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.     

The role of IFN-γ in regulation of IFN-γ-inducible protein 10 (IP-10) expression in lung epithelial cell and peripheral blood mononuclear cell co-cultures

Background

Epithelial to mesenchymal transition (EMT) in alveolar epithelial cells (AECs) has been widely observed in patients suffering interstitial pulmonary fibrosis. In vitro studies have also demonstrated that AECs could convert into myofibroblasts following exposure to TGF-β1. In this study, we examined whether EMT occurs in bleomycin (BLM) induced pulmonary fibrosis, and the involvement of bronchial epithelial cells (BECs) in the EMT. Using an α-smooth muscle actin-Cre transgenic mouse (α-SMA-Cre/R26R) strain, we labelled myofibroblasts in vivo. We also performed a phenotypic analysis of human BEC lines during TGF-β1 stimulation in vitro.

Methods

We generated the α-SMA-Cre mouse strain by pronuclear microinjection with a Cre recombinase cDNA driven by the mouse α-smooth muscle actin (α-SMA) promoter. α-SMA-Cre mice were crossed with the Cre-dependent LacZ expressing strain R26R to produce the double transgenic strain α-SMA-Cre/R26R. β-galactosidase (βgal) staining, α-SMA and smooth muscle myosin heavy chains immunostaining were carried out simultaneously to confirm the specificity of expression of the transgenic reporter within smooth muscle cells (SMCs) under physiological conditions. BLM-induced peribronchial fibrosis in α-SMA-Cre/R26R mice was examined by pulmonary βgal staining and α-SMA immunofluorescence staining. To confirm in vivo observations of BECs undergoing EMT, we stimulated human BEC line 16HBE with TGF-β1 and examined the localization of the myofibroblast markers α-SMA and F-actin, and the epithelial marker E-cadherin by immunofluorescence.

Results

βgal staining in organs of healthy α-SMA-Cre/R26R mice corresponded with the distribution of SMCs, as confirmed by α-SMA and SM-MHC immunostaining. BLM-treated mice showed significantly enhanced βgal staining in subepithelial areas in bronchi, terminal bronchioles and walls of pulmonary vessels. Some AECs in certain peribronchial areas or even a small subset of BECs were also positively stained, as confirmed by α-SMA immunostaining. In vitro, addition of TGF-β1 to 16HBE cells could also stimulate the expression of α-SMA and F-actin, while E-cadherin was decreased, consistent with an EMT.

Conclusion

We observed airway EMT in BLM-induced peribronchial fibrosis mice. BECs, like AECs, have the capacity to undergo EMT and to contribute to mesenchymal expansion in pulmonary fibrosis.     

Ambulatory oxygen: why do COPD patients not use their portable systems as prescribed? A qualitative study

Background

In mechanically ventilated preterm infants with respiratory distress syndrome (RDS), exogenous surfactant application has been demonstrated both to decrease DNA-synthesis but also and paradoxically to increase epithelial cell proliferation. However, the effect of exogenous surfactant has not been studied directly on alveolar type II cells (ATII cells), a key cell type responsible for alveolar function and repair.

Objective

The aim of this study was to investigate the effects of two commercially available surfactant preparations on ATII cell viability and DNA synthesis.

Methods

Curosurf^® and Alveofact^® were applied to two ATII cell lines (human A549 and mouse iMATII cells) and to primary rat ATII cells for periods of up to 24 h. Cell viability was measured using the redox indicator resazurin and DNA synthesis was measured using BrdU incorporation.

Results

Curosurf^® resulted in slightly decreased cell viability in all cell culture models. However, DNA synthesis was increased in A549 and rat ATII cells but decreased in iMATII cells. Alveofact^® exhibited the opposite effects on A549 cells and had very mild effects on the other two cell models.

Conclusion

This study showed that commercially available exogenous surfactants used to treat preterm infants with RDS can have profound effects on cell viability and DNA synthesis.     

Value of supplemental interventions to enhance the effectiveness of physical exercise during respiratory rehabilitation in COPD patients. A Systematic Review

Background

Oxygen toxicity is a major cause of lung injury. The base excision repair pathway is one of the most important cellular protection mechanisms that responds to oxidative DNA damage. Lesion-specific DNA repair enzymes include hOgg1, hMYH, hNTH and hMTH.

Methods

The above lesion-specific DNA repair enzymes were expressed in human alveolar epithelial cells (A549) using the pSF91.1 retroviral vector. Cells were exposed to a 95% oxygen environment, ionizing radiation (IR), or H₂O₂. Cell growth analysis was performed under non-toxic conditions. Western blot analysis was performed to verify over-expression and assess endogenous expression under toxic and non-toxic conditions. Statistical analysis was performed using the paired Student's t test with significance being accepted for p < 0.05.

Results

Cell killing assays demonstrated cells over-expressing hMYH had improved survival to both increased oxygen and IR. Cell growth analysis of A549 cells under non-toxic conditions revealed cells over-expressing hMYH also grow at a slower rate. Western blot analysis demonstrated over-expression of each individual gene and did not result in altered endogenous expression of the others. However, it was observed that O₂ toxicity did lead to a reduced endogenous expression of hNTH in A549 cells.

Conclusion

Increased expression of the DNA glycosylase repair enzyme hMYH in A549 cells exposed to O₂ and IR leads to improvements in cell survival. DNA repair through the base excision repair pathway may provide an alternative way to offset the damaging effects of O₂ and its metabolites.     

Legionella pneumophila infection induces programmed cell death,caspase activation,and release of high-mobility group box 1 protein in A549 alveolar epithelial cells: inhibition by methyl prednisolone

Background

Legionella pneumophila pneumonia often exacerbates acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Apoptosis of alveolar epithelial cells is considered to play an important role in the pathogenesis of ALI and ARDS. In this study, we investigated the precise mechanism by which A549 alveolar epithelial cells induced by L. pneumophila undergo apoptosis. We also studied the effect of methyl prednisolone on apoptosis in these cells.

Methods

Nuclear deoxyribonucleic acid (DNA) fragmentation and caspase activation in L. pneumophila-infected A549 alveolar epithelial cells were assessed using the terminal deoxyribonucleotidyl transferase-mediated triphosphate (dUTP)-biotin nick end labeling method (TUNEL method) and colorimetric caspase activity assays. The virulent L. pneumophila strain AA100jm and the avirulent dotO mutant were used and compared in this study. In addition, we investigated whether methyl prednisolone has any influence on nuclear DNA fragmentation and caspase activation in A549 alveolar epithelial cells infected with L. pneumophila.

Results

The virulent strain of L. pneumophila grew within A549 alveolar epithelial cells and induced subsequent cell death in a dose-dependent manner. The avirulent strain dotO mutant showed no such effect. The virulent strains of L. pneumophila induced DNA fragmentation (shown by TUNEL staining) and activation of caspases 3, 8, 9, and 1 in A549 cells, while the avirulent strain did not. High-mobility group box 1 (HMGB1) protein was released from A549 cells infected with virulent Legionella. Methyl prednisolone (53.4 μM) did not influence the intracellular growth of L. pneumophila within alveolar epithelial cells, but affected DNA fragmentation and caspase activation of infected A549 cells.

Conclusion

Infection of A549 alveolar epithelial cells with L. pneumophila caused programmed cell death, activation of various caspases, and release of HMGB1. The dot/icm system, a major virulence factor of L. pneumophila, is involved in the effects we measured in alveolar epithelial cells. Methyl prednisolone may modulate the interaction of Legionella and these cells.     

Mechanisms of the acute effects of inhaled ozone in humans

Ambient air ozone (O₃) is generated photochemically from oxides of nitrogen and volatile hydrocarbons. Inhaled O₃ causes remarkably reversible acute lung function changes and inflammation. Approximately 80% of inhaled O₃ is deposited on the airways. O₃ reacts rapidly with CC double bonds in hydrophobic airway and alveolar surfactant-associated phospholipids and cholesterol. Resultant primary ozonides further react to generate bioactive hydrophilic products that also initiate lipid peroxidation leading to eicosanoids and isoprostanes of varying electrophilicity. Airway surface liquid ascorbate and urate also scavenge O₃. Thus, inhaled O₃ may not interact directly with epithelial cells.Acute O₃–induced lung function changes are dominated by involuntary inhibition of inspiration (rather than bronchoconstriction), mediated by stimulation of intraepithelial nociceptive vagal C-fibers via activation of transient receptor potential (TRP) A1 cation channels by electrophile (e.g., 4-oxo-nonenal) adduction of TRPA1 thiolates enhanced by PGE₂-stimulated sensitization.Acute O₃-induced neutrophilic airways inflammation develops more slowly than the lung function changes. Surface macrophages and epithelial cells are involved in the activation of epithelial NFkB and generation of proinflammatory mediators such as IL-6, IL-8, TNFa, IL-1b, ICAM-1, E-selectin and PGE₂. O₃-induced partial depolymerization of hyaluronic acid and the release of peroxiredoxin-1 activate macrophage TLR4 while oxidative epithelial cell release of EGFR ligands such as TGFa or EGFR transactivation by activated Src may also be involved. The ability of lipid ozonation to generate potent electrophiles also provides pathways for Nrf2 activation and inhibition of canonical NFkB activation. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.     

Endothelium-dependent epithelial–mesenchymal transition of tumor cells: Exclusive roles of transforming growth factor β1 and β2

Background

Induction of epithelial–mesenchymal transition (EMT) is essential for the metastasis of tumor cells and maintaining their stemness. This study aimed to examine whether endothelial cells, which are most closely located to tumor cells in vivo, play a role in inducing EMT in tumor cells or not.

Methods

Concentrated culture medium of bovine aortic endothelial cells (BAECs) was applied to tumor cell lines (A549 and PANC-1) and epithelial cell line (NMuMg). Cadherin conversion, expressions of α-smooth muscle actin and ZO-1, actin fiber formation and cell migration were examined as hallmarks of the induction of EMT in these cell lines. Transforming growth factor β (TGFβ) antibodies were used to neutralize TGFβ₁, TGFβ₂ and TGFβ₃. Expression and release of TGFβ proteins in BAECs as well as in porcine and human endothelial cells were assessed by Western blotting and ELISA, respectively.

Results

Conditioned medium of BAEC induced EMT in the examined cell lines. All endothelial cells from various species and locations expressed TGFβ₁ and TGFβ₂ proteins and much lower level of TGFβ₃ protein. Conditioned medium from these endothelial cells contained TGFβ₁ and TGFβ₂, but TGFβ₃ could not be detected. Neutralizing antibody against each of TGFβ₁ or TGFβ₂ did not reverse endothelium-dependent EMT, but simultaneous neutralization of both TGFβ₁ and TGFβ₂ completely abolished it.

Conclusions

Endothelial cells may play a role in the induction and maintenance of EMT in tumor cells by constitutively releasing TGFβ₁ and TGFβ₂.

General significance

The present results provide a novel strategy of the inhibition of tumor metastasis by targeting vascular endothelium.     

AC-93253 iodide,a novel Src inhibitor,suppresses NSCLC progression by modulating multiple Src-related signaling pathways

Background

The tyrosine kinase Src is involved in the progression of many cancers. Moreover, inhibiting Src activity has been shown to obstruct several signaling pathways regulated by the EGFR. Thus, Src is a valuable target molecule in drug development. The purpose of this study was to identify compounds that directly or indirectly modulate Src to suppress lung cancer cell growth and motility and to investigate the molecular mechanisms underlying the effects of these compounds.

Methods

Human non-small cell lung cancer (NSCLC) cell lines (PC9, PC9/gef, A549, and H1975) with different EGFR statuses were tested by cytotoxicity and proliferation assays after AC-93253 iodide treatment. Src and Src-related protein expression in AC-93253 iodide-treated PC9, PC9/gef, and A549 cells were assessed by western blotting. The effects of AC-93253 iodide on cancer cell colony formation, invasion, and migration were assessed in PC9 and PC9/gef cells. The synergistic effects of gefitinib and AC-93253 iodide were evaluated by combination index (CI)-isobologram analysis in gefitinib-resistant cell lines. The efficacy of AC-93253 iodide in vivo was determined using nude mice treated with either the compound or the vehicle.

Results

Among the compounds, AC-93253 iodide exhibited the most potent dose-independent inhibitory effects on the activity of Src as well as on that of the Src-related proteins EGFR, STAT3, and FAK. Furthermore, AC-93253 iodide significantly suppressed cancer cell proliferation, colony formation, invasion, and migration in vitro and tumor growth in vivo. AC-93253 iodide sensitized tumor cells to gefitinib treatment regardless of whether the cells were gefitinib-sensitive (PC9) or resistant (H1975 and PC9/gef), indicating that it may exert synergistic effects when used in combination with established therapeutic agents. Our findings also suggested that the inhibitory effects of AC-93253 iodide on lung cancer progression may be attributable to its ability to modulate multiple proteins, including Src, PI3K, JNK, Paxillin, p130cas, MEK, ERK, and EGFR.

Conclusions

Our data suggest that AC-93253 iodide inhibits NSCLC cell growth and motility by regulating multiple Src-related pathways. Our findings may facilitate the development of therapeutic strategies and anti-tumor drugs that may be useful for treating lung cancer in the future.

     

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.     

Endosomal Deubiquitinating Enzymes Control Ubiquitination and Down-regulation of Protease-activated Receptor 2

The E3 ubiquitin ligase c-Cbl ubiquitinates the G protein-coupled receptor protease-activated receptor 2 (PAR₂), which is required for postendocytic sorting of activated receptors to lysosomes, where degradation terminates signaling. The mechanisms of PAR₂ deubiquitination and its importance in trafficking and signaling of endocytosed PAR₂ are unknown. We report that receptor deubiquitination occurs between early endosomes and lysosomes and involves the endosomal deubiquitinating proteases AMSH and UBPY. Expression of the catalytically inactive mutants, AMSH(D348A) and UBPY(C786S), caused an increase in PAR₂ ubiquitination and trapped the receptor in early endosomes, thereby preventing lysosomal trafficking and degradation. Small interfering RNA knockdown of AMSH or UBPY also impaired deubiquitination, lysosomal trafficking, and degradation of PAR₂. Trapping PAR₂ in endosomes through expression of AMSH(D348A) or UBPY(C786S) did not prolong the association of PAR₂ with β-arrestin2 or the duration of PAR₂-induced ERK2 activation. Thus, AMSH and UBPY are essential for trafficking and down-regulation of PAR₂ but not for regulating PAR₂ dissociation from β-arrestin2 or PAR₂-mediated ERK2 activation.Ubiquitination of certain G protein-coupled receptors (GPCRs)³ is an essential signal for their postendocytic trafficking to lysosomes, which prevents uncontrolled signaling during chronic stimulation. Agonists stimulate ubiquitination of the β₂-adrenergic receptor (β₂AR), chemokine (CXC motif) receptor 4, and protease-activated receptor 2 (PAR₂), and the E3 ubiquitin ligases that mediate ubiquitination of these GPCRs and associated proteins, such as β-arrestins, have been identified (1–3). Although ubiquitination of these receptors is not required for endocytosis, ubiquitin-resistant mutant receptors show diminished postendocytic sorting to lysosomes and impaired down-regulation. However, despite of the reversible nature of this post-translational modification, little is known about the role of deubiquitinating proteases (DUBs) in the postendocytic trafficking and signaling of GPCRs.Our understanding of the role of DUBs in postendocytic receptor trafficking mostly derives from studies of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). Two endosomal DUBs, AMSH (associated molecule with the Src homology 3 domain of STAM (signal-transducing adapter molecule)) and UBPY (ubiquitin-specific protease Y) (also known as USP8), regulate deubiquitination and postendocytic trafficking of EGFR (4). AMSH belongs to the JAMM (JAB1/MPN/Mov34) family of metalloproteases and shows specificity for Lys⁶³- over Lys⁴⁸-linked ubiquitin chains (5, 6). UBPY is a cysteine protease of the ubiquitin-specific protease (USP) family and does not discriminate between Lys⁴⁸- and Lys⁶³-linked ubiquitin (7, 8). Activated EGFR recruits the E3 ligase c-Cbl, which ubiquitinates the receptor (9). Ubiquitinated EGFR then interacts with the Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate)-STAM complex in early endosomes (10). Hrs-STAM forms part of the ESCRT (endosomal sorting complex required for transport)-I, -II, -III complex that sorts ubiquitinated receptors in multivesicular bodies (MVBs) to intralumenal vesicles that eventually fuse with lysosomes, where degradation occurs (11). Before receptors are incorporated into the intralumenal vesicles, they are deubiquitinated, which serves to maintain levels of free ubiquitin (11). AMSH and UBPY interact directly with STAM through a common binding site within its Src homology 3 domain (12–14). The balance of EGFR ubiquitination by c-Cbl and deubiquitination by AMSH and UBPY controls the postendocytic trafficking and down-regulation of the EGFR. c-Cbl promotes lysosomal degradation of the EGFR (9), AMSH opposes c-Cbl action and promotes EGFR recycling (5), and UBPY is required for lysosomal sorting and degradation of EGFR (8, 15–17). The role of AMSH and UBPY in regulating deubiquitination, trafficking, and signaling of GPCRs in endosomes is largely unknown. A recent study has shown, however, that AMSH and UBPY regulate the down-regulation of the δ-opioid receptor (DOR), a GPCR that is ubiquitinated and degraded following activation (18). Expression of catalytically inactive mutants of AMSH or UBPY or knockdown of AMSH or UBPY levels using siRNA inhibits down-regulation of DOR. Interestingly, the roles of AMSH and UBPY in DOR down-regulation appear to be nonredundant, since depletion of either DUB produced comparable effects, and simultaneous depletion of both DUBs did not have additional consequences (18). Different DUBs, USP20 and -33, have been recently shown to reverse agonist-induced ubiquitination of the β₂AR (19).We examined the roles of AMSH and UBPY in the ubiquitination, postendocytic trafficking, and lysosomal degradation of PAR₂. We also determined whether AMSH and UBPY regulate PAR₂ association with β-arrestins in endosomes and control β-arrestin-mediated extracellular signal-regulated kinase (ERK) activation. PAR₂ is a receptor for multiple serine proteases that are generated during injury and inflammation (20). Activated PAR₂ promotes inflammation and pain, and PAR₂ contributes to inflammatory diseases of the airway, joints, and intestine. PAR₂ levels are elevated during inflammation, due to increased mRNA expression or perhaps decreased receptor degradation, which amplifies the proinflammatory actions of proteases (21). Given the irreversible nature of proteolytic activation, and since the internalized receptor probably signals by the β-arrestin-dependent recruitment of mitogen-activated protein kinase (MAPK) to endosomes (22), termination of PAR₂ signaling requires receptor degradation in lysosomes, which in turn is ubiquitination-dependent (3, 23). It is therefore important to understand mechanisms of PAR₂ ubiquitination and lysosomal targeting and also how these processes can be reversed. We have reported that activated PAR₂ is monoubiquitinated at multiple sites by the E3 ligase c-Cbl and targeted to lysosomes by an Hrs-dependent pathway (3, 24). Nothing is known about the mechanism and function of PAR₂ deubiquitination. Herein, we examined the role of AMSH and UBPY in regulating the deubiquitination, lysosomal trafficking, and degradation of PAR₂, the interaction of PAR₂ with β-arrestin2, and β-arrestin-mediated ERK2 activation. We demonstrate that endosomal DUBs are key regulators of GPCR down-regulation.     

Protease Signaling to G Protein-Coupled Receptors: Implications for Inflammation and Pain

Proteases, like thrombin, trypsin, cathepsins, or tryptase, can signal to cells by cleaving in a specific manner, a family of G protein-coupled receptors, the protease-activated receptors (PARs). Proteases cleave the extracellular N-terminal domain of PARs to reveal tethered ligand domains that bind to and activate the receptors. Recent evidence has supported the involvement of PARs in inflammation and pain. Activation of PAR₁, PAR₂, and PAR₄ either by proteinases or by selective agonists causes inflammation inducing most of the cardinal signs of inflammation: swelling, redness, and pain. Recent studies suggest a crucial role for the different PARs in innate immune response. The role of PARs in the activation of pain pathways appears to be dual. Subinflammatory doses of PAR₂ agonists induce hyperalgesia and allodynia, and PAR₂ activation has been implicated in the generation of inflammatory hyperalgesia. In contrast, subinflammatory doses of PAR₁ or PAR₄ increase nociceptive threshold, inhibiting inflammatory hyperalgesia, thereby acting as analgesic mediators. PARs have to be considered as an additional subclass of G protein-coupled receptors that are active participants to inflammation and pain responses and that could constitute potential novel therapeutic targets.     

MicroRNA-147 Induces a Mesenchymal-To-Epithelial Transition (MET) and Reverses EGFR Inhibitor Resistance

Background

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer progression and may promote resistance to therapy. An analysis of patients (n = 71) profiled with both gene expression and a global microRNA assessment (∼415 miRs) identified miR-147 as highly anti-correlated with an EMT gene expression signature score and postulated to reverse EMT (MET).

Methods and Findings

miR-147 was transfected into colon cancer cells (HCT116, SW480) as well as lung cancer cells (A-549). The cells were assessed for morphological changes, and evaluated for effects on invasion, motility, and the expression of key EMT markers. Resistance to chemotherapy was evaluated by treating cells with gefitinib, an EGFR inhibitor. The downstream genes regulated by miR-147 were assayed using the Affymetrix GeneChip U133 Plus2.0 platform. miR-147 was identified to: 1. cause MET primarily by increasing the expression of CDH1 and decreasing that of ZEB1; 2. inhibit the invasion and motility of cells; 3. cause G1 arrest by up-regulating p27 and down-regulating cyclin D1. miR-147 also dramatically reversed the native drug resistance of the colon cancer cell line HCT116 to gefitinib. miR-147 significantly repressed Akt phosphorylation, and knockdown of Akt with siRNA induced MET. The morphologic effects of miR-147 on cells appear to be attenuated by TGF-B1, promoting a plastic and reversible transition between MET and EMT.

Conclusion

miR-147 induced cancer cells to undergo MET and induced cell cycle arrest, suggesting a potential tumor suppressor role. miR-147 strikingly increased the sensitivity to EGFR inhibitor, gefitinib in cell with native resistance. We conclude that miR-147 might have therapeutic potential given its ability to inhibit proliferation, induce MET, as well as reverse drug sensitivity.     

Induction of release and up-regulated gene expression of interleukin (IL)-8 in A549 cells by serine proteinases

Background  

Hypersecretion of cytokines and serine proteinases has been observed in asthma. Since protease-activated receptors (PARs) are receptors of several serine proteinases and airway epithelial cells are a major source of cytokines, the influence of serine proteinases and PARs on interleukin (IL)-8 secretion and gene expression in cultured A549 cells was examined.     

Normal Human Lung Epithelial Cells Inhibit Transforming Growth Factor-β Induced Myofibroblast Differentiation via Prostaglandin E2

Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with very few effective treatments. The key effector cells in fibrosis are believed to be fibroblasts, which differentiate to a contractile myofibroblast phenotype with enhanced capacity to proliferate and produce extracellular matrix. The role of the lung epithelium in fibrosis is unclear. While there is evidence that the epithelium is disrupted in IPF, it is not known whether this is a cause or a result of the fibroblast pathology. We hypothesized that healthy epithelial cells are required to maintain normal lung homeostasis and can inhibit the activation and differentiation of lung fibroblasts to the myofibroblast phenotype. To investigate this hypothesis, we employed a novel co-culture model with primary human lung epithelial cells and fibroblasts to investigate whether epithelial cells inhibit myofibroblast differentiation.

Measurements and Main Results

In the presence of transforming growth factor (TGF)-β, fibroblasts co-cultured with epithelial cells expressed significantly less α-smooth muscle actin and collagen and showed marked reduction in cell migration, collagen gel contraction, and cell proliferation compared to fibroblasts grown without epithelial cells. Epithelial cells from non-matching tissue origins were capable of inhibiting TGF-β induced myofibroblast differentiation in lung, keloid and Graves’ orbital fibroblasts. TGF-β promoted production of prostaglandin (PG) E₂ in lung epithelial cells, and a PGE₂ neutralizing antibody blocked the protective effect of epithelial cell co-culture.

Conclusions

We provide the first direct experimental evidence that lung epithelial cells inhibit TGF-β induced myofibroblast differentiation and pro-fibrotic phenotypes in fibroblasts. This effect is not restricted by tissue origin, and is mediated, at least in part, by PGE₂. Our data support the hypothesis that the epithelium plays a crucial role in maintaining lung homeostasis, and that damaged and/ or dysfunctional epithelium contributes to the development of fibrosis.     

The effects of interleukin-8 on airway smooth muscle contraction in cystic fibrosis

Background

Clara cells are the epithelial progenitor cell of the small airways, a location known to be important in many lung disorders. Although migration of alveolar type II and bronchiolar ciliated epithelial cells has been examined, the migratory response of Clara cells has received little attention.

Methods

Using a modification of existing procedures for Clara cell isolation, we examined mouse Clara cells and a mouse Clara-like cell line (C22) for adhesion to and migration toward matrix substrate gradients, to establish the nature and integrin dependence of migration in Clara cells.

Results

We observed that Clara cells adhere preferentially to fibronectin (Fn) and type I collagen (Col I) similar to previous reports. Migration of Clara cells can be directed by a fixed gradient of matrix substrates (haptotaxis). Migration of the C22 cell line was similar to the Clara cells so integrin dependence of migration was evaluated with this cell line. As determined by competition with an RGD containing-peptide, migration of C22 cells toward Fn and laminin (Lm) 511 (formerly laminin 10) was significantly RGD integrin dependent, but migration toward Col I was RGD integrin independent, suggesting that Clara cells utilize different receptors for these different matrices.

Conclusion

Thus, Clara cells resemble alveolar type II and bronchiolar ciliated epithelial cells by showing integrin mediated pro-migratory changes to extracellular matrix components that are present in tissues after injury.     

High CO2 levels impair alveolar epithelial function independently of pH

Background

In patients with acute respiratory failure, gas exchange is impaired due to the accumulation of fluid in the lung airspaces. This life-threatening syndrome is treated with mechanical ventilation, which is adjusted to maintain gas exchange, but can be associated with the accumulation of carbon dioxide in the lung. Carbon dioxide (CO₂) is a by-product of cellular energy utilization and its elimination is affected via alveolar epithelial cells. Signaling pathways sensitive to changes in CO₂ levels were described in plants and neuronal mammalian cells. However, it has not been fully elucidated whether non-neuronal cells sense and respond to CO₂. The Na,K-ATPase consumes ∼40% of the cellular metabolism to maintain cell homeostasis. Our study examines the effects of increased pCO₂ on the epithelial Na,K-ATPase a major contributor to alveolar fluid reabsorption which is a marker of alveolar epithelial function.

Principal Findings

We found that short-term increases in pCO₂ impaired alveolar fluid reabsorption in rats. Also, we provide evidence that non-excitable, alveolar epithelial cells sense and respond to high levels of CO₂, independently of extracellular and intracellular pH, by inhibiting Na,K-ATPase function, via activation of PKCζ which phosphorylates the Na,K-ATPase, causing it to endocytose from the plasma membrane into intracellular pools.

Conclusions

Our data suggest that alveolar epithelial cells, through which CO₂ is eliminated in mammals, are highly sensitive to hypercapnia. Elevated CO₂ levels impair alveolar epithelial function, independently of pH, which is relevant in patients with lung diseases and altered alveolar gas exchange.     

Smoking cessation and bronchial epithelial remodelling in COPD: a cross-sectional study

Background

Chronic Obstructive Pulmonary Disease (COPD) is associated with bronchial epithelial changes, including squamous cell metaplasia and goblet cell hyperplasia. These features are partially attributed to activation of the epidermal growth factor receptor (EGFR). Whereas smoking cessation reduces respiratory symptoms and lung function decline in COPD, inflammation persists. We determined epithelial proliferation and composition in bronchial biopsies from current and ex-smokers with COPD, and its relation to duration of smoking cessation.

Methods

114 COPD patients were studied cross-sectionally: 99 males/15 females, age 62 ± 8 years, median 42 pack-years, no corticosteroids, current (n = 72) or ex-smokers (n = 42, median cessation duration 3.5 years), postbronchodilator FEV₁ 63 ± 9% predicted. Squamous cell metaplasia (%), goblet cell (PAS/Alcian Blue⁺) area (%), proliferating (Ki-67⁺) cell numbers (/mm basement membrane), and EGFR expression (%) were measured in intact epithelium of bronchial biopsies.

Results

Ex-smokers with COPD had significantly less epithelial squamous cell metaplasia, proliferating cell numbers, and a trend towards reduced goblet cell area than current smokers with COPD (p = 0.025, p = 0.001, p = 0.081, respectively), but no significant difference in EGFR expression. Epithelial features were not different between short-term quitters (<3.5 years) and current smokers. Long-term quitters (≥3.5 years) had less goblet cell area than both current smokers and short-term quitters (medians: 7.9% vs. 14.4%, p = 0.005; 7.9% vs. 13.5%, p = 0.008; respectively), and less proliferating cell numbers than current smokers (2.8% vs. 18.6%, p < 0.001).

Conclusion

Ex-smokers with COPD had less bronchial epithelial remodelling than current smokers, which was only observed after long-term smoking cessation (>3.5 years).

Trial registration

NCT00158847