Articles in PresS. Am J Physiol Lung Cell Mol Physiol (April 17, 2015). doi:10.1152/ajplung.00396.2014 MesoMT is PI3K and NFκB dependent 1
Mesomesenchymal Transition of Pleural Mesothelial Cells is PI3K and NFκB-dependent
2
Shuzi Owens1, Ann Jeffers1, Jake Boren2, Yoshikazu Tsukasaki2, Kathleen Koenig1, Mitsuo
3
Ikebe2, Steven Idell1, Torry A. Tucker1.
4 5
1
Texas Lung Injury Institute, 2Department of Cellular and Molecular Biology. The University of
Texas Health Science Center at Tyler, Tyler Texas
6 7
Please address correspondence to:
8
Torry A. Tucker, Ph.D.
9
Assistant Professor of Cellular and Molecular Biology
10
The Texas Lung Injury Institute
11
The University of Texas Health Science Center at Tyler
12
11937 US HWY 271
13
Biomedical Research Building, Lab C-5
14
Tyler, TX 75708
15
Tele: 903-877-7010, FAX: 903-877-7316
16
Email: [email protected]
17 18 19 20
Keywords: pleural mesothelial cells, thrombin, plasmin, TGF-β, PI3K, NFκB, mesomesenchymal transition, mesothelial cell plasticity
21
Support: NIH HL115466 and Texas Lung Injury Institute
22
1
Copyright © 2015 by the American Physiological Society.
MesoMT is PI3K and NFκB dependent 23
ABSTRACT
24
Pleural organization follows acute injury and is characterized by pleural fibrosis, which may
25
involve the visceral and parietal pleural surfaces. This process affects patients with complicated
26
parapneumonic pleural effusions, empyema and other pleural diseases prone to pleural fibrosis
27
and loculation.
28
mesenchymal transition (MesoMT), by which PMCs acquire a profibrotic phenotype
29
characterized by cellular enlargement and elongation, increased expression of α-smooth muscle
30
actin (α-SMA), and matrix proteins including collagen (Col)-1. While MesoMT contributes to
31
pleural fibrosis and lung restriction in mice with carbon black/bleomycin-induced pleural injury
32
and procoagulants and fibrinolytic proteases strongly induce MesoMT in vitro, the mechanism
33
by which this transition occurs remains unclear. We found that thrombin and plasmin potently
34
induce MesoMT in vitro as does TGF-β. Further, these mediators of MesoMT activate PI3K/Akt
35
and NFκB signaling pathways. Inhibition of PI3K/Akt signaling prevented TGF-β, thrombin-
36
and plasmin-mediated induction of the MesoMT phenotype exhibited by primary human (H)
37
PMCs. Similar effects were demonstrated through blockade of the NFκB signaling cascade
38
using two distinctly different NFκB inhibitors, SN50 and Bay-11 7085. Conversely, expression
39
of constitutively active Akt induced MT in HPMCs while the process was blocked by PX866 and
40
AKT8. Further, thrombin mediated MesoMT is dependent on PAR-1 expression, which is linked
41
to PI3K/Akt signaling downstream. These are the first studies to demonstrate that PI3K/Akt
42
and/or NFκB signaling is critical for induction of MesoMT.
Pleural mesothelial cells (PMCs) undergo a process called mesothelial
43 44 45 46 2
MesoMT is PI3K and NFκB dependent 47 48
INTRODUCTION
49
Pleural organization is characterized by the formation of a pleural rind with the accumulation
50
of fibrotic tissue within the visceral and parietal pleura. The process generally occurs in
51
association with organization of exudative pleural effusions that are likewise prone to loculation
52
(5). When severe, pleural fibrosis (PF) can cause restrictive lung disease and fibrothorax that
53
occur with respiratory impairment and morbidity. Along these lines, PF can result from
54
pneumonia with complicated parapneumonic pleural effusions, empyema, trauma, malignant
55
pleural effusions that loculate, asbestos-related pleural injury and occur in some patients that
56
have undergone coronary artery bypass graft surgery, (12, 21). PF also contributes to increased
57
mortality of patients with co-morbidities including cardiac disease (4, 7). Fibrosing pleuritis is
58
characterized by infiltration of the pleural space by proliferating, alpha smooth muscle actin (α-
59
SMA) expressing myofibroblasts.
60
extracellular matrix proteins such as collagen and fibronectin. In a recent publication (19), we
61
reported that pleural fibrosis induced by carbon black and bleomycin (CBB) in mice is
62
characterized by the presence of a visceral pleural rind composed of thickened mesothelium and
63
submesothelial tissue. This thickened rind contributes to reduced lung volume, decreased lung
64
compliance and lung restriction that closely approximates fibrosing pleural injury and
65
fibrothorax in humans (19).
These myofibroblasts promote the accumulation of
66
Our work and that of others has shown that pleural mesothelial cells (PMCs) contribute to the
67
progression of PF by undergoing a process called mesothelial (Meso) mesenchymal transition
68
(MT) (1, 2, 13, 14, 19), through which PMCs assume characteristics of myofibroblasts.
69
MesoMT is characterized by mesenchymal changes in cell morphology. Further, increased
3
MesoMT is PI3K and NFκB dependent 70
expression of α-SMA and collagen 1 (Col-1) are hallmarks of myofibroblasts (1, 2, 14, 19).
71
Both α-SMA and Col-1 were upregulated in mesothelial cells undergoing MesoMT in vivo in a
72
CBB-mediated model of pleural injury in which thrombin and plasmin were upregulated within
73
the injured pleural compartment (19). Further, these same factors promote MT in human (H)
74
PMCs. Although transforming growth factor β-1 (TGF-β) has been shown to potently promote
75
MT in diverse cell types including PMCs (11, 14, 17, 19, 22, 23), the mechanism by which
76
thrombin and plasmin contribute to this process in PMCs has not, to our knowledge, been
77
previously investigated.
78
Recently, we reported that expression of tissue factor pathway inhibitor is PI3K/Akt and
79
NFκB dependent and that these signaling pathways are linked in HPMCs (19). We therefore
80
inferred that PI3K/Akt/NFκB signaling could contribute to the progression of MesoMT, a
81
possibility further supported by prior reports linking these pathways to mesenchymal transition
82
in alternative cell types (10, 15, 18). In this study we show for the first time both PI3K/Akt and
83
NFκB signaling are activated by thrombin and plasmin in HPMCs undergoing MesoMT.
84
Further, blockade of either of these pathways effectively blocks the induction of MT in HPMCs.
85
METHODS
86
Primary Pleural Mesothelial Cell Isolation and Culture:
87
Permission to collect and use HPMCs was granted through an exempt protocol approved by the
88
Institutional Human Subjects Review Board of the University of Texas Health Science Center at
89
Tyler. HPMCs were cultured in a humidified incubator at 37°C in 5% CO2/95% air. These cells
90
were isolated from pleural fluids collected from patients with congestive heart failure or post
91
coronary bypass pleural effusions as previously described (6). HPMCs were maintained in LHC-
92
8 culture media (Life Technologies, Grand Island NY) containing 5% fetal bovine serum (Life 4
MesoMT is PI3K and NFκB dependent 93
Technologies), 2% antibiotic-antimycotic (Life Technologies) and 1% L-glutamine (Life
94
Technologies) as previously described (20). The cells are passaged for up to five times before
95
discontinuing use.
96
Cell Treatments
97
Cells were treated with TGF-β (5 ng/ml, R&D, Minneapolis MN), plasmin (6 nM, Sekisui,
98
Lexington MA) and thrombin (7 nM, Enzyme Research Laboratories, South Bend IN) as
99
previously described (8, 19). Briefly, cells were serum starved for 16-24 h prior to treatment.
100
Serum starved cells were treated with TGF-β, plasmin and thrombin for 48 h. Cells were then
101
lysed, cleared and protein concentration determined (8, 19). Lysates were then probed for
102
changes in α-SMA (Cat. No. MAB1420; R&D), Col-1 (1310-08, Southern Biotech, Birmingham
103
AL), phosphorylated Akt (Cat. No. 4060, Cell Signaling, Danvers MA) and total Akt (Cat. No.
104
4685, Cell Signaling) and β-actin (1:12000, Cat. No. ab6276, Abcam, Cambridge MA).
105
Phosphorylated P65 analyses were performed in cells treated with plasmin and thrombin for 15
106
minutes and then lysed, resolved on SDS-PAGE and immunoblotted for phosphorylated P65
107
(Cat. No. 3033, Cell Signaling). All antibodies with the exception of β-actin were used at a
108
concentration of 1:1000.
109
Inhibitor studies
110
Cells were treated with varying doses of PI3K/Akt and NFκB inhibitors prior to treatment with
111
inducers of MesoMT. PI3K/Akt signaling was blocked with the PI3Kinase inhibitor, PX866 (1 -
112
0.05 μM, a kind gift from Scott Peterson, Oncothyreon) and the pan Akt inhibitor, Akt VIII
113
(AKT8, 2 – 0.25 μM, Calbiochem, Billerica MA) as previously described (8). NFκB signaling
114
was blocked using SN50 (20 – 1 μM, Calbiochem), as previously described (8), and Bay-11-
5
MesoMT is PI3K and NFκB dependent 115
7085 (10 μM, Calbiochem) according to manufacturer’s instructions. Briefly cells were serum-
116
starved in the presence of the inhibitor for 16-24 h. Cells were washed and then treated with
117
PBS, TGF-β, plasmin or thrombin in the presence of inhibitors and allowed to incubate for
118
another 48 h to assess their impact on the progression of MesoMT.
119
Adenoviral Transduction
120
HPMCs were adenovirally transduced as previously described (8). Briefly, cells were transduced
121
with β-galactosidase (LacZ) or myristoylated Akt (Myr-Akt), a constitutively active isoform of
122
Akt, at a concentration of 2 multiplicities of infection (MOI) for 24 h. Cells were then placed in
123
serum-free media for 48 h and then assayed for markers of MesoMT.
124
Immunofluorescence
125
Immunofluorescence analyses were performed as previously described (20). Serum starved
126
HPMCs were treated with PBS, TGF-β (5 ng/ml), plasmin (6 nM) or thrombin (7 nM). Cells
127
were fixed in ice cold methanol for 30 minutes. Cells were then washed and blocked with 2%
128
donkey serum in PBS. Alexa-488-conjugated α-SMA antibody (1 µg/ml, Cat. No IC1420G;
129
R&D) or phosphorylated p65 (1:100, Cell Signaling) and 4',6-diamidino-2-phenylindole (DAPI,
130
Life Technologies) for nuclear staining, in 2% donkey serum were next applied to cells o/n. Cells
131
were then washed, mounted onto slides and imaged as previously described (20).
132
PAR siRNA Transfection
133
HPMCs were transfected with PAR-1 siRNAs (200 nM) using Lipofectamine 3000 (Life
134
Technologies) as previously described (8). Briefly, HPMCs were transfected with PAR-1 siRNA
135
(5’ AGA UUA GUC UCC AUC AAU 3’, Eurofins, Huntsville AL) for 6 h at 37◦C in serum free
6
MesoMT is PI3K and NFκB dependent 136
media. Cells then recovered in LHC8 complete medium for 48 h. They were then serum starved
137
(16 h) and used in subsequent experiments.
138
Flow cytometry.
139
Flow cytometry analyses were performed as previously described (8, 20). Briefly cells were
140
fixed with 4% formalin for 30 minutes in ice. For PAR-1 analyses, after which serum starved
141
cells were incubated with an alexafluor 488-conjugated mouse anti-human PAR-1 antibody (sc-
142
13503 AF488, 10 µg/ml, Santa Cruz Biotechnology, Dallas TX). For calretinin analyses, cells
143
were first permeabilized with saponin and then incubated with a rabbit anti-human calretinin
144
antibody (C7479, 10 µg/ml, Sigma-Aldrich, St. Louis MO). Cells were then labeled with
145
dyLight 647-conjugated donkey anti-rabbit antibody and then analyzed on the Millipore Guava
146
easyCyte HT flow cytometer.
147
Statistics
148
All statistics were performed using the Student t-Test. A p-value of less than 0.05 was
149
considered significant.
150
RESULTS
151
TGF-β, plasmin and thrombin induce MesoMT.
152
Prior to use in experiments, HPMC cultures were assayed for expression of the mesothelial
153
cell marker calretinin (Figure 1A). FACs analyses confirmed that the cells were mesothelial
154
cells and that greater than 95% of the cells expressed the mesothelial cell marker calretinin. We
155
previously showed that TGF-β, thrombin and plasmin induce biomarkers of MT in M and
156
HPMCs and was associated with phenotypic changes indicative of MesoMT (19) and sought to
157
confirm the expression of these biomarkers for all subsequent signaling analyses. These findings 7
MesoMT is PI3K and NFκB dependent 158
were extended in HPMCs that were visually analyzed for changes in α-SMA expression and
159
localization (Figure 1B). Plasmin and thrombin treated HPMCs demonstrated increased α-SMA
160
expression and the presence of filamentous actin when compared to PBS-treated cells. TGF-β,
161
which is known to induce MesoMT (14, 19), was used a positive control. The conditioned media
162
(CM) for Col-1 expression and cell lysates were next subjected to SDS-PAGE followed by
163
Western blot analyses. Col-1 and α-SMA expression were increased by either plasmin or TGF-β
164
treatment (Figure 1C) while thrombin induced α-SMA. Further, thrombin and plasmin promoted
165
Akt phosphorylation (Figure 1C) and P65 phosphorylation in HPMCs (Figure 1D). Because our
166
HPMCs are partially activated, we sought to confirm our findings using unperturbed, primary
167
rabbit (R) PMCs. RPMCS were treated with PBS and TGF-β (Figure 1E) for 48h. TGF-β
168
treated cells demonstrated increased Col-1 and α-SMA while the tight junction marker, zona
169
occludins-1 (ZO-1), was decreased. These findings demonstrated that TGF-β, plasmin and
170
thrombin induced phenotypic and molecular changes that characterize MesoMT in the primary
171
cells used.
172
MesoMT of HPMCs is PI3K/Akt dependent.
173
As TGF-β, plasmin, and thrombin were all implicated as potent activators of the
174
PI3K/Akt signaling pathway (19), we next sought to determine the role of PI3K in MesoMT.
175
Because we previously demonstrated that the PI3K inhibitor, PX866 reduced thrombin-
176
mediated-Akt phosphorylation in HPMCs (8), we next assayed the effect of PX866 on thrombin-
177
mediated MesoMT. RNA was isolated from thrombin-treated cells (7 nM, 24 h) in the presence
178
of varying concentrations of PX866 (1.0 - 0.05 µM) and analyzed by qPCR. While thrombin
179
increased α-SMA mRNA expression by HPMCs, PI3K inhibition significantly blunted α-SMA
180
induction (p
Mesomesenchymal Transition of Pleural Mesothelial Cells is PI3K and NFκB-dependent
2
Shuzi Owens1, Ann Jeffers1, Jake Boren2, Yoshikazu Tsukasaki2, Kathleen Koenig1, Mitsuo
3
Ikebe2, Steven Idell1, Torry A. Tucker1.
4 5
1
Texas Lung Injury Institute, 2Department of Cellular and Molecular Biology. The University of
Texas Health Science Center at Tyler, Tyler Texas
6 7
Please address correspondence to:
8
Torry A. Tucker, Ph.D.
9
Assistant Professor of Cellular and Molecular Biology
10
The Texas Lung Injury Institute
11
The University of Texas Health Science Center at Tyler
12
11937 US HWY 271
13
Biomedical Research Building, Lab C-5
14
Tyler, TX 75708
15
Tele: 903-877-7010, FAX: 903-877-7316
16
Email: [email protected]
17 18 19 20
Keywords: pleural mesothelial cells, thrombin, plasmin, TGF-β, PI3K, NFκB, mesomesenchymal transition, mesothelial cell plasticity
21
Support: NIH HL115466 and Texas Lung Injury Institute
22
1
Copyright © 2015 by the American Physiological Society.
MesoMT is PI3K and NFκB dependent 23
ABSTRACT
24
Pleural organization follows acute injury and is characterized by pleural fibrosis, which may
25
involve the visceral and parietal pleural surfaces. This process affects patients with complicated
26
parapneumonic pleural effusions, empyema and other pleural diseases prone to pleural fibrosis
27
and loculation.
28
mesenchymal transition (MesoMT), by which PMCs acquire a profibrotic phenotype
29
characterized by cellular enlargement and elongation, increased expression of α-smooth muscle
30
actin (α-SMA), and matrix proteins including collagen (Col)-1. While MesoMT contributes to
31
pleural fibrosis and lung restriction in mice with carbon black/bleomycin-induced pleural injury
32
and procoagulants and fibrinolytic proteases strongly induce MesoMT in vitro, the mechanism
33
by which this transition occurs remains unclear. We found that thrombin and plasmin potently
34
induce MesoMT in vitro as does TGF-β. Further, these mediators of MesoMT activate PI3K/Akt
35
and NFκB signaling pathways. Inhibition of PI3K/Akt signaling prevented TGF-β, thrombin-
36
and plasmin-mediated induction of the MesoMT phenotype exhibited by primary human (H)
37
PMCs. Similar effects were demonstrated through blockade of the NFκB signaling cascade
38
using two distinctly different NFκB inhibitors, SN50 and Bay-11 7085. Conversely, expression
39
of constitutively active Akt induced MT in HPMCs while the process was blocked by PX866 and
40
AKT8. Further, thrombin mediated MesoMT is dependent on PAR-1 expression, which is linked
41
to PI3K/Akt signaling downstream. These are the first studies to demonstrate that PI3K/Akt
42
and/or NFκB signaling is critical for induction of MesoMT.
Pleural mesothelial cells (PMCs) undergo a process called mesothelial
43 44 45 46 2
MesoMT is PI3K and NFκB dependent 47 48
INTRODUCTION
49
Pleural organization is characterized by the formation of a pleural rind with the accumulation
50
of fibrotic tissue within the visceral and parietal pleura. The process generally occurs in
51
association with organization of exudative pleural effusions that are likewise prone to loculation
52
(5). When severe, pleural fibrosis (PF) can cause restrictive lung disease and fibrothorax that
53
occur with respiratory impairment and morbidity. Along these lines, PF can result from
54
pneumonia with complicated parapneumonic pleural effusions, empyema, trauma, malignant
55
pleural effusions that loculate, asbestos-related pleural injury and occur in some patients that
56
have undergone coronary artery bypass graft surgery, (12, 21). PF also contributes to increased
57
mortality of patients with co-morbidities including cardiac disease (4, 7). Fibrosing pleuritis is
58
characterized by infiltration of the pleural space by proliferating, alpha smooth muscle actin (α-
59
SMA) expressing myofibroblasts.
60
extracellular matrix proteins such as collagen and fibronectin. In a recent publication (19), we
61
reported that pleural fibrosis induced by carbon black and bleomycin (CBB) in mice is
62
characterized by the presence of a visceral pleural rind composed of thickened mesothelium and
63
submesothelial tissue. This thickened rind contributes to reduced lung volume, decreased lung
64
compliance and lung restriction that closely approximates fibrosing pleural injury and
65
fibrothorax in humans (19).
These myofibroblasts promote the accumulation of
66
Our work and that of others has shown that pleural mesothelial cells (PMCs) contribute to the
67
progression of PF by undergoing a process called mesothelial (Meso) mesenchymal transition
68
(MT) (1, 2, 13, 14, 19), through which PMCs assume characteristics of myofibroblasts.
69
MesoMT is characterized by mesenchymal changes in cell morphology. Further, increased
3
MesoMT is PI3K and NFκB dependent 70
expression of α-SMA and collagen 1 (Col-1) are hallmarks of myofibroblasts (1, 2, 14, 19).
71
Both α-SMA and Col-1 were upregulated in mesothelial cells undergoing MesoMT in vivo in a
72
CBB-mediated model of pleural injury in which thrombin and plasmin were upregulated within
73
the injured pleural compartment (19). Further, these same factors promote MT in human (H)
74
PMCs. Although transforming growth factor β-1 (TGF-β) has been shown to potently promote
75
MT in diverse cell types including PMCs (11, 14, 17, 19, 22, 23), the mechanism by which
76
thrombin and plasmin contribute to this process in PMCs has not, to our knowledge, been
77
previously investigated.
78
Recently, we reported that expression of tissue factor pathway inhibitor is PI3K/Akt and
79
NFκB dependent and that these signaling pathways are linked in HPMCs (19). We therefore
80
inferred that PI3K/Akt/NFκB signaling could contribute to the progression of MesoMT, a
81
possibility further supported by prior reports linking these pathways to mesenchymal transition
82
in alternative cell types (10, 15, 18). In this study we show for the first time both PI3K/Akt and
83
NFκB signaling are activated by thrombin and plasmin in HPMCs undergoing MesoMT.
84
Further, blockade of either of these pathways effectively blocks the induction of MT in HPMCs.
85
METHODS
86
Primary Pleural Mesothelial Cell Isolation and Culture:
87
Permission to collect and use HPMCs was granted through an exempt protocol approved by the
88
Institutional Human Subjects Review Board of the University of Texas Health Science Center at
89
Tyler. HPMCs were cultured in a humidified incubator at 37°C in 5% CO2/95% air. These cells
90
were isolated from pleural fluids collected from patients with congestive heart failure or post
91
coronary bypass pleural effusions as previously described (6). HPMCs were maintained in LHC-
92
8 culture media (Life Technologies, Grand Island NY) containing 5% fetal bovine serum (Life 4
MesoMT is PI3K and NFκB dependent 93
Technologies), 2% antibiotic-antimycotic (Life Technologies) and 1% L-glutamine (Life
94
Technologies) as previously described (20). The cells are passaged for up to five times before
95
discontinuing use.
96
Cell Treatments
97
Cells were treated with TGF-β (5 ng/ml, R&D, Minneapolis MN), plasmin (6 nM, Sekisui,
98
Lexington MA) and thrombin (7 nM, Enzyme Research Laboratories, South Bend IN) as
99
previously described (8, 19). Briefly, cells were serum starved for 16-24 h prior to treatment.
100
Serum starved cells were treated with TGF-β, plasmin and thrombin for 48 h. Cells were then
101
lysed, cleared and protein concentration determined (8, 19). Lysates were then probed for
102
changes in α-SMA (Cat. No. MAB1420; R&D), Col-1 (1310-08, Southern Biotech, Birmingham
103
AL), phosphorylated Akt (Cat. No. 4060, Cell Signaling, Danvers MA) and total Akt (Cat. No.
104
4685, Cell Signaling) and β-actin (1:12000, Cat. No. ab6276, Abcam, Cambridge MA).
105
Phosphorylated P65 analyses were performed in cells treated with plasmin and thrombin for 15
106
minutes and then lysed, resolved on SDS-PAGE and immunoblotted for phosphorylated P65
107
(Cat. No. 3033, Cell Signaling). All antibodies with the exception of β-actin were used at a
108
concentration of 1:1000.
109
Inhibitor studies
110
Cells were treated with varying doses of PI3K/Akt and NFκB inhibitors prior to treatment with
111
inducers of MesoMT. PI3K/Akt signaling was blocked with the PI3Kinase inhibitor, PX866 (1 -
112
0.05 μM, a kind gift from Scott Peterson, Oncothyreon) and the pan Akt inhibitor, Akt VIII
113
(AKT8, 2 – 0.25 μM, Calbiochem, Billerica MA) as previously described (8). NFκB signaling
114
was blocked using SN50 (20 – 1 μM, Calbiochem), as previously described (8), and Bay-11-
5
MesoMT is PI3K and NFκB dependent 115
7085 (10 μM, Calbiochem) according to manufacturer’s instructions. Briefly cells were serum-
116
starved in the presence of the inhibitor for 16-24 h. Cells were washed and then treated with
117
PBS, TGF-β, plasmin or thrombin in the presence of inhibitors and allowed to incubate for
118
another 48 h to assess their impact on the progression of MesoMT.
119
Adenoviral Transduction
120
HPMCs were adenovirally transduced as previously described (8). Briefly, cells were transduced
121
with β-galactosidase (LacZ) or myristoylated Akt (Myr-Akt), a constitutively active isoform of
122
Akt, at a concentration of 2 multiplicities of infection (MOI) for 24 h. Cells were then placed in
123
serum-free media for 48 h and then assayed for markers of MesoMT.
124
Immunofluorescence
125
Immunofluorescence analyses were performed as previously described (20). Serum starved
126
HPMCs were treated with PBS, TGF-β (5 ng/ml), plasmin (6 nM) or thrombin (7 nM). Cells
127
were fixed in ice cold methanol for 30 minutes. Cells were then washed and blocked with 2%
128
donkey serum in PBS. Alexa-488-conjugated α-SMA antibody (1 µg/ml, Cat. No IC1420G;
129
R&D) or phosphorylated p65 (1:100, Cell Signaling) and 4',6-diamidino-2-phenylindole (DAPI,
130
Life Technologies) for nuclear staining, in 2% donkey serum were next applied to cells o/n. Cells
131
were then washed, mounted onto slides and imaged as previously described (20).
132
PAR siRNA Transfection
133
HPMCs were transfected with PAR-1 siRNAs (200 nM) using Lipofectamine 3000 (Life
134
Technologies) as previously described (8). Briefly, HPMCs were transfected with PAR-1 siRNA
135
(5’ AGA UUA GUC UCC AUC AAU 3’, Eurofins, Huntsville AL) for 6 h at 37◦C in serum free
6
MesoMT is PI3K and NFκB dependent 136
media. Cells then recovered in LHC8 complete medium for 48 h. They were then serum starved
137
(16 h) and used in subsequent experiments.
138
Flow cytometry.
139
Flow cytometry analyses were performed as previously described (8, 20). Briefly cells were
140
fixed with 4% formalin for 30 minutes in ice. For PAR-1 analyses, after which serum starved
141
cells were incubated with an alexafluor 488-conjugated mouse anti-human PAR-1 antibody (sc-
142
13503 AF488, 10 µg/ml, Santa Cruz Biotechnology, Dallas TX). For calretinin analyses, cells
143
were first permeabilized with saponin and then incubated with a rabbit anti-human calretinin
144
antibody (C7479, 10 µg/ml, Sigma-Aldrich, St. Louis MO). Cells were then labeled with
145
dyLight 647-conjugated donkey anti-rabbit antibody and then analyzed on the Millipore Guava
146
easyCyte HT flow cytometer.
147
Statistics
148
All statistics were performed using the Student t-Test. A p-value of less than 0.05 was
149
considered significant.
150
RESULTS
151
TGF-β, plasmin and thrombin induce MesoMT.
152
Prior to use in experiments, HPMC cultures were assayed for expression of the mesothelial
153
cell marker calretinin (Figure 1A). FACs analyses confirmed that the cells were mesothelial
154
cells and that greater than 95% of the cells expressed the mesothelial cell marker calretinin. We
155
previously showed that TGF-β, thrombin and plasmin induce biomarkers of MT in M and
156
HPMCs and was associated with phenotypic changes indicative of MesoMT (19) and sought to
157
confirm the expression of these biomarkers for all subsequent signaling analyses. These findings 7
MesoMT is PI3K and NFκB dependent 158
were extended in HPMCs that were visually analyzed for changes in α-SMA expression and
159
localization (Figure 1B). Plasmin and thrombin treated HPMCs demonstrated increased α-SMA
160
expression and the presence of filamentous actin when compared to PBS-treated cells. TGF-β,
161
which is known to induce MesoMT (14, 19), was used a positive control. The conditioned media
162
(CM) for Col-1 expression and cell lysates were next subjected to SDS-PAGE followed by
163
Western blot analyses. Col-1 and α-SMA expression were increased by either plasmin or TGF-β
164
treatment (Figure 1C) while thrombin induced α-SMA. Further, thrombin and plasmin promoted
165
Akt phosphorylation (Figure 1C) and P65 phosphorylation in HPMCs (Figure 1D). Because our
166
HPMCs are partially activated, we sought to confirm our findings using unperturbed, primary
167
rabbit (R) PMCs. RPMCS were treated with PBS and TGF-β (Figure 1E) for 48h. TGF-β
168
treated cells demonstrated increased Col-1 and α-SMA while the tight junction marker, zona
169
occludins-1 (ZO-1), was decreased. These findings demonstrated that TGF-β, plasmin and
170
thrombin induced phenotypic and molecular changes that characterize MesoMT in the primary
171
cells used.
172
MesoMT of HPMCs is PI3K/Akt dependent.
173
As TGF-β, plasmin, and thrombin were all implicated as potent activators of the
174
PI3K/Akt signaling pathway (19), we next sought to determine the role of PI3K in MesoMT.
175
Because we previously demonstrated that the PI3K inhibitor, PX866 reduced thrombin-
176
mediated-Akt phosphorylation in HPMCs (8), we next assayed the effect of PX866 on thrombin-
177
mediated MesoMT. RNA was isolated from thrombin-treated cells (7 nM, 24 h) in the presence
178
of varying concentrations of PX866 (1.0 - 0.05 µM) and analyzed by qPCR. While thrombin
179
increased α-SMA mRNA expression by HPMCs, PI3K inhibition significantly blunted α-SMA
180
induction (p
