Thalidomide Inhibits TGF-β1-induced Epithelial to Mesenchymal Transition in Alveolar Epithelial Cells via Smad-Dependent and Smad-Independent Signaling Pathways
Scientific REPORTS |
Thalidomide Inhibits TGF-?1-
OPEN Recent evidence indicates that the epithelial to mesenchymal transition (EMT) in primary alveolar cells (AECs) plays an important role in idiopathic pulmonary fibrosis (IPF). In vivo models have suggested that thalidomide (THL) has anti-fibrotic effects against pulmonary fibrosis, but the underlying mechanism of this effect is not clear. This study investigated whether THL regulates alveolar EMT and the possible mechanisms underlying this process. CCL-149 cells were treated with TGF-?1 in the presence of THL at the indicated concentrations. EMT was assessed by changes in cell morphology and in phenotypic markers. Signaling pathways involved in EMT were characterized by western blot analysis. THL inhibited the TGF-?1 induction of ?-SMA, vimentin, MMP-2/-9 and collagen type IV expression and restored the morphological changes in primary alveolar epithelial cells caused by TGF?1. TGF-?1 induction of ?-SMA expression was partially dependent on the activation of p38, JNK, ERK, Akt, Smad 2 and Smad3. Moreover, THL inhibited TGF-?1-induced phosphorylation of p38, JNK, ERK, Akt, GSK3?, Smad 2 and Smad3 without altering the total expression levels of those proteins. These findings indicate that TGF-?1-induced EMT in alveolar epithelial cells is inhibited by THL via both Smaddependent and non-Smad-dependent signaling pathways and suggests therapeutic approaches for targeting this process in pulmonary fibrosis.
Recent evidence indicates that the epithelial to mesenchymal transition (EMT) plays an important role in the
development of pulmonary fibrosis1. Several mediators, including cytokines and growth factors, are thought to
induce EMT, and TGF-? has been shown to be the major inducer of EMT in carcinogenesis and fibrosis2. A
previous study reported that alveolar epithelial cells (AECs) undergo EMT when exposed to TGF-?1, indicating
that AECs may serve as a novel source of myofibroblasts in pulmonary fibrosis3. Following exposure to TGF-?1,
AECs undergo EMT as evidenced by decreased expression of epithelial markers (AQP5, ZO-1 and E-Cadherin),
increased expression of mesenchymal markers (?-SMA, vimentin and desmin) and a transition to a fibroblast-like
morphology4. In addition, fibrosis- or TGF-?1-induced EMT is mainly mediated by Smad-dependent (Smad2/3)
and Smad-independent pathways (MAPK, PI3 kinase, Ras and Wnt signaling pathways, etc.)5. Thus, researchers
are investigating the possibility that the regulation of Smad-independent and Smad-dependent pathways in AECs
could result in reduced fibrogenesis through the attenuation of TGF-?1-induced EMT6,7. Although the role of
EMT in organ fibrosis has recently been questioned by lineage-tracing studies8,9, experimental evidence suggests
an important role of EMT in the pathogenesis of pulmonary fibrosis10,11.
Thalidomide (THL) has been recognized as a therapeutic agent for multiple myeloma (MM). Recent
studies have shown that thalidomide has anti-fibrotic effects in animal models of bleomycin-induced12 and
paraquat-induced pulmonary fibrosis13. Although the anti-fibrotic effects of thalidomide in pulmonary fibrosis
have been demonstrated, the exact mechanism of this effect remains poorly understood. The results of previous
studies have suggested that the anti-fibrotic effects of thalidomide on pulmonary fibrosis might be related to
suppression of the ERK1/2 signaling pathway14. Liu et al. reported that thalidomide is able to inhibit JNK signaling
in an animal model of pulmonary fibrosis15. In addition, a study by Liang et al. suggested that thalidomide has an
anti-fibrotic effect that is caused by suppression of TGF-?1-induced p38 and Smad3 signaling16. Considering the
anti-fibrotic effect of thalidomide in pulmonary fibrosis, the possible mechanisms of that effect and the
characteristics of EMT in pulmonary fibrosis, we hypothesized that thalidomide inhibits TGF-?1-induced EMT in AECs.
In the present study, we induced EMT in vitro to investigate the potential effects of thalidomide on alveolar EMT
and the possible mechanisms of this effect.
Thalidomide reversed TGF-?1-induced EMT in AECs. Stimulation with 5 ng/mL TGF-?1 for 48
h resulted in apparent changes in cellular morphology and changes in the expression of cellular markers. As
seen in Fig.?1A, treatment with thalidomide restored the morphological changes observed in cells after
TGF?1 stimulation. The majority of TGF-?1-treated cells in the thalidomide-treated groups showed an epithelial
morphology instead of a fibroblast-like morphology. Western-blot assays showed that thalidomide significantly
inhibited the TGF-?1-induced increases in ?-SMA and vimentin in a dose-dependent manner (p < 0.05 for both
?-SMA and vimentin). Treatment with thalidomide significantly prevented the TGF-?1-induced decrease in
the epithelial markers ZO-1 and E-Cadherin (p < 0.05 for both ZO-1 and E-Cadherin) (Fig.?1B). In addition,
ELISAs showed that pretreatment with TGF-?1 significantly increased the secretion of MMP-2, MMP-9 and
collagen type IV in CCL-149 cells. However, thalidomide abrogated these changes in a dose-dependent manner
(p < 0.05) (Fig.?2). Moreover, immunofluorescence microscopy demonstrated localized expression of the
epithelial marker E-Cadherin and the mesenchymal marker ?-SMA in CCL-149 cells (Fig.?3). Cells in the control group
expressed significant amounts of E-Cadherin and low levels of ?-SMA on their cell membranes. TGF-?1
markedly reduced the expression of E-Cadherin and increased the expression of ?-SMA. However, cells concurrently
treated with TGF-?1 and thalidomide retained high levels of expression of the epithelial marker E-Cadherin
without a significant increase in the expression of the mesenchymal marker ?-SMA.
Thalidomide inhibited TGF-?1-induced phosphorylation of p38, JNK and ERK. TGF-?1-induced
EMT is associated with the activation of numerous signaling pathways. It is well known that the MAPKs,
including p38, JNK, and ERK, can be activated by TGF-?1 in pulmonary epithelial cells. In this study, we first confirmed
the role of p38, JNK, and ERK in TGF-?1-induced EMT. When cells were treated with inhibitors specific for these
proteins, including SB203580 (p38 inhibitor), PD098059 (JNK inhibitor) and SP600125 (ERK inhibitor), the
activation of p38, JNK and ERK by TGF-?1 and the expression of the mesenchymal marker ?-SMA was
significantly inhibited (p < 0.05) (Fig.?4A). Importantly, we observed that thalidomide inhibited the activation of these
MAPKs after TGF-?1 treatment in a dose-dependent manner (p < 0.05) without altering the total expression
levels of those proteins (Fig.?4B). These results indicate that thalidomide might inhibit TGF-?1-induced EMT in
pulmonary epithelial cells through the inhibition of MAPK signaling pathways.
Thalidomide inhibited TGF-?1-induced phosphorylation of Akt and GSK-3?. A previous study
confirmed that TGF-?1 induces the phosphorylation of Akt and its downstream protein GSK-3? in primary
AECs17. In this study, we also found that TGF-?1 induced phosphorylation of Akt in CCL-149 cells in vitro. In
addition, treatment with the PI3-K/Akt specific inhibitor LY294002 significantly inhibited the TGF-?1-induced
phosphorylation of Akt and the subsequent induction of ?-SMA expression (Fig.?5A). Furthermore, TGF-?1
treatment also induced the phosphorylation of GSK-3? in AECs. These results suggest that the Akt/GSK-3?
pathway is involved in TGF-?1-induced EMT in CCL-149 cells. However, treatment with thalidomide inhibited
TGF-?1-induced Akt and GSK-3? phosphorylation (Fig.?5B and C) in a dose-dependent manner (p < 0.05).
In addition, as shown above, thalidomide also inhibited TGF-?1-induced production of ?-SMA and vimentin
and restored the morphological changes caused by TGF-?1. Thus, we conclude that the Akt/GSK-3? pathway is
involved in thalidomide-mediated anti-EMT effects.
Thalidomide inhibited TGF-?1-induced phosphorylation of Smad 2/3. Smad transcription factors
are well-known mediators of TGF-?1 signaling. A previous study showed that Smads are activated by TGF-?1 and
regulate EMT-related changes in pulmonary epithelial cells18. To confirm the role of Smad2/3 in TGF-?1-induced
EMT, Smad2 and Smad3 were knocked down using siRNAs. As seen in Fig.?6A, the knockdown of Smad2
and Smad3 proteins was comparable in cells with or without TGF-?1 treatment. We also investigated whether
co-treatment with Smad2 and Smad3 siRNAs enhanced the knockdown of target genes in this study. However,
no significant changes were observed in cells co-treated with Smad2/3 siRNAs compared to cells treated with
single siRNAs (data not shown). Moreover, we investigated the effects of the knockdown of Smad2 and Smad3
on TGF-?1-induced ?-SMA expression. The TGF-?1-induced increase in ?-SMA expression was significantly
attenuated by knockdown of both Smad2 and Smad3. Furthermore, the TGF-?1-induced increase in ?-SMA
expression was completely inhibited in cells transfected with both Smad2 and Smad3 siRNAs (Fig.?6B). These
results confirm that both Smad2 and Smad3 are involved in the TGF-?1-induced increase in ?-SMA
expression in CCL-149 cells. Importantly, cells concurrently treated with TGF-?1 and thalidomide showed significantly
lower levels of ?-SMA expression and decreased phosphorylation of Smad2/3 compared to cells treated with
TGF-?1 in the absence of thalidomide (Fig.?5C). These results indicate that the inhibitory effect of thalidomide on
TGF-?1-induced EMT is associated with the activation of both Smad2 and Smad3.
This study is the first to demonstrate that thalidomide inhibits TGF-?1-induced alveolar epithelial to
mesenchymal transition (EMT) in CCL-149 cells. Thalidomide significantly reduced the expression of mesenchymal
markers, increased the expression of epithelial markers after TGF-?1 stimulation, and reverted the TGF-?1-induced
morphological changes in alveolar epithelial cells. We also found that the inhibitory effects of thalidomide on
TGF-?1-induced EMT may be related to inhibition of the TGF-?1-mediated signaling pathways Smad2/3, Akt/
GSK-3? and MAPK.
Currently, increasing attention has been given to the process of EMT and its possible role in the
pathogenesis of pulmonary fibrosis. Numerous studies have reported EMT as a response to TGF-?1 treatment in vitro in
alveolar epithelial cells19,20. The mechanism of TGF-?1-induced EMT is complicated because TGF-?1 signals are
transmitted through multiple pathways, including the Smad, MAPK and PI3K pathways. The TGF-?1 signal itself
is predominantly transduced by the Smad proteins, including Smad2, Smad3 and Smad421. Binding of TGF-?1
to its receptors results in the phosphorylation of Smad proteins, causing translocation of the Smad complex into
the nucleus where it regulates the expression of target genes. Although both Smad2 and Smad3 are important
for the regulation of TGF-?1 signals, the mechanisms of action and the functions of these two Smads are
different. Experimental Smad3-deficient mouse models of EMT and fibrosis demonstrated that Smad3 signaling
is important in fibrosis22; however, the role of Smad2 in TGF-?1-induced EMT remains unclear. In the present
study, we observed that the TGF-?1-induced phosphorylation of Smad2 and Smad3 and the TGF-?1-induced
increase in ?-SMA production were partially dependent on Smad2 and Smad3 and were reversed by knockdown
of both Smad2 and Smad3. These results are consistent with those of previous reports23. Moreover, the results of
our study suggest that thalidomide inhibits TGF-?1-induced EMT in pulmonary epithelial cells by blocking the
phosphorylation of both Smad2 and Smad3. However, previous studies demonstrated that Smad3, but not Smad2,
is required for the fibrotic response to TGF-?124,25. In addition, a study of alterations in gene expression during
EMT in IHAEo? cells showed that only collagen I gene expression was Smad2/3 dependent; all other alterations
in gene expression during EMT appeared to be Smad independent18. The discrepancies between the findings of
these studies may be due to the cell types used. However, the Smad dependency of EMT changes in CCL-149 cells
(other than those studied here) requires further investigation.
In addition to the Smad pathways, TGF-? utilizes several non-Smad pathways, such as the MAP kinase
and phosphatidylinositol-3-kinase/AKT pathways, to regulate a wide array of cellular functions. To date, the
best-characterized non-Smad pathway utilized by TGF-? is the p38 MAPK and JNK signaling cascade. It is
well known that TGF-? can rapidly activate p38 MAPK and JNK through MKK3/626 and MKK427, respectively.
In addition, the p38 MAPK and JNK pathways play very important roles in TGF-?-induced EMT. Inhibiting
p38 or JNK activity using specific inhibitors or gene silencing can inhibit TGF-?-induced changes in cell
morphology and expression of phenotypic markers18,28. Similar to p38 and JNK, the ERK pathway is another
non-Smad signaling pathway necessary for TGF-?-induced EMT29. Liang et al. reported that thalidomide
treatment significantly inhibited TGF-?-induced p38 phosphorylation but did not have a significant effect on
ERK or JNK phosphorylation24. An experimental in vivo pulmonary fibrosis rat model demonstrated that
thalidomide reduced the degree of bleomycin-induced pulmonary fibrosis by down-regulating the expression of
p-JNK and ?-SMA15. Moreover, Choe et al. reported that the anti-fibrotic effect of thalidomide on pulmonary
fibrosis might be related to its inhibition of TGF-?1-induced activation of the ERK1/2 signaling pathway14.
In the present study, we found that TGF-?1 activated these MAPK cascades and that the TGF-?1-induced
increase in the expression of mesenchymal markers, including ?-SMA and vimentin, was inhibited by p38-,
JNK- and ERK-specific inhibitors. This suggests that the TGF-?1-induced increase in the expression of ?-SMA
and vimentin is mediated by the activation of these MAPKs. We also observed that thalidomide treatment
resulted in a significant reduction in the levels of phosphorylated p38, JNK and ERK in CCL-149 cells,
indicating that the inhibitory effect of thalidomide on TGF-?1-induced EMT is associated with the suppression of
MAPK signaling pathways.
Furthermore, several studies have suggested that the PI3K/Akt pathway is another non-Smad pathway that
contributes to TGF-?1-induced EMT. Inhibition of Akt activity attenuates TGF-?1-induced EMT in kidney
epithelial cells30. In this study, we observed that thalidomide inhibited TGF-?1-induced phosphorylation of
Akt and that a specific PI3K inhibitor inhibited Akt activation and ?-SMA production after TGF-?1
stimulation. This suggests that the attenuation of TGF-?1-induced EMT by thalidomide is associated with the
PI3K/Akt pathway. In addition, the activation of GSK-3?, a downstream effector of Akt, was suppressed by
thalidomide treatment. Previous studies have shown that inactivation of GSK-3? could lead to the stabilization
of key mediators of EMT, including SNAI1 and ?-catenin31,32. These findings lead us to hypothesize that the
inhibitory effect of thalidomide on TGF-?1-induced EMT may be partly mediated by inhibition of the PI3-K/
In a bleomycin model of pulmonary fibrosis, approximately one third of fibroblasts are of epithelial origin33.
EMT may also contribute to fibroblastic foci and fibrotic development in idiopathic pulmonary fibrosis (IPF)34,35.
However, Rock et al. suggested that epithelial cells do not generate myofibroblasts in the context of pulmonary
fibrosis9. These contradictory results may be due to differences in the study methods, the pulmonary fibrosis
model, or the cell lines. Collectively, the role of EMT in the development of pulmonary fibrosis still needs to
be confirmed in future studies. Our study provides some insight into the mechanism by which thalidomide is
able to prevent EMT in a pulmonary epithelial cell line. Finally, there were three main limitations in this study.
First, although we identified that both Smad-dependent and non-Smad-dependent pathways were involved in
TGF-?1-induced EMT in pulmonary epithelial cell lines, we failed to determine which pathway plays the main
role in this pathological process. Second, further studies are required to investigate the relationship between the
Smad-dependent and non-Smad-dependent pathways during TGF-?1-induced EMT in pulmonary epithelial
cells. Third, we used a fetal rat pulmonary epithelial cell line (CCL-149) instead of a human pulmonary epithelial
cell line (such as A549) in this study. Thus, the different characteristics between species should be considered.
The results of this study demonstrate that treatment with thalidomide inhibits TGF-?1-induced ?-SMA and
vimentin production and TGF-?1-induced cell morphological changes in pulmonary epithelial cells by
suppressing both Smad-dependent and non-Smad-dependent pathways. Thus, thalidomide may be valuable as a
therapeutic agent for the inhibition of lung fibrosis.
Materials and Methods
Antibodies and Reagents. TGF-?1 was purchased from R&D Systems (Minneapolis, MN, USA).
TGF?1 receptor kinase inhibitor (SB431542) was purchased from Selleck (Houston, TX, USA). PI3K/Akt inhibitor
(LY294002) was purchased from Cell Signaling Technology (Beverly, MA, USA). JNK inhibitor (SP600125) and
ERK inhibitor (PD098059) were purchased from Millipore Co. (Billerica, MA, USA). Thalidomide was purchased
from Sigma-Aldrich (St. Louis, MO, USA). Anti-rat p38 (ab7952), p-p38 (ab4822), Smad2/3 (ab63672) and
p-Smad2/3 (ab63399) were purchased from Abcam (Shanghai, China). Anti-rat ERK (BS1121) and E-Cadherin
(BS1097) were purchased from Bioworld (Minneapolis, USA). Anti-rat p-ERK (#4370), AKT (#9272), p-AKT
(#2965s), GSK3? (#9315) and p-GSK3? (#9336) were purchased from Cell Signaling (Beverly, MA, USA).
Antirat JNK (sc-571), p-JNK (sc-81502) and ?-SMA (sc-71626) were purchased from Santa Cruz (CA, USA). Anti-rat
vimentin (3634?100) was purchased from Biovision (Mountain View, CA, USA). Anti-rat ZO-1 was purchased
from Invitrogen (Carlsbad, CA, USA). Anti-?-actin and HRP-labeled secondary antibodies were purchased from
Boster (Wuhan, Hubei, China). Commercial ELISA kits for matrix metalloproteinase (MMP)-2 and MMP-9 were
purchased from R&D Systems (Shanghai, China). ELISA kit for collagen type IV was purchased from BlueGene
Biotech (Shanghai, China).
Cell culture and treatments. Rat lung epithelial cells (CCL-149) were purchased from Wuhan Biofavor
Biotech Service CO., LTD (Wuhan, Hubei, China). Cells were cultured in DMEM/F12 supplemented with 10%
fetal bovine serum (Invitrogen, San Diego, CA) and 1% streptomycin-penicillin solution. Cultures were
maintained in a humidified 5% CO2 incubator at 37?C. After 24 hours of serum starvation, cells were maintained
in growth media supplemented with 5 ng/mL TGF-?1 (dissolved in 10 mM citric acid, pH = 3.0) for 48 h to
induce EMT36. In the majority of experiments, cells were maintained in media supplemented with TGF-?1 with
or without thalidomide (1, 10, or 100 ?g/mL) for 48 h to investigate the effects of thalidomide on EMT in
CCL149 epithelial cells. To investigate pathways possibly involved in the effect of thalidomide on EMT, the cells
were preincubated for 1 h with SB431542 (5 ?M), LY294002 (3 ?M), SB203580 (20 ?M), SP600125 (10 ?M) and
PD098059 (40 ?M) before treatment with exogenous TGF-?1 with or without thalidomide. The concentrations
of thalidomide and inhibitors were determined according to the results of our pilot study. Cells were harvested
at the indicated time points for protein extraction and western blotting and the cell culture media was collected
for ELISA analysis.
Smad2 and Smad3 siRNA transfection. Pre-designed Smad2 (Ambion, Austin, TX) and Smad3
(Thermo, PA, USA) siRNAs were transfected into cells using siPORT? NeoFX? Transfection Agent (Ambion) at
a concentration of 20 nM. The siRNA-transfection reagent complexes were prepared as described by the
manufacturer. Cells were treated with or without 5 ng/mL TGF-?1 in the presence or absence of different concentrations
of thalidomide for 48 h. The efficiency of siRNA gene silencing was evaluated with western blots for Smad2 and
Smad3. The effect of siRNA transfection on TGF-?1-induced ?-SMA production was analyzed by western
blotting as described below.
Western blot assay. Cells were lysed in RIPA (P0013B, Beyotime Biotechnology, Shanghai, China) and
protein concentrations were determined using the BCA protein assay kit (P0010, Beyotime Biotechnology, Shanghai,
China). Equal amounts of proteins (50 ?g) were resolved by 10% SDS-PAGE and the proteins were transferred
to Hybond ECL membranes (Amersham, Buckinghamshire, UK). The membranes were incubated with
primary antibodies at 4?C overnight. The primary antibodies used included anti-rat p38 (1:300), phosphorylated
p38 (1:1000), vimentin (1:300), ERK (1:600), phosphorylated ERK (1:1200), Smad2/3 (1:300), phosphorylated
Smad2/3 (1:600), E-Cadherin (1:600), ?-SMA (1:300), JNK (1:300), phosphorylated JNK (1:300), AKT (1:800),
phosphorylated AKT (1:800), GSK3? (1:1000), phosphorylated GSK3? (1:1000) and ZO-1 (1:250). After washing
with TBST, the membranes were probed with HRP-labeled secondary antibodies. The membranes were visualized
using an enhanced chemiluminescence system (Kodak, Rochester, NY, USA). ?-Actin was used as a loading
control. To normalize the intensities of different membranes, standard samples were performed on every blot. Then,
the densitometric data of Western blots of the three independent experiments were combined.
ELISA. As described above, the cell culture media was collected for the detection of MMP-2, MMP-9 and
collagen type IV secreted from CCL-149 cells in response to pretreatment with TGF-?1 and thalidomide. Culture
media was centrifuged at 3000 rpm for 20 min. Quantification of MMP-2, MMP-9 and collagen type IV was
performed using commercial ELISA kits according to the manufacturer?s instructions.
Immunofluorescence staining. Immunofluorescence staining was performed as previously described37.
Briefly, cells cultured on coverslips were washed thrice with PBS and fixed with paraformaldehyde (4%) for 15
min. Following three washes with PBS, the cells were blocked with 0.1% Triton X-100 and 2% normal serum for
30 min at room temperature (RT) and then incubated with specific primary antibodies, including those against
?-SMA (BM0002, Boster, Wuhan, China) and E-Cadherin (sc-7870, Santa Cruz, CA, USA), at 4?C overnight.
The cells were then stained with the appropriate secondary antibodies (Boster, Wuhan, China). The cells were
also stained with 4,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei and were washed with PBST four
times for 5 min each time. The slides were viewed with an epi-fluorescence microscope (Nikon Eclipse 80i, Nikon,
Tokyo, Japan) equipped with a digital camera (DS-Ri1, Nikon, Tokyo, Japan).
Statistical analysis. Data are expressed as the means ? SD. Data were analyzed using the statistical analysis
software SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). Comparisons of several means were performed using
one-way and repeated measure two-way analysis of variance followed by the Tukey-Kramer test to identify
significant differences between groups. All P values were two-tailed, and a P value of less than 0.05 was considered
This study was financially supported by the Hubei Province Health and Family Planning Research Project
(WJ2015mb100) and the Emergency Diagnostic and Therapeutic Center of Central China (Wuhan, Hubei,
X.L.Z., H.H.C. and Y.Z. designed this study. X.L.Z., P.X., C.J., S.J. and J.S. performed cell culture, western blotting
and immunofluorescence staining. B.X. and L.L. performed siRNA transfections. X.L.Z., P.X. and H.H.C.
collected data and performed statistical analyses. X.L.Z. and H.H.C. wrote the manuscript. All authors reviewed
Competing Interests: The authors declare that they have no competing interests.
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