TGF-β potentiates airway smooth muscle responsiveness to bradykinin

The molecular mechanisms by which bradykinin induces excessive airway obstruction in asthmatics remain unknown. Transforming growth factor (TGF)-β has been involved in regulating airway inflammation and remodeling in asthma, although it is unknown whether TGF-β can modulate bradykinin-associated bronchial hyperresponsiveness. To test whether TGF-β directly modulates airway smooth muscle (ASM) responsiveness to bradykinin, isolated murine tracheal rings were used to assess whether TGF-β alters ASM contractile responsiveness to bradykinin. Interestingly, we found TGF-β-treated murine rings (12.5 ng/ml, 18 h) exhibited increased expression of bradykinin 2 (B2) receptors and became hyperreactive to bradykinin, as shown by increases in maximal contractile responses and receptor distribution. We investigated the effect of TGF-β on bradykinin-evoked calcium signals since calcium is a key molecule regulating ASM excitation-contraction coupling. We reported that TGF-β, in a dose- (0.5–10 ng/ml) and time- (2–24 h) dependent manner, increased mRNA and protein expression of the B2 receptor in cultured human ASM cells. Maximal B2 receptor protein expression that colocalized with CD44, a marker of membrane cell surface, occurred after 18 h of TGF-β treatment and was further confirmed using fluorescence microscopy. TGF-β (2.5 ng/ml, 18 h) also increased bradykinin-induced intracellular calcium mobilization in fura-2-loaded ASM cells. TGF-β-mediated enhancement of calcium mobilization was not attenuated with indomethacin, a cyclooxygenase inhibitor. These data demonstrate for the first time that TGF-β may play a role in mediating airway hyperresponsiveness to bradykinin seen in asthmatics by enhancing ASM contractile responsiveness to bradykinin, possibly as a result of increased B2 receptor expression and signaling.

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TIMP-1 Induces α-Smooth Muscle Actin in Fibroblasts to Promote Urethral Scar Formation

Cellular Physiology and Biochemistry ◽

10.1159/000374028 ◽

2015 ◽

Vol 35 (6) ◽

pp. 2233-2243 ◽

Cited By ~ 15

Author(s):

Yinglong Sa ◽

Chao Li ◽

Hongbin Li ◽

Hailin Guo

Keyword(s):

Smooth Muscle ◽

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Human Fibroblasts ◽

Mapk Signaling ◽

Scar Formation ◽

Dependent Manner ◽

Muscle Actin ◽

Mesenchymal Transition

Background/Aims: Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been reported to upregulate in urethral scar. However, the underlying molecular mechanisms remain undefined. Methods: Here, we studied levels of TIMP-1 and α-smooth muscle actin (α-SMA) in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Then we either overexpressed TIMP-1, or inhibited TIMP-1 by lentiviruses carrying a transgene or a short hairpin small interfering RNA for TIMP-1 in human fibroblasts. We examined the effects of modulation of TIMP-1 on α-SMA, and on epithelial-mesenchymal transition (EMT)-related genes. We also studied the underlying mechanisms. Results: We detected significantly higher levels of TIMP-1 and α-smooth muscle actin (α-SMA) in the fibroblasts isolated from urethral scar tissues, compared to the fibroblasts isolated from normal urethra. Moreover, the levels of TIMP-1 and α-SMA strongly correlated. Moreover, we found that TIMP-1 significantly increased levels of α-SMA, transforming growth factor β 1 (TGFβ1), Collagen I and some other key factors related to an enhanced EMT, suggesting that TIMP-1 may induce transformation of fibroblasts into myofibroblasts to promote tissue EMT to enhance the formation of urethral scar. Moreover, increases in TIMP-1 also induced an increase in fibroblast cell growth and cell invasion, in an ERK/MAPK-signaling-dependent manner. Conclusion: Our study thus highlights a pivotal role of TIMP-1 in urethral scar formation.

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Mechanisms of induction of airway smooth muscle hyperplasia by transforming growth factor-β

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00068.2007 ◽

2007 ◽

Vol 293 (1) ◽

pp. L245-L253 ◽

Cited By ~ 73

Author(s):

Shaoping Xie ◽

Maria B. Sukkar ◽

Razao Issa ◽

Nadia M. Khorasani ◽

Kian Fan Chung

Keyword(s):

Smooth Muscle ◽

Growth Factor ◽

P38 Mapk ◽

Airway Smooth Muscle ◽

Transforming Growth Factor ◽

Dominant Negative ◽

Negative Regulator ◽

Dependent Manner ◽

Mitogenic Signaling ◽

Tgf Β1

Airway smooth muscle (ASM) hyperplasia is a characteristic feature of the asthmatic airway, but the underlying mechanisms that induce ASM hyperplasia remain unknown. Because transforming growth factor (TGF)-β is a potent regulator of ASM cell proliferation, we determined its expression and mitogenic signaling pathways in ASM cells. We obtained ASM cells by laser capture microdissection of bronchial biopsies and found that ASM cells from asthmatic patients expressed TGF-β1 mRNA and protein to a greater extent than nonasthmatic individuals using real-time RT-PCR and immunohistochemistry, respectively. TGF-β1 stimulated the growth of nonconfluent and confluent ASM cells either in the presence or absence of serum in a time- and concentration-dependent manner. The mitogenic activity of TGF-β1 on ASM cells was inhibited by selective inhibitors of TGF-β receptor I kinase (SD-208), phosphatidylinositol 3-kinase (PI3K, LY-294002), ERK (PD-98059), JNK (SP-600125), and NF-κB (AS-602868). On the other hand, p38 MAPK inhibitor (SB-203580) augmented TGF-β1-induced proliferation. To study role of the Smads, we transduced ASM cells with an adenovirus vector-expressing Smad4, Smad7, or dominant-negative Smad3 and found no involvement of these Smads in TGF-β1-induced proliferation. Dexamethasone caused a dose-dependent inhibition in TGF-β1-induced proliferation. Our findings suggest that TGF-β1 may act in an autocrine fashion to induce ASM hyperplasia, mediated by its receptor and several kinases including PI3K, ERK, and JNK, whereas p38 MAPK is a negative regulator. NF-κB is also involved in the TGF-β1 mitogenic signaling, but Smad pathway does not appear important.

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TGF-β1 stimulates IL-8 release, COX-2 expression, and PGE2release in human airway smooth muscle cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2000.279.1.l201 ◽

2000 ◽

Vol 279 (1) ◽

pp. L201-L207 ◽

Cited By ~ 75

Author(s):

Choong Yi Fong ◽

Linhua Pang ◽

Elaine Holland ◽

Alan J. Knox

Keyword(s):

Smooth Muscle ◽

Airway Smooth Muscle ◽

Transforming Growth Factor ◽

Airway Smooth Muscle Cells ◽

Dependent Manner ◽

Human Airway Smooth Muscle ◽

Human Airway ◽

Cox Inhibitor ◽

Cox 2 ◽

Tgf Β1

We have recently shown that endogenous prostanoids are critical in bradykinin-stimulated interleukin (IL)-8 release from human airway smooth muscle (ASM) cells. In this study, we tested the ability of transforming growth factor (TGF)-β1 to stimulate IL-8 release, cyclooxygenase (COX)-2 expression and PGE2 generation in cultured human ASM cells and explored the role of COX products and COX-2 induction on IL-8 release. TGF-β1 stimulated IL-8 release, COX-2 induction, and PGE2 generation in a concentration- and time-dependent manner. Maximal IL-8 release was achieved with 10 ng/ml of TGF-β1 after 16 h of incubation, which was inhibited by the transcription inhibitor actinomycin D and the corticosteroid dexamethasone but was not affected by the nonselective COX inhibitor indomethacin and the selective COX-2 inhibitor NS-398 despite their inhibition on TGF-β1-induced PGE2 release. These results show for the first time that TGF-β1 stimulates IL-8 release, COX-2 induction, and PGE2 generation in human ASM cells and that PGE2 generation is not critical for TGF-β1-induced IL-8 release. These findings suggest that TGF-β1 may play an important role in the pathophysiology of asthma.

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Histamine stimulates proliferation of airway smooth muscle and induces c-fos expression

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1990.259.6.l365 ◽

1990 ◽

Vol 259 (6) ◽

pp. L365-L371 ◽

Cited By ~ 23

Author(s):

R. A. Panettieri ◽

P. A. Yadvish ◽

A. M. Kelly ◽

N. A. Rubinstein ◽

M. I. Kotlikoff

Keyword(s):

Cell Proliferation ◽

Smooth Muscle ◽

Protein Expression ◽

Myosin Heavy Chain ◽

Airway Smooth Muscle ◽

Heavy Chain ◽

Thymidine Incorporation ◽

Dependent Manner ◽

Altered Expression ◽

Mrna Induction

Although chronic severe asthma is characterized by increased smooth muscle mass in the airways, the physiological stimuli that promote airway smooth muscle (ASM) proliferation (hyperplasia) or increase ASM protein expression (hypertrophy) are unknown. We examined the effects of histamine, an autocoid associated with airway hyperresponsiveness, on protein synthesis, myosin heavy chain expression, and cell proliferation in cultured canine ASM cells. In confluent ASM cells, histamine significantly increased incorporation of [35S]-methionine in protein. Maintenance of the proportion of smooth muscle-specific myosin heavy chain to total myosin heavy chain suggested a nonspecific increase in contractile protein expression. DNA synthesis, as measured by [3H]thymidine incorporation, was significantly increased by histamine in a concentration-dependent manner. Cell proliferation paralleled [3H]thymidine incorporation; histamine significantly increased cell numbers at 24 and 48 h of stimulation. Because growth of mesenchymal-derived cells is associated with transcription of c-fos mRNA, we examined whether histamine altered expression of this proto-oncogene. Histamine-treated cells showed marked increases in expressions of steady-state c-fos mRNA, with a time course of mRNA induction similar to cells exposed to platelet-derived growth factor or serum, known smooth muscle and fibroblast cell mitogens. Therefore, histamine is an ASM mitogen with an action similar to other mesenchymal cell growth factors and may play a role in the hyperplasia of ASM in asthma.

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ET-1 induces mitogenesis in ovine airway smooth muscle cells via ETA and ETB receptors

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.272.5.l1021 ◽

1997 ◽

Vol 272 (5) ◽

pp. L1021-L1024

Author(s):

P. Carratu ◽

M. Scuri ◽

J. L. Styblo ◽

A. Wanner ◽

M. K. Glassberg

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Airway Smooth Muscle ◽

Muscle Cells ◽

Receptor Expression ◽

Airway Smooth Muscle Cells ◽

Receptor Subtypes ◽

Dependent Manner ◽

Bq 610 ◽

20 Nm

The proliferation of airway smooth muscle cells is a characteristic feature of asthma. Endothelin (ET)-1, a member of a family of three isopeptides (ET-1, ET-2, and ET-3), functions as a spasmogen and mitogen for airway smooth muscle cells. Two types of ET receptors have been identified in mammalian species (ETA and ETB). Because the respective roles of ETA and ETB receptors in ET-1-induced mitogenesis are not known, we determined the effect of two selective ETA and ETB antagonists (BQ-610 and BQ-788) on ET-1-induced mitogenesis of cultured ovine airway smooth muscle cells. Both BQ-610 and BQ-788 inhibited ET-1-induced mitogenesis in a concentration-dependent manner, with BQ-788 exhibiting more potent antagonism [half-maximal inhibitory concentration (IC50) = 3.5 nM, slope of 0.49] compared with BQ-610 (IC50 = 20 nM, slope of 0.27). The combined ETA-ETB antagonist, bosentan, also inhibited ET-1-induced mitogenesis (IC50 = 20 nM, slope of 0.60). The effects of BQ-788 and bosentan appear to be mediated via the same receptor (ETB), as their slopes are comparable. These observations suggest that both receptor subtypes are utilized in ET-1-induced proliferation of ovine airway smooth muscle. ET receptor expression may be important in the increase in airway smooth muscle mass seen in the airways of patients with bronchial asthma.

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ROS and PDFG-β receptors are critically involved in indoxyl sulfate actions that promote vascular smooth muscle cell proliferation and migration

AJP Cell Physiology ◽

10.1152/ajpcell.00206.2009 ◽

2009 ◽

Vol 297 (2) ◽

pp. C389-C396 ◽

Cited By ~ 69

Author(s):

Hidehisa Shimizu ◽

Yuichi Hirose ◽

Fuyuhiko Nishijima ◽

Yoshiharu Tsubakihara ◽

Hitoshi Miyazaki

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Nadph Oxidase ◽

Protein Expression ◽

Receptor Expression ◽

Indoxyl Sulfate ◽

Receptor Protein ◽

Uremic Toxin ◽

Diphenylene Iodonium ◽

Pdgf Stimulation

Patients with chronic renal failure are at greater risk of developing atherosclerosis than healthy individuals, and recent data suggest that the putative uremic toxin indoxyl sulfate (IS) promotes the pathogenesis of atherosclerosis. The present study examined the effects of IS on vascular smooth muscle cells (VSMCs) with respect to reactive oxygen species (ROS), platelet-derived growth factor (PDGF) receptors, and mitogen-activated protein kinases (MAPKs). IS induced the migration and proliferation of VSMCs and synergistically enhanced their PDGF-induced migration as well as proliferation. The effects of PDGF were promoted after a 24-h incubation with IS despite the absence of IS during PDGF stimulation. Intracellular ROS levels were increased in the presence of IS, and PDGF-dependent ROS production was augmented by a prior 24-h incubation with IS even in the absence of IS during PDGF stimulation. These data suggest that IS increases the sensitivity of VSMCs to PDGF. IS also phosphorylated PDGF-β-receptors and upregulated PDGF-β receptor but not α-receptor protein expression in the absence of exogenous PDGF. The NADPH oxidase inhibitor diphenylene iodonium blocked IS-dependent increase in receptor expression. Administration of IS to nephrectomized rats also elevated receptor protein expression in arterial VSMCs. Inhibitors of NADPH oxidase, PDGF-β receptors, extracellular-regulated protein kinase (ERK), and p38 MAPK all inhibited IS-induced VSMCs migration and proliferation. Taken together, these findings indicate that IS induces the migration as well as proliferation of VSMCs through PDGF-β receptors and that ROS generation is critically involved in this process, which promotes the development of atherosclerosis.

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l-Thyroxine promotes a proliferative airway smooth muscle phenotype in the presence of TGF-β1

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00071.2014 ◽

2015 ◽

Vol 308 (3) ◽

pp. L301-L306 ◽

Cited By ~ 9

Author(s):

Bart G. J. Dekkers ◽

Saeideh Naeimi ◽

I. Sophie T. Bos ◽

Mark H. Menzen ◽

Andrew J. Halayko ◽

...

Keyword(s):

Smooth Muscle ◽

Thyroid Hormones ◽

Protein Expression ◽

Airway Smooth Muscle ◽

Transforming Growth Factor ◽

Airway Remodeling ◽

Contractile Protein ◽

Cell Phenotype ◽

Minor Effect ◽

Tgf Β1

Hypothyroidism may reduce, whereas hyperthyroidism may aggravate, asthma symptoms. The mechanisms underlying this relationship are largely unknown. Since thyroid hormones have central roles in cell growth and differentiation, we hypothesized that airway remodeling, in particular increased airway smooth muscle (ASM) mass, may be involved. To address this hypothesis, we investigated the effects of triiodothyronine (T3) and l-thyroxine (T4) in the absence and presence of the profibrotic transforming growth factor (TGF)-β1 on human ASM cell phenotype switching. T3 (1–100 nM) and T4 (1–100 nM) did not affect basal ASM proliferation. However, when combined with TGF-β1 (2 ng/ml), T4 synergistically increased the proliferative response, whereas only a minor effect was observed for T3. In line with a switch from a contractile to a proliferative ASM phenotype, T4 reduced the TGF-β1-induced contractile protein expression by ∼50%. Cotreatment with T3 reduced TGF-β1-induced contractile protein expression by ∼25%. The synergistic increase in proliferation was almost fully inhibited by the integrin αvβ3 antagonist tetrac (100 nM), whereas no significant effects of the thyroid receptor antagonist 1–850 (3 μM) were observed. Inhibition of MEK1/2, downstream of the integrin αvβ3, also inhibited the T4- and TGF-β1-induced proliferative responses. Collectively, the results indicate that T4, and to a lesser extent T3, promotes a proliferative ASM phenotype in the presence of TGF-β1, which is predominantly mediated by the membrane-bound T4 receptor αvβ3. These results indicate that thyroid hormones may enhance ASM remodeling in asthma, which could be of relevance for hyperthyroid patients with this disease.

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Muscarinic receptor stimulation augments TGF-β1-induced contractile protein expression by airway smooth muscle cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00400.2011 ◽

2012 ◽

Vol 303 (7) ◽

pp. L589-L597 ◽

Cited By ~ 28

Author(s):

Tjitske A. Oenema ◽

Marieke Smit ◽

Lyanne Smedinga ◽

Kurt Racké ◽

Andrew J. Halayko ◽

...

Keyword(s):

Smooth Muscle ◽

Protein Expression ◽

Muscarinic Receptor ◽

Airway Smooth Muscle ◽

Transforming Growth Factor ◽

Glycogen Synthase ◽

Airway Remodeling ◽

Contractile Protein ◽

Airway Smooth Muscle Cells ◽

Receptor Stimulation

Acetylcholine (ACh) is the primary parasympathetic neurotransmitter in the airways. Recently, it was established that ACh, via muscarinic receptors, regulates airway remodeling in animal models of asthma and chronic obstructive pulmonary disease (COPD). The mechanisms involved are not well understood. Here, we investigated the functional interaction between muscarinic receptor stimulation and transforming growth factor (TGF)-β1 on the expression of contractile proteins in human airway smooth muscle (ASM) cells. ASM cells expressing functional muscarinic M2 and M3 receptors were stimulated with methacholine (MCh), TGF-β1, or their combination for up to 7 days. Western blot analysis revealed a strong induction of sm-α-actin and calponin by TGF-β1, which was increased by MCh in ASM cells. Immunocytochemistry confirmed these results and revealed that the presence of MCh augmented the formation of sm-α-actin stress fibers by TGF-β1. MCh did not augment TGF-β1-induced gene transcription of contractile phenotype markers. Rather, translational processes were involved in the augmentation of TGF-β1-induced contractile protein expression by muscarinic receptor stimulation, including phosphorylation of glycogen synthase kinase-3β and 4E-binding protein 1, which was enhanced by MCh. In conclusion, muscarinic receptor stimulation augments functional effects of TGF-β1 in human ASM cells on cellular processes that underpin ASM remodeling in asthma and COPD.

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Coptisine, a protoberberine alkaloid, relaxes mouse airway smooth muscle via blockade of VDLCCs and NSCCs

Bioscience Reports ◽

10.1042/bsr20190534 ◽

2020 ◽

Vol 40 (2) ◽

Cited By ~ 1

Author(s):

Nana Wen ◽

Lu Xue ◽

Yongle Yang ◽

Shunbo Shi ◽

Qing-Hua Liu ◽

...

Keyword(s):

Smooth Muscle ◽

Muscle Contraction ◽

Airway Smooth Muscle ◽

Molecular Mechanisms ◽

Force Measurement ◽

Dependent Manner ◽

Active Constituent ◽

High K ◽

Voltage Dependent

Abstract Background/Aims: Recently, effective and purified ingredients of traditional Chinese medicine (TCM) were extracted to play crucial roles in the treatment of pulmonary diseases. Our previous research focused on TCM drug screening aimed at abnormal airway muscle contraction during respiratory diseases. Coptisine, an effective ingredient extracted from bitter herbs has shown a series of antioxidant, antibacterial, cardioprotective and neuroprotective pharmacological properties. In the current study, we questioned whether coptisine could also participate in asthma treatment through relaxing abnormal contracted mouse airway smooth muscle (ASM). The present study aimed to characterize the relaxant effects of coptisine on mouse ASM and uncover the underlying molecular mechanisms. Methods: To investigate the role of coptisine on pre-contracted mouse ASM, a series of biological techniques, including force measurement and patch-clamp experiments were employed. Results: Coptisine was found to inhibit high K+ or acetylcholine chloride (ACh)-induced pre-contracted mouse tracheal rings in a dose-dependent manner. Further research demonstrated that the coptisine-induced mouse ASM relaxation was mediated by alteration of calcium mobilization via voltage-dependent L-type Ca2+ channels (VDLCCs) and non-selective cation channels (NSCCs). Conclusion: Our data showed that mouse ASM could be relaxed by coptisine via altering the intracellular Ca2+ concentration through blocking VDLCCs and NSCCs, which suggested that this pharmacological active constituent might be classified as a potential new drug for the treatment of abnormal airway muscle contraction.

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