Matrix metalloproteinase expression and activity in human airway smooth muscle cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Elshaw SR
PROVIDER: S-EPMC1575191 | biostudies-other | 2004 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Defining mechanisms by which differentiated, contractile smooth muscle cells become proliferative and secretory in response to mechanical and environmental stress is crucial for determining the contribution of airway smooth muscle (ASM) to inflammatory responses that result in airway disease. Regulation by microRNAs (miRNAs) has emerged as an important post-transcriptional mechanism regulating gene expression that may modulate ASM phenotype, but little is known about the expression and functions of miRNA in smooth muscle. In the present study we used microarrays to determine whether miRNAs in human ASM cells are altered by a proinflammatory stimulus. In ASM cells exposed to IL-1beta, TNF-alpha, and IFN-gamma, we found 11 miRNAs to be significantly down-regulated. We verified decreased expression of miR-25, miR-140*, mir-188, and miR-320 by quantitative PCR. Analysis of miR-25 expression indicates that it has a broad role in regulating ASM phenotype by modulating expression of inflammatory mediators such as RANTES, eotaxin, and TNF-alpha; genes involved in extracellular matrix turnover; and contractile proteins, most notably myosin heavy chain. miRNA binding algorithms predict that miR-25 targets Krüppel-like factor 4 (KLF4), a potent inhibitor of smooth muscle-specific gene expression and mediator of inflammation. Our study demonstrates that inhibition of miR-25 in cytokine-stimulated ASM cells up-regulates KLF4 expression via a post-transcriptional mechanism. This provides novel evidence that miR-25 targets KLF4 in ASM cells and proposes that miR-25 may be an important mediator of ASM phenotype.
| S-EPMC2848741 | biostudies-literature
Project description:ObjectiveVascular injury causes neointimal hypertrophy, which is characterized by redox-mediated matrix degradation and smooth muscle cell (SMC) migration and proliferation. We hypothesized that, as compared to the adjacent medial SMCs, neointimal SMCs produce increased superoxide via NADPH oxidase, which induces redox-sensitive intracellular signaling to activate matrix metalloproteinase-9 (MMP-9).Methods and resultsTwo weeks after balloon injury, rat aorta developed a prominent neointima, containing increased expression of NADPH oxidase and reactive oxygen species (ROS) as compared to the medial layer. Next, SMCs were isolated from either the neointima or the media and studied in culture. Neointimal-derived SMCs exhibited increased Nox1 expression and ROS levels as compared to medial SMCs. Neointimal SMCs had higher cell growth rates than medial SMCs. ROS-dependent ERK1/2 phosphorylation was greater in neointimal SMCs. MMP-9 activity, as detected by gel zymography, was greater in neointimal SMCs under resting and stimulated conditions and was prevented by expression of an antisense to Nox1 or treatment with an ERK1/2 inhibitor.ConclusionsFollowing vascular injury, the increased expression of Nox1 in SMCs within the neointima initiates redox-dependent phosphorylation of ERK1/2 and subsequent MMP-9 activation.
| S-EPMC3369242 | biostudies-literature
Project description:Pentraxin-3 (PTX3) is a multifunctional protein involved in both innate and adaptive immunity. Glucocorticoid (GC) is the first-line therapy to mitigate airway inflammation in asthma. Previous pieces of evidence showed that GC has divergent effects on PTX3 production in various cell types. The molecular mechanisms controlling PTX3 expression in HASMC are, however, not yet characterized. In this study, we demonstrate that the synthetic GC, dexamethasone (DEX) increases the expression of PTX3 both at the protein and mRNA levels. We also found that such an effect of DEX was dependent on de novo protein synthesis and the GC receptor (GR). While DEX increases PTX3 mRNA stability, it did not affect its promoter activity. Interestingly, HASMC pre-treated with p42/p44 ERK inhibitor, but not with p38 or JNK-MAPK inhibitors, significantly interfered with DEX-induced PTX3 secretion. Taken together, our data suggest that GC regulates PTX3 expression in HASMC through transcriptional and post-transcriptional mechanisms in a GR and ERK-dependent manner.
| S-EPMC6706008 | biostudies-literature
Project description:Leukotriene B4 (LTB4) increases in induced sputum and exhaled breath condensate in people with asthma. Furthermore, the T(H)2-type immune response and airway hyperresponsiveness induced by ovalbumin sensitization is markedly suppressed in LTB4 receptor (BLT) 1 null mice. These studies suggest that LTB4 may contribute to asthma pathophysiology. However, the direct effects of LTB4 on human airway smooth muscle (ASM) have not been studied.We sought to determine the expression of LTB4 receptors on human ASM and its functional role in mediating responses of human ASM cells, and the effect of LTB4 on these cells.Immunohistochemistry, RT-PCR, Western blotting, and flow cytometry were used to determine the expression of LTB4 receptors. To determine the effect of LTB4 on human ASM cells, cell proliferation was assessed by counting cells, and chemokinesis was assessed by gold particle phagokinesis assay.We confirmed expression of both BLT1 and BLT2 in human ASM cells in bronchial tissue and in cell culture. LTB4 markedly induced cyclin D1 expression, proliferation, and chemokinesis of human ASM cells. LTB4 also induced phosphorylation of both p42/p44 mitogen-activated protein kinase (MAPK) and downstream PI3 kinase effector, Akt1. However, we observed no induction of c-Jun N-terminal kinase or p38 MAPK. Notably, LTB4-induced migration and proliferation of ASM cells were inhibited by the BLT1 specific antagonist, U75302, and by inhibitors of p42/p44 MAPK phosphorylation (U1026), and PI3 kinase (LY294002).These observations are the first to suggest a role for a LTB4-BLT1 signaling axis in ASM responses that may contribute to the pathogenesis of airway remodeling in asthma.
| S-EPMC4301732 | biostudies-literature
Project description:Previous studies have shown that enhanced accumulation of contractile proteins such as smooth muscle myosin light chain kinase (smMLCK) plays a major role in human airway smooth muscle cells (HASM) cell hypercontractility and hypertrophy. Furthermore, serum IgE levels play an important role in smooth muscle hyperreactivity. However, the effect of IgE on smMLCK expression has not been investigated. In this study, we demonstrate that IgE increases the expression of smMLCK at mRNA and protein levels. This effect was inhibited significantly with neutralizing abs directed against Fc?RI but not with anti-Fc?RII/CD23. Furthermore, Syk knock down and pharmacological inhibition of mitogen activated protein kinases (MAPK) (ERK1/2, p38, and JNK) and phosphatidylinositol 3-kinase (PI3K) significantly diminished the IgE-mediated upregulation of smMLCK expression in HASM cells. Taken together, our data suggest a role of IgE in regulating smMLCK in HASM cells. Therefore, targeting the Fc?RI activation on HASM cells may offer a novel approach in controlling the bronchomotor tone in allergic asthma.
| S-EPMC3983085 | biostudies-literature
Project description:Background:Airway remodeling is an important feature of chronic obstructive pulmonary disease (COPD) that is associated with disease severity and irreversible airflow limitation. An extensive alteration of the extracellular matrix (ECM) surrounding the airway smooth muscle (ASM) bundle is one of the pathological manifestations of airway remodeling, which contributes to the decline in lung function. Tiotropium, a long-acting inhaled muscarinic receptor antagonist, has been confirmed to play a role in preventing airway remodeling including ECM deposition beyond bronchodilation in vivo, but the relationship between ASM cell (ASMC) relaxation and ECM production remains unclear. Purpose:In this study, we attempted to investigate the influence of tiotropium on ECM production by ASMCs and the underlying mechanism. Methods:Tiotropium was added 30 minutes before the addition of methacholine to primary cultured human ASMCs. Protein expression was analylized by Western Blot and mRNA abundance was determined by real-time PCR. Results:We found that tiotropium reduced collagen I protein expression, and the mRNA abundance of collagen I, fibronectin, and versican. ?-catenin signaling was inactivated by inhibiting glycogen synthase kinase 3? (GSK3?) phosphorylation in this process. Tiotropum inhibited the amount of active ?-catenin and its transcription activity. Furthermore, overexpression of active ?-catenin by adenoviruses carrying the S33Y mutant resisted the suppressive effect of tiotropium on collagen I protein expression. However, silencing ?-catenin by specific small interfering RNA enhanced the negative effect of tiotropium. Conclusion:These findings suggest that relaxation of ASMCs by tiotropium can prevent ECM production through ?-catenin signaling.
| S-EPMC5939907 | biostudies-literature
Project description:BACKGROUND:Matrix metalloproteinase 9 (MMP-9) is involved in extracellular matrix degradation and remodeling. An increase in MMP-9 expression by vascular component cells plays an important role in atherosclerotic plaque formation and rupture. Resveratrol, a polyphenolic substance, was suggested to play a role in preventing the progress of atherosclerotic disease. The aim of this study was to investigate the effect of resveratrol on MMP-9 and tissue inhibitors of metalloproteinases (TIMPs) in vascular smooth muscle cells (VSMCs) after treatment with H2O2. METHODS:Cultured VSMCs were pre-treated with 0.2 mM of H2O2 before stimulation with different concentration of resveratrol. Expression of MMP-9, TIMP-1, and TIMP-3 genes were measured using real-time polymerase chain reaction (PCR) method, and MMP-9 protein level was detected using western blot analysis. RESULTS:Resveratrol at 120 ?mol/l concentration reduced the elevated level of MMP-9 induced by H2O2 in VSMCs as 1.85 ± 0.35 folds (P < 0.050) and 8.70 ± 1.20 folds (P < 0.050) after 24 and 48 hours, respectively. Resveratrol increased the diminished level of TIMP-1 induced by H2O2 as 2.5 ± 0.48 folds following the treatment with 120 ?mol/l after 48 hours (P < 0.050). CONCLUSION:Resveratrol as an antioxidant can decrease MMP-9 production, not only by suppressing MMP-9 expression, but also by augmenting TIMP-1 production. Altogether, resveratrol as an antioxidant can regulate the MMP-9/TIMP-1 balance, and may be considered as a preservative agent in the treatment and prevention of atherosclerosis.
| S-EPMC6312567 | biostudies-literature
Project description:β-adrenergic receptor (βAR) activation promotes relaxation of both vascular and airway smooth muscle cells (VSMCs and ASMCs, respectively), though the signaling mechanisms have not been fully elucidated. We previously found that the activity of Kv7.5 voltage-activated potassium channels in VSMCs is robustly enhanced by activation of βARs via a mechanism involving protein kinase A (PKA)-dependent phosphorylation. We also found that enhancement of Kv7 channel activity in ASMCs promotes airway relaxation. Here we provide evidence that Kv7.5 channels are natively expressed in primary cultures of human ASMCs and that they conduct currents which are robustly enhanced in response to activation of the βAR/cyclic adenosine monophosphate (cAMP)/PKA pathway. MIT Scansite software analysis of putative PKA phosphorylation sites on Kv7.5 identified 8 candidate serine or threonine residues. Each residue was individually mutated to an alanine to prevent its phosphorylation and then tested for responses to βAR activation or to stimuli that elevate cAMP levels. Only the mutation of serine 53 (S53A), located on the amino terminus of Kv7.5, significantly reduced the increase in Kv7.5 current in response to these stimuli. A phospho-mimic mutation (S53D) exhibited characteristics of βAR-activated Kv7.5. Serine-to-alanine mutations of 6 putative PKA phosphorylation sites on the Kv7.5 C-terminus, individually or in combination, did not significantly reduce the enhancement of the currents in response to forskolin treatment (to elevate cAMP levels). We conclude that phosphorylation of S53 on the amino terminus of Kv7.5 is essential for PKA-dependent enhancement of channel activity in response to βAR activation in vascular and airway smooth muscle cells.
| S-EPMC6121446 | biostudies-literature
Project description:Airway smooth muscle (ASM) cells play a critical role in the pathophysiology of asthma due to their hypercontractility and their ability to proliferate and secrete inflammatory mediators. microRNAs (miRNAs) are gene regulators that control many signaling pathways and thus serve as potential therapeutic alternatives for many diseases. We have previously shown that miR-708 and miR-140-3p regulate the MAPK and PI3K signaling pathways in human ASM (HASM) cells following TNF-? exposure. In this study, we investigated the regulatory effect of these miRNAs on other asthma-related genes. Microarray analysis using the Illumina platform was performed with total RNA extracted from miR-708 (or control miR)-transfected HASM cells. Inhibition of candidate inflammation-associated gene expression was further validated by qPCR and ELISA. The most significant biologic functions for the differentially expressed gene set included decreased inflammatory response, cytokine expression and signaling. qPCR revealed inhibition of expression of CCL11, CXCL10, CCL2 and CXCL8, while the release of CCL11 was inhibited in miR-708-transfected cells. Transfection of cells with miR-140-3p resulted in inhibition of expression of CCL11, CXCL12, CXCL10, CCL5 and CXCL8 and of TNF-?-induced CXCL12 release. In addition, expression of RARRES2, CD44 and ADAM33, genes known to contribute to the pathophysiology of asthma, were found to be inhibited in miR-708-transfected cells. These results demonstrate that miR-708 and miR-140-3p exert distinct effects on inflammation-associated gene expression and biological function of ASM cells. Targeting these miRNA networks may provide a novel therapeutic mechanism to down-regulate airway inflammation and ASM proliferation in asthma.
| S-EPMC4801396 | biostudies-literature
Project description:Lung function declines as people age and their lungs become stiffer. With an increasing elderly population, understanding mechanisms that contribute to these structural and functional changes in the aging lung is important. Part of the aging process is characterized by thicker, more fibrotic airways, and senile emphysema caused by changes in lung parenchyma. There is also senescence, which occurs throughout the body with aging. Here, using human airway smooth muscle (ASM) cells from patients in different age groups, we explored senescence pathways and changes in intracellular calcium signaling and extracellular matrix (ECM) deposition to elucidate potential mechanisms by which aging leads to thicker and stiffer lungs. Senescent markers p21, γH2AX, and β-gal, and some senescence-associated secretory proteins (SASP) increased with aging, as shown by staining and biochemical analyses. Agonist-induced intracellular Ca2+ responses, measured using fura-2 loaded cells and fluorescence imaging, increased with age. However, biochemical analysis showed that expression of the following markers decreased with age: M3 muscarinic receptor, TRPC3, Orai1, STIM1, SERCA2, MMP2 and MMP9. In contrast, collagen III, and fibronectin deposition increased with age. These data show that senescence increases in the aging airways that is associated with a stiffer but surprisingly greater intracellular calcium signaling as a marker for contractility. ASM senescence may enhance fibrosis in a feed forward loop promoting remodeling and altered calcium storage and buffering.
| S-EPMC8321097 | biostudies-literature