Pulmonary artery smooth muscle cell HIF-1 regulates endothelin expression 1
via microRNA-543 2
Ching-Chia Wang1,4, Lihua Ying1,2, Elizabeth A Barnes1.2, Eloa S. Adams,1,6 Francis Y. 3
Kim1,5, Karl W. Engel1, Cristina M. Alvira1,3, David N. Cornfield1,2,3 4
5
Center for Excellence in Pulmonary Biology1, Division of Pulmonary, Asthma and 6
Sleep Medicine2 and Critical Care Medicine3, Department of Pediatrics, Stanford 7
University Medical School, Stanford, CA 94305; and Department of Pediatrics, 8
National Taiwan University Children Hospital, National Taiwan University Medical 9
College, Taipei, Taiwan4, Milwaukee Children’s Hospital, Medical College of 10
Wisconsin, Milwaukee, WI5, Kaiser Oakland, Oakland, CA6. 11
12
Address for Correspondence: David N. Cornfield, M.D. 13
Center for Excellence in Pulmonary Biology 14
Stanford University School of Medicine 15
770 Welch Road, Suite 350 16
Stanford, CA 94305 17
Phone: (650)-725-8325 18
Fax: (650)-498-5560 19
Email: [email protected] 20
Running Title: HIF-1 constrains endothelin expression via microRNA 21
Author Contributions: 22
Concept and Design: ESA, CMA, DNC, LY 23
Acquisition, Analysis, and Interpretation of the Data: CW, ESA, EAB, FYK, LY, CMA, 24
DNC 25
Composing the first draft of the Manuscript: CW, LY 26
Revising the Manuscript for Important Intellectual Content: LY, CMA, DNC 27
Word count: 28
Abstract 239 words 29
Body 2945 30
31
Keywords: hypoxia-inducible factor-1, ET-1, miRNA, pulmonary arterial 32 hypertension, TWIST 33
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Abstract 35
Pulmonary artery smooth muscle cells (PASMC) express endothelin (ET-1) which 36
modulates the pulmonary vascular response to hypoxia. Although cross-talk between 37
hypoxia-inducible factor-1HIF-1 an O2-sensitive transcription factor, and ET-1 38
is established, the cell-specific relationship between HIF-1 and ET-1 expression 39
remains incompletely understood. We tested the hypotheses that in PASMC: (i) 40
HIF-1α expression constrains ET-1 expression; and (ii) a specific microRNA (miRNA) 41
links HIF-1α and ET-1 expression. In human PASMC (hPASMC), depletion of HIF-1 42
with siRNA, increased ET-1 expression at both the mRNA and protein level (p<0.01). 43
In HIF-1-/- murine (m)PASMC, ET-1 gene and protein expression was increased 44
(p<0.0001) compared to HIF-1+/+ cells. miRNA profiles were screened in hPASMC 45
transfected with siRNA-HIF-1 and RNA hybridization performed on the Agilent 46
human miRNA microarray. With HIF-1depletion, miRNA-543 increased by 2.4 fold 47
(p<0.01). In hPASMC, miRNA-543 overexpression increased ET-1 gene (p<0.01) and 48
protein (p<0.01) expression, decreased TWIST gene expression (p<0.05) and 49
increased ET-1 gene and protein expression, compared to NTC (p<0.01). Moreover, 50
we evaluated low passage hPASMC from control and pulmonary arterial hypertension 51
(IPAH) patients. Compared to controls, protein expression of HIF-1 and TWIST1 52
was decreased (p<0.05) and miRNA-543, and ET-1 expression increased (p<0.001), 53
in hPASMC from IPAH patients. Thus, in PASMC, loss of HIF-1 increases 54
miRNA-543 which decreases Twist expression, leading to an increase in PASMC 55
ET-1 expression. This previously undescribed link between HIF-1, and ET-1 via 56
miRNA-543 mediated Twist suppression, represents another layer of molecular 57
regulation that might determine pulmonary vascular tone. 58
59
60
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Introduction 61
At all points in mammalian life, oxygen (O2) tension plays a central role in 62
determining pulmonary vascular tone. During mammalian lung development in the 63
intrauterine environment pulmonary blood flow is constrained and O2 tension is low (1, 64
36). With the increase in O2 tension that accompanies the onset of air-breathing life, 65
pulmonary blood flow increases approximately 10 fold, as pulmonary artery pressure 66
falls to less than half of systemic levels over the first 24 hours of postnatal life(12). 67
Throughout air-breathing life, low pulmonary vascular tone is maintained even as the 68
pulmonary circulation responds to compromised ventilation with vasoconstriction to 69
prevent intrapulmonary shunting (44). If, however, pulmonary vascular tone is 70
persistently increased, as occurs in a number of disease states, the pulmonary 71
circulation remodels and right heart failure, and even death, can occur(40). 72
Though the physiology of pulmonary blood flow is relatively well described, the 73
molecular mechanisms that underlie the developmental regulation of pulmonary 74
vascular tone across fetal, neonatal and adult life remain incompletely understood. 75
Multiple lines of evidence point to a central role for the O2-sensitive transcription 76
factor, hypoxia-inducible factor-1 (HIF-1), in the regulation of pulmonary vascular 77
tone (5, 15, 22, 35). In pulmonary artery smooth muscle cells (PASMC) derived from 78
the fetal circulation, HIF-1 protein is relatively O2-insensitive, owing perhaps to 79
developmental differences in prolyl hydroxylase expression (35). However, in PASMC 80
derived from the mature pulmonary circulation, HIF-1 protein expression is highly 81
O2-sensitive (35, 50). In the context of the regulation of pulmonary vascular tone, 82
cell-specific loss of HIF-1 has been reported to either mitigate (5) or accentuate (24) 83
hypoxia-induced pulmonary hypertension. In mice wherein HIF-1 was deleted in 84
smooth muscle cells using an inducible myosin heavy chain promoter, hypoxic 85
pulmonary hypertension is attenuated (5). In contrast, data from mice with a 86
constitutive SMC-specific deletion of HIF-1 indicates that HIF-1 plays a role in 87
maintaining low pulmonary vascular resistance by constraining myosin chain 88
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phosphorylation (24), and augmenting expression of a calcium sensitive subunit of the 89
calcium-sensitive K+ channel (2). Though data derived from human tissues generally 90
has demonstrated an overall increase in HIF-1 expression(27), including in PASMC 91
(9, 42), our lab evaluated PASMC derived from patients with pulmonary arterial 92
hypertension, demonstrated a decrease in HIF-1 expression, and an increase in 93
myosin light chain phosphorylation, kinase activity and contractility (6). Arguably, the 94
divergent findings may derive from dynamic changes in HIF-1 expression in PASMC 95
in association with increases in pulmonary vascular tone and remodeling. 96
Endothelin-1 (ET-1), a vasoactive polypeptide and known downstream target of 97
HIF-1also plays a central role in regulating pulmonary vascular tone(3). Though 98
there is wide appreciation for the role of pulmonary endothelial cell derived ET-1, the 99
physiologic significance of ET-1 produced by PASMC is less well established. Data 100
from our laboratory demonstrated that ET-1 produced by PASMC potentiates the 101
pulmonary vascular response to hypoxia (23). However, the relationship between 102
HIF-1and ET-1 in PASMC is complex with clear evidence that HIF-1 can increase 103
ET-1 expression even as an increase in ET-1 expression can augment HIF-1 protein 104
expression by either increasing synthesis and decreasing prolyl hydroxylase 105
mediated degradation (33) or alternatively by suppressing proteasome-dependent 106
degradation(28). Whether HIF-1 might constrain ET-1 expression in PASMC 107
remains unknown. 108
HIF-1 functions as a key regulator of the response to hypoxia by regulating 109
proteins involved in essential biological processes such as erythropoiesis, cell-cycle, 110
angiogenesis, metabolism and bioenergetics (37). Recent data demonstrates that 111
miRNAs can also mediate the hypoxic response via either HIF-dependent or 112
-independent mechanisms. miRNAs, short noncoding RNA molecules of 21–24 113
nucleotides in length (7), regulate gene expression by binding the 3’UTR of mRNA 114
targets (18). For example, under hypoxic conditions miR-210, a HIF-1 target, 115
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promotes cell cycle progression and limits apoptosis (14). Similarly, miR-191, a 116
HIF-1 target, promotes cell migration and proliferation under hypoxic conditions (31). 117
Given that endogenous miRNAs can downregulate gene expression at the 118
post-transcriptional level through mRNA degradation or promotion of targeted mRNA 119
degradation, we hypothesized that miRNAs might mechanistically link ET-1 and 120
HIF-1. We tested the hypotheses that in PASMC: (i) HIF-1α expression constrains 121
ET-1 expression; and (ii) a specific miRNA links HIF-1α and ET-1 expression. 122
123
Methods 124
Primary mouse PASMC isolation. Primary mouse PASMC (mPASMC) were isolated 125
from SM22-HIF-1-/- and littermate control mice using a modified 126
elastase/collagenase digestion protocol (23, 24). PA tissue was digested in dispersion 127
medium containing 40μmol/l CaCl2, 0.5mg/ml elastase obtained from Worthington 128
Biochemical (Lakewood, NJ), 0.5mg/ml collagenase (Worthington Biochemical), 129
0.2mg/ml soybean trypsin inhibitor (Worthington Biochemical), and 2 mg/ml albumin 130
obtained from Sigma-Aldrich (St. Louis, MO) for 20 min at 37°C. After filtration with 131
100-μm cell strainers, cells were incubated with Dynabeads purchased from Thermo 132
Fisher Scientific (Waltham, MA) coated with anti-CD31 and anti-CD102 antibodies 133
obtained from BD Biosciences (San Jose, CA) for 15 min, in order to deplete 134
endothelial cells expressing CD31 and CD102. Remaining SMC were collected 135
through centrifugation at 225g for 6 min at 4°C and cultured in DMEM obtained from 136
Thermo Fisher containing 10% fetal bovine serum (FBS) with antibiotic solution 137
obtained from Thermo Fisher Scientific (Waltham, MA). To confirm isolation of 138
PASMC, cells were stained for -smooth muscle actin (-SMA) with an antibody 139
obtained from Sigma-Aldrich (catalog number A2547, St. Louis, MO) at 1:400 dilution 140
using immunofluorescence. 141
Cell culture. In vitro studies were performed with mPASMC isolated from control and 142
transgenic C57BL/6J mice with selective deletion of HIF-1 in SMC (SM22-HIF-1-143
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-/-) (24). hPASMC were purchased from Lonza (Basel, Switzerland) and grown 144
according to the manufacturer’s protocol. Cells from passages 4–8 were used for all 145
experiments. 146
siRNA transfection. hPASMC were transfected at 50–70% confluence using 147
Lipofectamine RNAiMAX purchased from Thermo Fischer Scientific, according to the 148
manufacturer’s protocol. Briefly, siHIF-1 purchased from Thermo Scientific 149
Dharmacon or siNTC purchased from Thermo Scientific Dharmacon was transfected 150
at a final concentration of 50nM. Twenty-four hours post-transfection, cells were 151
re-fed with fresh media. After an additional 24 h, cells were harvested and used for 152
ET-1, HIF-1α and miRNA studies. 153
HIF-1 overexpression. The HIF-1α expression plasmid, HA-HIF-1 (CA), 154
containing the double mutant P402A/P564A thereby preventing hydroxylation and 155
permitting constitutive activation(13), was a kind gift from Dr. A.J. Giaccia, Stanford 156
University. An empty vector, pcDNA3, served as a transfection control. hPASMC were 157
transfected by Lipofectamine LTX/PLUS method Thermo Fisher Scientific, per the 158
manufacturer’s instructions. In brief, cells at 50-80% confluence were transfected with 159
10μg of DNA per 100mm plate. 24h post-transfection, cells were re-fed with fresh 160
media. After an additional 24h, cells were harvested and used for ET-1, HIF-1 and 161
miRNA studies. 162
miRNA microarray assay. Total RNA from hPASMC was prepared using Trizol 163
Reagent (Invitrogen, Carlsbad, CA, USA). RNA hybridization was then performed on 164
the Agilent (Santa Clara, CA) human miRNA microarray (v3) with 15k features. 165
miRNAs with significantly different expression profiles (1.2 fold change and p<0.05) 166
were selected through statistical analysis using GeneSpring (Agilent) Software. 167
Analysis was performed using Pathway Studio and MedScan obtained from Ariadne 168
Genomics of Elsevier (Amsterdam, NL) to predict target genes. 169
miRNA overexpression and inhibition. Synthesized miRNA mimics were purchased 170
from GE-Dharmacon (Lafayette, CO) to overexpress miRNA-543 or locked nucleic 171
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acid (LNA) inhibitors from Exiqon Qiagen (Woburn, MA) to deplete miRNA-543 and 172
each control, consisting of the parental vector without a miR target (32), were 173
purchased. hPASMC were transfected by Lipofectamine RNAiMAX method from 174
Thermo Fisher Scientific per the manufacturer’s instructions. In brief, cells at 50-80% 175
confluence were transfected with 50nM of miRNA mimics or 50nM of LNA inhibitors 176
per 100mm plate. 6 h post-transfection, cells were re-fed with fresh media. After a 177
total of 48 h, cells were harvested and used for ET-1, HIF-1 and miRNA studies. 178
Quantitative RT-PCR. To determine ET-1, HIF-1 TWIST1 and miRNA mRNA 179
expression, total RNA was isolated from cultured hPASMC and mPASMC using the 180
RNeasy Mini Kit purchased from Qiagen (Hilden, FDR). First-strand cDNA was 181
synthesized using SuperScript III Reverse Transcriptase obtained from Thermo 182
Fischer Scientific and cDNA for miRNA analysis was synthesized using specific 183
stem-loop reverse transcription primers according to the TaqMan miRNA Assay 184
protocol. Products were subsequently amplified on the C1000 Thermal Cycler CFX 185
384 Real-Time System obtained from Bio-Rad Laboratories (Hercules, CA) using 186
PCR Universal Master Mix obtained from Thermo Fischer Scientific. Quantitative 187
RT-PCR was performed using the following cycle: 95°C for 10 min, 40 cycles of 95°C 188
for 15s and 60°C for 60 s, 60°C for 5 min, followed by a dissociation curve analysis. 189
The relative expression levels of ET-1 and HIF-1 were normalized to 18S ribosomal 190
RNA and miRNAs were normalized to U6 small nuclear RNA (snRNA) expression. All 191
mRNA expression levels were analyzed using the ∆∆CT method. 192
ET-1 ELISA. Secreted ET-1 was measured using an ET-1 colorimetric immunometric 193
ELISA kit purchased from Enzo Life Sciences (Farmingdale, New York) (4, 23, 45). 194
Briefly, media from transfected hPASMC or mPASMC were plated in duplicate and 195
incubated for 1 h at room temperature. To measure intracellular ET-1 protein in 196
hPASMC and mPASMC cells were harvested and lysed. Cell lysate samples were 197
plated in duplicate, and incubated for 24 h at 4°C. Optical density was measured at 198
450 nm, with the concentration of ET-1 in samples calculated from a standard curve of 199
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recombinant ET-1. Final data were normalized to protein concentration. 200
Human PASMC from control and patients with Pulmonary Arterial Hypertension 201
(IPAH). Primary PASMC isolated from control (n=3) and IPAH (n=3) patients were 202
provided by the Pulmonary Hypertensive Breakthrough Initiative (PHBI). Funding for 203
the PHBI is provided by the Cardiovascular Medical Research and Education Fund 204
(CMREF, University of Pennsylvania, Philadelphia, USA). Isolated PASMC were 205
cultured in smooth muscle cell basic media (Lonza, Mapleton, Illinois, USA) 206
containing 5% FBS with 0.1% insulin, 0.2% hFGF-B, 0.1% hEGF, and 0.1% 207
gentamicin/amphotericin (GA-1000). PASMC between passages 2-6 were used for 208
this study. Cells were evaluated expression of -SMA, MHC11, SM-22, and calponin 209
(all markers of vascular SMC) to ensure identity as SMC (6). 210
Western immunoblotting. For Western immunoblotting, protein content was 211
quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA). 212
10g of protein/sample were subjected to SDS-PAGE analysis. Immobilon-P 213
(Millipore-Sigma, St. Louis, MO) membranes were incubated with primary antibodies 214
to detect HIF-1 (10006421, Cayman Chemical, Ann Arbor, MI), -actin (A5441, 215
Sigma, St. Louis, MO), and TWIST (sc-81417, Santa Cruz Biotechnology, Inc., Dallas, 216
TX), and then incubated with horseradish peroxidase (HRP)-conjugated secondary 217
antibodies, followed by detection with ECL reagents (GE Healthcare Life Sciences, 218
Marlborough, MA). Graph represents quantification of protein expression by 219
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densitometry with results represented relative to -actin. 220
Immunoprecipitation assay. Isolated PASMC were harvested and lysed with 0.5% 221
NP‐ 40 buffer (6). Protein content was standardized as determined by BCA assay. 222
10μg of lysates were pre‐ cleared for 1h at 4°C with 50L of protein A–Sepharose 223
beads and then incubated with 50L of TWIST antibodies (sc-81417) overnight at 224
4°C. Addition of 20L of protein A–Sepharose beads with incubation for 4h at 4°C 225
followed. Samples were washed with PBS and analyzed by SDS–PAGE, transferred 226
to immobilon membrane and detected by ECL using the TWIST antibody (sc-81417). 227
The whole cell lysate samples included 30μg of total cell lysate. Moreover, the TWIST 228
antibody used in the course of this study has been previously validated (29, 39). 229
Statistical analysis. Results are expressed as means ± SEM. Statistical significance 230
was assessed with Student’s t-test and ANOVA where appropriate. A p value of < 0.05 231
was taken as the threshold level for statistical significance. All experiments were 232
repeated a minimum of 3 times (with most being repeated ≥ 5 times). 233
234
Results 235
HIF-1 depletion or overexpression increases hPASMC ET-1 expression and 236
secretion. 237
Initially, we assessed whether deletion of HIF-1 modulates ET-1 expression levels in 238
hPASMC. siRNA directed against HIF-1 effectively depleted HIF-1 gene (Fig. 1A) 239
and protein (Fig. 1B) expression and increased ET-1 gene (Fig. 1C) expression 240
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compared with NTC siRNA-transfected SMC. With HIF-1 depletion, ET-1 protein did 241
not change in the (Fig. 1D) cell lysates but increased significantly in the media (Fig. 242
1E) compared with NTC siRNA-transfected SMC. We then overexpressed HIF-1 by 243
transfecting hPASMC with a plasmid containing a mutant form of HIF-1 that is 244
resistant to oxygen-mediated degradation (13). Consistent with multiple prior reports 245
(11, 20) in other cell types, overexpression of HIF-1 in hPASMC (Fig. 2A) also 246
increased ET-1 gene expression (Fig. 2B), had no effect on protein expression in cell 247
lysate (Fig. 2C) but significantly increased ET-1 protein expression in the media (Fig. 248
2D), compared with SMC transfected with empty vector. These results suggest that 249
either a decrease or increase in HIF-1 expression can increase ET-1 expression in 250
PASMC. 251
Deletion of HIF-1 increases ET-1 expression in mPASMC. 252
To provide further proof-of-concept in a distinct, and complementary experimental 253
model, we measured ET-1 expression and secretion in mPASMC isolated from mice 254
with SMC specific deletion of HIF-1 (HIF-1-/-) and WT mice (HIF-1+/+) (24). As 255
shown in Fig. 3A, HIF-1 was virtually absent in HIF-1-/- mPASMC. Consistent with 256
our results in hPASMC, in HIF-1-/- mPASMC, ET-1 gene expression (Fig. 3B) and 257
protein expression (Fig. 3C) in the media were significantly increased relative to 258
HIF-1 expressing mPASMC (HIF-1+/+). 259
HIF-1 depletion in hPASMC alters expression of specific miRNA molecules. 260
To determine whether silencing of HIF-1α dysregulates miRNAs which modulate ET-1 261
expression, we performed miRNA expression analysis using the Agilent human 262
miRNAs microarray (v3) on hPAMSC transfected with NTC or HIF-1 siRNA. 263
Differentially expressed miRNA are included in Table 1. miRNA-543, with the most 264
dynamic expression profile (2.4 fold change and p<0.05), was selected through 265
statistical analysis using GeneSpring Software (Table 1). Analysis performed with 266
Pathway Studio and MedScan software suggested that miRNA-543 might link ET-1 267
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and HIF-1 (43). 268
miRNA-543 expression is inversely proportional to HIF-1α expression. 269
We next sought to directly demonstrate that HIF-1 expression modulates PASMC 270
miRNA-543 expression. As shown in Fig. 4, knockdown of HIF-1 using siRNA in 271
hPASMC increased miRNA-543 expression compared with NTC siRNA-transfected 272
SMC. 273
Overexpression of miRNA-543 increases, and depletion of miRNA-543 274
decreases, HIF-1 and ET-1 expression 275
We next overexpressed miRNA-543 in hPASMC and measured HIF-1 and ET-1 276
expression. We confirmed that transfection with the miRNA-543 mimic increased 277
miRNA-543 expression by over 150 fold, compared to hPASMC transfected with the 278
control mimic (Fig. 5A). As depicted in Fig. 5B, miRNA-543 overexpression, 279
increased ET-1 gene expression by approximately 5-fold, and increased ET-1 protein 280
expression in the media by approximately 70% (Fig. 5C). Conversely, transfection of 281
hPASMC with differing concentrations of the miR-543 LNAs effectively depleted 282
miRNA-543 (Fig. 5D). Transfection of hPASMC with 50nM resulted in a significant 283
reduction in ET-1 (Fig. 5E) gene and protein (Fig. 5F) expression compared to 284
hPASMC treated with the control(32), a vector that did not contain any 285
oligonucleotide. 286
Overexpression of miRNA-543 decreases TWIST1 and increases ET-1 287
expression. 288
Given that miRNA molecules limit expression of target molecules through 289
post-transcriptional modification, we next sought to discover how increased 290
miRNA-543 resulted in increased ET-1 expression. Computational analysis of the 291
TWIST1 3’ UTR, identified TWIST1 as a high probability target of miR-543, and 292
miR-543 mimics repress expression in a luciferase 3’ UTR TWIST1 construct (17). 293
Moreover, in the context of osteosarcoma, TWIST inversely correlates with ET-1 294
expression (53). In keeping with these data, overexpression of miRNA-543 in 295
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hPASMC significantly decreased TWIST1 expression (Fig. 6A). Moreover, with 296
effective silencing of TWIST1 with siRNA (Fig. 6B), ET-1 gene expression increased 297
by approximately 3-fold (Fig. 6C). Although ET-1 protein expression did not change in 298
the cell lysate (Fig. 6D), it increased 2-fold in the conditioned media (Fig. 6E). 299
in hPASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) 300
compared to control patients, HIF-1 and TWIST1 are decreased and 301
miRNA-543 and ET-1 are increased. 302
To determine whether the observations in cell culture and murine models have fidelity 303
with human pathobiology, we measured expression HIF-1, TWIST1 and ET-1 in 304
hPASMC derived from controls (n=3) and patients with idiopathic pulmonary arterial 305
hypertension (IPAH) (n=3; Table 2). HIF-1306
expression were decreased in PASMC from patients with IPAH, compared to control 307
patients. Consistent with our in vitro and murine studies, TWIST1 gene (Fig. 7C) and 308
protein (Fig. 7D) expression were significantly decreased. Moreover, miRNA-543 309
expression was increased in hPASMC from patients with IPAH compared to controls 310
(Fig. 7E). Finally, as further proof of principle, we demonstrated that ET-1 expression 311
was significantly increased in the cell media of hPASMC from patients with IPAH, as 312
compared to control patients (Fig. 7F). 313
314
Discussion 315
The present report is the first to demonstrate that deletion of HIF-1in PASMC 316
increases ET-1 gene and protein expression. Specifically, these results demonstrate 317
that in PASMC the transcription factor HIF-1 modulates expression of a powerful 318
pulmonary vasoconstrictor, ET-1, via a specific miRNA molecule, miRNA-543. The 319
strength of the evidence is buttressed by observations in both human and murine 320
systems wherein with HIF-1 depletion, ET-1 expression is increased. In hPASMC 321
HIF-1 depletion increases miRNA-543 expression as well. Our data demonstrate 322
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that in hPASMC an increase in miRNA-543 decreased TWIST1 gene expression, and 323
increased both ET-1 gene and protein expression. Consistent with this construct, 324
depletion of miRNA-543 in hPASMC increased TWIST1 gene expression, and 325
decreased ET-1 gene and protein expression. Further evidence for the biologic 326
relevance of the findings derive from experiments undertaken in low passage 327
hPASMC from control patients and from patients with IPAH. The findings were entirely 328
consistent with those from the in vitro and murine experiments. Specifically, HIF-1 329
and TWIST1 were decreased while miRNA-543 and ET-1 were increased. Taken 330
together, these results provide evidence of a previously undescribed mechanistic link 331
wherein a decrease in HIF-1 expression can result in an increase in ET-1, molecules 332
that play a central role in the regulation of both physiologic and pathophysiologic 333
pulmonary vascular tone. 334
Although HIF-1 is a molecule that determines the cellular response to hypoxia in 335
multiple ways, including cell proliferation, migration, metabolism and hypertrophy (26, 336
30, 35, 38, 51), increasing evidence suggests a cell-specific role in the pulmonary 337
vasculature. For example, mice globally haploinsufficient for HIF-1 demonstrated an 338
attenuated response to both acute and chronic hypoxia(51). Similarly, in mice with 339
conditional, cell specific deletion of HIF-1 in myosin heavy chain expressing cells, 340
hypoxia-induced pulmonary hypertension was mitigated (5). Conversely, in another 341
report wherein HIF-1 was deleted in SM-22 expressing cells, pulmonary vascular 342
tone was increased in both normoxic and hypoxic conditions, and myosin light chain 343
phosphorylation in PASMC was increased, even in the absence of vascular 344
remodeling (24). Even more recent reports indicate that overexpression of HIF-1 in 345
pulmonary artery endothelial cells leads to dramatic elevations in pulmonary artery 346
pressures and marked vascular remodeling (10, 15, 40). Thus, the present report 347
adds to the accumulating evidence for a cell-specific role for HIF-1 in the pulmonary 348
circulation. 349
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There is a large body of evidence, predominately in endothelial cells, indicating 350
that ET-1 is a downstream target of HIF-1, which is upregulated during hypoxia (11, 351
20, 46). Moreover, there is a significant body of evidence supporting the notion that in 352
PASMC, an increase in ET-1 leads to an increase in HIF-1 expression through either 353
an increase in synthesis, a decrease in hydroxylation (33) or a decrease in 354
proteosomal degradation(28). However, the present data focus on a mechanism 355
whereby a decrease in HIF-1 expression can drive ET-1 expression in PASMC or 356
stated more directly, HIF-1 expression might normally act to constrain ET-1 357
expression. Previously, we demonstrated that PASMC ET-1 expression modulates the 358
pulmonary vascular response, including tone, structural remodeling, cell proliferation 359
and migration, to chronic hypoxia (23). In this report, we outline an interaction 360
between molecules with physiologically significant roles in the pulmonary circulation 361
via miRNA-543. The observation that in both human and murine PASMC either 362
HIF-1 overexpression or depletion can increase ET-1 expression, via distinct 363
pathways, argues for the importance of closely regulated HIF-1 expression. The 364
physiologic significance of the observation is amplified by the marked increase in ET-1 365
secretion by HIF-1-/- cells. That ET-1 expression is increased in the media but not the 366
cell lysate of SMC is consistent with ET-1 expression in endothelial cells wherein ET-1 367
is synthesized and continuously released. In endothelial cells ET-1 is predominately 368
regulated at the transcriptional level(41). 369
Previous studies have demonstrated a role for miRNA-543, located on human 370
chromosome 14, in tumorigenesis and metastasis (21). Recent studies have showed 371
that miRNA-543 can suppress tumorigenesis and metastasis by inhibiting focal 372
adhesion kinase and TWIST in endometrial cancer cell lines (8). However, in 373
osteosarcoma cells TWIST expression is inversely correlated with ET-1 expression 374
(53). Moreover, miRNA-543 decreases Twist expression by directly targeting the 3’–375
UTR of TWIST (8). This represents the first report of a role for miRNA-543 mediated 376
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15
regulation of ET-1 expression in PASMC. Given the capacity of ET-1 to modulate 377
vascular development (46, 47), the data in the present report suggest the potential of 378
a pro-angiogenic role for miRNA-543 via augmented ET-1 expression (52). 379
The results have implications for the regulation of pulmonary vascular tone under 380
both physiologic and pathophysiologic conditions. Data from our laboratory indicate 381
that in PASMC, HIF-1 is present under both hypoxic and normoxic conditions (35). In 382
a murine model of SM-22-specific deletion of HIF-1, compared to controls, 383
pulmonary vascular tone is increased under normoxic conditions and hypoxia-induced 384
pulmonary hypertension is exaggerated relative to controls(24). As PASMC do 385
express ET-1(23), the loss of HIF-1 may increase pulmonary vascular tone through 386
direct effects that entail an increase in myosin light chain phosphorylation(6, 24), as 387
well as via indirect effects wherein ET-1 production is increased via miR-543 mediated 388
suppression of the transcription factor TWIST. Thus, the result of decreased HIF-1 in 389
PASMC, such as in the present report and a prior report from our lab in PASMC from 390
patients with pulmonary hypertension(6), is an increase pulmonary vascular tone. 391
Conversely, with well-preserved HIF-1 expression in PASMC, myosin light chain 392
phosphorylation and ET-1 production is constrained, thereby maintaining the low tone 393
of the normal pulmonary circulation. 394
To our knowledge this represents the initial report of the association between 395
miRNA-543, Twist expression and ET-1 in the pulmonary vasculature (Fig. 7). These 396
data detail a relationship between HIF-1 and miRNA-543 expression in PASMC, 397
where in the absence of HIF-1, miRNA-543 and ET-1 expression both increase, with 398
Twist linking the two molecules. These results underscore the importance of PASMC 399
HIF-1 in the regulation of pulmonary vascular tone under not only hypoxic, but also 400
normoxic conditions. An important limitation of the present series is that the 401
experiments were conducted in normoxia and a hypoxic environment may well alter 402
the findings. Notwithstanding that caveat, the biologic fidelity between the findings 403
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from in vitro studies, murine models and the hPASMC derived from patients increases 404
the likelihood that the present findings possess clinically relevant implications for both 405
pulmonary vascular development and the regulation of pulmonary vascular tone. 406
Modulation of either miRNA-543 or Twist expression might represent a viable 407
therapeutic strategy to address pathophysiologic elevations of pulmonary vascular 408
tone. 409
TWIST is a direct transcriptional target of HIF-1 (49). In the context of cancer, an 410
increase in HIF-1 expression can promote epithelial-mesenchymal transition (EMT) 411
and metastatic disease(48, 49). Recent data indicate that HIF-1 regulates TWIST 412
expression by directly binding to the hypoxia response element in the proximal region 413
of the TWIST promoter. In the presence of both HIF-1 and TWIST expression, 414
squamous cell cancers of the head and neck are more likely to metastasize providing 415
evidence that HIF-1 signals, in part, via TWIST (48, 49). Conversely, data in the 416
present report indicate that loss of HIF-1 can decrease TWIST expression, albeit 417
indirectly. With loss of HIF-1 expression, miR-543 expression increases, which in 418
turn, represses TWIST expression. The relatively recent reports demonstrating a role 419
for TWIST in EMT (34), myogenesis(25), inflammation (16) and BMP signaling (19), 420
processes all involved in the pathogenesis of pulmonary hypertension, suggest that 421
manipulating miR-543, a molecule that links HIF-1 to TWIST, might represent a 422
worthwhile and novel therapeutic strategy. 423
424
425
Acknowledgments 426
Funding: The work has been supported by the National Institutes of Health (HL060784 427
(DNC), HL0706280 (DNC), and HL122918 (CMA). Contributing investigators have also 428
been supported by Burroughs Welcome Fund Preterm Birth Initiative (DNC) and the 429
Stanford Child Health Research Institute Tashia and John Morgridge Faculty Scholar 430
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17
Award (CMA). 431
432
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618
619
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Figure legends 620
Fig. 1. HIF-1 depletion increases human pulmonary artery smooth muscle cell 621
(hPASMC) ET-1 expression and secretion. hPASMC were transfected with 622
nontargeted control small interfering RNA (siNTC) (n=6) or siRNA directed against 623
HIF-1. In hPASMC transfected with siHIF-1(n=6) (A) HIF-1mRNA was 624
decreased; (B) Expression of HIF-1 in hPASMC by Western immunoblot after 625
transfection with NTC siRNA or HIF-1 siRNA. -actin serves as a loading control 626
(n=3); (C) HIF-1depletion markedly increased ET-1 mRNA (n=6) compared to cells 627
transfected with siNTC; ET-1 protein expression did not change in (D) cell lysates of 628
hPASMC transfected with siHIF-1(n=9) but did increase significantly in the (E) 629
media (n=9). Results are presented as means ± SEM. **p<0.01, ***p<0.001, 630
****p<0.0001 by unpaired Student’s t-test, NTC siRNA vector versus HIF-1 siRNA 631
632
Fig. 2. HIF-1 overexpression increases human pulmonary artery smooth muscle cell 633
(hPASMC) ET-1 expression and secretion. Compared to hPASMC transfected with 634
empty vector (pcDNA3) (n=6), hPASMC, transfected with constitutively active (CA) 635
HIF-1demonstrated: an increase in (A) HIF-1 (n=4) and (B) ET-1 mRNA (n=6) and 636
ET-1 protein expression did not change in the (C) cell lysates (n=3) but increased in 637
the (D) media (n=3). Results are shown as means ± SEM. ***p<0.001, ***p<0.0001, 638
by unpaired Student’s t-test, control vector versus HIF-1 or CA-HIF-1. 639
640
Fig. 3. In murine PASMC (mPASMC)., HIF-1 deletion increases ET-1 expression. 641
(A) HIF-1 mRNA expression in mPASMC isolated from SM22-HIF-1α-/- (n=3) and 642
WT (n=3) mice. (B) Expression of ET-1 mRNA (n=3) in isolated mPASMC. (C) 643
Expression of ET-1 protein (n=3) in isolated mPASMC. Results are presented as 644
means ± SEM. HIF-1+/+ vs. HIF-1-/-. **p<0.01, by unpaired Student’s t-test, 645
HIF-1+/+ vs. HIF-1-/- 646
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24
647
Fig. 4. In human PASMC (hPASMC), HIF-1 depletion increases miRNA-543 648
expression. In hPASMC with HIF-1 depletion (n=3) compared to siNTC (n=3). 649
Results are presented as means ± SEM. *p<0.05, by non-parametric Mann-Whitney U 650
test, siHIF-1 vs. siNTC). 651
652
Fig. 5. Overexpression of miRNA-543 increases, and depletion decreases, ET-1 653
expression in hPASMC. With miRNA-543 overexpression in hPASMC using a 654
constitutively active miRNA-543 mimetic (n=6), (A) miRNA-543 expression was 655
dramatically increased compared to miR-543 mRNA expression in hPASMC 656
transfected with a negative control (n=6). In the presence of miRNA-543 657
overexpression in hPASMC, (B) ET-1 mRNA (n=4) and (C) protein expression (n=3) 658
were all increased compared to control (*p<0.05, by non-parametric Mann-Whitney U 659
test, miRNA-543 mimic (n=3) vs. negative control (n=3)). (D) hPASMC were 660
transfected with negative control (n=6) or locked nucleic acid miRNA-543 inhibitor 661
(12.5, 25, 50, and 100nM) (n=6). Inhibition decreased miRNA-543 expression. 662
Inhibition of miRNA-543 expression by miRNA-543 inhibitor (50nm) decreased (E) 663
ET-1 mRNA expression (n=18), and (F) ET-1 protein expression (n=3) in the 664
conditioned media. Results are presented as means ± SEM. *p<0.05, **p<0.01, 665
****p<0.0001, by non-parametric Mann-Whitney U test, miRNA-543 inhibitor vs. 666
control 667
668
Fig. 6. TWIST1 depletion increases human pulmonary artery smooth muscle cell 669
(hPASMC) ET-1 expression and secretion. With miRNA-543 overexpression in 670
hPASMC using a constitutively active miRNA-543 mimetic, (A) TWIST mRNA 671
expression (n=8) decreased. (B) siRNA directed against TWIST effectively decreased 672
mRNA (n=5); (C) ET-1 mRNA (n=5) was increased while protein expression (n=5) 673
was unchanged in the (E) cell lysate but increased significantly, compared to control in 674
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25
the (E) conditioned media (n=5). Results are presented as means ± SEM. **p<0.01, 675
***<0.001, ****p<0.0001, by non-parametric Mann-Whitney U test, NTC vector versus 676
TWIST1 mimic or siRNA. 677
678
Fig. 7. In PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) 679
(n=3) compared to control (n=3) patients, HIF-1 and TWIST1 are decreased and 680
miRNA-543 and ET-1 are increased. (A) HIF-1 mRNA expression in PASMC isolated 681
from control and patients with IPAH; (B) Western immunoblot of HIF-1 protein 682
expression in PASMC from control and patients with IPAH with -actin loading control. 683
Graph represents means ± SEM, *** p < 0.001, IPAH vs. Control; (C) TWIST1 684
mRNA expression in mPASMC from control and patients with IPAH, Graph represents 685
means ± SEM, *p < 0.05, IPAH vs. Control; (D) Western immunoblot of TWIST1 686
protein expression in PASMC from control and IPAH patients with -actin loading 687
control, Graph represents means ± SEM (n=3). ***p < 0.001, IPAH vs. Control. 688
Expression of TWIST1 in whole cell lysates (WCLs) in isolated PASMC and 689
immunoprecipitated TWIST in control PASMC by Western immunoblot; (E) 690
miRNA-543 expression in mPASMC isolated from controls and patients with IPAH. *p 691
< 0.05, IPAH vs. Control; (F) ET-1 protein expression did not change in the cell media 692
(n=3) of hPASMC from control and IPAH patients. Results are shown as means ± 693
SEM. *p<0.05, by unpaired Student’s t-test. 694
695
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Fig. 8. Schematic representation of the proposed HIF-1 and ET-1 interaction. In the 696
pulmonary artery smooth muscle cell, HIF-1 is constitutively active leading to a low 697
level of endothelin (EDNI) gene expression. With a decrease in HIF-1 expression, 698
miRNA-543 expression increases which serves to decrease TWIST1 expression and 699
nuclear accumulation to thereby derepress endothelin gene expression and augment 700
secretion of the peptide endothelin (ET-1), a powerful vasoconstrictor molecule. 701
Under hypoxic conditions, HIF-1 protein is stable and translocates into the nucleus, 702
and increases EDNI transcription and ET-1 secretion. 703
704
705
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Table 1. 706
hPASMC HIF-1 depletion alters expression of specific microRNA (miRNA) 707
molecules. RNA hybridization was performed on the Agilent human miRNAs 708
microarray (v3) with 15k features (Agilent). miRNA-543 demonstrated a divergent 709
expression profile after HIF-1 depletion (2.4 fold change and p<0.05) and was 710
selected through statistical analysis using GeneSpring Software. 711
712
713
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28
714
Table 1. 715
716
717
718
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29
719
720
721
722
Table 2.
Demographic characteristics of patients. Isolated PASMC were derived from
the above listed control and IPAH patients (initial passage number 1-2).
Subsequent studies were performed on cells between passage numbers 2-6.
HT, head trauma; IH, intracranial hemorrhage; IPAH, idiopathic pulmonary
arterial hypertension.
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Figure 1
NTC siRNA
HIF-1α siRNA
HIF
-1α
RN
A E
xpre
ssio
n (n
orm
aliz
ed to
18s
)
****
1.0
0.5
0.0
****
ET-
1 R
NA
Exp
ress
ion
(nor
mal
ized
to 1
8s)
0NTC
siRNA HIF-1α siRNA
cell media
ET-
1 P
rote
in E
xpre
ssio
n
2
3
0
4
NTC siRNA
HIF-1α siRNA
cell lysate
NTC siRNA
HIF-1α siRNA
ET-
1 P
rote
in E
xpre
ssio
n
1.5
1.0
0.5
0.0
2.0
A
1
C D
1.5
**
2
3
5
4
1
HIF-1α
β-actin
NTC siRNA
HIF-1α siRNA
***
HIF
-1α
Pro
tein
Exp
ress
ion
(nor
mal
ized
to β
-act
in)
2.0
1.0
0.5
0
1.5
NTC siRNA
HIF-1α siRNA
B
E
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Figure 2
HIF
-1α
RN
A E
xpre
ssio
n (n
orm
aliz
ed to
18s
)
2
3
0
4
1
Vector HA-HIF-1α
cell lysate 1.5
1.0
0.5
0.0 Vector HA-
HIF-1α
ET-
1 P
rote
in E
xpre
ssio
n
20
15
10
5
0Vector HA-
HIF-1α
ET-
1 R
NA
Exp
ress
ion
(nor
mal
ized
to 1
8s)
**** ***
cell media
3
1
2
0
4
Vector HA-HIF-1α
ET-
1 P
rote
in E
xpre
ssio
n
***
A B
C D
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Figure 3
**
1.5
1.0
0.5
0.0
HIF
-1α
RN
A E
xpre
ssio
n (n
orm
aliz
ed to
18s
)
10
15
0
20
5
25 **
ET-
1 R
NA
Exp
ress
ion
(nor
mal
ized
to 1
8s)
A B
ET-
1 P
rote
in E
xpre
ssio
n
1.0
1.5
0
2.0
0.5
2.5 *
cell media C
HIF-1α+/+ HIF-1α-/- HIF-1α+/+ HIF-1α-/- HIF-1α+/+ HIF-1α-/-
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Figure 4
miR
-543
Exp
ress
ion
(nor
mal
ized
to U
6) *
2
4
0
6
NTC siRNA
HIF-1α siRNA
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Figure 5 m
iR-5
43 E
xpre
ssio
n (n
orm
aliz
ed to
U6)
100
250
0
300
Control miR-543 mimic
****
ET-
1 E
xpre
ssio
n (n
orm
aliz
ed to
18s
)
4
6
0
8
2
10 *
A
ET-
1 P
rote
in E
xpre
ssio
n
*
10
15
0
20
5
Control miR-543 mimic
1.0
1.5
0
0.5
Control
miR-543 inhibitor
12.5nm 25nm
50nm
miR
-543
Exp
ress
ion
(nor
mal
ized
to U
6)
ET-
1 R
NA
Exp
ress
ion
(nor
mal
ized
to 1
8s)
1.0
2.0
0
0.5
Control miR-543 inhibitor
* 4
6
0
2
Control miR-543 inhibitor
ET-
1 P
rote
in E
xpre
ssio
n
E
cell media
cell media
100nm
**
****
1.5
B C
D F
Control miR-543 mimic
+ +
+ +
+
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NTC siRNA
TWIST1 siRNA
***
TWIS
T1 R
NA
Exp
ress
ion
(nor
mal
ized
to β
-act
in)
ET-
1 R
NA
Exp
ress
ion
(nor
mal
ized
to β
-act
in)
**
2
3
0
4
1
NTC siRNA
TWIST1 siRNA
cell lysate
ET-
1 P
rote
in E
xpre
ssio
n
1.5
1.0
0.5
0.0
2
3
0
1
NTC siRNA
TWIST1 siRNA
ET-
1 P
rote
in E
xpre
ssio
n cell media
***
Control miR-543 mimic
TWIS
T1 R
NA
Exp
ress
ion
(nor
mal
ized
to β
-act
in)
**
2.0
1.0
0.5
0.0
1.5
A
E
B
C D
1.5
1.0
0.5
0.0
NTC siRNA
TWIST1 siRNA
Figure 6
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control
TWIS
T1 e
xpre
ssio
n (n
orm
aliz
ed to
18s
)
* 1.0
1.5
0
0.5
IPAH
Figure 7 H
IF-1α
Exp
ress
ion
(nor
mal
ized
to 1
8s)
1.0
1.5
0
0.5
IPAH Control
A!
100kDa HIF-1α
IPAH 3 4 1 2 6 5
Control
50kDa β-actin
37kDa
B!
Control IPAH
1.0
1.5
0
0.5
***
HIF
-1α
Pro
tein
Exp
ress
ion
(nor
mal
ized
to β
-act
in)
miR
-543
Exp
ress
ion
(nor
mal
ized
to U
6)
Control
2.0
1.0
0.5
0
1.5
*
IPAH
1.0
1.5
0
0.5
Control IPAH TWIS
T1 P
rote
in E
xpre
ssio
n (n
orm
aliz
ed to
β-a
ctin
)
***
50kDa
37kDa
TWIST1 37kDa
β-actin
25kDa
IPAH 3 4 1 2 6 5
Control D!
25kDa TWIST1
TWIST IP Control IPAH
WCLs
37kDa
E!
ET-
1 P
rote
in E
xpre
ssio
n cell media
2
0
4
6 *
Control IPAH
F!
C!
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Figure 8
TWIST1
HIF-1αMir-543
PulmonaryArterySmoothCellMembrane
ET-1
HIF-1α
HIF1α HIF1β
HRE
Nucleus
Hypoxia
EDN1VEGF
ET-1
O2
O2
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