Tumstatin regulates the angiogenic and inflammatory potential of airway smooth muscle extracellular matrix

The extracellular matrix (ECM) creates the microenvironment of the tissue; an altered ECM in the asthmatic airway may be central in airway inflammation and remodelling. Tumstatin is a collagen IV‐derived matrikine reduced in the asthmatic airway wall that reverses airway inflammation and remodelling in small and large animal models of asthma. This study hypothesized that the mechanisms underlying the broad asthma‐resolving effects of tumstatin were due to autocrine remodelling of the ECM. Neutrophils and endothelial cells were seeded on decellularized ECM of non‐asthmatic (NA) or asthmatic (A) airway smooth muscle (ASM) cells previously exposed to tumstatin in the presence or absence of a broad matrix metalloproteinase inhibitor, Marimastat. Gene expression in NA and A ASM induced by tumstatin was assessed using RT‐PCR arrays. The presence of tumstatin during ECM deposition affected neutrophil and endothelial cell properties on both NA and A ASM‐derived matrices and this was only partly due to MMP activity. Gene expression patterns in response to tumstatin in NA and A ASM cells were different. Tumstatin may foster an anti‐inflammatory and anti‐angiogenic microenvironment by modifying ASM‐derived ECM. Further work is required to examine whether restoring tumstatin levels in the asthmatic airway represents a potential novel therapeutic approach.     

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.     

The phosphoinositide 3'-kinase p110δ modulates contractile protein production and IL-6 release in human airway smooth muscle

Transforming growth factor (TGF) β1 increases pro‐inflammatory cytokines and contractile protein expression by human airway smooth muscle (ASM) cells, which could augment airway inflammation and hyperresponsiveness. Phosphoinositide 3′ kinase (PI3K) is one of the signaling pathways implicated in TGFβ1 stimulation, and may be altered in asthmatic airways. This study compared the expression of PI3K isoforms by ASM cells from donors with asthma (A), chronic obstructive pulmonary disease (COPD), or neither disease (NA), and investigated the role of PI3K isoforms in the production of TGFβ1 induced pro‐inflammatory cytokine and contractile proteins in ASM cells. A cells expressed higher basal levels of p110δ mRNA compared to NA and COPD cells; however COPD cells produced more p110δ protein. TGFβ1 increased 110δ mRNA expression to the same extent in the three groups. Neither the p110δ inhibitor IC87114 (1, 10, 30 µM), the p110β inhibitor TGX221 (0.1, 1, 10 µM) nor the PI3K pan inhibitor LY294002 (3, 10 µM) had any effect on basal IL‐6, calponin or smooth muscle α‐actin (α‐SMA) expression. However, TGFβ1 increased calponin and α‐SMA expression was inhibited by IC87114 and LY294002 in all three groups. IC87114, TGX221, and LY294002 reduced TGFβ1 induced IL‐6 release in a dose related manner in all groups of ASM cells. PI3K p110δ is important for TGFβ1 induced production of the contractile proteins calponin and α‐SMA and the proinflammatory cytokine IL‐6 in ASM cells, and may therefore be relevant as a potential therapeutic target to treat both inflammation and airway remodeling. J. Cell. Physiol. 227: 3044–3052, 2012. © 2011 Wiley Periodicals, Inc.     

Inhibition of connective tissue growth factor/CCN2 expression in human dermal fibroblasts by interleukin‐1α and β

Connective tissue growth factor (CTGF/CCN2) is a matricellular protein induced by transforming growth factor (TGF)‐β and intimately involved with tissue repair and overexpressed in various fibrotic conditions. We previously showed that keratinocytes in vitro downregulate TGF‐β‐induced expression of CTGF in fibroblasts by an interleukin (IL)‐1 α‐dependent mechanism. Here, we investigated further the mechanisms of this downregulation by both IL‐1α and β. Human dermal fibroblasts and NIH 3T3 cells were treated with IL‐1α or β in presence or absence of TGF‐β1. IL‐1 suppressed basal and TGF‐β‐induced CTGF mRNA and protein expression. IL‐1α and β inhibited TGF‐β‐stimulated CTGF promoter activity, and the activity of a synthetic minimal promoter containing Smad 3‐binding CAGA elements. Furthermore, IL‐1α and β inhibited TGF‐β‐stimulated Smad 3 phosphorylation, possibly linked to an observed increase in Smad 7 mRNA expression. In addition, RNA interference suggested that TGF‐β activated kinase1 (TAK1) is necessary for IL‐1 inhibition of TGF‐β‐stimulated CTGF expression. These results add to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated, which in turn may have implications for the pathogenesis of fibrotic conditions involving the skin. J. Cell. Biochem. 110: 1226–1233, 2010. Published 2010 Wiley‐Liss, Inc.     

Tryptase activates TGFbeta in human airway smooth muscle cells via direct proteolysis

Transforming growth factor β (TGFβ) is a key remodelling factor in asthma. It is produced as a latent complex and the main limiting step in TGFβ bioavailability is its activation. Mast cell tryptase has been shown to stimulate the release of functionally active TGFβ from human airway smooth muscle (ASM) cells [P. Berger, P.O. Girodet, H. Begueret, O. Ousova, D.W. Perng, R. Marthan, A.F. Walls, J.M. Tunon de Lara, Tryptase-stimulated human airway smooth muscle cells induce cytokine synthesis and mast cell chemotaxis, FASEB J. 17 (2003) 2139-2141]. The aim of this study was to determine if tryptase could cause TGFβ activation as well as expression in ASM cells via its receptor, proteinase-activated receptor 2 (PAR2). Tryptase caused TGFβ activation without affecting levels of total TGFβ. This effect was inhibited by the selective tryptase inhibitor FUT175 and leupeptin but not mimicked by the PAR2 activating peptide SLIGKV-NH₂. Furthermore, the ASM cells used in the study did not express PAR2. The results indicate that tryptase activates TGFβ via a PAR2-independent proteolytic mechanism in human ASM cells and may help understanding the role of tryptase in asthma.     

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.     

Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connective tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-β-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G_q/11 protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP₃) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G_q/11 protein and inositol-1,4,5-trisphosphate-induced Ca²⁺ mobilization in human ASMCs.     

Vasoactive peptides upregulate mRNA expression and secretion of vascular endothelial growth factor in human airway smooth muscle cells

Airway remodeling and associated angiogenesis are documented features of asthma, of which the molecular mechanisms are not fully understood. Angiotensin (ANG)II and endothelin (ET)-1 are potent vasoconstricting circulatory hormones implicated in asthma. We investigated the effects of ANG II and ET-1 on human airway smooth muscle (ASM) cells proliferation and growth and examined the mRNA expression and release of the angiogenic peptide, vascular endothelial growth factor (VEGF). Serum deprived (48 h) human ASM cells were incubated with ANG II (100 nM) or ET-1 (10nM) for 30 min, 1, 2, 4, 8, 16, and 24 h and the endogenous synthesis of VEGF was examined in relation to control cells receiving serum free culture medium. ET-1 induced time dependent DNA biosynthesis as determined by [³H]-thymidine incorporation assay. Using northern blot hybridization, we detected two mRNA species of 3.9 and 1.7 kb encoding VEGF in the cultured smooth muscle cells. Both ANG II and ET-1 induced the mRNA expression (two-to threefold) and secretion (1.8-to 2.8-fold) of VEGF reaching maximal levels between 4–8 h of incubation. Induced expression and release of VEGF declined after 8 h of ANG II incubation while levels remained elevated in the case of ET-1. The conditioned medium derived from ET-1-treated ASM cells induced [³H]-thymidine incorporation and cell number in porcine pulmonary artery endothelial as well as human umbilical vein endothelial cells. Moreover, the VEGF tyrosine kinase receptor inhibitor blocked the conditioned medium induced mitogenesis in endothelial cells. Our results suggest a potential role for ANG II and ET-1 in ASM cell growth and upregulation of VEGF that may participate in endothelial cell proliferation via paracrine mechanisms and thus causing pathological angiogenesis and vascular remodelling seen during asthma.     

EPO modified MSCs can inhibit asthmatic airway remodeling in an animal model

There was no effective measures can be obtained at present to reverse or prevent airway remodeling. We investigated the therapeutic effect of Erythropoietin (EPO) gene modified mesenchymal stem cells (MSCs) on asthmatic airway remodeling and the possible underlied molecular mechanisms. EPO gene was transfected into MSCs via lentivirus vector. The transfected cells (EPO‐MSCs) were identified by flow cytometry and the EPO secreting function was detected by PCR and Western blot. MSCs or EPO‐MSCs were administrated to albumin (OVA)‐induced chronic asthmatic mouse model via tail veins. The asthmatic phenotype was analyzed. Number of cells in bronchoalveolar lavage fluid (BALF) was counted using a hemocytometer. Histological findings of airways were evaluated by microscopic examination. The concentrations of interleukin 4(IL‐4), interleukin 5(IL‐5), and interleukin 13(IL‐13) in lung homogenate were determined by ELISA. The activation state of transforming growth factor‐β 1 (TGF‐β1), Transforming growth factor beta‐activated kinase 1 (TAK1), and p38 Mitogen Activated Protein Kinase (p38MAPK) signaling was detected by Real‐Time PCR and Western blotting. EPO‐MSCs were successfully constructed. EPO‐MSCs showed a more potently suppressive effect on local asthmatic airway inflammation and the level of IL‐4, IL‐5, and IL‐13 in lung tissue than MSCs. Moreover, the numbers of goblet cells, the thicknesses of smooth muscle layer, collagen density, percentage of proliferating cell nuclear antigen positive (PCNA⁺) mesenchymal cells, and von Willebrand factor positive(vWF⁺) vessels were also significantly inhibited by EPO‐MSCs. Furthermore, EPO‐MSCs could downregulate the expression of TGF‐β1, TAK1, and p38MAPK in lung tissue both in mRNA level and in protein level. EPO gene modified MSCs may more efficiently attenuate asthmatic airway remodeling, which maybe related with the downregulation of TGF‐β1‐TAK1‐p38MAPK pathway activity.     

Proinflammatory cytokines upregulate mRNA expression and secretion of vascular endothelial growth factor in cultured human airway smooth muscle cells

Airflow obstruction in chronic airway disease is associated with airway and pulmonary vascular remodeling, of which the molecular mechanisms are poorly understood. Paracrine actions of angiogenic factors released by resident or infiltrating inflammatory cells following activation by proinflammatory cytokines in diseased airways could play a major role in the airway vascular remodeling process. Here, the proinflammatory cytokines interleukin (IL)-1β, and tumor necrosis factor (TNF)-α were investigated on cell cultures of human airway smooth muscle (ASM) for their effects on mRNA induction and protein release of the angiogenic peptide, vascular endothelial growth factor (VEGF). IL-1β (0.5 ng/mL) and TNF-α (10ng/mL) each increased VEGF mRNA (3.9 and 1.7 kb) expression in human ASM cells, reaching maximal levels between 16 and 24 and 4 and 8h, respectively. Both cytokines also induced a time-dependent release of VEGF, which was not associated with increased ASM growth. Preincubation of cells with 1μM dexamethasone abolished enhanced release of VEGF by TNF-α. The data suggest that human ASM cells express and secrete VEGF in response to proinflammatory cytokines and may participate in paracrine inflammatory mechanisms of vascular remodeling in chronic airway disease.     

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

MicroRNA‐503 promotes angiotensin II‐induced cardiac fibrosis by targeting Apelin‐13

Cardiac fibrosis is a major cause of heart failure. MicroRNAs (miRs) are important epigenetic regulators of cardiac function and cardiovascular diseases, including cardiac fibrosis. This study aimed to explore the role of miR‐503 and its mechanisms in regulating cardiac fibrosis. miR‐503 was found up‐regulated in the mouse LV tissues subjected to transverse aortic constriction (TAC) and in neonatal cardiac fibroblasts (CFs) cultured with Angiotension II. The role of miR‐503 in regulating CF cell proliferation and/or collagen production in mice neonatal CFs were determined using an MTT assay and RT‐PCR respectively. Forced expression of miR‐503 increased the cellular proliferation and collagen production in mice neonatal CFs. The effects were abrogated by cotransfection with AMO‐503 (a specific inhibitor of miR‐503). Injection of antagomiR‐503 elevated cardiac function and inhibited the expression of connective tissue growth factor (CTGF) and transforming growth factor (TGF)‐β in the TAC mice. Additional analysis revealed that Apelin‐13 is a direct target of miR‐503, as the overexpression of miR‐503 decreased the protein and mRNA expression levels of Apelin‐13. In the CFs with pre‐treatment of AngII, we transfected AMO‐503 into the cells treated with siRNA‐APLN. siRNA‐APLN abolished the effects of AMO‐503 on the production of collagen I and III and the expression of TGF‐β and CTGF. Furthermore, pre‐treatment of CFs with Apelin‐13 (1–100 nmol/l) inhibited angiotensin II‐mediated collagen production and activation of CTGF and TGF‐β. So we conclude that miR‐503 promotes cardiac fibrosis via miR‐503‐Apelin‐13‐TGF‐β‐CTGF‐collagen production pathway. Thus, miR‐503 is a promising therapeutic target for reducing cardiac fibrosis.     

Latent adenoviral infection induces production of growth factors relevant to airway remodeling in COPD

Previous studies showed an association between latent adenoviral infection with expression of the adenoviral E1A gene and chronic obstructive pulmonary disease (COPD). The present study focuses on how the adenoviral E1A gene could alter expression of growth factors by human bronchial epithelial (HBE) cells. The data show that connective tissue growth factor (CTGF) and transforming growth factor (TGF)-beta 1 mRNA and protein expression were upregulated in E1A-positive HBE cells. Upregulation of CTGF in this in vitro model was independent of TGF-beta secreted into the growth medium. Comparison of E1A-positive with E1A-negative HBE cells showed that both expressed cytokeratin but only E1A-positive cells expressed the mesenchymal markers vimentin and alpha-smooth muscle actin. We conclude that latent infection of epithelial cells by adenovirus E1A could contribute to airway remodeling in COPD by the viral E1A gene, inducing TGF-beta 1 and CTGF expression and shifting cells to a more mesenchymal phenotype.     

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.     

Caveolin-1 is required for contractile phenotype expression by airway smooth muscle cells

Airway smooth muscle cells exhibit phenotype plasticity that underpins their ability to contribute both to acute bronchospasm and to the features of airway remodelling in chronic asthma. A feature of mature, contractile smooth muscle cells is the presence of abundant caveolae, plasma membrane invaginations that develop from the association of lipid rafts with caveolin-1, but the functional role of caveolae and caveolin-1 in smooth muscle phenotype plasticity is unknown. Here, we report a key role for caveolin-1 in promoting phenotype maturation of differentiated airway smooth muscle induced by transforming growth factor (TGF)-β(1). As assessed by Western analysis and laser scanning cytometry, caveolin-1 protein expression was selectively enriched in contractile phenotype airway myocytes. Treatment with TGF-β(1) induced profound increases in the contractile phenotype markers sm-α-actin and calponin in cells that also accumulated abundant caveolin-1; however, siRNA or shRNAi inhibition of caveolin-1 expression largely prevented the induction of these contractile phenotype marker proteins by TGF-β(1). The failure by TGF-β(1) to adequately induce the expression of these smooth muscle specific proteins was accompanied by a strongly impaired induction of eukaryotic initiation factor-4E binding protein(4E-BP)1 phosphorylation with caveolin-1 knockdown, indicating that caveolin-1 expression promotes TGF-β(1) signalling associated with myocyte maturation and hypertrophy. Furthermore, we observed increased expression of caveolin-1 within the airway smooth muscle bundle of guinea pigs repeatedly challenged with allergen, which was associated with increased contractile protein expression, thus providing in vivo evidence linking caveolin-1 expression with accumulation of contractile phenotype myocytes. Collectively, we identify a new function for caveolin-1 in controlling smooth muscle phenotype; this mechanism could contribute to allergic asthma.     

Regulatory mechanisms of TGF‐β1‐induced fibrogenesis of human alveolar epithelial cells

Pulmonary fibrosis is characterized by an extensive activation of fibrogenic cells and deposition of extracellular matrix (ECM). Transforming growth factor (TGF)‐β1 plays a pivotal role in the pathogenesis of pulmonary fibrosis, probably through the epithelial‐ to‐mesenchymal transition (EMT) and ECM production. The present study investigates potential mechanism by which TGF‐β1 induces EMT and ECM production in the fibrogenesis of human lung epithelial cells during pulmonary fibrosis. The expression of EMT phenotype and other proteins relevant to fibrogenesis were measured and the cell bio‐behaviours were assessed using Cell‐IQ Alive Image Monitoring System. We found that TGF‐β1‐induced EMT was accompanied with increased collagen I deposition, which may be involved in the regulation of connective tissue growth factor (CTGF) and phosphoinositide 3‐kinase (PI3K) signalling pathway. Treatment with PI3K inhibitors significantly attenuated the TGF‐β1‐ induced EMT, CTGF expression and collagen I synthesis in lung epithelial cells. The interference of CTGF expression impaired the basal and TGF‐β1‐stimulated collagen I deposition, but did not affect the process of EMT. Our data indicate that the signal pathway of TGF‐β1/PI3K/CTGF plays an important role in the fibrogenesis of human lung epithelial cells, which may be a novel therapeutic approach to prevent and treat pulmonary fibrosis.     

Regulation of TGF-beta 1-induced connective tissue growth factor expression in airway smooth muscle cells

Transforming growth factor (TGF)-beta may play an important role in airway remodeling, and the fibrogenic effect of TGF-beta may be mediated through connective tissue growth factor (CTGF) release. We investigated the role of MAPKs and phosphatidylinositol 3-kinase (PI3K) and the effects of inflammatory cytokines on TGF-beta-induced CTGF expression in human airway smooth muscle cells (ASMC). We examined whether Smad signal was involved in the regulatory mechanisms. TGF-beta 1 induced a time- and concentration-dependent expression of CTGF gene and protein as analyzed by real-time RT-PCR and Western blot. Inhibition of ERK and c-jun NH(2)-terminal kinase (JNK), but not of p38 MAPK and PI3K, blocked the effect of TGF-beta 1 on CTGF mRNA and protein expression and on Smad2/3 phosphorylation. T helper lymphocyte 2-derived cytokines, IL-4 and IL-13, attenuated TGF-beta 1-stimulated mRNA and protein expression of CTGF and inhibited TGF-beta 1-stimulated ERK1/2 and Smad2/3 activation in ASMC. The proinflammatory cytokines tumor necrosis factor-alpha and IL-1 beta reduced TGF-beta 1-stimulated mRNA expression of CTGF but did not inhibit TGF-beta-induced Smad2/3 phosphorylation. TGF-beta 1-stimulated CTGF expression is mediated by mechanisms involving ERK and JNK pathways and is downregulated by IL-4 and IL-13 through modulation of Smad and ERK signals.