Interaction between a fluoroquinolone derivative KG022 and RNAs: Effect of base pairs 3' adjacent to the bulged residues.

RNA-targeted small molecules are a promising modality in drug discovery. Recently, we found that a fluoroquinolone derivative, KG022, can bind to RNAs with bulged C or G. To clarify the RNA specificity of KG022, we analyzed the effect of the base pair located at the 3'side of the bulged residue. It was found that KG022 prefers G-C and A-U base pairs at the 3'side. Solution structures of the complexes of KG022 with the four RNA molecules with bulged C or G and G-C or A-U base pairs at the 3'side of the bulged residue were determined to find that the fluoroquinolone moiety is located between two purine bases, and this may be the mechanism of the specificity. This work provides an important example of the specificity of RNA-targeted small molecules.

Interaction between a fluoroquinolone derivative and RNAs with a single bulge.

Interaction analysis between small molecules and RNA as well as structure determination of RNA-small molecule complexes will be the clues to search for compounds that bind to specific mRNA or non-coding RNA in drug discovery. In this study, the RNA-binding ability of a fluoroquinolone derivative, KG022, was examined against single-residue bulge-containing hairpin RNAs as RNA models. Nuclear magnetic resonance analysis indicated that KG022 interacts with the RNAs in the vicinity of the bulge residue, with preferring C and G as the bulge residues. The solution structures of the RNA-KG022 complexes showed that the KG022 binds to the RNAs at the bulge-out regions. Each substituent in KG022 interacts with specific position of RNAs around the bulge-out region probably contributing the specificity of the binding. This work provides a novel member for the RNA-targeted small molecules.

Structure of macrophage migration inhibitory factor in complex with methotrexate.

Methotrexate (MTX) is an anticancer and anti-rheumatoid arthritis drug that is considered to block nucleotide synthesis and the cell cycle mainly by inhibiting the activity of dihydrofolate reductase (DHFR). Using affinity-matrix technology and X-ray analysis, the present study shows that MTX also interacts with macrophage migration inhibitory factor (MIF). Fragment molecular-orbital calculations quantified the interaction between MTX and MIF based on the structure of the complex and revealed the amino acids that are effective in the interaction of MTX and MIF. It should be possible to design new small-molecule compounds that have strong inhibitory activity towards both MIF and DHFR by structure-based drug discovery.

Structure basis 1/2SLPI and porcine pancreas trypsin interaction.

SLPI (secretory leukocyte protease inhibitor) is a 107-residue protease inhibitor which inhibits various serine proteases, including elastase, cathepsin G, chymotrypsin and trypsin. SLPI is obtained as a multiple inhibitor in lung defense and in chronic airway infection. X-ray crystal structures have so far reported that they are full-length SLPIs with bovine α-chymotrypsin and 1/2SLPI (recombinant C-terminal domain of SLPI; Arg58-Ala107) with HNE (human neutrophil elastase). To understand the role of this multiple inhibitory mechanism, the crystal structure of 1/2SLPI with porcine pancreas trypsin was solved and the binding modes of two other complexes compared. The Leu residue surprisingly interacts with the S1 site of trypsin, as with chymotrypsin and elastase. The inhibitory mechanism of 1/2SLPI using the wide primary binding site contacts (from P2' to P5) with various serine proteases is discussed. These inhibitory mechanisms have been acquired in the evolution of the protection system for acute inflammatory diseases.

The size of motoneurons of the gastrocnemius muscle in rats with diabetes.

Alterations in the number and size of motoneurons were studied in the medial gastrocnemius (MG) motor nucleus of diabetic rats (12 or 22 weeks after injection of storeptozotocin) and age-matched controls. Each group contained 6 animals. MG motoneurons were retrogradely labeled by dextran-fluorescein and the number and size of cell bodies were examined. Significantly fewer labeled MG motoneurons were found in the 22-week diabetic rats as compared with age-matched control animals. The mean soma diameter of MG motoneurons was significantly smaller in the 12- and 22-week diabetic animals. Furthermore the soma size for 22-week diabetic animals was smaller than for 12-week diabetic animals. The distribution of average soma diameters in the MG nucleus of control animals was bimodal; cells with larger average diameter were presumed to be alpha-motoneurons and those with smaller diameters were presumed to be gamma. Compared to control animals, the number of smaller MG motoneurons was reduced in 12 week diabetic animals. By 22 weeks, diabetic animals had no small MG motoneurons and the size distribution became unimodal. We conclude that there is a significant decrease in the absolute number and size of MG motoneurons in diabetic rats, with the possibility that the decrease occurred predominantly among the smaller gamma-motoneurons.

CDP/cut is an osteoblastic coactivator of the vitamin D receptor (VDR).

Vitamin D plays an important role in regulating bone and calcium metabolism. The actions of vitamin D are mediated through the nuclear vitamin D receptor (VDR), and gene disruption of the VDR in mice causes skeletal disorders. However, the precise role of the VDR in each stage of osteoblastogenesis is not well understood. To address this issue, we used a biochemical approach to identify an osteoblast-specific coregulator of the VDR. Using a GST-fused VDR ligand-binding domain as bait, proteins associated with liganded VDR were purified from nuclear extracts of HOS osteoblastic cells and compared with those of HeLa cells. Among the interactants identified by mass fingerprinting, CCAAT displacement protein (CDP) was found as a novel ligand-dependent VDR interactant in HOS cells, together with other previously reported DRIP/TRAP complex components. Further biochemical analysis showed that complex formation between the VDR and CDP was distinct from the previously known DRIP/TRAP complex and the p160 family coactivator complexes. Transient expression of CDP potentiated VDR-mediated transcriptional activation in HOS cells. Furthermore, modulation of CDP expression levels in osteoblastic SaM-1 cells affected vitamin D-dependent osteoblast differentiation before the maturation (mineralization) stage. These findings suggest that CDP is a novel differentiation stage-specific coactivator of the VDR in osteoblasts.

Increased rate of force development of elbow flexors by antagonist conditioning contraction.

The effects of isometric antagonist conditioning contraction (ACC) at various durations and intensities on the contractile force, electromyographic (EMG) amplitude, and their rates of rise of elbow flexor muscles were examined in healthy participants. In particular, we focused on the change in the maximum rate of initial force development of agonists (dFagonist/dt(max)), which was evaluated by subtracting antagonist force decaying from apparent initial force development. While the ACC caused no statistically significant effect on the average force during elbow flexion, dFagonist/dt(max) was significantly increased by the ACC of short durations (1-2s) and large intensities. Similarly, the ACC did not affect the root mean square EMG amplitude of biceps brachii during elbow flexion, but significantly increased the maximum rate of rise of the absolute EMG amplitude (dE/dt(max)). These results suggested that facilitating effects of the ACC could be observed in the initial phase of agonist action in healthy participants, and ACC of shorter durations might be more effective. The increased dE/dt(max) suggested that increased neural activities might contribute to the antagonist conditioned facilitation of force development.

Prebeta1-HDL is elevated in the fasting state, but markedly reduced postprandially in poorly controlled type 2 diabetic patients.

BACKGROUND: Prebeta1-HDL is a minor HDL subfraction that is an initial acceptor of cellular free cholesterol. Prebeta1-HDL is elevated in hypertriglyceridemia, which is exaggerated with postprandial hyperglycemia. We investigated whether the prebeta1-HDL concentration changes postprandially in type 2 diabetic patients and blood glucose (BG) control reduces this change. METHODS: We examined 9 healthy controls and 20 diabetic patients with poor BG control. Seven blood samples (30 min before and 2 h after each meal, and at midnight) were obtained daily in the poor (poor-GC: n=20) and improved (imp-GC: n=11) glycemic control phases of diabetic patients after intensive insulin therapy and a low-calorie diet. RESULTS: The prebeta1-HDL concentration did not change postprandially in the controls. However, the fasting prebeta1-HDL concentration in the poor-GC phase was 28.3% higher than in the controls (25.4+/-6.8 vs 19.8+/-6.9 mg/l ApoAI, p<0.05) and decreased markedly after breakfast (20.9+/-7.7 mg/l ApoAI, p<0.01). In the imp-GC phase, the prebeta1-HDL concentration showed no morning surge, as in the controls. CONCLUSIONS: Type 2 diabetic patients in the poor-GC phase have high prebeta1-HDL levels in the morning, followed by a gradual reduction until midnight. BG control diminishes this postprandial change. Glucose metabolism may be involved in modulating reverse cholesterol transport in type 2 diabetic patients.

Complex of human neutrophil elastase with 1/2SLPI.

SLPI (secretory leukocyte protease inhibitor) is a 107-residue non-glycosylated protease inhibitor, which inhibits a wide range of serine proteases, trypsin, chymotrypsin, neutrophil elastase, chymase and cathepsin G. X-ray crystallographic analyses have shown that SLPI comprises two separate domains of similar architecture [Grütter, Fendrich, Huber & Bode (1988), EMBO J. 7, 345-351] and the C-terminal domain interacts with bovine alpha-chymotrypsin. In order to understand SLPI's multiple functions against various serine proteases, the complex HNE (human neutrophil elastase) has been co-crystallized with 1/2SLPI (recombinant C-terminal domain of SLPI; Arg58-Ala107), which has a biological activity similar to full SLPI. The 1/2SLPI and HNE complex structure was solved at 1.7 A resolution, and compared with the interaction mechanism of elafin, which is a specific inhibitor of elastase. It was found that P1 Leu72i and six hydrogen bonds between the main chains in the primary contact region have sufficient ability to inhibit HNE and PPE (porcine pancreatic elastase), and P5 Tyr68i is important in increasing the selectivity of 1/2SLPI against HNE. The mechanisms of the functions of SLPI are relatively unknown, but the current study could help understand the selectivity of SLPI against HNE and PPE.

Relationship between insulin-like growth factor-I and brain natriuretic peptide in patients with acromegaly after surgery.

BACKGROUND: Increased cardiac insulin-like growth factor (IGF)-I production is associated with physiological cardiac hypertrophy in athletes, and IGF-I has been recognized as a cardioprotective agent in experimental animal studies. On the other hand, acromegaly which is characterized by an excess of IGF-I has been linked to impaired cardiac function. METHODS AND RESULTS: Both the relationship between the serum levels of IGF-I and brain natriuretic peptide (BNP), which is released from the cardiac ventricles in response to ventricular stress, and that between IGF-I and the concentrations of the plasma amino-terminal propeptide of procollagen type III (P-III-P), which is associated with myocardial fibrosis, were evaluated in 19 patients after surgical treatment for acromegaly. Echocardiography revealed that left ventricular systolic function and dimensions were within normal range in all patients. Significant inverse correlations were found between IGF-I and the BNP (r=-0.5, p=0.02) and P-III-P levels (r=-0.62, p=0.005). CONCLUSION: We observed an inverse significant relationship between IGF-I and both the BNP and P-III-P value in surgically treated acromegaly patients. These observations suggest that appropriate levels of IGF-I have beneficial cardioprotective effects after surgery in patients with acromegaly.

Potentiation of knee extensor contraction by antagonist conditioning contraction at several intensities.

The purpose of this study was to examine the effect of graded conditioning contractions of the antagonist knee flexor muscles on the output characteristics of knee extensor muscles in healthy humans. Eight male university students performed maximum isometric contractions of knee extensors, preceded by isometric conditioning contractions of the antagonist knee flexors. The developed force and electromyographic (EMG) amplitudes of the knee extensors after the conditioning contraction were measured and compared with those of simple knee extension without conditioning. The forces of the conditioning flexor contraction were set at three levels: low (20% of maximum voluntary contraction: MVC), moderate (60% of MVC), and high (100% of MVC). The EMG amplitudes of the vastus medialis, vastus lateralis, and rectus femoris muscle were recorded and the root mean square amplitudes were calculated. The strongest enhancement of the extension force was obtained by moderate intensity conditioning contraction (108.95+/-1.87% of simple knee extension), although high intensity conditioning also induced a significant increase (105.41+/-2.69%). Low intensity conditioning did not cause a significant enhancement of the contraction force (103.17+/-2.99%). Similarly, the EMG amplitudes were significantly increased by moderate and/or high conditioning. These results suggest that antagonist conditioning contraction of moderate intensities is sufficient and may be optimal to potentiate knee extensor contraction.

Tissue factor pathway inhibitor is highly susceptible to chymase-mediated proteolysis.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that primarily inhibits the extrinsic pathway of blood coagulation. It is synthesized by various cells and its expression level increases in inflammatory environments. Mast cells and neutrophils accumulate at sites of inflammation and vascular disease where they release proteinases as well as chemical mediators of these conditions. In this study, the interactions between TFPI and serine proteinases secreted from human mast cells and neutrophils were examined. TFPI inactivated human lung tryptase, and its inhibitory activity was stronger than that of antithrombin. In contrast, mast cell chymase rapidly cleaved TFPI even at an enzyme to substrate molar ratio of 1:500, resulting in markedly decreased TFPI anticoagulant and anti-(factor Xa) activities. N-terminal amino-acid sequencing and MS analyses of the proteolytic fragments revealed that chymase preferentially cleaved TFPI at Tyr159-Gly160, Phe181-Glu182, Leu89-Gln90, and Tyr268-Glu269, in that order, resulting in the separation of the three individual Kunitz domains. Neutrophil-derived proteinase 3 also cleaved TFPI, but the reaction was much slower than the chymase reaction. In contrast, alpha-chymotrypsin, which shows similar substrate specificities to those of chymase, resulted in a markedly lower level of TFPI degradation. These data indicate that TFPI is a novel and highly susceptible substrate of chymase. We propose that chymase-mediated proteolysis of TFPI may induce a thrombosis-prone state at inflammatory sites.

The human airway trypsin-like protease modulates the urokinase receptor (uPAR, CD87) structure and functions.

The human airway trypsin-like protease (HAT) is a respiratory epithelium-associated, type II transmembrane serine protease, which is also detected as an extracellular enzyme in lung fluids during airway inflammatory disorders. We have evaluated its capacity to affect the urokinase-type plasminogen activator receptor (uPAR), a membrane glycolipid-anchored, three-domain (D1D2D3) glycoprotein that plays a crucial role in innate immunity and inflammation by supporting cell migration and matrix degradation, with structure and biological properties that can be regulated via limited endoproteolysis. With the use of immunoblotting, flow immunocytometry, and ELISA analyses applied to a recombinant uPAR protein and to uPAR-expressing monocytic and human bronchial epithelial cells, it was shown that exposure of uPAR to soluble HAT in the range of 10-500 nM resulted in the proteolytic processing of the full-length (D1D2D3) into the truncated (D2D3) species, with cleavage occurring in the D1 to D2 linker sequence after arginine residues at position 83 and 89. Using immunohistochemistry, we found that both HAT and uPAR were expressed in the human bronchial epithelium. Moreover, transient cotransfection in epithelial cells showed that membrane coexpression of the two partners produced a constitutive and extensive shedding of the D1 domain, occurring for membrane-associated HAT concentrations in the nanomolar range. Because the truncated receptor was found to be unable to bind two of the major uPAR ligands, the adhesive matrix protein vitronectin and the serine protease urokinase, it thus appears that proteolytic regulation of uPAR by HAT is likely to modulate cell adherence and motility, as well as tissue remodeling during the inflammatory response in the airways.

Inhibitory effect of statins on renal epithelial-to-mesenchymal transition.

BACKGROUND/AIM: Recent studies have suggested that statins may play a role in the protection against renal failure which is independent of cholesterol reduction. Activation of RhoGTPases is a key step in renal tubular cells' epithelial-to-mesenchymal transition (EMT) which contributes to renal interstitial fibrosis. We hypothesized that statins could act by inhibiting the synthesis of the isoprenoids, such as geranylgeranyl pyrophosphate, which is essential for membrane attachment and biological activity of RhoGTPases, RhoA and Rac1. METHODS: Human proximal tubular epithelial cells (HK2) were used to examine the inhibitory effect of statins on EMT induced with medium conditioned by activated peripheral blood mononuclear cells. RESULTS: Our study demonstrates that the statins lovastatin, simvastatin, and pravastatin inhibit HK2 cells to undergo EMT. Inhibition of EMT in HK2 cells with these statins resulted in a reduction of RhoA and Rac1 activation in both the cytoplasmic and membrane-bound forms, in preservation of the expression of the epithelial cell markers E-cadherin and cytokeratin-19, and in a decrease in Fn-EDA expression, a marker for the myofibroblast phenotype. The decreased levels of activated RhoA and Rac1 in both the cytoplasmic and membrane fractions of the cells were reversed by geranylgeranyl pyrophosphate and mevalonate, and thus attributable to the inhibition of isoprenylation of RhoGTPases by statins. CONCLUSION: This phenomenon could explain the beneficial effect of statins on EMT and on renal fibrosis prevention.

Culture serum-induced conversion from agonist to antagonist of a Vitamin D analog, TEI-9647.

The nuclear receptor for Vitamin D (VDR) mediates many of the effects of Vitamin D in target tissues by regulating gene expression. The transactivation function of ligand-bound VDR in target tissues is thought to depend on the tissue-type and the cellular-environment, but the molecular basis for these differences has not been fully understood. In this study, during characterization of TEI-9647 as a synthetic ligand for the VDR, we found that depletion of serum from the culture medium converted TEI-9647 from an antagonist to an agonist of VDR-mediated transactivation, whereas it retained antagonistic activity in the presence of serum. Consistent with these results, using a mammalian two-hybrid system, we found that TEI-9647 recruited different coactivators to the VDR in the presence and absence of serum. These findings suggest that an unknown serum factor modulates the transactivation function of the VDR.

Development of affinity chromatography using a bioactive peptide as a ligand.

By repeatedly introducing hydrophilic polyethylene glycol (PEG) spacer (2) onto affinity resin bearing a bioactive peptide (1/2 secretory leukocyte protease inhibitor, 1/2SLPI) as a ligand, the adsorption of nonspecific binding proteins was effectively reduced and the purification efficacy of elastase, which is one of the target molecules for 1/2SLPI, from a protein mixture was improved. Moreover, using this resin, we also successfully detected L-plastin, as an endogenous target molecule for SLPI, from HL-60 cell lysate.

Metalloprotease-dependent amphiregulin release mediates tumor necrosis factor-alpha-induced IL-8 secretion in the human airway epithelial cell line NCI-H292.

Tumor necrosis factor-alpha (TNF-alpha) is a potent multifunctional cytokine that plays a central role in the pathogenesis of many inflammatory diseases. Interleukin-8 (IL-8) is a principle neutrophil chemoattractant and activator in humans. The alveolar macrophage-derived TNF-alpha initiates lung inflammation through its ability to stimulate IL-8 synthesis in airway epithelial cells. Since recent studies demonstrated that the stimulation of epidermal growth factor receptor (EGFR) could induce IL-8 secretion, the involvement of EGFR in TNF-alpha-induced IL-8 secretion in airway epithelium-like NCI-H292 cells was investigated in this study. TNF-alpha and epidermal growth factor (EGF) stimulated IL-8 secretion in a time- and concentration-dependent manner. Inhibition of the EGFR by either an anti-EGFR neutralizing antibody or by its specific inhibitor AG1478 (1 microM) blocked TNF-alpha-induced IL-8 secretion. In addition, TNF-alpha stimulated tyrosine phosphorylation of the EGFR within 5 min after stimulation. Further, TNF-alpha-induced IL-8 secretion was completely inhibited by the neutralizing antibody against amphiregulin (AR), an EGFR ligand, suggesting that TNF-alpha-induced IL-8 secretion was mediated by the AR-EGFR pathway. Furthermore, TNF-alpha stimulated the release of AR in a concentration-dependent manner. Finally, both AR and IL-8 release-induced by TNF-alpha were eliminated by pretreatment with either GM6001, a broad-spectrum inhibitor for metalloprotease, or TAPI-1, relatively selective inhibitor for TNF-alpha converting enzyme (TACE). These findings indicate that metalloprotease-mediated AR shedding and subsequent activation of EGFR play a critical role in TNF-alpha-induced IL-8 secretion from the human airway epithelium-like NCI-H292 cells, and that TACE is one of the most possible candidates for metalloprotease responsible for TNF-alpha-induced AR shedding.

Human airway trypsin-like protease induces amphiregulin release through a mechanism involving protease-activated receptor-2-mediated ERK activation and TNF alpha-converting enzyme activity in airway epithelial cells.

Human airway trypsin-like protease (HAT), a serine protease found in the sputum of patients with chronic airway diseases, is an agonist of protease-activated receptor-2 (PAR-2). Previous results have shown that HAT enhances the release of amphiregulin (AR); further, it causes MUC5AC gene expression through the AR-epidermal growth factor receptor pathway in the airway epithelial cell line NCI-H292. In this study, the mechanisms by which HAT-induced AR release can occur were investigated. HAT-induced AR gene expression was mediated by extracellular signal-regulated kinase (ERK) pathway, as pretreatment of cells with ERK pathway inhibitor eliminated the effect of HAT on AR mRNA. Both HAT and PAR-2 agonist peptide (PAR-2 AP) induced ERK phosphorylation; further, desensitization of PAR-2 with a brief exposure of cells to PAR-2 AP resulted in inhibition of HAT-induced ERK phosphorylation, suggesting that HAT activates ERK through PAR-2. Moreover, PAR-2 AP induced AR gene expression subsequent to protein production in the cellular fraction through the ERK pathway indicating that PAR-2-mediated activation of ERK is essential for HAT-induced AR production. However, in contrast to HAT, PAR-2 AP could not cause AR release into extracellular space; it appears that activation of PAR-2 is not sufficient for HAT-induced AR release. Finally, HAT-induced AR release was eliminated by blockade of tumour necrosis factor alpha-converting enzyme (TACE) by the TAPI-1 and RNA interference, suggesting that TACE activity is necessary for HAT-induced AR release. These observations show that HAT induces AR production through the PAR-2 mediated ERK pathway, and then causes AR release by a TACE-dependent mechanism.

Species differences in angiotensin II generation and degradation by mast cell chymases.

Although chymases are known to exhibit species differences in regard to angiotensin (Ang) II generation and degradation, their properties have never been compared under the same experimental conditions. We analyzed the processing of Ang I by chymases of a variety of species (human chymase, dog chymase, hamster chymase-1, rat mast cell protease-1 [rMCP-1], mouse mast cell protease-4 [mMCP-4]) at physiological ionic strength and under neutral pH conditions. Human chymase generated Ang II from Ang I without further degradation, whereas the chymases of other species generated Ang II, followed by degradation at the Tyr4-Ile5 site in a time-dependent manner. Kinetic analysis showed that in terms of Ang II generating activity (analyzed by cleavage of the Phe8-His9 bond using the model peptide Ang(5-10), Ile5-His6-Pro7-Phe8-His9-Leu10), the chymases ranked as follows: dog > human > hamster > mouse > rat (kcat/Km: 18, 11, 0.69, 0.059, 0.030 microM-1min-1), and that in terms of Ang II degrading activity (i.e., cleavage of the Tyr4-Ile5 bond of Ang II), the order was hamster > rat > mouse > dog (kcat/Km: 5.4, 4.8, 0.39, 0.29 microM-lmin-1). These results suggest species differences in the contribution of chymases to local Ang II generation and degradation.

TGF-beta1 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-beta1 could induce epithelial mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-beta1-mediated EMT. METHODS: A549 cells were examined for evidence of EMT after treatment with TGF-beta1. 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-beta1 treatment in the presence or absence of MEK inhibitors. The role of Smad2 in TGF-beta1-mediated EMT was investigated using siRNA. RESULTS: The data showed that TGF-beta1, but not TNF-alpha or IL-1beta, 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-beta1-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-beta1 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.