asthma.

Bronchial Epithelium–Derived IL-8 and RANTES Increased Bronchial Smooth Muscle Cell Migration and Proliferation by Krüppel-like Factor 5 in Areca Nut–Mediated Airway Remodeling

This study is first to analyze the inflammatory cytokines, produced by the bronchial epithelium after exposure to areca nut extract (ANE), which contribute to airway remodeling by increasing human bronchial smooth muscle cells (BSMC) migration and proliferation. We treated human bronchial epithelial cell lines BEAS-2B and HBE135-E6E7 (HBE) with ANE, saliva-reacted ANE (sANE), and the areca alkaloids arecoline and then harvested the conditioned medium (CM) that was added to BSMC. Exposure of BEAS-2B and HBE to ANE, sANE, and arecoline increased interleukin 8 (IL-8) and Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES) production. Cultures of BSMC with ANE-, sANE-, and arecoline-BEAS-2B-CM and -HBE-CM increased BSMC proliferation and migration. Induction of BSMC proliferation and migration by sANE-BEAS-2B-CM and -HBE-CM was associated with increased phosphorylation of Raf, MEK1/2, and extracellular signal regulated kinase (ERK)1/2 and the upregulation of krüppel-like factor 5 (KLF5), cyclin D, and integrin-linked kinase. Blocking ERK1/2 by a specific inhibitor significantly decreased BSMC proliferation and migration by inhibiting KLF5 enhancement. KLF5 knockdown also decreased sANE-BEAS-2B-CM, sANE-HBE-CM, and recombinant human interleukin 8/recombinant human RANTES-mediated BSMC proliferation and migration, suggesting that KLF5 was involved in the regulation of BSMC proliferation and migration. Our study suggests that inhibition of IL-8 and RANTES or IL-8/RANTES-mediated mitogen-activated protein kinase/KLF5 signaling is an attractive therapeutic target for areca nut-induced asthma.

The Effect of IL-17 on Glucocorticoid Receptor [GR] Translocation

RATIONALE: Severe asthma, associated with increased IL-17 expression, is characterized by resistance to CS. The function of GR is regulated by phosphorylation of N-terminal serine residues. We hypothesized that IL-17, a pro-inflammatory cytokine produced by TH17 cells that activates intracellular signaling kinases, would impair the translocation of GR induced by dex.METHODS: Human bronchial smooth muscle cells were transfected with pEGFP-hGRα plasmid, which expresses hGR coupled to green fluorescent protein. The transfected cells were treated with IL-17 for 15 min, 2 hours, 4 hours and 6 hours. GR function was then assessed by stimulating the cells with dex for 30 minutes. Cells were fixed and GR-GFP translocation was analyzed using MetaMorph.RESULTS: IL-17 produced a time- and dose-dependent alteration of GR function. Short (<2hr) exposures produced limited dex-independent GR translocation and blunted dex-induced GR function. In contrast, in cells treated ≥4 hours with IL-17 dex-induced GR translocation was entirely abrogated, demonstrating complete steroid resistance.CONCLUSIONS: IL-17 blunts and blocks dex-induced GR function in a dose and time dependent fashion. Early induction of dex-independent GR translocation is consistent with GR activation seen with other inflammatory cytokines, prototypically TNF-α. IL-17 may be a molecular mechanism by which steroid resistance in severe asthma is produced. Supported by HL69130, NIAID T35 (L Soong PhD). RATIONALE: Severe asthma, associated with increased IL-17 expression, is characterized by resistance to CS. The function of GR is regulated by phosphorylation of N-terminal serine residues. We hypothesized that IL-17, a pro-inflammatory cytokine produced by TH17 cells that activates intracellular signaling kinases, would impair the translocation of GR induced by dex. METHODS: Human bronchial smooth muscle cells were transfected with pEGFP-hGRα plasmid, which expresses hGR coupled to green fluorescent protein. The transfected cells were treated with IL-17 for 15 min, 2 hours, 4 hours and 6 hours. GR function was then assessed by stimulating the cells with dex for 30 minutes. Cells were fixed and GR-GFP translocation was analyzed using MetaMorph. RESULTS: IL-17 produced a time- and dose-dependent alteration of GR function. Short (<2hr) exposures produced limited dex-independent GR translocation and blunted dex-induced GR function. In contrast, in cells treated ≥4 hours with IL-17 dex-induced GR translocation was entirely abrogated, demonstrating complete steroid resistance. CONCLUSIONS: IL-17 blunts and blocks dex-induced GR function in a dose and time dependent fashion. Early induction of dex-independent GR translocation is consistent with GR activation seen with other inflammatory cytokines, prototypically TNF-α. IL-17 may be a molecular mechanism by which steroid resistance in severe asthma is produced. Supported by HL69130, NIAID T35 (L Soong PhD).

Human Bronchial Smooth Muscle Cells Express Adenylyl Cyclase Isoforms 2, 4, and 6 in Distinct Membrane Microdomains

Adenylyl cyclases (AC) are important regulators of airway smooth muscle function, because β-adrenergic receptor (AR) agonists stimulate AC activity and increase airway diameter. We assessed expression of AC isoforms in human bronchial smooth muscle cells (hBSMC). Reverse transcriptase-polymerase chain reaction and immunoblot analyses detected expression of AC2, AC4, and AC6. Forskolin-stimulated AC activity in membranes from hBSMC displayed Ca(2+)-inhibited and G(βγ)-stimulated AC activity, consistent with expression of AC6, AC2, and AC4. Isoproterenol-stimulated AC activity was inhibited by Ca(2+) but unaltered by G(βγ), whereas butaprost-stimulated AC activity was stimulated by G(βγ) but unaffected by Ca(2+) addition. Using sucrose density centrifugation to isolate lipid raft fractions, we found that only AC6 localized in lipid raft fractions, whereas AC2 and AC4 localized in nonraft fractions. Immunoisolation of caveolae using caveolin-1 antibodies yielded Ca(2+)-inhibited AC activity (consistent with AC6 expression), whereas the nonprecipitated material displayed G(βγ)-stimulated AC activity (consistent with expression of AC2 and/or AC4). Overexpression of AC6 enhanced cAMP production in response to isoproterenol and beraprost but did not increase responses to prostaglandin E(2) or butaprost. β(2)AR, but not prostanoid EP(2) or EP(4) receptors, colocalized with AC5/6 in lipid raft fractions. Thus, particular G protein-coupled receptors couple to discreet AC isoforms based, in part, on their colocalization in membrane microdomains. These different cAMP signaling compartments in airway smooth muscle cells are responsive to different hormones and neurotransmitters and can be regulated by different coincident signals such as Ca(2+) and G(βγ).

Cigarette Smoke Extract-induced Reduction in Migration and Contraction in Normal Human Bronchial Smooth Muscle Cells

The proliferation, migration, cytokine release, and contraction of airway smooth muscle cells are key events in the airway remodeling process that occur in lung disease such as asthma, chronic obstruction pulmonary disease, and cancer. These events can be modulated by a number of factors, including cigarette smoke extract (CSE). CSE-induced alterations in the viability, migration, and contractile abilities of normal human airway cells remain unclear. This study investigated the effect of CSE on cell viability, migration, tumor necrosis factor (TNF)-α secretion, and contraction in normal human bronchial smooth muscle cells (HBSMCs). Treatment of HBSMCs with 10% CSE induced cell death, and the death was accompanied by the generation of reactive oxygen species (ROS). CSE-induced cell death was reduced by N-acetyl-l-cysteine (NAC), an ROS scavenger. In addition, CSE reduced the migration ability of HBSMCs by 75%. The combination of NAC with CSE blocked the CSE-induced reduction of cell migration. However, CSE had no effect on TNF-α secretion and NF-κB activation. CSE induced an increase in intracellular Ca2+ concentration in 64% of HBSMCs. CSE reduced the contractile ability of HBSMCs, and the ability was enhanced by NAC treatment. These results demonstrate that CSE treatment induces cell death and reduces migration and contraction by increasing ROS generation in normal HBSMCs. These results suggest that CSE may induce airway change through cell death and reduction in migration and contraction of normal HBSMCs.

Rosiglitazone reverses salbutamol-induced β2-adrenoceptor tolerance in airway smooth muscle

β₂-Adrenoceptor agonists are important therapeutic agents in the treatment of asthma and chronic obstructive pulmonary disease. The regular use of these drugs has been associated with proasthmatic-like changes that limit their efficacy and increase the risk of severe adverse reactions. We investigated whether the peroxisome-proliferator-activated receptor (PPAR)γ agonist rosiglitazone modulated salbutamol-induced β₂-adrenoceptor desensitization in vivo and in vitro. An in vivo model of homologous β₂-adrenoceptor desensitization, established in guinea-pigs by administering salbutamol continuously, was used to study the ability of rosiglitazone to prevent β₂-adrenoceptor tolerance. In vitro experiments on human bronchial smooth muscle cells were performed to increase the clinical relevance of the study. In tracheal smooth muscle tissues from desensitized animals, we observed a decrease in the protective effect of salbutamol on carbachol-induced contraction, a hyperresponsiveness to cholinergic stimuli, a modest underexpression of β₂-adrenoceptor gene and a marked decrease in β-adrenoceptor number, relative to control values. Treatment with rosiglitazone preserved salbutamol relaxant activity, mitigated carbachol hyperresponsiveness and partially restored β₂-adrenoceptor binding sites in tracheal tissues from homologously desensitized animals. The highly selective PPARγ agonist, GW1929, reproduced the effect of rosiglitazone, in vivo. In vitro β₂-adrenoceptor desensitization decreased salbutamol-mediated cAMP production, without affecting forskolin responses and β₂-adrenoceptor expression. Rosiglitazone and 15-deoxy-Δ¹²(,)¹⁴-prostaglandin J₂ restored salbutamol sensitivity in homologously desensitized cells. These data suggest a potential pharmacodynamic interaction between PPARγ agonists and salbutamol on airway smooth muscle responsiveness, supporting the therapeutic potential of this combination in chronic airway disease.