TGF-β1 induced epithelial to mesenchymal transition (EMT) in human bronchial epithelial cells is enhanced by IL-1β but not abrogated by corticosteroids

Background

Chronic persistent asthma is characterized by ongoing airway inflammation and airway remodeling. The processes leading to airway remodeling are poorly understood, and there is increasing evidence that even aggressive anti-inflammatory therapy does not completely prevent this process. We sought to investigate whether TGFβ₁ stimulates bronchial epithelial cells to undergo transition to a mesenchymal phenotype, and whether this transition can be abrogated by corticosteroid treatment or enhanced by the pro-inflammatory cytokine IL-1β.

Methods

BEAS-2B and primary normal human bronchial epithelial cells were stimulated with TGFβ₁ and expression of epithelial and mesenchymal markers assessed by quantitative real-time PCR, immunoblotting, immunofluorescence microscopy and zymography. In some cases the epithelial cells were also incubated with corticosteroids or IL-1β. Results were analyzed using non-parametric statistical tests.

Results

Treatment of BEAS-2B or primary human bronchial epithelial cells with TGFβ₁ significantly reduced the expression level of the epithelial adherence junction protein E-cadherin. TGFβ₁ then markedly induced mesenchymal marker proteins such as collagen I, tenascin C, fibronectin and α-smooth muscle actin mRNA in a dose dependant manner. The process of mesenchymal transition was accompanied by a morphological change towards a more spindle shaped fibroblast cell type with a more motile and invasive phenotype. Corticosteroid pre-treatment did not significantly alter the TGFβ₁ induced transition but IL-1β enhanced the transition.

Conclusion

Our results indicate, that TGFβ₁ can induce mesenchymal transition in the bronchial epithelial cell line and primary cells. Since asthma has been strongly associated with increased expression of TGFβ₁ in the airway, epithelial to mesenchymal transition may contribute to the contractile and fibrotic remodeling process that accompanies chronic asthma.     

Effects of PPARγ ligands on TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells

Background

Transforming growth factor β1 (TGF-β1)-mediated epithelial mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to lung fibrosis. Since PPARγ ligands have been shown to inhibit fibroblast activation by TGF-β1, we assessed the ability of the thiazolidinediones rosiglitazone (RGZ) and ciglitazone (CGZ) to regulate TGF-β1-mediated EMT of A549 cells, assessing changes in cell morphology, and expression of cell adhesion molecules E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell marker), and collagen 1α1 (COL1A1), CTGF and MMP-2 mRNA.

Methods

Serum-deprived A549 cells (human AEC cell line) were pre-incubated with RGZ and CGZ (1 - 30 μM) in the absence or presence of the PPARγ antagonist GW9662 (10 μM) before TGFβ-1 (0.075-7.5 ng/ml) treatment for up to 72 hrs. Changes in E-cadherin, N-cadherin and phosphorylated Smad2 and Smad3 levels were analysed by Western blot, and changes in mRNA levels including COL1A1 assessed by RT-PCR.

Results

TGFβ-1 (2.5 ng/ml)-induced reductions in E-cadherin expression were associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin, MMP-2, CTGF and COL1A1 were evident in predominantly elongated fibroblast-like cells. Neither RGZ nor CGZ prevented TGFβ1-induced changes in cell morphology, and PPARγ-dependent inhibitory effects of both ligands on changes in E-cadherin were only evident at submaximal TGF-β1 (0.25 ng/ml). However, both RGZ and CGZ inhibited the marked elevation of N-cadherin and COL1A1 induced by TGF-β1 (2.5 ng/ml), with effects on COL1A1 prevented by GW9662. Phosphorylation of Smad2 and Smad3 by TGF-β1 was not inhibited by RGZ or CGZ.

Conclusions

RGZ and CGZ inhibited profibrotic changes in TGF-β1-stimulated A549 cells independently of inhibition of Smad phosphorylation. Their inhibitory effects on changes in collagen I and E-cadherin, but not N-cadherin or CTGF, appeared to be PPARγ-dependent. Further studies are required to unravel additional mechanisms of inhibition of TGF-β1 signalling by thiazolidinediones and their implications for the contribution of EMT to lung fibrosis.     

MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor β1: a novel role of miR-382

We reported previously an approach for identifying microRNA (miRNA)-target pairs by combining miRNA and proteomic analyses. The approach was applied in the present study to examine human renal epithelial cells treated with transforming growth factor β1 (TGFβ1), a model of epithelial–mesenchymal transition important for the development of renal interstitial fibrosis. Treatment of human renal epithelial cells with TGFβ1 resulted in upregulation of 16 miRNAs and 18 proteins and downregulation of 17 miRNAs and 16 proteins. Of the miRNAs and proteins that exhibited reciprocal changes in expression, 77 pairs met the sequence criteria for miRNA–target interactions. Knockdown of miR-382, which was up-regulated by TGFβ1, attenuated TGFβ1-induced loss of the epithelial marker E-cadherin. miR-382 was confirmed by 3′-untranslated region reporter assay to target five genes that were downregulated at the protein level by TGFβ1, including superoxide dismutase 2 (SOD2). Knockdown of miR-382 attenuated TGFβ1-induced downregulation of SOD2. Overexpression of SOD2 ameliorated TGFβ1-induced loss of the epithelial marker. The study provided experimental evidence in the form of reciprocal expression at the protein level for a large number of predicted miRNA-target pairs and discovered a novel role of miR-382 and SOD2 in the loss of epithelial characteristics induced by TGFβ1.     

Bone morphogenetic proteins enhance an epithelial-mesenchymal transition in normal airway epithelial cells during restitution of a disrupted epithelium

Background

Mechanisms of airway repair are poorly understood. It has been proposed that, following injury, progenitor populations such as club cells (Clara) become undifferentiated, proliferate and re-differentiate to re-epithelialise the airway. The exact phenotype of such cells during repair is unknown however. We hypothesised that airway epithelial cells (AECs) undergo some degree of epithelial-mesenchymal transition (EMT) in order to migrate over a denuded airway and effect re-epithelialisation. Furthermore, based on our previous findings that BMP signalling is an early event in AECs following injury in vivo and that BMP4 down-regulates E-cadherin expression and enhances migration in AECs in vitro, we hypothesised that BMPs could play a role in inducing such a phenotypic switch.

Methods

Normal AECs were isolated from mouse lungs and analysed in a model of a disrupted epithelium. EMT marker expression and BMP signalling were examined by immunofluorescence, Western blotting and RT-PCR.

Results

Following generation of a wound area, AECs at the wound edge migrated and acquired a mesenchymal-like morphology. E-cadherin expression was reduced in migrating cells while vimentin and α-smooth muscle actin (α-SMA) expression was increased. Re-expression of membrane E-cadherin was subsequently observed in some cells in the wound area following re-establishment of the monolayer. A transient increase in the incidence of nuclear phosphorylated Smad1/5/8 was observed in migrating cells compared with confluent cells, indicating active BMP signalling during migration. BMP antagonists noggin and gremlin inhibited cell migration, confirming the involvement of BMP signalling in migration and indicating autocrine signalling, possibly involving BMP7 or BMP4 which were expressed in AECs. Exogenous BMP2, BMP4 and BMP7 induced a mesenchymal-like morphology in AECs, enhanced the rate of cell migration and increased α-SMA protein expression in AECs.

Conclusions

Following disruption of an intact epithelium, migrating AECs at the wound edge acquire an EMT-like phenotype involving altered expression of E-cadherin, vimentin and α-SMA. BMP signalling is involved in AEC migration and is likely to mediate the switch towards an EMT-like phenotype by altering protein expression to facilitate cell migration and wound closure. We propose therefore that acquisition of an EMT-like phenotype by AECs is a normal aspect of wound repair. Furthermore, we suggest that diseases involving fibrosis may arise because the EMT phase of repair is prolonged by chronic injury/inflammation, rather than being caused by it, as is the current paradigm.     

Norcantharidin Inhibits Renal Interstitial Fibrosis by Blocking the Tubular Epithelial-Mesenchymal Transition

Epithelial–mesenchymal transition (EMT) is thought to contribute to the progression of renal tubulointerstitial fibrosis. Norcantharidin (NCTD) is a promising agent for inhibiting renal interstitial fibrosis. However, the molecular mechanisms of NCTD are unclear. In this study, a unilateral ureteral obstruction (UUO) rat model was established and treated with intraperitoneal NCTD (0.1 mg/kg/day). The UUO rats treated with NCTD showed a reduction in obstruction-induced upregulation of α-SMA and downregulation of E-cadherin in the rat kidney (P<0.05). Human renal proximal tubule cell lines (HK-2) stimulated with TGF-β₁ were treated with different concentrations of NCTD. HK-2 cells stimulated by TGF-β₁ in vitro led to downregulation of E-cadherin and increased de novo expression of α-SMA; co-treatment with NCTD attenuated all of these changes (P<0.05). NCTD reduced TGF-β₁-induced expression and phosphorylation of Smad2/3 and downregulated the expression of Snail1 (P<0.05). These results suggest that NCTD antagonizes tubular EMT by inhibiting the Smad pathway. NCTD may play a critical role in preserving the normal epithelial phenotype and modulating tubular EMT.     

Regional Differences in Susceptibiity of Bronchial Epithelium to Mesenchymal Transition and Inhibition by the Macrolide Antibiotic Azithromycin

Objective

Dysregulated repair following epithelial injury is a key forerunner of disease in many organs, and the acquisition of a mesenchymal phenotype by the injured epithelial cells (epithelial to mesenchymal transition, EMT) may serve as a source of fibrosis. The macrolide antibiotic azithromycin and the DNA synthesis inhibitor mycophenolate are in clinical use but their mechanism of action remains unknown in post-transplant bronchiolitis obliterans syndrome (BOS). Here we determined if regional variation in the EMT response to TGFβ1 underlies the bronchiolocentric fibrosis leading to BOS and whether EMT could be inhibited by azithromycin or mycophenolate.

Methods/Results

We found that small and large airway epithelial cells from stable lung transplant patients underwent EMT when stimulated with TGFβ1, however mesenchymal protein expression was higher and loss of epithelial protein expression more complete in small airway epithelial cells. This regional difference was not mediated by changes in expression of the TGFβRII or Smad3 activation. Azithromycin potentially inhibited EMT in both small and large airway epithelial cells by inhibiting Smad3 expression, but not activation.

Conclusion

Collectively, these observations provide a biologic basis for a previously unexplained but widely observed clinical phenomena, and a platform for the development of new approaches to fibrotic diseases.     

BMP-7 Does Not Protect against Bleomycin-Induced Lung or Skin Fibrosis

Bone morphogenic protein (BMP)-7 is a member of the BMP family which are structurally and functionally related, and part of the TGFβ super family of growth factors. BMP-7 has been reported to inhibit renal fibrosis and TGFβ1-induced epithelial-mesenchymal transition (EMT), in part through negative interactions with TGFβ1 induced Smad 2/3 activation. We utilized in vivo bleomycin-induced fibrosis models in the skin and lung to determine the potential therapeutic effect of BMP-7. We then determined the effect of BMP-7 on TGFβ1-induced EMT in lung epithelial cells and collagen production by human lung fibroblasts. We show that BMP-7 did not affect bleomycin-induced fibrosis in either the lung or skin in vivo; had no effect on expression of pro-fibrotic genes by human lung fibroblasts, either at rest or following exposure to TGFβ1; and did not modulate TGFβ1 -induced EMT in human lung epithelial cells. Taken together our data indicates that BMP-7 has no anti-fibrotic effect in lung or skin fibrosis either in vivo or in vitro. This suggests that the therapeutic options for BMP-7 may be confined to the renal compartment.     

TRIP-1 via AKT modulation drives lung fibroblast/myofibroblast trans-differentiation

Background

Myofibroblasts are the critical effector cells in the pathogenesis of pulmonary fibrosis which carries a high degree of morbidity and mortality. We have previously identified Type II TGFβ receptor interacting protein 1 (TRIP-1), through proteomic analysis, as a key regulator of collagen contraction in primary human lung fibroblasts—a functional characteristic of myofibroblasts, and the last, but critical step in the process of fibrosis. However, whether or not TRIP-1 modulates fibroblast trans-differentiation to myofibroblasts is not known.

Methods

TRIP-1 expression was altered in primary human lung fibroblasts by siRNA and plasmid transfection. Transfected fibroblasts were then analyzed for myofibroblast features and function such as α-SMA expression, collagen contraction ability, and resistance to apoptosis.

Results

The down-regulation of TRIP-1 expression in primary human lung fibroblasts induces α-SMA expression and enhances resistance to apoptosis and collagen contraction ability. In contrast, TRIP-1 over-expression inhibits α-SMA expression. Remarkably, the effects of the loss of TRIP-1 are not abrogated by blockage of TGFβ ligand activation of the Smad3 pathway or by Smad3 knockdown. Rather, a TRIP-1 mediated enhancement of AKT phosphorylation is the implicated pathway. In TRIP-1 knockdown fibroblasts, AKT inhibition prevents α-SMA induction, and transfection with a constitutively active AKT construct drives collagen contraction and decreases apoptosis.

Conclusions

TRIP-1 regulates fibroblast acquisition of phenotype and function associated with myofibroblasts. The importance of this finding is it suggests TRIP-1 expression could be a potential target in therapeutic strategy aimed against pathological fibrosis.     

TGF-β1 induces human alveolar epithelial to mesenchymal cell transition (EMT)

Background

Fibroblastic foci are characteristic features in lung parenchyma of patients with idiopathic pulmonary fibrosis (IPF). They comprise aggregates of mesenchymal cells which underlie sites of unresolved epithelial injury and are associated with progression of fibrosis. However, the cellular origins of these mesenchymal phenotypes remain unclear. We examined whether the potent fibrogenic cytokine TGF-β1 could induce epithelial mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-β1-mediated EMT.

Methods

A549 cells were examined for evidence of EMT after treatment with TGF-β1. EMT was assessed by: morphology under phase-contrast microscopy; Western analysis of cell lysates for expression of mesenchymal phenotypic markers including fibronectin EDA (Fn-EDA), and expression of epithelial phenotypic markers including E-cadherin (E-cad). Markers of fibrogenesis, including collagens and connective tissue growth factor (CTGF) were also evaluated by measuring mRNA level using RT-PCR, and protein by immunofluorescence or Western blotting. Signaling pathways for EMT were characterized by Western analysis of cell lysates using monoclonal antibodies to detect phosphorylated Erk1/2 and Smad2 after TGF-β1 treatment in the presence or absence of MEK inhibitors. The role of Smad2 in TGF-β1-mediated EMT was investigated using siRNA.

Results

The data showed that TGF-β1, but not TNF-α or IL-1β, induced A549 cells with an alveolar epithelial type II cell phenotype to undergo EMT in a time-and concentration-dependent manner. The process of EMT was accompanied by morphological alteration and expression of the fibroblast phenotypic markers Fn-EDA and vimentin, concomitant with a downregulation of the epithelial phenotype marker E-cad. Furthermore, cells that had undergone EMT showed enhanced expression of markers of fibrogenesis including collagens type I and III and CTGF. MMP-2 expression was also evidenced. TGF-β1-induced EMT occurred through phosphorylation of Smad2 and was inhibited by Smad2 gene silencing; MEK inhibitors failed to attenuate either EMT-associated Smad2 phosphorylation or the observed phenotypic changes.

Conclusion

Our study shows that TGF-β1 induces A549 alveolar epithelial cells to undergo EMT via Smad2 activation. Our data support the concept of EMT in lung epithelial cells, and suggest the need for further studies to investigate the phenomenon.     

Dioxin Receptor Expression Inhibits Basal and Transforming Growth Factor β-induced Epithelial-to-mesenchymal Transition

Recent studies have emphasized the role of the dioxin receptor (AhR) in maintaining cell morphology, adhesion, and migration. These novel AhR functions depend on the cell phenotype, and although AhR expression maintains mesenchymal fibroblasts migration, it inhibits keratinocytes motility. These observations prompted us to investigate whether AhR modulates the epithelial-to-mesenchymal transition (EMT). For this, we have used primary AhR^+/+ and AhR^−/− keratinocytes and NMuMG cells engineered to knock down AhR levels (sh-AhR) or to express a constitutively active receptor (CA-AhR). Both AhR^−/− keratinocytes and sh-AhR NMuMG cells had increased migration, reduced levels of epithelial markers E-cadherin and β-catenin, and increased expression of mesenchymal markers Snail, Slug/Snai2, vimentin, fibronectin, and α-smooth muscle actin. Consistently, AhR^+/+ and CA-AhR NMuMG cells had reduced migration and enhanced expression of epithelial markers. AhR activation by the agonist FICZ (6-formylindolo[3,2-b]carbazole) inhibited NMuMG migration, whereas the antagonist α-naphthoflavone induced migration as did AhR knockdown. Exogenous TGFβ exacerbated the promigratory mesenchymal phenotype in both AhR-expressing and AhR-depleted cells, although the effects on the latter were more pronounced. Rescuing AhR expression in sh-AhR cells reduced Snail and Slug/Snai2 levels and cell migration and restored E-cadherin levels. Interference of AhR in human HaCaT cells further supported its role in EMT. Interestingly, co-immunoprecipitation and immunofluorescence assays showed that AhR associates in common protein complexes with E-cadherin and β-catenin, suggesting the implication of AhR in cell-cell adhesion. Thus, basal or TGFβ-induced AhR down-modulation could be relevant in the acquisition of a motile EMT phenotype in both normal and transformed epithelial cells.     

Low-Dose Paclitaxel Ameliorates Pulmonary Fibrosis by Suppressing TGF-β1/Smad3 Pathway via miR-140 Upregulation

Abnormal TGF-β1/Smad3 activation plays an important role in the pathogenesis of pulmonary fibrosis, which can be prevented by paclitaxel (PTX). This study aimed to investigate an antifibrotic effect of the low-dose PTX (10 to 50 nM in vitro, and 0.6 mg/kg in vivo). PTX treatment resulted in phenotype reversion of epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AECs) with increase of miR-140. PTX resulted in an amelioration of bleomycin (BLM)-induced pulmonary fibrosis in rats with reduction of the wet lung weight to body weight ratios and the collagen deposition. Our results further demonstrated that PTX inhibited the effect of TGF-β1 on regulating the expression of Smad3 and phosphorylated Smad3 (p-Smad3), and restored the levels of E-cadherin, vimentin and α-SMA. Moreover, lower miR-140 levels were found in idiopathic pulmonary fibrosis (IPF) patients, TGF-β1-treated AECs and BLM-instilled rat lungs. Through decreasing Smad3/p-Smad3 expression and upregulating miR-140, PTX treatment could significantly reverse the EMT of AECs and prevent pulmonary fibrosis of rats. The action of PTX to ameliorate TGF-β1-induced EMT was promoted by miR-140, which increased E-cadherin levels and reduced the expression of vimentin, Smad3 and p-Smad3. Collectively, our results demonstrate that low-dose PTX prevents pulmonary fibrosis by suppressing the TGF-β1/Smad3 pathway via upregulating miR-140.     

miR-218 regulates focal adhesion kinase–dependent TGFβ signaling in fibroblasts

Scarring, which occurs in essentially all adult tissue, is characterized by the excessive production and remodeling of extracellular matrix by α-smooth muscle actin (SMA)–expressing myofibroblasts located within connective tissue. Excessive scarring can cause organ failure and death. Oral gingivae do not scar. Compared to dermal fibroblasts, gingival fibroblasts are less responsive to transforming growth factor β (TGFβ) due to the reduced expression, due to the reduced expression and activity of focal adhesion kinase (FAK) by this cell type. Here we show that, compared with dermal fibroblasts, gingival fibroblasts show reduced expression of miR-218. Introduction of pre–miR-218 into gingival fibroblasts elevates FAK expression and, via a FAK/src-dependent mechanism, results in the ability of TGFβ to induce α-SMA. The deubiquitinase cezanne is a direct target of miR-218 and has increased expression in gingival fibroblasts compared with dermal fibroblasts. Knockdown of cezanne in gingival fibroblasts increases FAK expression and causes TGFβ to induce α-smooth muscle actin (α-SMA). These results suggest that miR-218 regulates the ability of TGFβ to induce myofibroblast differentiation in fibroblasts via cezanne/FAK.     

Curcumin Inhibits Transforming Growth Factor-β1-Induced EMT via PPARγ Pathway,Not Smad Pathway in Renal Tubular Epithelial Cells

Tubulointerstitial fibrosis (TIF) is the final common pathway in the end-stage renal disease. Epithelial-to-mesenchymal transition (EMT) is considered a major contributor to the TIF by increasing the number of myofibroblasts. Curcumin, a polyphenolic compound derived from rhizomes of Curcuma, has been shown to possess potent anti-fibrotic properties but the mechanism remains elusive. We found that curcumin inhibited the EMT as assessed by reduced expression of α-SMA and PAI-1, and increased E-cadherin in TGF-β1 treated proximal tubular epithelial cell HK-2 cells. Both of the conventional TGF-β1/Smad pathway and non-Smad pathway were investigated. Curcumin reduced TGF-β receptor type I (TβR-I) and TGF-β receptor type II (TβR II), but had no effect on phosphorylation of Smad2 and Smad3. On the other hand, in non-Smad pathway curcumin reduced TGF-β1-induced ERK phosphorylation and PPARγ phosphorylation, and promoted nuclear translocation of PPARγ. Further, the effect of curcumin on α-SMA, PAI-1, E-cadherin, TβR I and TβR II were reversed by ERK inhibitor U0126 or PPARγ inhibitor BADGE, or PPARγ shRNA. Blocking PPARγ signaling pathway by inhibitor BADGE or shRNA had no effect on the phosphorylation of ERK whereas the suppression of ERK signaling pathway inhibited the phosphorylation of PPARγ. We conclude that curcumin counteracted TGF-β1-induced EMT in renal tubular epithelial cells via ERK-dependent and then PPARγ-dependent pathway.     

DEK‐targeting aptamer DTA‐64 attenuates bronchial EMT‐mediated airway remodelling by suppressing TGF‐β1/Smad,MAPK and PI3K signalling pathway in asthma

This study is to investigate the inhibitory effects and mechanisms of DEK‐targeting aptamer (DTA‐64) on epithelial mesenchymaltransition (EMT)‐mediated airway remodelling in mice and human bronchial epithelial cell line BEAS‐2B. In the ovalbumin (OVA)‐induced asthmatic mice, DTA‐64 significantly reduced the infiltration of eosinophils and neutrophils in lung tissue, attenuated the airway resistance and the proliferation of goblet cells. In addition, DTA‐64 reduced collagen deposition, transforming growth factor 1 (TGF‐β1) level in BALF and IgE levels in serum, balanced Th1/Th2/Th17 ratio, and decreased mesenchymal proteins (vimentin and α‐SMA), as well as weekend matrix metalloproteinases (MMP‐2 and MMP‐9) and NF‐κB p65 activity. In the in vitro experiments, we used TGF‐β1 to induce EMT in the human epithelial cell line BEAS‐2B. DEK overexpression (ovDEK) or silencing (shDEK) up‐regulated or down‐regulated TGF‐β1 expression, respectively, on the contrary, TGF‐β1 exposure had no effect on DEK expression. Furthermore, ovDEK and TGF‐β1 synergistically promoted EMT, whereas shDEK significantly reduced mesenchymal markers and increased epithelial markers, thus inhibiting EMT. Additionally, shDEK inhibited key proteins in TGF‐β1‐mediated signalling pathways, including Smad2/3, Smad4, p38 MAPK, ERK1/2, JNK and PI3K/AKT/mTOR. In conclusion, the effects of DTA‐64 against EMT of asthmatic mice and BEAS‐2B might partially be achieved through suppressing TGF‐β1/Smad, MAPK and PI3K signalling pathways. DTA‐64 may be a new therapeutic option for the management of airway remodelling in asthma patients.     

Phosphatidylinositol 3-Kinase Class II α-Isoform PI3K-C2α Is Required for Transforming Growth Factor β-induced Smad Signaling in Endothelial Cells

We have recently demonstrated that the PI3K class II-α isoform (PI3K-C2α), which generates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphates, plays crucial roles in angiogenesis, by analyzing PI3K-C2α knock-out mice. The PI3K-C2α actions are mediated at least in part through its participation in the internalization of VEGF receptor-2 and sphingosine-1-phosphate receptor S1P₁ and thereby their signaling on endosomes. TGFβ, which is also an essential angiogenic factor, signals via the serine/threonine kinase receptor complex to induce phosphorylation of Smad2 and Smad3 (Smad2/3). SARA (Smad anchor for receptor activation) protein, which is localized in early endosomes through its FYVE domain, is required for Smad2/3 signaling. In the present study, we showed that PI3K-C2α knockdown nearly completely abolished TGFβ1-induced phosphorylation and nuclear translocation of Smad2/3 in vascular endothelial cells (ECs). PI3K-C2α was necessary for TGFβ-induced increase in phosphatidylinositol 3,4-bisphosphates in the plasma membrane and TGFβ receptor internalization into the SARA-containing early endosomes, but not for phosphatidylinositol 3-phosphate enrichment or localization of SARA in the early endosomes. PI3K-C2α was also required for TGFβ receptor-mediated formation of SARA-Smad2/3 complex. Inhibition of dynamin, which is required for the clathrin-dependent receptor endocytosis, suppressed both TGFβ receptor internalization and Smad2/3 phosphorylation. TGFβ1 stimulated Smad-dependent VEGF-A expression, VEGF receptor-mediated EC migration, and capillary-like tube formation, which were all abolished by either PI3K-C2α knockdown or a dynamin inhibitor. Finally, TGFβ1-induced microvessel formation in Matrigel plugs was greatly attenuated in EC-specific PI3K-C2α-deleted mice. These observations indicate that PI3K-C2α plays the pivotal role in TGFβ receptor endocytosis and thereby Smad2/3 signaling, participating in angiogenic actions of TGFβ.     

Fibroblast-myofibroblast transition is differentially regulated by bronchial epithelial cells from asthmatic children

Background

Airway remodeling is a proposed mechanism that underlies the persistent loss of lung function associated with childhood asthma. Previous studies have demonstrated that human lung fibroblasts (HLFs) co-cultured with primary human bronchial epithelial cells (BECs) from asthmatic children exhibit greater expression of extracellular matrix (ECM) components compared to co-culture with BECs derived from healthy children. Myofibroblasts represent a population of differentiated fibroblasts that have greater synthetic activity. We hypothesized co-culture with asthmatic BECs would lead to greater fibroblast to myofibroblast transition (FMT) compared to co-culture with healthy BECs.

Methods

BECs were obtained from well-characterized asthmatic and healthy children and were proliferated and differentiated at an air-liquid interface (ALI). BEC-ALI cultures were co-cultured with HLFs for 96 hours. RT-PCR was performed in HLFs for alpha smooth muscle actin (α-SMA) and flow cytometry was used to assay for α-SMA antibody labeling of HLFs. RT-PCR was also preformed for the expression of tropomyosin-I as an additional marker of myofibroblast phenotype. In separate experiments, we investigated the role of TGFβ₂ in BEC-HLF co-cultures using monoclonal antibody inhibition.

Results

Expression of α-SMA by HLFs alone was greater than by HLFs co-cultured with healthy BECs, but not different than α-SMA expression by HLFs co-cultured with asthmatic BECs. Flow cytometry also revealed significantly less α-SMA expression by healthy co-co-cultures compared to asthmatic co-cultures or HLF alone. Monoclonal antibody inhibition of TGFβ₂ led to similar expression of α-SMA between healthy and asthmatic BEC-HLF co-cultures. Expression of topomyosin-I was also significantly increased in HLF co-cultured with asthmatic BECs compared to healthy BEC-HLF co-cultures or HLF cultured alone.

Conclusion

These findings suggest dysregulation of FMT in HLF co-cultured with asthmatic as compared to healthy BECs. Our results suggest TGFβ₂ may be involved in the differential regulation of FMT by asthmatic BECs. These findings further illustrate the importance of BEC-HLF cross-talk in asthmatic airway remodeling.