TRIP-1 regulates TGF-β1-induced epithelial-mesenchymal transition of human lung epithelial cell line A549

Ricardo E. Perez, Angels Navarro, Mohammad H. Rezaiekhaligh, Sherry M. Mabry, Ikechukwu I. Ekekezie

Abstract

Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells undergo conversion to a mesenchymal phenotype contributing to wound repair by fibrosis and to cancer cell acquisition of invasive ability. Recently, we showed that type II TGF-β receptor interacting protein-1 (TRIP-1), a protein identified as a phosphorylation target of the TGF-β type II receptor kinase and as a functional component of eukaryotic translation initiator factor 3 (eiF3) multiprotein complex, is a novel modulator of fibroblast collagen contraction, an important step in wound repair stimulated by TGF-β1 action. TGF-β1 drives EMT, but it is not known whether TRIP-1 expression influences EMT induction. To investigate whether TRIP-1 plays a role in EMT induction we studied the effect of downregulating TRIP-1 expression in the well-characterized A549 model of TGF-β1 induction of EMT. Here we report that short hairpin RNA (shRNA)-mediated depletion of TRIP-1 gene transcripts in A549 cells promotes EMT as assessed by changes in phenotypic markers, morphology, and migrative ability. Knockdown of TRIP-1 dramatically increased A549 responsiveness to TGF-β1 induction of EMT. Mechanistically, a pathway involving increased TGF-β type II receptor level, enhanced Smad3 phosphorylation, and the transcription factor SLUG is implicated. Altogether, the findings point to regulation of endogenous TRIP-1 protein expression as a potential strategy to target EMT, and related invasive behavior, in cancer cells.

  • transforming growth factor-β
  • type II transforming growth factor-β receptor interacting protein-1

excessive scar formation or fibrosis may complicate wound healing after injury, and fibroblasts are the chief cells involved. In the lungs, such aberrant repair by fibrosis causes great morbidity and mortality due to disruption of lung structure and gas exchange functions. The excessive fibrogenesis stems from 1) unchecked activation of interstitial fibroblasts, which leads to an increase in number of wound fibroblasts, and 2) enhanced transdifferentiation into the myofibroblast phenotype, which is avid at matrix synthesis (2, 23, 37, 40). Moreover, it has been shown that this increase in numbers of fibroblasts/myofibroblasts at fibrotic foci derives from 1) recruitment of fibrocytes and bone marrow progenitors in circulation (8, 18, 19, 24) as well as 2) induction of lung epithelial cells, which then undergo epithelial-mesenchymal transition (EMT) (11, 14, 26, 35). In fact, in a study of bleomycin-induced lung injury in mice it was reported that as many as 30% of fibroblasts in the fibrotic foci derive from EMT of type II lung epithelial cells (31).

EMT is a biological process by which epithelial cells undergo phenotypic conversion to a fibroblast-like morphology with variable acquisition of their functional attributes. The process was initially identified for its involvement in normal tissue development as occurs during embryogenesis and organogenesis (9, 21, 29). However, evolving knowledge has since identified EMT and its regulatory factors as key contributors to wound healing by fibrosis (12) and to tumor cell acquisition of invasive behavior (6, 32). There is also emerging recognition consistent with current understanding about cell plasticity that the EMT process may occur to varying extents, and may be reversible depending on the nature of the cell and the conditions. In the EMT process, epithelial cells undergo phenotypic conversion to mesenchymal cells by reprogramming their gene signature. A central event is transcriptional repression of key epithelial molecules, notably the cell-cell adhesion protein E-cadherin, the loss of which disrupts the adherens junction of cell-cell attachment, leading to a change in cell shape from cytoskeletal rearrangement. Complementary to the epithelial gene repression is simultaneous induced expression of mesenchymal genes coding for N-cadherin and vimentin and of matrix proteins as well as matrix-degrading enzymes (25, 33).

Among the plethora of cytokines and growth factors present in wound healing, IL-1β, TNF-α, and transforming growth factor (TGF)-β1 are notable for their ability to influence EMT in lung epithelial cells, with TGF-β1 being the single most potent inducer. Moreover, TGF-β signaling is strongly linked to wound fibrosis (27, 30). As an experimental model, it has aptly been demonstrated by investigators that treatment of primary lung type II epithelial cells or the human lung type II epithelial-like cell line A549 with TGF-β1 reproducibly results in EMT (13, 34, 36, 38, 39). Regulation of TGF-β1 signaling is a very complex event and occurs at multiple levels. TGF-β1 may signal through the Smad group of intracellular transduction proteins or in Smad-independent pathways such as via ERK-MAP kinase activation—both of which have been implicated in TGF-β1-induced EMT in lung epithelial cells (4).

Type II TGF-β receptor interacting protein-1 (TRIP-1) is a WD-40-containing endogenous protein that has been identified in in vitro studies as a phosphorylation target of the TGF-β type II receptor kinase (3) and also as a functional component of eukaryotic translation initiator factor 3 (eiF3) multiprotein complex (1). We showed recently (20) that TRIP-1 is a novel modulator of fibroblast collagen contraction, an important step in wound repair and an event stimulated by TGF-β1 action. TGF-β1 action drives EMT, but it is not known whether TRIP-1 expression influences EMT induction.

Here we report that short hairpin RNA (shRNA)-mediated depletion of TRIP-1 gene transcripts in A549 cells promotes EMT as assessed by changes in phenotypic markers, morphology, and migrative ability. Knockdown of TRIP-1 dramatically increases A549 responsiveness to TGF-β1 induction of EMT. Mechanistically, a pathway involving increased TGF-β type II receptor level, enhanced Smad3 phosphorylation, and the transcription factor SLUG is implicated. Altogether, the findings point to regulation of endogenous TRIP-1 protein expression as a potential strategy to target EMT, and related invasive behavior, in cancer cells.

MATERIALS AND METHODS

Cells and reagents.

The human lung adenocarcinoma cell line A549 was purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultured in DMEM (Mediatech, Manassas, VA) containing 10% fetal bovine serum (FBS) and 100 U/ml each of penicillin and streptomycin and maintained at 37°C in an atmosphere of 5% CO2. Cells were used between passages 25 and 40. TGF-β1 was purchased from PeproTech (Rocky Hill, NJ). The TGF-β type I receptor inhibitor SB-431452 was purchased from Sigma-Aldrich (St. Louis, MO).

Small interfering RNA transient transfection.

A549 cells were transiently transfected with a final concentration of 50 nM of negative control (catalog no. 4611) or human eiF3 (TRIP-1, siRNA ID no. 13735) small interfering RNA (siRNA) with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. siRNAs were purchased from Applied Biosystems/Ambion (Austin, TX). Transfections were allowed to incubate for 48 h, at which time cells were washed three times with serum-free medium, placed in 0.1% FBS-DMEM, and treated for 48 h with or without 5 ng/ml TGF-β1. Cells were collected, lysed, and prepared for Western blot analysis.

Generation of cell lines.

The human TRIP-1 shRNA was generated with the BLOCK-iT RNAi Designer tool (Invitrogen). The sequence used for TRIP-1 was 5′-GGTCCATTACGCAGATTAAGT-3′, and a shRNA sequence for LacZ provided by the manufacturer was used as control. These sequences were inserted into pENTR/H1/TO and transfected into A549 cells with Lipofectamine (Invitrogen) according to manufacturer's protocols. Stable cell lines were generated by selection using 1 mg/ml Zeocin for 7–10 days, generating either A549-shLacZ or A549-shTRIP-1, and individual and pool clones were selected.

Immunoblotting.

In experiments that incorporated TGF-β1 treatment, 0.1% FBS-DMEM medium was used. Cells were rinsed three times with serum-free medium and then placed in 0.1% FBS medium. Total cell lysates were prepared by washing cells with cold phosphate-buffered saline (PBS, Mediatech) followed by resuspension in lysis buffer containing 40 mM Tris·HCl (pH 7.9), 0.5 M KCl, 0.4 mM EDTA, 20% glycerol, and 0.2% Tween 20, with complete mini protease inhibitor EDTA-Free cocktail tablet (Roche Applied Science, Indianapolis, IN), 10 mM sodium fluoride, 0.1 mM sodium orthovanadate, 10 mM β-glycerol phosphate, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 10% horse serum in PBS-0.1% Tween 20 (PBS-T) and incubated with primary antibodies overnight, followed by incubation with the corresponding horseradish peroxide-conjugated secondary antibody [Thermo Fisher (Rockford, IL), Cell Signaling Technology (Danvers, MA), and Santa Cruz Biotechnology (Santa Cruz, CA), respectively] for 1 h. Proteins were detected with the West Pico chemiluminescence system (Pierce Thermo Scientific). Primary antibodies used were goat anti-β-actin (I-19, 1:2,000), rabbit anti-TGF-β RI (V-22, 1:500), rabbit anti-TGF-β RII (L-21, 1:500), and mouse monoclonal anti-Smad3 (38-Q, 1:500) from Santa Cruz Biotechnology; mouse monoclonal anti-E-cadherin (1:1,000) and mouse anti-N-cadherin (1:1,000) from BD Biosciences (San Jose, CA); rabbit phospho-Smad3 (Ser423/425) (1:1,000) from Cell Signaling Technology; rabbit anti-occludin (1:500) from Invitrogen; and mouse monoclonal anti-vimentin (clone V9, 1:500) and mouse monoclonal anti-α-tubulin (B-5-1-2, 1:10,000) from Sigma-Aldrich.

RNA isolation and real-time PCR.

Total RNA isolation was performed with TRIzol reagent according to the manufacturer's protocol. First-strand cDNA synthesis was carried out with SuperScript III reverse transcriptase followed by ribonuclease H treatment. The following primer sequences were used: TRIP-1 forward 5′-AGATGGGCTACCAGTGCTTTGTGA-3′, reverse 5′-TGCAAGGGATCTTCATGTAGGGCT-3′; E-cadherin forward 5′-CCACCAAAGTCACGCTGAATAC-3′, reverse 5′-GGAGTTGGGAAATGTGAGCAA-3′; and GAPDH forward 5′-TGACAACTTTGGTATCGTGGAAGG-3′, reverse 5′-AGGGATGATGTTCTGGAGAGCC-3′. Reactions were run on a real-time PCR system (iCycler MyiQ System; Bio-Rad Laboratories, Hercules, CA), using iQ SYBR Green (Bio-Rad) for gene detection. Relative gene expression was determined by normalizing to GAPDH by the ΔCT method (where CT is threshold cycle).

Cell counting assay.

Cells were plated on 60-mm dishes at a seeding density of 100,000 cells/dish. Cells were then counted each day for a total of 3 days with a Beckman Coulter Z1 Particle Counter (Beckman Coulter, Brea, CA) after trypsinization, and the average cell numbers from triplicate measurements were plotted.

Cell proliferation assay.

Cells were seeded in a 24-well plate at a density of 50,000 cells/well. The cell proliferation reagent WST-1 (Roche Applied Science, Indianapolis, IN) was added to each well according to manufacturer's instructions. The absorbance of the dye after incubation was measured at a wavelength of 450 nm with background subtraction at 690 nm with a BioTek Synergy HTTR microplate reader (BioTek Instruments, Winooski, VT). Experiments were performed with six wells per cell type, and the average absorbance from the six wells was plotted.

Wounding assay.

A549-shLacZ and A549-shTRIP-1 were trypsinized and plated on 12-well plates at a density of 200,000 or 350,000 cells/well in complete medium and allowed to reach confluence (usually 1–2 days). At that time, cells were rinsed three times in warm serum-free DMEM and starved in DMEM-1% FBS medium overnight, and then a vertical line across the middle of the wells (wound) was created with a 200-μl pipette tip (average wound width was 300 μm). Medium was replaced with either DMEM-1% FBS or DMEM-1% FBS plus 5 ng/ml TGF-β1. Pictures were taken immediately after the wound (at 0 h) and at 24 h after the wound with an Olympus IX50 microscope and QCapture software controlling a cooled charge-coupled device (CCD) camera (×40 magnification). Three pictures were taken per well at each time point, and six wells per condition were analyzed with ImageJ Software. Wounded area per field was individually assessed and averaged per each well, and the percentage of area healed was calculated for each well on an individual basis. To determine scale, a picture was taken of a micrometer and calibration was performed with the Analysis tool in ImageJ. Experiments were performed with six wells per cell type, and condition wells were individually analyzed.

Statistical analysis.

Results are expressed as the means ± SD of data obtained. Statistical analysis was performed with Student's t-test for paired comparisons and ANOVA with Tukey-Kramer posttest for other data. A value of P < 0.05 was considered significant.

RESULTS

Loss of TRIP-1 induces A549 cell epithelial-mesenchymal transition.

TRIP-1 is a phosphorylation target of the TGF-β type II receptor kinase (3). We have previously shown (20) a correlation between TRIP-1 and the ability of human lung fibroblasts to contract collagen, comparing fetal versus adult fibroblasts. We were interested in investigating whether TRIP-1 plays a role in EMT induction. A549 cells, a type II epithelial-like cell line, were transiently transfected with either control or TRIP-1 siRNA at a final concentration of 50 nM. After 48 h, these cells were rinsed three times with serum-free medium and then incubated with or without 5 ng/ml TGF-β1 for 48 h in 0.1% FBS-DMEM. Transfection with TRIP-1 siRNA showed a 50% reduction of TRIP-1 protein level compared with transfection with control siRNA. A549 cells transfected with TRIP-1 siRNA showed a significant decrease in the epithelial marker E-cadherin and an increase in the mesenchymal marker N-cadherin compared with control cells (Fig. 1A). The addition of TGF-β1 showed further decrease of E-cadherin in control cells and a complete loss of E-cadherin expression in cells transfected with TRIP-1 siRNA. N-cadherin levels were further increased by TGF-β1 treatment in TRIP-1 siRNA cells, while no induction was seen in control cells. This result shows that TRIP-1 plays a role in TGF-β1-induced EMT in A549 cells. To better understand the role that TRIP-1 plays in TGF-β1-induced EMT in A549 cells, we stably transfected A549 cells with either pENTR/H1/TO-shTRIP-1, which expresses a shRNA for human TRIP-1, or pENTR/H1/TO-shLacZ, which was used as control cells. These cells were stably selected by using 1 mg/ml Zeocin. To determine the quantity by which the shRNA for TRIP-1 reduced the levels found in A549, quantitative real-time PCR (RT-PCR) was performed for an individual cell clone (shTRIP-1 B8) and the pooled clones (shTRIP-1 Pool). Clones were processed, and clone B8 and pooled clones were found to have a reduction of TRIP-1 mRNA of 3.4- and 4.3-fold, respectively, compared with control cells (Fig. 1B). While culturing these cells, we noticed a change in the morphology of the A549-shTRIP-1 cells compared with their control, A549-shLacZ. These cells had lost their epithelial cobblestone morphology and had adopted a more mesenchymal shape. The A549-shTRIP-1 clones were longer in shape and were less connected to each other than the A549-shLacZ. In Fig. 1C we show how the A549-shTRIP-1 cells differ in morphology compared with A549-shLacZ.

Fig. 1.

Type II transforming growth factor-β (TGF-β) receptor interacting protein-1 (TRIP-1) knockdown induces an epithelial-mesenchymal transition (EMT) response in A549 cells. A: transient transfection of A549 cells with either control or TRIP-1 small interfering RNA (siRNA) (50 nM final concentration). Cells were treated for 48 h with or without 5 ng/ml TGF-β1. E-cadherin, N-cadherin, and TRIP-1 levels were assessed by Western blot analysis. α-Tubulin was used as a loading control. Experiments were performed 3 times; a representative blot is shown. B: relative mRNA expression of TRIP-1 in a clone of A549-shTRIP-1 and a pool of clones compared with control cells A549-shLacZ. Values are fold change of TRIP-1; bars show SD. ***P < 0.05. C: morphological differences between A549-shTRIP-1 cells and their control cells, A549-shLacZ. A549-shTRIP-1 cells display looser cell-cell contact and more of an elongated fibroblast morphology than A549-shLacZ, which exhibit the traditional epithelial cobblestone shape.

TGF-β1-induced EMT is regulated by TRIP-1.

The loss of the epithelial marker E-cadherin is a key indicator in EMT. The morphological difference seen between A549-shTRIP-1 and A549-shLacZ along with the findings in the transient transfection experiment led to an interest in determining how E-cadherin expression had been modified by TRIP-1 knockdown. To verify whether E-cadherin mRNA levels were affected by the loss of TRIP-1, we extracted mRNA from A549-shTRIP-1 clones and their control cells. TRIP-1 knockdown led to the reduction of E-cadherin mRNA expression (2.0- and 1.8-fold, respectively) (Fig. 2A). To further understand the mechanism by which E-cadherin levels are repressed, mRNA levels of two known E-cadherin repressors, Snail and Slug, were measured. A549-shTRIP-1 cells exhibited a 2.5-fold increase in Slug mRNA levels compared with those found in A549-shLacZ, while there was no significant change in Snail mRNA expression between the cell lines (Fig. 2B). Previous studies have shown that TGF-β1 can induce EMT in A549 cells (10, 13). Thus our interest was to determine whether TRIP-1 is involved in the regulation of TGF-β1-induced EMT. TRIP-1 knockdown cells and their controls were removed from 10% FBS and placed in 0.1% FBS for experiments involving TGF-β1 treatment. A549-shLacZ and A549-shTRIP-1 clones were treated with 0.5 ng/ml TGF-β1 for 24 h. EMT was measured by the expression of the epithelial marker E-cadherin along with the mesenchymal markers N-cadherin and vimentin (Fig. 2C). E-cadherin protein expression was significantly decreased in the A549-shTRIP-1 B8 clone and similarly decreased in the A549-shTRIP-1 Pool clone at basal conditions compared with A549-shLacZ. Treatment with TGF-β1 further decreased the levels of E-cadherin in all three cell types but decreased E-cadherin significantly more in A549-shTRIP-1 clones. The mesenchymal marker vimentin was induced by treatment with TGF-β1 in A549-shTRIP-1 clones (1.5- and 1.4-fold, respectively) and not in A549-shLacZ, while N-cadherin expression was induced in all cell types. These results demonstrate that lack of TRIP-1 increases the sensitivity of A549 cells to TGF-β1-induced EMT. To really ascertain how sensitive these A549 cells have become to TGF-β1 treatment, we treated A549-shLacZ and A549-shTRIP-1 cells (from this point forward all experiments were done with clone B8) with TGF-β1 concentrations ranging from 0 to 5 ng/ml. E-cadherin protein expression was significantly decreased in A549-shTRIP1 at a TGF-β1 concentration of 0.05 ng/ml, while N-cadherin expression was significantly increased at a TGF-β1 concentration of 0.25 ng/ml in A549-shTRIP1, compared with A549-shLacZ (Fig. 3, A and B). Along with E-cadherin, the expression of occludin, an epithelial marker, was reduced in A549-shTRIP-1 cells at the same concentration of TGF-β1, as was E-cadherin (Fig. 3A). These results demonstrate that lack of TRIP-1 enhances A549 cell sensitivity to TGF-β1 induction of EMT. Next, we were interested in investigating the sensitivity of A549-shTRIP-1 cells for TGF-β; therefore, lower concentrations of TGF-β1 (0–0.05 ng/ml) were used to treat A549-shLacZ and A549-shTRIP-1 in the same manner as before. E-cadherin expression decreased with 0.001 ng/ml TGF-β1 in A549-shTRIP-1, while no significant change was observed in A549-shLacZ until 0.01 ng/ml TGF-β1 (Fig. 3, C and D). These results further demonstrate the hypersensitivity of A549-shTRIP-1 to TGF-β1 and the possible regulation of TGF-β1-induced EMT by TRIP-1.

Fig. 2.

E-cadherin expression is regulated by TRIP-1 expression. A: knockdown of TRIP-1 leads to a reduction of E-cadherin mRNA levels in A549-shTRIP-1 clones compared with A549-shLacZ. Values are fold change of E-cadherin; bars show SD. **,***P < 0.05. B: Snail and Slug mRNA expression in A549-shLacZ and A549-shTRIP-1 B8 clones. Values are mean relative expression; bars show SD. **P < 0.05. C: Western blot analysis of EMT markers E-cadherin, N-cadherin, and vimentin in A549-shLacZ and A549-shTRIP-1 clones treated with or without 0.5 ng/ml TGF-β1. β-Actin was used as a loading control. Cells were treated for 24 h. Fold changes are compared with A549-shLacZ 0 and presented below each blot. A representative blot from 3 experiments is shown.

Fig. 3.

Reduced levels of TRIP-1 make A549 cells more sensitive to TGF-β1 treatment. A: A549-LacZ and A549-shTRIP-1 cells were treated with 0, 0.05, 0.25, 1, and 5 ng/ml TGF-β1 for 24 h. E-cadherin, N-cadherin, and occludin levels were compared after TGF-β1 treatment by Western blot. B: quantification of E-cadherin and N-cadherin normalized against actin. Bars show SD. C: to determine the sensitivity of A549-shTRIP-1 cells to TGF-β1, cells were treated with 0, 0.001, 0.005, 0.01, or 0.05 ng/ml for 24 h. Western blot for E-cadherin was performed. D: quantification of E-cadherin levels normalized against actin. Bars show SD. *P < 0.05. Thirty micrograms of total protein was used, and β-actin was used as a loading control for all Western blots. Blots are representative of 3 experiments.

TRIP-1 expression affects TGF-β-Smad pathway.

Since phosphorylation of Smad3 is a direct target of the type I TGF-β receptor, to further characterize the role of TRIP-1 in TGF-β-induced EMT we first looked at the expression of TGF-β receptor types I and II in A549-shTRIP-1 and control cells. In Fig. 4A, Western blot analysis showed similar expression of the type I receptor between cell lines, but there was an increase in expression of the type II receptor in A549-shTRIP-1 cultured in either complete (10% FBS) or starvation (0.1% FBS) medium. We then looked at the effect of TRIP-1 expression on Smad3 phosphorylation. TRIP-1 knockdown-engineered A549 cell lines were treated with 5.0 ng/ml TGF-β1 for short periods of time (30, 60, and 120 min). Western blot analysis of phospho-Smad3 in A549-shTRIP-1 cells revealed a higher level of phosphorylation compared with A549-shLacZ (Fig. 4, B and C). To further investigate the role of Smad3 in the regulation of E-cadherin by TRIP-1, we used a TGF-β receptor type I kinase inhibitor, SB-431542, that has been shown previously to inhibit Smad2 phosphorylation in A549 cells as well as inhibit Smad3 nuclear localization in malignant glioma cell lines (7, 10). A549-shLacZ and A549-shTRIP-1 B8 cells were treated with 10 μM SB-431542 for 24 h. After treatment, Western blot analysis showed that inhibition of the TGF-β type I receptor kinase resulted in an increase of E-cadherin protein expression in A549-shTRIP1 cells (1.4-fold change compared with A549-shTRIP1 cells treated with DMSO) and no change of expression in A549-shLacZ (Fig. 4D). We were also interested in seeing whether the TGF-β receptor I inhibitor had any affect on the phosphorylation of Smad3 in the A549-shTRIP1 cells. After 24-h treatment with SB-431542, control and TRIP-1 knockdown cells were treated with 5 ng/ml TGF-β1 for 60 min. Phosphorylation of Smad3 was inhibited in both A549-shLacZ and A549-shTRIP1 by the TGF-β receptor I inhibitor (Fig. 4E). These results suggest that TRIP-1 is a negative regulator of the TGF-β-Smad pathway.

Fig. 4.

TRIP-1 regulates TGF-β receptor type II and Smad3 activation. A: Western blot analysis of TGF-β receptor (TGFβR) types I and II in A549-shLacZ and A549-shTRIP-1 grown in either 10% FBS- or 0.1% FBS-DMEM. B: TRIP-1 knockdown cells along with their control cells were treated with 5 ng/ml TGF-β1 for 30, 60, and 120 min. Phospho (P)-Smad3 and total Smad3 levels were assessed by Western blot analysis. Fold changes for total Smad3 normalized to α-tubulin are compared between corresponding time points and presented below blot. C: quantification of P-Smad3 levels normalized against α-tubulin. Bars show SD. *P < 0.05. D: A549-shTRIP1 cells were treated with SB-431542 for 24 h along with A549-shLacZ cells. E-cadherin level was determined by Western blot analysis. Actin was used as a loading control. Fold changes are compared within each cell type between treated and untreated and presented below blot. E: P-Smad3 expression after 24-h treatment with SB-431542 and 5.0 ng/ml TGF-β1 for 60 min. α-Tubulin was used as loading control. For all Western blots, 30 μg of total protein was used. Blots are representative of 3 experiments.

Cell motility and proliferation in A549 cells are regulated by TRIP-1 expression.

We investigated the biological function of TRIP-1 knockdown by wound closure and cell proliferation assay. To determine their proliferation rates, two forms of quantification were used: cell number assay and cell proliferation reagent WST-1. For the cell number experiment, 100,000 cells were plated onto a 60-mm dish and allowed to grow for up to 3 days. After 3 days in culture, A549-shTRIP1 clones had a reduction in cell number of 52.3% (Fig. 5A). This result was validated by incubation with the cell proliferation reagent WST-1, which measures the cleavage of the tetrazolium salt WST-1 into formazan at the mitochondrial membrane. According to this assay, A549-shTRIP1 clones had a metabolic rate reduction of 52.8% compared with that of A549-shLacZ (Fig. 5B). As shown in Fig. 5C and quantified in Fig. 5D, A549-shTRIP-1 cells recovered 84% wounded area compared with 52% in A549-shLacZ after 24 h in 1% FBS-DMEM. Wound closure was accelerated in 1% FBS-DMEM plus 5 ng/ml TGF-β1, up to 94% in A549-shTRIP-1 and 83% in A549-shLacZ. Cells with decreased expression of TRIP-1 exhibited an increase in cell motility compared with their control.

Fig. 5.

Effect on cellular proliferation and wound closure by TRIP-1 in lung epithelial cells treated with or without TGF-β1. Cellular proliferation measured by cell number assay (A) and the metabolic assay WST-1 (B) showed cell proliferation rates to be lowered in cells with reduced levels of TRIP-1 compared with control. Cell number assay was performed in triplicate, while WST-1 assay was performed with an n = 6. Error bars for cell number assay are too small to be visible. A450 − A690, absorbance at wavelength of 450 nm with background subtraction at 690 nm. C: representative example of wound healing assay at time 0, 24 h in 1% FBS-DMEM, and 24 h in 1% FBS-DMEM + 5 ng/ml TGF-β1 in A549-shLacZ or A549-shTRIP-1. Magnification ×40. Experiments were performed with 6 wells per cell type, and condition wells were individually analyzed. D: summary of wound assay results. Values are % of area of wound healed; bars show SD. *,***P < 0.01.

DISCUSSION

TRIP-1 is a WD-40-containing protein identified as a phosphorylation target of the TGF-β type II receptor kinase (3) and a functional component of initiator of elongation factor 3 multiprotein complex (1). We have also reported that TRIP-1 is a negative regulator of fibroblast collagen contraction. In the present work, we used the well-characterized A549 model of TGF-β1 induction of EMT to investigate the effect of TRIP-1 expression on EMT. EMT is a biological process by which epithelial cells undergo phenotypic conversion to a mesenchymal phenotype. It is employed in normal tissue development (9, 21, 29) and in pathological processes such as wound repair by fibrosis as well as by cancer cells to acquire invasive behavior (6, 12, 15, 25, 32, 33). Here we report for the first time that downregulation of TRIP-1 expression in A549 cells results in a loss of expression of epithelial markers along with a gain in expression of mesenchymal markers. Moreover, the cells show a change to spindled morphology and increased migrative ability as assessed by wound healing assay. Loss of TRIP-1 resulted in increased responsiveness to EMT induction by TGF-β1. Taken together, the results point to regulation of endogenous TRIP-1 protein expression as a potential strategy to target EMT, and related invasive behavior, in cancer cells.

Expression of the adhesion protein E-cadherin, which associates in complex with β-catenin to form the adherens junction of cell-cell attachment, is considered crucial to maintenance of the epithelial phenotype. Loss of E-cadherin disrupts the adherens junction, allowing individual cell motility rather than movement en masse, as is typical of epithelial cells, and is a hallmark feature of cells that have undergone EMT (25, 33). We found that A549 cells with downregulated TRIP-1 expression when cultured in serum starvation conditions (0.1% FBS) express less E-cadherin protein compared with control A549 cells. Treatment with minute concentrations of TGF-β1 down to the order of 0.001 ng/ml was sufficient to result in a dramatic and near complete loss of E-cadherin protein expression in these cells with marked transition to a spindled mesenchymal morphology. Indeed, the increased sensitivity to TGF-β1 of these cells is such that culture in medium containing 10% FBS results in complete loss of E-cadherin. The cells do not survive if cultured in the absence of serum.

Aside from loss of E-cadherin, we also detected a significant loss in occludin expression in the TRIP-1 knockdown cells at baseline and upon treatment with TGF-β1. Like E-cadherin, occludin is an epithelial marker, and their loss along with a concurrent gain in several mesenchymal markers as we detected signify EMT.

Mesenchymal cells express N-cadherin and vimentin, which are classified as mesenchymal markers and are used to assess transition of epithelial cells to the mesenchymal phenotype. We detected that basal levels of these markers are present in A549 cells, consistent with their nature, since A549 is an adenocarcinoma cell line derived from human lung alveolar type II cells. However, it is used extensively in studies as a model to study functions of normal type II cells because of many features shared in common (5). We found that N-cadherin and vimentin expression were increased in the TRIP-1 knockdown cells concomitant with the reduction in E-cadherin expression upon treatment with TGF-β1. Gain in N-cadherin expression, like loss of E-cadherin expression, was responsive to TGF-β1 treatment in a concentration- and time-dependent fashion but required relatively higher concentrations of TGF-β1 treatment.

TGF-β1 is a multieffector cytokine with diverse functions that include regulation of cell growth, extracellular matrix remodeling, and fibrosis (16). Moreover, it is linked to EMT of lung epithelial cells contributing to fibrotic repair and epithelial cancer acquisition of invasive behavior and to transdifferentiation of fibroblasts into the myofibroblast phenotype that has enhanced ECM synthetic ability as observed in idiopathic pulmonary fibrosis (6, 25, 33). TGF-β also has critical roles in lung development, as both excessive and reduced TGF-β1 activity have been shown to impair mouse lung development or result in fibrosis. Although signals occur through multiple pathways (4, 17), an important mechanism by which TGF-β1 signals EMT in A549 cells is through binding to its type II cell surface receptors to cause phosphorylation of the Smad group of intracellular signaling proteins (13, 34, 36, 38, 39). Choy and Derynck (3) have reported modulation of Smad pathway by TRIP-1, and in their study of osteoblasts Sheu et al. (28) observed the same in addition to a cell differentiation effect of TRIP-1. Binding of TGF-β1 ligand to the type II receptor leads to recruitment, phosphorylation, and consequent activation of the type I receptor, which then directly phosphorylates Smad2 and Smad3. Phosphorylated Smad2/3 associates with Smad4 to translocate into the nucleus, where in interactions with coactivators or corepressors they activate or suppress target gene transcription (4, 17). We found an increase in the level of TGF-β type II receptor expression in our TRIP-1 knockdown A549 cells relative to control and no difference in the type I receptor levels. Also, these cells upon treatment with TGF-β1 exhibit a more robust activation of the Smad pathway as reflected in phospho-Smad3 level compared with control. It has been reported that TGF-β type II receptor through nontranscriptional mechanisms can mediate dissolution of epithelial tight cell junctions (22), a crucial and early event in the EMT process. However, repression of E-cadherin expression via the transcription factor SLUG also results in loss of tight junctions. Interestingly, we observed under basal culture conditions (i.e., without TGF-β treatment) that TRIP-1 knockdown cells express significantly higher levels of not only the TGF-β type II receptor but also the transcription factor SLUG. SLUG has been implicated in TGF-β1/Smad3 pathway-driven EMT. Blocking the Smad pathway with a chemical inhibitor targeting TGF-β receptor I kinase, which is the upstream activator of Smad3, resulted in some rescue of E-cadherin protein expression in the TRIP-1 knockdown cells. This suggests, at the very least, the existence of a pathway of TRIP-1 regulation of EMT induction via modulation of TGF-β type II receptor level and enhancement of Smad activation/transduction in these cells.

Although TGF-β type II receptor protein level was increased in our TRIP-1 knockdown cells, we did not detect a change in the mRNA level, suggesting that nontranscriptional mechanisms are involved in this effect of loss of TRIP-1. Indeed, aside from its being an associative partner of TGF-β receptor II kinase, TRIP-1 was also reported to be a component of the human eIF3 complex. eIF3 is involved in protein synthesis, making it likely that the effect seen on TGF-β type II receptor protein abundance from loss of TRIP-1 is due to a change in the type II receptor protein translation. The levels of TGF-β pathway components are also controlled via ubiquitin-mediated proteosomal degradation. If loss of TRIP-1 does disrupt degradation of the TGF-β type II receptor protein, it could explain the altered level observed.

An important characteristic of cells that have undergone EMT is acquisition of a more migratory phenotype. We found that our A549 cells lacking TRIP-1 have decreased proliferation but repair an epithelial monolayer wound much faster than controls. Thus loss of TRIP-1, aside from promoting a change in phenotypic markers and morphology, confers a more migratory phenotype to A549 cells, all of which are consistent with characteristics of cells that have undergone EMT.

In summary, our results indicate that loss of TRIP-1 in A549 cells not only has a pro-EMT effect but dramatically increases the ability of the cells to undergo EMT in response to TGF-β1 treatment. The findings are based on assessment of changes in phenotypic markers, morphology, and migrative ability. Mechanistically, a pathway involving increased TGF-β type II receptor level, enhanced Smad3 phosphorylation, and the transcription factor SLUG is implicated. Taken together, the results point to regulation of endogenous TRIP-1 protein expression as a potential strategy to target EMT, and related invasive behavior, in cancer cells. Further studies are warranted to ascertain whether the present observations with TRIP-1 extend to other epithelial cell types.

GRANTS

This work was supported by a Children's Mercy Hospital Physician Scientist Award to I. I. Ekekezie.

DISCLOSURES

No conflicts of interest, financial or otherwise are declared by the author(s).

ACKNOWLEDGMENTS

The authors thank Christine Concepción for her secretarial assistance in preparation of this manuscript.

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View Abstract