Upregulation of a disintegrin and metalloproteinase-33 by VEGF in human airway smooth muscle cells: Implications for asthma

Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodeling. Features of airway remodeling include increased airway smooth muscle (ASM) mass. A disintegrin and metalloproteinase (ADAM)–33 has been identified as playing a role in the pathophysiology of asthma. ADAM-33 is expressed in ASM cells and is suggested to play a role in the function of these cells. However, the regulation of ADAM-33 is not fully understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodeling in asthmatics. Although VEGF was initially thought of as an endothelial-specific growth factor, recent reports have found that VEGF can promote proliferation of other cell types, including ASM cells. To investigate the precise mechanism of VEGF's effect on ASM cell proliferation, we tested the expression of ADAM-33, phospho-extracellularsignal-regulated kinase 1/2 (ERK1/2), and phospho-Akt in VEGF-stimulated ASM cells. We found that VEGF up-regulates ADAM-33 mRNA and protein levels in a dose- and time-dependent manner as well as phosphorylation of ERK1/2 and Akt. We also found that VEGF-induced ASM cell proliferation is inhibited by both ADAM-33 knockdown and a selective VEGF receptor 2 (VEGFR2) inhibitor (SU1498). Furthermore, VEGF-induced ADAM-33 expression and ASM cell proliferation were suppressed by inhibiting ERK1/2 activity, but not by inhibiting Akt activity. Collectively, our findings suggest that VEGF enhances ADAM-33 expression and ASM cell proliferation by activating the VEGFR2/ERK1/2 signaling pathway, which might be involved in the pathogenesis of airway remodeling. Further elucidation of the mechanisms underlying these observations might help develop therapeutic strategies for airway diseases associated with smooth muscle hyperplasia such as asthma.     

PDGF-induced airway smooth muscle proliferation is associated with Human antigen R activation and could be weakened by AMPK activation

CyclinD1 over-expression is the key pathogenetic event underlying airway smooth muscle (ASM) proliferation. Human antigen R (HuR) is a ubiquitously expressed RNA-binding protein, and is known to regulate the expression of multiple cell cycle regulators. The aim of the study is to investigate whether HuR might also be involved in ASM proliferation. In cultured ASM cells, PDGF treatment induced a significant elevation of HuR expression at both mRNA and protein levels. Immunofluorescence analysis demonstrated PDGF might promote HuR translocation from nucleus to cytoplasma as well. RNA-interference of HuR effectively decreased PDGF-induced cyclinD1 over-expression in ASM cells. Furthermore, AMPK activation by AICAR could effectively decrease PDGF-induced HuR cytoplasmatic translocation, cyclinD1 expression and ASM cells proliferation. In conclusion, altered expression and activity of HuR might participate in PDGF-induced ASM cells cyclinD1 expression and proliferation. The effectiveness of AMPK activation indicated a novel intervention method for airway remodeling.     

The Pro-Proliferative Effects of Nicotine and Its Underlying Mechanism on Rat Airway Smooth Muscle Cells

Recent studies have shown that nicotine, a major component of cigarette smoke, can stimulate the proliferation of non-neuronal cells. Cigarette smoking can promote a variety of pulmonary and cardiovascular diseases, such as chronic obstructive pulmonary disease (COPD), atherosclerosis, and cancer. A predominant feature of COPD is airway remodeling, which includes increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodeling in COPD have not yet been fully elucidated. Here, we show that nicotine induces a profound and time-dependent increase in DNA synthesis in rat airway smooth muscle cells (RASMCs) in vitro. Nicotine also significantly increased the number of RASMCs, which was associated with the increased expression of Cyclin D1, phosphorylation of the retinoblastoma protein (RB) and was dependent on the activation of Akt. The activation of Akt by nicotine occurred within minutes and depended upon the nicotinic acetylcholine receptors (nAchRs). Activated Akt increased the phosphorylation of downstream substrates such as GSK3β. Our data suggest that the binding of nicotine to the nAchRs on RASMCs can regulate cellular proliferation by activating the Akt pathway.     

Heat shock protein 60 involvement in vascular smooth muscle cell proliferation

AimHeat shock protein 60 (Hsp60) is a mediator of stress-induced vascular smooth muscle cell (VSMC) proliferation. This study will determine, first, if the mitochondrial or cytoplasmic localization of Hsp60 is critical to VSMC proliferation and, second, the mechanism of Hsp60 induction of VSMC proliferation with a focus on modification of nucleocytoplasmic trafficking.Methods and resultsHsp60 was overexpressed in primary rabbit VSMCs with or without a mitochondrial targeting sequence (AdHsp60^mito-). Both interventions induced an increase in VSMC PCNA expression and proliferation. The increase in VSMC PCNA expression and growth was not observed after siRNA-mediated knockdown of Hsp60 expression. Nuclear protein import in VSMC was measured by fluorescent microscopy using a microinjected fluorescent import substrate. Nuclear protein import was stimulated by both AdHsp60 and AdHsp60^mito- treatments. AdHsp60 treatment also induced increases in nucleoporin (Nup) 62, Nup153, importin-α, importin-β and Ran expression as well as cellular ATP levels compared to control. AdHsp60^mito- treatment induced an up-regulation in importin-α, importin-β and Ran expression compared to control. Hsp60 knockdown did not change nuclear protein import nor the expression of any nuclear transport receptors or nucleoporins. Both heat shock treatment and Hsp60 overexpression promoted the interaction of Ran with Hsp60.ConclusionsVSMC proliferation can be modulated via an Hsp60 dependent, cytosol localized mechanism that in part involves a stimulation of nuclear protein import through an interaction with Ran. This novel cellular signaling role for Hsp60 may be important in growth-based vascular pathologies like atherosclerosis and hypertension.     

Role of extracellular matrix and its regulators in human airway smooth muscle biology

Altered extracellular matrix (ECM) deposition contributing to airway wall remodeling is an important feature of asthma and chronic obstructive pulmonary disease (COPD). The molecular mechanisms of this process are poorly understood. One of the key pathological features of these diseases is thickening of airway walls. This thickening is largely to the result of airway smooth muscle (ASM) cell hyperplasia and hypertrophy as well as increased deposition of ECM proteins such as collagens, elastin, laminin, and proteoglycans around the smooth muscle. Many growth factors and cytokines, including fibroblast growth factor (FGF)-1, FGF-2, and transforming growth factor (TGF)-α₁, that are released from the airway wall have the potential to contribute to airway remodeling, revealed by enhanced ASM proliferation and increased ECM protein deposition. TGF-α₁ and FGF-1 stimulate mRNA expression of collagen I and III in ASM cells, suggesting their role in the deposition of extracellular matrix proteins by ASM cells in the airways of patients with chronic lung diseases. Focus is now on the bidirectional relationship between ASM cells and the ECM. In addition to increased synthesis of ECM proteins, ASM cells can be involved in downregulation of matrix metalloproteinases (MMPs) and upregulation of tissue inhibitors of metalloproteinases (TIMPs), thus eventually contributing to the alteration in ECM. In turn, ECM proteins promote the survival, proliferation, cytokine synthesis, migration, and contraction of human airway smooth muscle cells. Thus, the intertwined relationship of ASM and ECM and their response to stimuli such as chronic inflammation in diseases such as asthma and COPD contribute to the remodeling seen in airways of patients with these diseases.     

Mechanisms of nucleophosmin (NPM)-mediated regulated cell death elucidated by Hsp70 during renal ischemia

Nucleophosmin (NPM), a nucleolar-based protein chaperone, promotes Bax-mediated mitochondrial injury and regulates cell death during acute kidney injury. However, the steps that transform NPM from an essential to a toxic protein during stress are unknown. To localize NPM-mediated events causing regulated cell death during ischemia, wild type (WT) and Hsp70 mutant proteins with characterized intracellular trafficking defects that restrict movement to either the nucleolar region (M45) or cytosol (985A) were expressed in primary murine proximal tubule epithelial cells (PTEC) harvested from Hsp70 null mice. After ischemia in vitro, PTEC survival was significantly improved and apoptosis reduced in rank order by selectively overexpressing WT?>?M45?>?985A Hsp70 proteins. Only Hsp70 with nuclear access (WT and M45) inhibited T95 NPM phosphorylation responsible for NPM translocation and also reduced cytosolic NPM accumulation. In contrast, WT or 985A?>?M45 significantly improved survival in Hsp70 null PTEC that expressed a cytosol-restricted NPM mutant, more effectively bound NPM, and also reduced NPM-Bax complex formation required for mitochondrial injury and cell death. Hsp70 knockout prevented the cytoprotective effect of suppressing NPM in ischemic PTEC and also increased cytosolic NPM accumulation after acute renal ischemia in vivo, emphasizing the inhibitory effect of Hsp70 on NPM-mediated toxicity. Distinct cytoprotective mechanisms by wild type and mutant Hsp70 proteins identify dual nuclear and cytosolic events that mediate NPM toxicity during stress-induced apoptosis and are rational targets for therapeutic AKI interventions. Antagonizing these early events in regulated cell death promotes renal cell survival during experimental AKI.

     

Mast cell chymase modifies cell-matrix interactions and inhibits mitogen-induced proliferation of human airway smooth muscle cells

The hallmarks of chronic, severe asthma include prominent airway inflammation and airway smooth muscle (ASM) hypertrophy and hyperplasia. One of the factors that contribute to the injury and repair process within the airway is activation of proteases and turnover of extracellular matrix components. Mast cells, which are present in increased numbers in the asthmatic airway, are a rich source of the neutral protease chymase, which can degrade several basement membrane components. Recent data suggest that proteases also play a critical role in regulating the expression of CD44, the primary receptor for the matrix glycosaminoglycan hyaluronan. In this study we investigated the effects of chymase treatment on human ASM cell function. We found that chymase degraded the smooth muscle cell pericellular matrix. This was accompanied by an increased release of fibronectin and soluble CD44, but not soluble ICAM-1 or soluble hyaluronan, into the conditioned medium. In addition, chymase inhibited T cell adhesion to ASM and dramatically reduced epidermal growth factor-induced smooth muscle cell proliferation. These data suggest that the local release of mast cell chymase may have profound effects on ASM cell function and airway remodeling.     

Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor alpha by phosphorylation.

The small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) form large oligomers which can act as molecular chaperones in vitro and protect cells from heat shock and oxidative stress when overexpressed. In addition, mammalian sHsps are rapidly phosphorylated by MAPKAP kinase 2/3 at two or three serine residues in response to various extracellular stresses. Here we analyze the effect of sHsp phosphorylation on its quaternary structure, chaperone function, and protection against oxidative stress. We show that in vitro phosphorylation of recombinant sHsp as well as molecular mimicry of Hsp27 phosphorylation lead to a significant decrease of the oligomeric size. We demonstrate that both phosphorylated sHsps and the triple mutant Hsp27-S15D,S78D,S82D show significantly decreased abilities to act as molecular chaperones suppressing thermal denaturation and facilitating refolding of citrate synthase in vitro. In parallel, Hsp27 and its mutants were analyzed for their ability to confer resistance against oxidative stress when overexpressed in L929 and 13.S.1.24 cells. While wild type Hsp27 confers resistance, the triple mutant S15D,S78D,S82D cannot protect against oxidative stress effectively. These data indicate that large oligomers of sHsps are necessary for chaperone action and resistance against oxidative stress whereas phosphorylation down-regulates these activities by dissociation of sHsp complexes to tetramers.     

Macrophage migration inhibitory factor (MIF) promotes rat airway muscle cell proliferation and migration mediated by ERK1/2 and FAK signaling

Macrophage migration inhibitory factor (MIF) is an inflammatory mediator that contributes to asthmatic airway remodeling; however, little is known regarding the effects of MIF on airway smooth muscle cells (ASMCs). In the present study, we found that an enhanced expression of MIF promoted ASMC proliferation, increased the population of cells in the S/G2 phase, downregulated P21 expression, and upregulated cyclin D1, cyclin D3, and Cdk6 expression. In addition, the apoptosis of ASMCs was significantly decreased in response to MIF overexpression, compared with the negative control. Moreover, MIF facilitated the migration of ASMCs by upregulating the expression of matrix metalloproteinase (MMP)‐2. Finally, we showed that MIF increased the phosphorylation of extracellular regulated protein kinases (ERK) 1/2 and focal adhesion kinase (FAK), which are associated with proliferation and migration. In conclusion, this study demonstrated that MIF overexpression promotes the proliferation and migration of ASMCs by upregulating the activity of the ERK1/2 and FAK signaling pathways in these cells, further indicating that inhibition of MIF may prove to be an effective strategy for treating asthma patients with airway remodeling.     

Vascular endothelial growth factor-induced secretion of fibronectin is ERK dependent

In severe asthma, cytokines and growth factors contribute to the proliferation of smooth muscle cells and blood vessels, and to the increased extracellular matrix deposition that constitutes the process of airway remodeling. Vascular endothelial growth factor (VEGF), which regulates vascular permeability and angiogenesis, also modulates the function of nonendothelial cell types. In this study, we demonstrate that VEGF induces fibronectin secretion by human airway smooth muscle (ASM) cells. In addition, stimulation of ASM with VEGF activates ERK, but not p38MAPK, and fibronectin secretion is ERK dependent. Both ERK activation and fibronectin secretion appear to be mediated through the VEGF receptor flt-1, as evidenced by the effects of the flt-1-specific ligand placenta growth factor. Finally, we demonstrate that ASM cells constitutively secrete VEGF, which is increased in response to PDGF, transforming growth factor-beta, IL-1beta, and PGE(2). We conclude that ASM-derived VEGF, through modulation of the extracellular matrix, may play an important role in airway remodeling seen in asthma.     

Invited review: the circle of life: cell cycle regulation in airway smooth muscle.

Severe asthma is characterized by increased airway smooth muscle (ASM) mass, due predominantly to ASM hyperplasia. Diverse stimuli, which include growth factors, plasma- or inflammatory cell-derived mediators, contractile agonists, cytokines, and extracellular matrix proteins, induce ASM proliferation. Mitogens act via receptor tyrosine kinase, G protein-coupled receptors, or cytokine receptors, to activate p21ras and stimulate two parallel signaling pathways in ASM cells, namely, the extracellular signal-regulated kinase (ERK) or the phosphatidylinositol 3-kinase (PI3K) pathways. ERK and PI3K regulate cell cycle protein expression and thus modulate cell cycle traversal. ERK activation and downstream effectors of PI3K, such as Rac1 and Cdc42, stimulate expression of cyclin D1, a key regulator of G(1) progression in the mammalian cell cycle. In addition, PI3K activates 70-kDa ribosomal S6 kinase, an enzyme that also regulates the translation of many cell cycle proteins, including the elongation factor E2F. The present review examines the mitogens and critical signal transduction pathways that stimulate ASM cell proliferation. Further study in this area may reveal new therapeutic targets to abrogate ASM hyperplasia in diseases such as asthma and chronic obstructive pulmonary disease.     

shRNA targeting β1-integrin suppressed proliferative aspects and migratory properties of airway smooth muscle cells

Dysfunction of airway smooth muscle (ASM) is an essential feature of airway remodeling in chronic asthma. However, the precise mechanisms of this pathological process have not been well studied. In previous study, we found that β1-integrin, which was dramatically upregulated in ASM cells in an asthmatic mouse model, was associated with the cell proliferation. In this study, we employed short hairpin RNA (shRNA) targeting β1-integrin to assess the effect of down-regulation of this receptor on the proliferative aspects and migratory properties of ASM cells in vitro. The cells were treated with shRNA expression vectors directed against β1-integrin, control vectors that included the blank control, empty vector without shRNA, and mismatched shRNA, respectively. The mRNA and protein expressions of β1-integrin were determined by real-time PCR and Western blotting. Cell proliferation was measured by BrdU ELISA and cell cycle by fluorescence-activated cell sorter. Cell apoptosis was detected by Annexin V-PE/7-AAD staining. Cell migration assays were evaluated by transwell assay and expression of IL-6 and IL-8 by ELISA. The results revealed that shRNA targeting β1-integrin significantly decreased the mRNA and protein expressions of β1-integrin, enhanced the proportion of cells in G0/G1 phase, decreased the proportion in S phase, promoted cell apoptosis, inhibited cell proliferation, migration, IL-6 and IL-8 secretion in vitro. In conclusion, the overexpression of β1-integrin in ASM cells is essential for airway dysfunction development because it promotes proliferative aspects and migratory properties of ASM cells. Importantly, shRNA targeting β1-integrin may provide a new approach to preventing airway remodeling in chronic asthma.     

Extracellular matrix proteins differentially regulate airway smooth muscle phenotype and function

Changes in the ECM and increased airway smooth muscle (ASM) mass are major contributors to airway remodeling in asthma and chronic obstructive pulmonary disease. It has recently been demonstrated that ECM proteins may differentially affect proliferation and expression of phenotypic markers of cultured ASM cells. In the present study, we investigated the functional relevance of ECM proteins in the modulation of ASM contractility using bovine tracheal smooth muscle (BTSM) preparations. The results demonstrate that culturing of BSTM strips for 4 days in the presence of fibronectin or collagen I depressed maximal contraction (E(max)) both for methacholine and KCl, which was associated with decreased contractile protein expression. By contrast, both fibronectin and collagen I increased proliferation of cultured BTSM cells. Similar effects were observed for PDGF. Moreover, PDGF augmented fibronectin- and collagen I-induced proliferation in an additive fashion, without an additional effect on contractility or contractile protein expression. The fibronectin-induced depression of contractility was blocked by the integrin antagonist Arg-Gly-Asp-Ser (RGDS) but not by its negative control Gly-Arg-Ala-Asp-Ser-Pro (GRADSP). Laminin, by itself, did not affect contractility or proliferation but reduced the effects of PDGF on these parameters. Strong relationships were found between the ECM-induced changes in E(max) in BTSM strips and their proliferative responses in BSTM cells and for E(max) and contractile protein expression. Our results indicate that ECM proteins differentially regulate both phenotype and function of intact ASM.     

Assays for in vitro monitoring of proliferation of human airway smooth muscle (ASM) and human pulmonary arterial vascular smooth muscle (VSM) cells

Vascular and airway remodeling, which are characterized by airway smooth muscle (ASM) and pulmonary arterial vascular smooth muscle (VSM) proliferation, contribute to the pathology of asthma, pulmonary hypertension, restenosis and atherosclerosis. To evaluate the proliferation of VSM and ASM cells in response to mitogens, we perform a [3H]thymidine incorporation assay. The proliferation protocol takes approximately 48 h and includes stimulating cells synchronized in G0/G1 phase of the cell cycle with agonists, labeling cells with [3H]thymidine and examining levels of [3H]thymidine incorporation by scintillation counting. Although using radiolabeled [3H]thymidine incorporation is a limitation, the greatest benefit of the assay is providing reliable and statistically significant data.     

Some properties of complexes formed by small heat shock proteins with denaturated actin

The effects of a recombinant small heat shock protein with an apparent molecular weight of 27 kDa (Hsp27) and both wild type (Hsp27-wt) and S15D, S78D, S82D mutants (Hsp27-3D), which mimic the naturally occurring phosphorylation of this protein, on the thermal denaturation and aggregation of F-actin were studied. It has been shown that at a Hsp27/actin weight ration of 1/4, both Hsp27-wt and Hsp27-3D do not affect the thermal denaturation of F-actin, but efficiently prevent its further aggregation by forming soluble complexes with denatured actin. Formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. Hsp27-wt is known to exist as a high-molecular-weight oligomer, whereas Hsp27-3D forms only small dimers or tetramers. However, the complexes formed by Hsp27-wt and Hsp27-3D with denatured actin did not differ in their size, as measured by dynamic light scattering, and demonstrated the same hydrodynamic radius of 17–18 nm. On the other hand, the sedimentation coefficients of these complexes differed: they ranged 10–45 S in the case of Hsp27-3D and 18–60 S in case of Hsp27-wt. Thus, the initial oligomeric state of Hsp27 does not significantly affect its capacity to form small soluble complexes with denatured actin.     

Multiple beta 1 integrins mediate enhancement of human airway smooth muscle cytokine secretion by fibronectin and type I collagen

Altered airway smooth muscle (ASM) function and enrichment of the extracellular matrix (ECM) with interstitial collagen and fibronectin are major pathological features of airway remodeling in asthma. We have previously shown that these ECM components confer enhanced ASM proliferation in vitro, but their action on its newly characterized secretory function is unknown. Here, we examined the effects of fibronectin and collagen types I, III, and V on IL-1beta-dependent secretory responses of human ASM cells, and characterized the involvement of specific integrins. Cytokine production (eotaxin, RANTES, and GM-CSF) was evaluated by ELISA, RT-PCR, and flow cytometry. Function-blocking integrin mAbs and RGD (Arg-Gly-Asp)-blocking peptides were used to identify integrin involvement. IL-1beta-dependent release of eotaxin, RANTES, and GM-CSF was enhanced by fibronectin and by fibrillar and monomeric type I collagen, with similar changes in mRNA abundance. Collagen types III and V had no effect on eotaxin or RANTES release but did modulate GM-CSF. Analogous changes in intracellular cytokine accumulation were found, but in <25% of the total ASM cell population. Function-blocking Ab and RGD peptide studies revealed that alpha2beta1, alpha5beta1, alphavbeta1, and alphavbeta3 integrins were required for up-regulation of IL-1beta-dependent ASM secretory responses by fibronectin, while alpha2beta1 was an important transducer for type I collagen. Thus, fibronectin and type I collagen enhance IL-1beta-dependent ASM secretory responses through a beta1 integrin-dependent mechanism. Enhancement of cytokine release from ASM by these ECM components may contribute to airway wall inflammation and remodeling in asthma.     

Rhinovirus infection induces extracellular matrix protein deposition in asthmatic and nonasthmatic airway smooth muscle cells

Airway remodeling, which includes increases in the extracellular matrix (ECM), is a characteristic feature of asthma and is correlated to disease severity. Rhinovirus (RV) infections are associated with increased risk of asthma development in young children and are the most common cause of asthma exacerbations. We examined whether viral infections can increase ECM deposition and whether this increased ECM modulates cell proliferation and migration. RV infection of nonasthmatic airway smooth muscle (ASM) cells significantly increased the deposition of fibronectin (40% increase, n = 12) and perlecan (80% increase, n = 14), while infection of asthmatic ASM cells significantly increased fibronectin (75% increase, n = 9) and collagen IV (15% increase, n = 9). We then treated the ASM cells with the Toll-like receptor (TLR) agonists polyinosinic:polycytidylic acid, imiquimod, and pure RV RNA and were able to show that the mechanism through which RV induced ECM deposition was via the activation of TLR3 and TLR7/8. Finally, we assessed whether the virus-induced ECM was bioactive by measuring the amount of migration and proliferation of virus-naive cells that seeded onto the ECM. Basically, ECM from asthmatic ASM cells induced twofold greater migration of virus-naive ASM cells than ECM from nonasthmatic ASM cells, and these rates of migration were further increased on RV-modulated ECM. Increased migration on the RV-modulated ECM was not due to increased cell proliferation, as RV-modulated ECM decreased the proliferation of virus-naive cells. Our results suggest that viruses may contribute to airway remodeling through increased ECM deposition, which in turn may contribute to increased ASM mass via increased cell migration.     

TAK1 plays a major role in growth factor-induced phenotypic modulation of airway smooth muscle

Increased airway smooth muscle (ASM) mass is a major feature of airway remodeling in asthma and chronic obstructive pulmonary disease. Growth factors induce a proliferative ASM phenotype, characterized by an increased proliferative state and a decreased contractile protein expression, reducing contractility of the muscle. Transforming growth factor-β-activated kinase 1 (TAK1), a mitogen-activated protein kinase kinase kinase, is a key enzyme in proinflammatory signaling in various cell types; however, its function in ASM is unknown. The aim of this study was to investigate the role of TAK1 in growth factor-induced phenotypic modulation of ASM. Using bovine tracheal smooth muscle (BTSM) strips and cells, as well as human tracheal smooth muscle cells, we investigated the role of TAK1 in growth factor-induced proliferation and hypocontractility. Platelet-derived growth factor- (PDGF; 10 ng/ml) and fetal bovine serum (5%)-induced increases in DNA synthesis and cell number in bovine and human cells were significantly inhibited by pretreatment with the specific TAK1 inhibitor LL-Z-1640-2 (5Z-7-oxozeaenol; 100 nM). PDGF-induced DNA synthesis and extracellular signal-regulated kinase-1/2 phosphorylation in BTSM cells were strongly inhibited by both LL-Z-1640-2 pretreatment and transfection of dominant-negative TAK1. In addition, LL-Z-1640-2 inhibited PDGF-induced reduction of BTSM contractility and smooth muscle α-actin expression. The data indicate that TAK1 plays a major role in growth factor-induced phenotypic modulation of ASM.