Hypoxia-responsive growth factors upregulate periostin and osteopontin expression via distinct...

Hypoxia-Responsive Growth Factors Upregulate Periostin and Osteopontin Expression

via Distinct Signaling Pathways in Rat Pulmonary Arterial Smooth Muscle Cells

Peng Li, Suzanne Oparil, Wenguang Feng, Yiu-Fai Chen

Vascular Biology and Hypertension Program

Division of Cardiovascular Disease

Department of Medicine

Birmingham, Alabama 35294

Running Head: Regulation of Periostin Expression in Pulmonary VSMCs

Word counts: Abastract: 250

Text: 5551

Figures: 8

Correspondence to: Yiu-Fai Chen, Ph.D.

1008 Zeigler Research Building

University of Alabama at Birmingham

UAB Station

Birmingham, Alabama 35294

(205) 934-2580 (Phone)

(205) 934-0424 (Fax)

E-mail: [email protected]

Articles in PresS. J Appl Physiol (April 30, 2004). 10.1152/japplphysiol.01311.2003

Copyright © 2004 by the American Physiological Society.

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ABSTRACT

This study tested the hypothesis that expression of the novel adhesion molecule periostin

(PN) and osteopontin (OPN), are increased in lung and in isolated pulmonary arterial smooth

muscle cells (PASMCs) in response to the stress of hypoxia and explored the signaling pathways

involved. Adult male rats were exposed to 10% O2 for 2 wks; and growth-arrested rat PASMCs

were incubated under 1% O2 for 24 hrs. Hypoxia increased PN and OPN mRNA expression in

rat lung. In PASMCs, hypoxia increased PN, but not OPN expression. The hypoxia-responsive

growth factors fibroblast growth factor-1 (FGF-1) and angiotensin II (ANG II) caused dose- and

time-dependent increases in PN and OPN expression in PASMCs. FGF-1-induced PN

expression was blocked by the FGFR-1 antagonist PD166866 and by inhibitors of PI3K

(LY294002, Wortmannin), p70S6K (rapamycin), MEK1/2 (U0126, PD98059) or p38MAPK

(SB203580), but not of JNK (SP600125). ANG II-induced PN expression was blocked by the AT1

receptor antagonist losartan and by inhibitors of PI3K and MEK1/2. In contrast, FGF-1-induced

OPN expression was blocked by inhibitors of JNK or MEK1/2, but not of PI3K, p70S6K or

p38MAPK. Activation of p70S6K and p38MAPK by anisomycin robustly stimulated PN, but not

OPN expression. This study is the first to demonstrate that growth factor-induced expression of

PN in PASMCs is mediated through PI3K/p70S6K, Ras/MEK1/2 and Ras/p38MAPK signaling

pathways, while the expression of OPN is mediated through Ras/MEK1/2 and Ras/JNK signaling

pathways. These differences in signaling suggest that PN and OPN may play different roles in

pulmonary vascular remodeling under pathophysiologic conditions.

Keywords: Pulmonary vascular remodeling, Pulmonary arterial smooth muscle cells, Periostin,

Osteopontin, Hypoxia

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INTRODUCTION

We have recently reported that expression of periostin (PN, a putative bone extracellular

matrix [ECM] protein previously named osteoblast-specific-factor 2, OSF-2) (5,6,10,18,23) is

greatly increased during remodelling of the cardiovascular system under stressful conditions, e.g.

pressure overload-induced cardiac hypertrophy and balloon-induced endoluminal injury of carotid

arteries (16,46). PN mRNA expression was increased 12- to 43-fold in hearts of wildtype and

atrial natriuretic peptide-deficient mice, respectively, with transverse aortic constriction

(TAC)-induced cardiac hypertrophy. This was the most dramatic response to TAC-induced

pressure overload of any of > 5,000 genes studied by microarray analysis (26).

Immunohistochemical analysis showed that PN is preferentially localized to the cardiac

fibroblasts, interstitium, and vascular smooth muscle cells (VSMCs) in these experimental

settings. Importantly, increased PN expression was associated with increased expression of

osteopontin (OPN), collagens, fibronectin, thrombospondin and MMPs (11,26), suggesting that

PN may participate in ECM remodelling in heart and blood vessels under stressful conditions (26).

The present study expanded these important findings to examine the regulation of PN and

OPN, in lungs of hypoxia-adapted rats and pulmonary arterial SMCs (PASMCs) exposed to

hypoxia or hypoxia-responsive growth factors in vitro. Based on our findings in the heart and our

previous experience with pulmonary vascular remodelling in the lung of hypoxia-adapted rats, we

postulated that PN and OPN expression would increase as part of a generalized ECM response to

hypoxic stress in the lung of adult rats and in PASMCs in vitro. Accordingly, this study tested the

primary hypothesis that PN and OPN are increased in lung and in isolated PASMCs in response

to normobaric hypoxia exposure. We further tested whether hypoxia-induced growth factors,

including the tyrosine kinase receptor-associated growth factor fibroblast growth factor-1 (FGF-1)

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(13) and the G-protein-coupled receptor-associated growth factor angiotensin II (ANG II) (19)

stimulate increased expression of ECM molecules in PASMCs. Finally, we used selective kinase

inhibitors to delineate the signaling pathways involved in FGF-1 and ANG II-induced PN and OPN

expression in PASMCs.

MATERIALS AND METHODS

Animals and cell culture

Male Sprague-Dawley rats were obtained from Charles River Breeding Laboratories

(Wilmington, MA) at age 8 wks. All animals used in this study were cared for and handled

according to the NIH Guide for the Care and Use of Laboratory Animals. For in vivo chronic

hypoxia studies, rats were exposed to hypoxia (10% O2, 1 atm) in an 800-liter model 818GBB

Plexiglas glove box (Plas Labs, Lansing, MI) beginning at age 8-9 wk for a total of 2 wks as

previously described (3,4,21).

For in vitro studies, PASMC were initially grown in DMEM (GIBCO BRL, Rockville, MD)

with 10% heat-inactivated low endotoxin fetal bovine serum (FBS) (GIBCO BRL), 2 mM

glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37oC in a humidified atmosphere

containing 95% air-5% CO2 and were passaged by harvesting with trypsin-EDTA and seeding in

6-cm cell culture dishes. Passaged cells were grown in DMEM with 10% FBS. PASMCs were

used for experiments between passage 4 and 8. To confirm the characteristics of SMCs in

culture, immunohistochemical staining of -actin was performed using specific -actin antibody

and peroxidase-labeled anti-IgM or anti-IgG antibodies.

Prior to each study, PASMCs were grown to 100% confluence and then made quiescent

by placing them in medium containing 0.1% FBS for 24 hrs. To examine the effects of hypoxia on

PN and OPN mRNA expression, PASMCs were transferred into an air-tight hypoxic chamber

(model 2710, Cell Culture Incubator, Queue Systems) containing 1%O2/5%CO2/94%N2 as

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previously described (13,22). For the study of signal transduction pathways, PASMCs were

pretreated with selective inhibitors for 45 min before addition of growth factors (FGF-1 or ANG II)

to the medium and incubation for an additional 24 hrs. For treatment with FGF-1, heparin was

added to the medium at a final concentration of 5 U/ml, because it protects FGF-1 against

enzymatic digestion and promotes FGF receptor binding (28).

Immunohistochemical Analysis of Periostin in lung

At 2 wks after initiation of hypoxic exposure, the lungs from hypoxia adapted and air

control rats were fixed in the distended state by infusion of 10% buffered formalin into the

pulmonary artery and trachea at 100 and 25 mmHg pressure, respectively, for 1 min, and then

placed in a bath of 4% paraformaldhyde for 24 hrs, paraffin embedded, and sectioned for

immunohistochemical examination of PN as previously described (4,26).

RNA extraction and Northern blot analysis

Lungs from 2-wk hypoxia adapted or air control rats or cultured PASMCs were

homogenized, and total RNA was extracted using the TRIZOL total RNA isolation reagent

(GibcoBRL, Life Technologies). Northern analysis was performed using a 32P-labeled selective

cDNA probes for PN and OPN which had been generated in our laboratory by reverse

transcription (RT) followed by the DNA polymerase chain reaction (PCR) using lung RNA as the

template, as previously described (13). A 32P-labeled 18S rRNA-oligonucleotide

(5=-ACGGTATCTGATCGTCTTCGAACC-3=) was used as the control probe to normalize data.

Autoradiographic signals were scanned with an optical densitometer (Model GS-670 Imaging

Densitometer, Bio-Rad, Hercules, CA). To estimate steady-state specific mRNA levels, PN and

OPN mRNA/18S rRNA ratios were determined by dividing the absorbance corresponding to the

specific cDNA probe hybridization by the absorbance corresponding to the 18S rRNA probe

hybridization. The results were expressed as the ratios of specific mRNA to 18S rRNA.

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Western blot analysis for Akt, GSK3, MEK1/2 and ERK1/2

To evaluate the effects of FGF-1 and ANG II on activities of the

phosphatidylinositol-3-kinase (PI3K)/Akt and the Ras/mitogen activated protein kinase

(MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathways, as well as selectivity of the

PI3K inhibitor LY294002 and the ERK kinase 1/2 (MEK1/2) inhibitor U0126, Western blot analysis

for phosphorylated Akt and GSK3 (an Akt substrate), as well as phosphorylated MEK1/2 and

ERK1/2 (p44/p42 MAPK), was performed using the PhosphoPlus Akt Antibody Kit and

Phospho-ERK1/2 Pathway Sample Kit (Cell Signaling Technology Inc., Beverly, MD),

respectively, as previously described (13). PASMCs were made quiescent by placing them in

medium containing 0.1% FBS for 24 hrs. For the study of signal transduction pathways, PASMCs

were pretreated with LY294002 (10 uM) or U0126 (5 uM) for 45 min before addition of FGF-1 (10

ng/ml) or ANG II (100 nM) to the medium and incubation for an additional 15 min.

After treatment with FGF-1 or ANG II, cells were lysed by the addition of 0.5 ml of ice-cold

lysis buffer containing (mM) NaCl 50, NaF 50, sodium pyrophosphate 50, EDTA 5, EGTA 5,

Na3VO4 2, phenylmethylsulfonyl fluoride 0.5, and HEPES 10 at pH 7.4 along with 0.1% Triton

X-100 and 10 ug/ml leupeptin, followed by immediate freezing on ethanol/dry ice. The cell lysates

were thawed on ice, scraped, sonicated, and centrifuged at 14,000x g at 4oC for 30 min. Cell

lysates (25 µg protein) were subjected to electrophoresis on a 10% SDS-polyacrylamide gel and

transferred to nitrocellulose membranes. The membranes were blocked for 2 hrs in 1% casein

(Hammarsten-prepared), 0.05% Tween, and 0.05% azide in PBS. Western blot analysis was

performed using anti-phospho-AKT (Ser473), anti-phospho-GSK3, anti-phospho-MEK1/2

(Ser217/221), anti-phospho-ERK1/2 (Thr202/Tyr204)-specific primary antibodies and a

horseradish peroxidase-conjugated goat anti-rabbit IgG. Immune complexes were detected by

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Phototop-HRP Western Detection Kit (Cell Signaling Technology Inc.). Autoradiograms exposed

in the linear range of film density were scanned using a densitometer (Model GS-670 Imaging

Densitometer, Bio-Rad).

Reagents

ANG II and the ANG II type 1 (AT-1) receptor antagonist losartan, human recombinant

FGF-1 and transcriptional inhibitor actinomycin D were purchased from Sigma Inc. (St. Louis,

MO). Selective rabbit anti-PN polyclonal antibody was provided by Drs. Lan Bo Chen and Meiru

Dai at Harvard Medical School (26). The FGF-1 receptor tyrosine kinase inhibitor PD-166866 was

provided by Parke-Davis Pharmaceutical Research, Division of Pfizer (17). The p38MAP and

p70S6 kinase activator anisomycin, the MEK1/2 inhibitors U0126 and PD98059, the PI3K

inhibitors LY-294002 and wortmannin, the p38MAP kinase inhibitor SB203580, the p70S6 kinase

inhibitor rapamycin, and the JNK (c-Jun NH2-terminal kinase) inhibitor SP600125 were purchased

from Calbiochem-Novabiochem Corp. (San Diego, CA).

Statistical analysis

Results were expressed as means ± SEM. Statistical analyses were carried out using the

SigmaStat package (Jandel Scientific Software, San Rafael, CA) on a PC. Statistical

comparisons of mRNA levels were performed with the one-way analysis of variance (ANOVA) or

unpaired t-test. If ANOVA results were significant, a post-hoc comparison among groups was

performed with the Newman-Keuls test. Differences were reported as significant if the P value

was < 0.05.

RESULTS

Effects of hypoxia on PN, and OPN expression in lungs and PASMCs

Chronic normobaric hypoxic exposure for 2 wks significantly increased steady state PN (a

2.2-fold increase) and OPN (a 2.7-fold increase) mRNA levels in rat lung (Figures 1A and 1B).

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Immunohistochemical analysis showed that 2-wk exposure to normobaric hypoxia increased PN

expression in pulmonary artery and bronchi of hypoxia-adapted rats compared with air controls

(Figure 2). PN was mainly expressed (brown stain) in medial SMCs and bronchial epithelial cells.

Exposure of growth-arrested PASMCs to 1% O2 (pO2=20-30 mmHg in culture media) for 6, 12 and

24 hrs resulted in a slight but significant increase in PN (a 40% increase) mRNA levels at 24 hrs,

but had no effect on OPN (Figures 1C and 1D). The effect of hypoxia on PN mRNA expression in

PASMCs in vitro was much less robust than that in intact lung in vivo. No significant cell death

(assessed by trypan blue [0.4%] exclusion) was observed in PASMCs exposed to hypoxia or

normoxia. These results suggest that other mediators, such as hypoxia-responsive growth

factors, may be needed to increase expression of PN and OPN in isolated PASMCs in vitro.

Dose- and time- dependent effects of FGF-1 and ANG II on PN and OPN expression in

PASMCs

Quantitative Northern blot analysis demonstrated that PN and OPN mRNA expression

increased in a dose-dependent fashion (range 0.1-20 ng/ml) in PASMCs treated with FGF-1 for

24 hrs (Figures 3A and 3B). The threshold concentration was 0.1 ng/ml (~ 6.7 pM), and the

largest effect was observed at doses of 10 and 20 ng/ml for PN (a 2.1-fold increase) and OPN (a

7.6-fold increase), respectively. The time course for enhancement of PN and OPN expression

with FGF-1 was examined at a concentration of 10 ng/ml. Significant increases in PN and OPN

mRNA levels in PASMCs were observed after 3 hrs of FGF-1 treatment (Figures 3C and 3D). PN

and OPN mRNA levels increased progressively over the 24 hr period of FGF-1 exposure.

Similarly, PN and OPN mRNA expression increased in a dose-dependent fashion (range

0.1-100 nM, or 0.1-100 ng/ml) in PASMCs treated with ANG II for 24 hrs (Figures 4A and 4B). The

threshold concentration was 0.1 nM (or 0.1 ng/ml), and the largest effect (a 3.2-fold increase of

PN and a 6.5-fold increase of OPN) was observed at the maximal dose of 100 nM. The time

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course for enhancement of PN and OPN gene expression with ANG II was examined at a

concentration of 100 nM. Significant increases in PN and OPN mRNA levels in PASMCs were

observed after 3 hrs of ANG II treatment (Figures 4C and 4D).

Effects of receptor inhibition on FGF-1 or ANG II-induced upregulation of PN and OPN

expression

PASMCs were pretreated with PD-166866 (5 µM), a potent and selective inhibitor of

FGF-1 receptor tyrosine kinase (17), or losartan (10 mM), a selective ANG II AT1 receptor

antagonist, for 45 min before FGF-1 (10 ng/ml) or ANG II (100 nM) was added to the medium for

an additional 24 hrs of incubation. PD-166866 or losartan completely blocked FGF-1 or ANG

II-induced upregulation of PN and OPN mRNA expression (Figures 5A-5D), indicating the

receptor dependence of growth factor stimulation. FGF-1-induced upregulation of PN and OPN

mRNA expression was also completely blocked by pretreatment of PASMCs with actinomycin D

(5 uM) (data not shown), indicating that transcription is essential to FGF-1-stimulated PN and

OPN mRNA expression.

Effects of signal transduction pathway inhibitors on FGF-1 or ANG II-induced upregulation

of PN and OPN expression

PASMCs were pre-treated with inhibitors for 45 min before FGF-1 (10 ng/ml) or ANG II

(100 nM) was added to the medium for an additional 24 hrs of incubation. LY294002 (5 uM) or

wortmannin (20 µM), PI3K inhibitors, as well as U0126 (5 uM) or PD98058 (20 uM), MEK1/2

inhibitors, completely blocked the FGF-1 or ANG II induced upregulation of PN mRNA expression

(Figures 5A and 5C). The MEK1/2 inhibitors U0126 (5 uM) or PD98059 (20 uM) also completely

blocked FGF-1 or ANG II-induced upregulation of OPN mRNA expression (Figures 5B and 5D).

In contrast, the PI3K inhibitors LY294002 (5 uM) or wortmannin (20 uM) did not alter the

stimulatory effect of FGF-1 or ANG II on OPN mRNA expression.

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The effects of signaling pathway inhibitors on PN and OPN mRNA expression in PASMCs

exposed to normoxia (control) or hypoxia for 24 hrs are shown in Figures 5E and 5F. PASMCs

were pretreated with inhibitors for 45 min before exposing them to hypoxia (1% O2) for an

additional 24 hrs. Inhibition of PI3K (by LY294002) and p38MAP kinase (by SB203580)

decreased PN mRNA expression; and inhibition of PI3K (by LY294002), MEK1/2 (by PD98059)

and JNK (by SP600125) decreased OPN mRNA expression in PASMCs exposed to either

normoxia or hypoxia.

Western blot analysis confirmed that FGF-1 or ANG II significantly increased activation of

PI3K and MEK1/2 within 15 min in PASMCs. The FGF-1 or ANG II-stimulated phosphorylation of

Akt and GSK3 was blocked by pretreatment with the selective PI3K inhibitor LY294002 (Figure

6A), and the FGF-1 or ANG II-stimulated phosphorylation of ERK1/2 was blocked by pretreatment

with the selective MEK1/2 inhibitor U0126 (Figure 6B). LY294002 did not block FGF-1 or ANG

II-stimulated phosphorylation of ERK1/2 (data not shown) and U0126 did not block FGF-1 or ANG

II-stimulated phosphorylation of Akt (data not shown) or MEK1/2 (Figure 6B), indicating the

selectivity of these signaling pathway inhibitors.

Effects of p70S6K and p38MAPK activation on PN and OPN expression in PASMCs

Quiescent PASMCs were treated with FGF-1 (10 ng/ml) or the p38MAPK and p70S6K

activator anisomycin (1 ug/ml) for 24 hrs with or without pretreatment (45 min) with selective

inhibitors of intracellular signaling, including rapamycin (100 nM, a p70S6K inhibitor), SB203580

(10 uM, a p38MAPK inhibitor), or SP600125 (20 uM, a JNK inhibitor). Both FGF-1 and

anisomysin induced PN expression were blocked by p70S6K and p38MAPK inhibitors, but not by

the JNK inhibitor (Figures 7A and 7B). In contrast, the FGF-1 induced OPN expression was

blocked by the JNK inhibitor, but not by p70S6K and p38 MAPK inhibitors (Figure 7C).

Anisomycin alone slightly but significantly decreased OPN expression in PASMCs, and

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rapamycin, SB203580 and SP600125 did not alter the effects of anisomycin on OPN expression

in PASMCs (Figure 7D).

DISCUSSION

The present study is the first to demonstrate increased expression of PN in lung and

pulmonary arteries of rats adapted to hypoxia as part of a generalized ECM response to hypoxic

stress, which also included increased OPN mRNA expression. We have previously reported that

hypoxia induces acute pulmonary vasoconstriction, chronic pulmonary hypertension, and

pulmonary vascular remodeling characterized by medial hypertrophy and marked increased

medial-wall thickness of small pulmonary arteries (50-100 um) in the rat (3,4). The present results

demonstrate localization of hypoxia-induced PN expression to PASMCs of hypertrophic

pulmonary arteries. This finding, coupled with the observation that PN expression increases in

parallel with OPN in lung of hypoxia adapted rats, suggests an involvement of PN in ECM

formation and pulmonary vascular hypertrophy and remodeling in response to chronic hypoxia

exposure.

Another novel finding of present study is the demonstration that the hypoxia-responsive

growth factors FGF-1 and ANG II stimulate PN expression in PASMCs in vitro. The rapid

increases in steady-state PN mRNA levels observed in response to low concentrations of FGF-1

and ANG II suggest that these growth factors, in addition to hypoxia per se, could be endogenous

physiological stimulators of PN gene expression in PASMCs. The FGFs (at least isoforms FGF-1,

-2, and -7) and ANG II are expressed in lung and play important roles in diverse aspects of

pulmonary development and growth, including epithelial cell, VSMC, and myofibroblast

proliferation, differentiation, angiogenesis and vascular constriction (14,19). It has been

hypothesized that these growth factors may contribute to hyperproliferation of PASMCs and

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muscularization of the pulmonary vasculature in hypoxia-adapted animals (14,19).

OPN is abundantly expressed in a range of lung diseases (16,25). Bronchial epithelial

cells and alveolar macrophages in normal lung express OPN (2), and its expression is greatly

amplified in epithelium, alveolar and interstitial macrophages, fibroblasts, and endothelial cells

and VSMCs in injured and diseased lungs (16). Microarray analysis (7) has demonstrated

increased OPN mRNA expression in the lungs of rats and mice exposed to chronic hypobaric

hypoxia for 1 and 3 wks. These data provide indirect evidence that OPN may play a role in the

pathogenesis of pulmonary vascular disease.

The current findings extend previous observations of the effects of hypoxia exposure on

OPN expression in vivo and in vitro by elucidating a role for hypoxia-responsive growth factors on

this process. While chronic hypoxia exposure per se increased OPN gene expression in lung of

hypoxia adapted rats, FGF-1 and ANG II, but not hypoxia per se, increased OPN mRNA

expression in PASMCs in vitro. This result is similar to our previous findings that ANP clearance

receptor (NPR-C) expression is downregulated in the lung of rats and mice exposed to hypoxia

but not in PASMCs cultured under hypoxic conditions (22). The absence of a direct effect of

hypoxia on OPN mRNA levels in PASMCs in vitro differs from the result of Sodhi et al. (20), who

found a biphasic increase in OPN mRNA and protein levels with exposure to 3% O2 (maximal at

2 hrs, no effect at 6 and 12 hrs, and slight increase at 24 hrs) in rat mesangial cells and aortic

SMCs in vitro. In the current study, we did not sample time points earlier than 6 hrs, but found no

effect of hypoxia (1% O2) on OPN expression in PASMCs at 6, 12 or 24 hrs. Apparent differences

between these results may relate to differences in tissues of origin of the SMCs (kidney/aorta vs.

lung), duration of hypoxia exposure or the severity of hypoxia. The observations that growth

factors time and dose-dependently induced increases in OPN mRNA expression in PASMCs in

vitro and that FGF-1, FGF-2 (13), and ANG II (19) expression increased in lung of rats adapted to

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chronic hypoxia provide additional evidence for a functional role for hypoxia-responsive growth

factors in stimulating ECM formation and remodeling in the hypoxic lung.

FGF-1 binds to any of 4 receptor isoforms (FGFR-1, -2, -3, and -4), and ANG II binds to

at least 2 ANG receptors (AT-1 and AT-2). FGFR-1 and AT-1 are the predominant receptor

isoforms in adult VSMCs (1,27). PD 166866 is a potent (nanomolar range) and highly selective

small molecule inhibitor of FGFR-1-dependent signaling that does not perturb signals induced by

PDGFR, EGFR, Src, MEK, PKC, or CDK4 (17), and losartan is highly potent and selective

non-peptide AT-1 receptor antagonist. The present study demonstrated that PD166866 or

losartan effectively inhibited PASMC expression of PN and OPN mRNA following FGF-1 or ANG

II treatment. This result indicates that FGF-1 or ANG II stimulation of PN or OPN mRNA

expression in PASMCs in vitro is mediated in an FGFR-1 or AT-1-dependent manner.

Multiple intracellular signaling pathways are activated by FGFs and ANG II (8,9) (Figure

8). Activation of the PI3K-mediated Akt and p706K, and Ras-mediated MEK1/2/ERK1/2,

p38MAPK, and JNK pathways are necessary for the mitogenic activity of FGFs and ANG II in

VSMCs (8,15,24). In this study, we investigated the involvement of PI3K-mediated and

Ras-mediated signaling pathways in the regulation of PN expression in PASMCs. Inhibition of

PI3K by LY294002 or wortmannin, p70S6K by rapamycin, MEK1/2 by U0128 or PD98059,

p38MAPK by SB203580, significantly inhibited FGF-1 or ANG II-induced PN mRNA expression.

However, inhibition of JNK by SP600125 had no effect on PN mRNA expression under FGF-1 or

anisomycin-treated conditions. These results suggest that the PI3K/p70S6K,

Ras/MEK1/2/ERK1/2 and Ras/p38MAPK pathways, but not the Ras/JNK pathway, are involved in

FGF-1 or ANG II-stimulated PN mRNA upregulation in PASMCs (Figure 8).

This study elucidates for the first time the signaling pathways by which FGF-1 and ANG

II stimulate OPN mRNA expression in PASMCs (Figure 8). We observed that FGF-1 and ANG II

13

potently and time- and dose-dependently stimulate expression of OPN mRNA in PASMCs in vitro

through the activation of the Ras/MEK1/2/ERK1/2 and Ras/JNK, but not the PI3K or p38MAPK

signaling pathways. These results are consistent with our previous observation that FGF

ligand-activated FGFR-1 plays a significant role in upregulation of OPN gene expression at the

transcriptional level via activation of FGFR-1 and subsequent activation of the

Src/Ras/MEK/MAPK pathway, but not the PI3K and p38MAPK pathways, in rat aortic SMCs in

vitro (12).

In summary, the present study provides the first evidence that the PN mRNA expression

is increased in lung of hypoxia-adapted animals and in cultured PASMCs exposed to hypoxia, and

that the hypoxia-responsive growth factors FGF-1 and ANG II play significant roles in upregulation

of PN and OPN expression at the transcriptional level via activation of FGFR-1 and AT-1

receptors, respectively. We also demonstrated that activation of the PI3K/Akt/p70S6K,

Ras/MEK1/2/ERK1/2 and Ras/p38MAPK signaling pathways, but not the Ras/JNK pathway, by

FGF-1 or ANG II increases PN gene expression in PASMCs in vitro. In contrast, the FGF-1 and

ANG II-stimulated OPN expression in PASMCs is mediated through activation of

Ras/MEK1/2/ERK1/2 and Ras/JNK, but not the PI3K/Akt/p70S6K or Ras/p38MAPK signaling

pathways. These differences in signaling suggest that PN and OPN may play different roles in

pulmonary vascular remodeling.

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ACKNOWLEDGMENTS

This work was supported in part by National Heart, Lung, and Blood Institute Grants

HL-44195, HL-50147, HL-45990, HL-07457 and HL56046.

15

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18. Sasaki H, Yu CY, Dai M, Tam C, Loda M, Auclair D, Chen LB, Elias A. Elevated serum

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19

FIGURE LEGENDS

Figure 1. (A and B), effects of 2-wk hypoxic exposure (10% O2, 1 atm) on steady-state periostin

(PN) and osteopontin (OPN) mRNA expression in rat lung in vivo. Air controls were

rats exposed to room air during the same period. (C and D), effects of 6 to 24-hr

hypoxic exposure (1% O2) on steady-state PN and OPN mRNA expression in

pulmonary arterial smooth muscle cells (PASMCs) in vitro. Growth arrested PASMCs

were exposed to hypoxia for 6, 12, and 24 hr before being harvested. Normoxic

controls were PASMCs cultured in 0.1% FBS medium for 24-hr and exposed to 21%

O2 for an additional 24 hrs. Numbers in parentheses are the number of rats or plates

of PASMCs contributing data to each group. Northern blot analysis was carried out

with 15 ug of total RNA extracted from each lung or plates of PASMCs. The Northern

blot membrane was probed with PN and OPN cDNAs, and 18S rRNA oligonucleotide

sequentially. The mRNA data were normalized by 18S rRNA to correct for variations in

RNA loading. Results are means±SE. * p<0.05 vs. respective air or normoxia control

groups by unpaired t-test (in vivo study) or 1-way ANOVA (in vitro study).

Figure 2. Representative light micrographs of small pulmonary arteries (PA; ~100 um) from rats

exposed to chronic hypoxia (10% O2) for 2 wks or room air. Lung sections were

immunostained (brown stain) with selective rabbit anti-PN polyclonal antibody.

Sections were counterstained with hematoxylin and eosin. BR-bronchi.

Figure 3. Dose- and time-dependent effects of FGF-1 on regulation of steady-state PN (A and

C) and OPN (B and D) mRNA expression in PASMCs in vitro. Growth arrested

PASMCs were exposed to different concentrations of FGF-1 for 24 hr (A and B) or 10

20

ng/ml FGF-1 for 3, 6, 12 and 24 hrs (C and D) before being harvested. Controls were

PASMCs cultured in 0.1% FBS medium for 24-hr and treated with vehicle for an

additional 24 hrs. The mRNA loading was normalized by 18S rRNA. Numbers in

parentheses are the number of plates contributing data to each group. Results are

means±SE. * p<0.05 vs. control group by 1-way ANOVA.

Figure 4. Dose- and time- dependent effects of ANG II on regulation of steady-state PN (A and

C) and OPN (B and D) mRNA expression in PASMCs. Growth arrested PASMCs were

exposed to different concentrations of ANG II for 24 hr (A and B) or 100 nM ANG II for

3, 6, 12 and 24 hrs (C and D) before being harvested. Controls were PASMCs

cultured in 0.1% FBS medium for 24-hr and treated with vehicle for an additional 24

hrs. The mRNA loading was normalized by 18S rRNA. Numbers in parentheses are

the number of plates contributing data to each group. Results are means±SE. *

p<0.05 vs. control group by 1-way ANOVA.

Figure 5. (A and B), effects of the FGF-1 receptor antagonist PD166866 and signaling pathway

inhibitors on the upregulation of PN and OPN mRNA expression in PASMCs by

FGF-1; (C and D), effects of the AT-1 receptor antagonist losartan and signaling

pathway inhibitors on the upregulation of PN and OPN mRNA expression in PASMCs

by ANG II; and (E and F), effects of signaling pathway inhibitors on PN and OPN

mRNA expression in PASMCs exposed to normoxia or hypoxia (1% O2) for 24 hrs.

Controls (or vehicles) were growth-arrested PASMCs cultured in 0.1% FBS medium.

PASMCs were pretreated with PD166866 (5 uM) or losartan (10 mM) for 45 min

before exposing to FGF-1 or ANG II for 24 hr. Other experimental groups were

21

PASMCs pretreated with the PI3K inhibitors LY294002 (LY, 10 uM) or wortmannin (20

uM), as well as the MEK1/2 inhibitors U-0126 (5 uM) or PD98059 (20 uM), for 45 min

before adding FGF-1 (10 ng/ml) (A and B) or ANG II (100 nM) (C and D) to the medium

or before exposing to hypoxia (E and F). An additional 24-hr incubation was carried

out before harvesting for analysis. The mRNA loading was normalized by 18S RNA.

Numbers in parentheses are the number of plates contributing data to each group.

Results are mean±SE. * p<0.05 vs. respective control groups; # p<0.05 vs. respective

FGF-1 or ANG II groups; and & p<0.05 vs. respective vehicle control groups by 1-way

ANOVA. Rapamycin (100 nM, a p70S6K inhibitor), SB203580 (SB, 10 uM, a p38

MAPK inhibitor), and SP600125 (20 uM, a JNK inhibitor).

Figure 6. A: effects of the selective PI3K inhibitor LY294002 (10 uM) on FGF-1 or ANG

II-stimulated PI3K in PASMCs. Control was growth-arrested PASMCs cultured in 0.1%

FBS medium. Quiescent PASMCs were stimulated with FGF-1 (10 ng/ml) or ANG II

(100 nM) for 15 min without or with pretreatment with LY294002 (10 uM, 45 min). Cell

lysates (25 ug) were size fractionated by SDS-PAGE, and Western blot analysis was

performed with or without anti-phospho-Akt or anti-phospho-GSK3 specific antibodies.

LY294002 selectively blocked the FGF-1 or ANG II-induced phosphorylation of Akt

and GSK3.

B: effects of the selective MEK1/2 inhibitor U0126 (5 uM) on FGF-1 or ANG

II-stimulated MAP kinase in PASMCs. Quiescent PASMCs were stimulated with

FGF-1 (10 ng/ml) or ANG II (100 nM) for 15 min without or with pretreatment with

U0126 (5 uM, 45 min). Cell lysates (25 ug) were size fractionated by SDS-PAGE, and

Western blot analysis was performed with anti-phospho-MEK1/2 or

22

anti-phospho-ERK1/2 specific antibodies. U0126 selectively blocked the FGF-1 or

ANG II-induced phosphorylation of ERK1/2, but not the phosphorylation of MEK1/2.

Figure 7. PN (A and B) and OPN (C and D) mRNA expression in PASMCs treated with FGF-1

(10 ng/ml) or the p38MAPK and p70S6K activator anisomycin (1 ug/ml) for 24 hrs in

the presence or absence of selective inhibitors intracellular signaling, including

rapamycin (100 nM, a p70S6K inhibitor), SB203580 (10 uM, a p38 MAPK inhibitor), or

SP600125 (20 uM, a JNK inhibitor). Quiescent PASMCs were pretreated with

inhibitors for 45 min before adding FGF-1 or anisomycin. Controls were PASMCs

cultured in 0.1% FBS medium for 24-hr and treated with vehicle for an additional 24

hrs. Representative Northern blots are shown at top. The mRNA loading was

normalized by 18S rRNA. Numbers in parentheses are the number of plates

contributing data to each group. Results are means±SE. * p<0.05 vs. control group;

and # p<0.05 vs. respective FGF-1 or anisomycin groups by 1-way ANOVA.

Figure 8. A simplified schematic illustration of the specific action of various signaling pathway

inhibitors and the intracellular signaling pathways that mediate FGF-1 or

ANGII-induced PN and OPN expression in cultured PASMCs. RTK - receptor tyrosine

kinase, GPCR - G-protein coupled receptor.

23

Figure 1

Air Hypoxia0

1

2

3

(6)

Ost

eopo

ntin

mR

NA

/18S

RN

A (6) *OsteopontinB.

Air Hypoxia0

1

2

3

(6)

Per

iost

inm

RN

A/1

8S R

NA

(6)*

PeriostinA.

6 hr 12 hr 24 hr0

1

2

3

(12)

Per

iost

inm

RN

A/1

8S R

NA

(5) (3)

(15)*

PeriostinC.

Hypoxia (1% O2)

Normoxia 6 hr 12 hr 24 hr0

1

2

3

(12)

Ost

eopo

ntin

mR

NA

/18S

RN

A

(5) (3)

OsteopontinD.

Hypoxia (1% O2)

Normoxia

(15)

Air Hypoxia0

1

2

3

(6)

Ost

eopo

ntin

mR

NA

/18S

RN

A (6) *OsteopontinB.

Air Hypoxia0

1

2

3

(6)

Per

iost

inm

RN

A/1

8S R

NA

(6)*

PeriostinA.

6 hr 12 hr 24 hr0

1

2

3

(12)

Per

iost

inm

RN

A/1

8S R

NA

(5) (3)

(15)*

PeriostinC.

Hypoxia (1% O2)

Normoxia 6 hr 12 hr 24 hr0

1

2

3

(12)

Ost

eopo

ntin

mR

NA

/18S

RN

A

(5) (3)

OsteopontinD.

Hypoxia (1% O2)

Normoxia

(15)

24

Air Control Hypoxia

Figure 2

PA

BR

PA

BR

25

Figure 3

0 0.1 1 10 200

2

4

6

8

(12)O

steo

pont

in m

RN

A/1

8S R

NA

FGF-1 (ng/ml)

(3)

(3)

(12)

(3)OsteopontinB.

0 0.1 1 10 200

1

2

3

(12)

Per

iost

in R

NA

/18S

RN

A

FGF-1 (ng/ml)

(3)

(3)

(12)(3)

PeriostinA.

Control 3 hr 6 hr 12 hr 24 hr0

1

2

3

(9)

Per

iost

in m

RN

A/1

8S R

NA

FGF-1 (10 ng/ml)

(3)

(3)(3)

(9)

PeriostinC.


2

4

6

8

(12)

Ost

eopo

ntin

mR

NA

/18S

RN

A

FGF-1 (10 ng/ml)

(3)

(3)(3)

(12)

OsteopontinD.

26

Figure 4

0 0.1 1 10 1000

2

4

6

8

10

(6)O

steo

pont

in m

RN

A/1

8S R

NA

ANG II (nM)

(3)(3)

(3)(6)

OsteopontinB.

0 0.1 1 10 1000

1

2

3

4

(6)

Per

iost

in m

RN

A/1

8S R

NA

ANG II (nM)

(3)(3)

(3)

(6)

PeriostinA.


1

2

3

4

(6)

Per

iost

in m

RN

A/1

8S R

NA

ANG II (100 nM)

(3)

(3)

(3)

(6)

PeriostinC.


2

4

6

8

10

(6)

Ost

eopo

ntin

mR

NA

/18S

RN

A

ANG II (100 nM)

(3)

(3)(3)(6)

OsteopontinD.

27

Figure 5

Control FGF-1 PD166866 LY WM U0126 PD980590

1

2

3

(9)

Per

iost

inm

RN

A/1

8S R

NA

FGF-1 (10 ng/ml)

(9)

(4)

(3)

Periostin

(4)

## #

A.

(3)#

(3)#


2

4

6

8

(9)

Ost

eopo

ntin

mR

NA

/18S

RN

A

FGF-1 (10 ng/ml)

(9)

(3)

(3)

Osteopontin

(4)#

B.

(3)#

(3)#

Control AII Losartan LY WM U0126 PD980590

1

2

3

4

5

(12)

Per

iost

inm

RN

A/1

8S R

NA

ANG II (100 nM)

(12)

(3) (3)

Periostin

(3)## #

C.

(3)#

(3)


4

8

12

16

(12)

Ost

eopo

ntin

mR

NA

/18S

RN

A

ANG II (100 nM)

(12)(3) (3)

Osteopontin

(3)

D.

(3)##

(3)

Vehicle LY PD98059 Rapamycin SB SP6001250

1

2

(9)

Per

iost

inm

RN

A/1

8S R

NA

(3)

(3)

(3)

(5)

E.

(5)

(3)&

*Normoxia Hypoxia

(3)(3) (5)

(3)(3)

Periostin

&

& &


1

2

(9)

Ost

eopo

ntin

mR

NA

/18S

RN

A

(3)

(3)

(3)(5)

F.

(5)(3)&

Normoxia Hypoxia

(3)(3)

(5)

(3)

(3)

Osteopontin

&& &

& &

#


1

2

3

(9)

Per

iost

inm

RN

A/1

8S R

NA

FGF-1 (10 ng/ml)

(9)

(4)

(3)

Periostin

(4)

## #

A.

(3)#

(3)#


2

4

6

8

(9)

Ost

eopo

ntin

mR

NA

/18S

RN

A

FGF-1 (10 ng/ml)

(9)

(3)

(3)

Osteopontin

(4)#

B.

(3)#

(3)#


1

2

3

4

5

(12)

Per

iost

inm

RN

A/1

8S R

NA

ANG II (100 nM)

(12)

(3) (3)

Periostin

(3)## #

C.

(3)#

(3)


4

8

12

16

(12)

Ost

eopo

ntin

mR

NA

/18S

RN

A

ANG II (100 nM)

(12)(3) (3)

Osteopontin

(3)

D.

(3)##

(3)


1

2

(9)

Per

iost

inm

RN

A/1

8S R

NA

(3)

(3)

(3)

(5)

E.

(5)

(3)&

*Normoxia Hypoxia

(3)(3) (5)

(3)(3)

Periostin

&

& &


1

2

(9)

Ost

eopo

ntin

mR

NA

/18S

RN

A

(3)

(3)

(3)(5)

F.

(5)(3)&

Normoxia Hypoxia

(3)(3)

(5)

(3)

(3)

Osteopontin

&& &

& &

#

28

Figure 6

Control FGF-1LY294002+ FGF-1 ANGII

LY294002+ ANGII

Phospho-Akt

Phospho-GSK3

Control FGF-1U0126

+ FGF-1 ANGIIU0126

+ ANGII

Phospho-ERK1/2

Phospho-MEK1/2

A.

B.

29

Figure 7

Control FGF-1 Rapamycin SB203580 SP6001250

1

2

3

4

(9)

Ost

eopo

ntin

mR

NA

/18S

RN

A

FGF-1 (10 ng/ml)

(9) (5) (5)

Osteopontin

(5)

*

#

* *


0.8

1.6

2.4

(9)

Per

iost

inm

RN

A/1

8S R

NA

FGF-1 (10 ng/ml)

(9)

(5)(5)

Periostin

(5)#

#

* *

Control Anisomycin Rapamycin SB203580 SP6001250

1

2

(9)

Ost

eopo

ntin

mR

NA

/18S

RN

A

Anisomycin (1 ug/ml)

(5)(5) (5)

Osteopontin

(5)

* * **

Control FGF-1 Rapamycin SB203580 SP600125 Control Anisomycin Rapamycin SB203580 SP600125

Anisomycin (1 ug/ml)FGF-1 (10 ng/ml)

- OPN -

- PN -


10

20

30

40

(9)P

erio

stin

mR

NA

/18S

RN

A


(5)

(5)

(5)

Periostin

(5)

*

*

*

*#

#


1

2

3

4

(9)

Ost

eopo

ntin

mR

NA

/18S

RN

A

FGF-1 (10 ng/ml)

(9) (5) (5)

Osteopontin

(5)

*

#

* *


0.8

1.6

2.4

(9)

Per

iost

inm

RN

A/1

8S R

NA

FGF-1 (10 ng/ml)

(9)

(5)(5)

Periostin

(5)#

#

* *


1

2

(9)

Ost

eopo

ntin

mR

NA

/18S

RN

A


(5)(5) (5)

Osteopontin

(5)

* * **

Control FGF-1 Rapamycin SB203580 SP600125 Control Anisomycin Rapamycin SB203580 SP600125

Anisomycin (1 ug/ml)FGF-1 (10 ng/ml)

- OPN -

- PN -


10

20

30

40

(9)P

erio

stin

mR

NA

/18S

RN

A


(5)

(5)

(5)

Periostin

(5)

*

*

*

*#

#

30

Figure 8

PI3K

GPCR

p70S6K

Src

Raf

MKK1/2

JNKERK1/2p38

Periostin expression Osteopontin expression

MKK3/6 MKK4/7

LY294002Wortmannin

Rapamycin

U0126 PD98059

SB203580 SP600125

( - )

Anisomycin

FGF AII

RTK

Ras

( - )( - )( + )

( - )

( - )

PI3K

GPCR

p70S6K

Src

Raf

MKK1/2

JNKERK1/2p38

Periostin expression Osteopontin expression

MKK3/6 MKK4/7

LY294002Wortmannin

Rapamycin

U0126 PD98059

SB203580 SP600125

( - )

Anisomycin

FGF AII

RTK

Ras

( - )( - )( + )

( - )

( - )

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