Introduction

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Mitogen-activated protein kinases (MAPKs) are a group of serine/threonine kinases that play an important role in vascular smooth-muscle growth and proliferation. There are three families of MAPKs that have been previously identified in various cell types. These families include p46-p54 Jun-N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), p44 extracellular signal-regulated protein kinase (ERK)₁ and p42ERK₂, and p38 high-osmolarity glycerol response 1 (HOG1) kinase. A new member of the family of MAPKs, big MAPK (BMK)-1, with the molecular weight of 110 kD, has recently been cloned (1, 2). MAPKs can be activated by a variety of stimuli, including mitogens, growth factors, hormones, oxidants, stress factors, etc. (3, 4). Evidence has been presented to show that activation of p44/p42 ERKs is a key step in the kinase cascade leading to cell proliferation in response to growth factors, agonists, and hormones (5). JNK has been shown to mediate signals in response to cytokines and environmental stress (8). Hyperosmolar stress and endotoxin are strong stimuli specifically for p38 kinase (12, 13). BMK-1 appears to participate in growth factor and redox-sensitive pathways, but not in pathways induced by agonists such as phorbol ester and angiotensin II (2, 14). Activation of MAPKs results in phosphorylation of several nuclear transcription factors, such as Egr-1, c-Jun, Elk-1, activated transcription factor (ATF)-2, and c-Fos, and in stimulation of cell growth and proliferation (3, 4).

A well documented phenomenon seen with chronic hypoxia is that low alveolar PO₂ causes pulmonary vasoconstriction that leads to pulmonary hypertension with significant arterial remodeling (15). A predominate feature seen with hypoxia-induced pulmonary hypertension is increases in thickness of intima, media, and adventitia layers of pulmonary artery (18, 19). The molecular/cellular mechanisms involved in hypoxic pulmonary arterial remodeling have been investigated for decades and significant progress has been made (19, 20). However, the role that MAPKs play in hypoxia-induced remodeling of pulmonary arteries has not been addressed. In the present study, the effects of hypoxia on activation of JNK, ERK, and p38 kinase were investigated. The study also investigated the signaling pathway of the upstream and downstream factors of MAPKs to determine the possible mechanisms that are involved in hypoxia-induced activation of MAPKs.

	Materials and Methods

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Materials

Reagents, except as specified below, were from Sigma (St. Louis, MO). Polyclonal anti-phospho-MAPK was purchased from New England Biolabs (Beverly, MA). Polyclonal anti-ERK₁, anti-JNK₁, anti-p38, anti-c-JUN/activator protein (AP)-1 antibodies, ATF-2, and protein A/G agarose macro beads were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-phospho- JNK, anti-phospho-ERK, and anti-phospho-p38 kinase were also purchased from Santa Cruz Biotechnology. Human recombinant c-Jun was from Promega (Madison, WI). Myelin basic protein (MBP) was from Sigma. Horseradish peroxidase (HRP)-conjugated antirabbit and antimouse immunoglobulin (Ig) G were purchased from Bio-Rad Laboratories (Hercules, CA). The Vectastain Elite ABC kit and peroxidase substrate kit were from Vector Labs, Inc. (Burlingame, CA). All reagents used for RNA isolation were of molecular biology grade. The RNA extraction solution, TRI reagent, which was used to extract total RNA, was purchased from Molecular Research Center, Inc. (Cincinnati, OH). The complementary DNAs (cDNAs) of c-jun, c-fos, egr-1, platelet-derived growth factor (PDGF) receptor B, and Chinese hamster ovary B (CHOB) were from American Type Culture Collection (Rockville, MD). Vascular endothelial growth factor (VEGF) cDNA was a generous gift from Genentech, Inc. (San Francisco, CA); [³²P]--deoxycytidine triphosphate (dCTP), [³²P]--adenosine triphosphate (ATP), and enhanced chemiluminescence (ECL) solution were purchased from Amersham Pharmacia Biotech (Piscataway, NJ).

Chronic Hypoxia Model and Pulmonary Artery Preparation

Male Sprague-Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, IN), weighing 275 to 300 g, were placed in a normobaric hypoxia chamber containing 10% O₂ (75 to 80 torr) for 1, 3, 7, or 14 d (11). The oxygen tension in the hypoxia chamber was monitored by a Beckman O₂ analyzer (Model C-2) and maintained at 15% (100 to 110 torr) during the first 8 h of exposure, then maintained at 10% (75 to 80 torr) for the remaining exposure time. Age- and size-paired rats that breathed room air served as the control group. Daily animal maintenance (feeding and cleaning) was carried out without interruption of the exposure through double ports in the chamber. At the end of the exposure period, each animal was anesthetized intraperitoneally with sodium pentobarbital (60 mg/kg body weight) and exsanguinated in the hypoxia chamber. Hearts and lungs were removed from the thoracic cavity and quickly frozen in liquid nitrogen.

Pulmonary arteries, including main trunk plus right and left branches, were isolated in ice-cold phosphate-buffered saline (PBS) containing 120 mM NaCl, 2.7 mM KCl, and 10 mM phosphate buffer. The isolated pulmonary arteries were subjected to measurement of tissue weight; or fixed in 10% formalin for histology study; or homogenized for MAPK activity assay, Western blot, and/or Northern blot analysis. To measure the wet tissue weight, the isolated pulmonary arteries were gently dried on paper towels and then weighed.

Hematoxylin and Eosin Staining and Immunohistochemistry

The pulmonary arteries of the main trunk plus the left and right branches from control rats (breathing room air for 14 d) and hypoxic rats (breathing 10% O₂ for 14 d) were isolated. Photographs of the fresh pulmonary arteries (Figure 1) were taken to show the tissue mass changes induced by hypoxia. The pulmonary arteries were fixed in 10% formalin for at least 24 h. Paraffin slices, 10 µm thick, were stained with hematoxylin and eosin. Pictures of the slices were taken under the microscope to show the structural changes of pulmonary arterial walls in response to hypoxia.

View larger version (69K): [in this window] [in a new window]   Figure 1.   Hypoxia-induced pulmonary arterial remodeling. Male Sprague-Dawley rats (275 to 300 g) were placed in a normobaric hypoxia chamber (PIO2 = 10% O2) for 14 d. Age- and size-matched controls breathed room air (14 d). (A) Pictures of extrapulmonary vessels (trunk plus right and left branches) from control and hypoxic rats (1:1 image ratio). (B) Microscopic study of pulmonary arterial walls from control and hypoxic rats. Bar: 250 microns.

To determine whether MAPK activation is associated with hypoxia-induced arterial remodeling, immunohistochemistry was used. Control and hypoxic lungs were removed and the fixative (3% formaldehyde in PBS) was infused into the lung via the trachea. The tissue was then immersed in fixative at 4°C for greater than 24 h. The tissues were dehydrated, embedded in paraffin, and sectioned at a thickness of 10 microns. Sections were mounted on slides, rehydrated, and immunostained with either rabbit anti-ERK₁ at 1:100 dilution or rabbit anti-phospho-MAPK at 1:50 dilution. Blocking, antibody treatment, and color development were done using the Vectastain Elite ABC kit and peroxidase substrate kit as per instructions (Vector Labs Inc.). Each slide had two sections, one of which was not treated with primary antibody to serve as a control. After color development, the sections were rinsed, counterstained with Harris Hematoxylin, dehydrated, and mounted. Digital images were obtained with a color camera and scaled and annotated with Photoshop.

Measurement of JNK, ERK, and p38 Tyrosine Phosphorylation

Frozen pulmonary arteries were homogenized in a cold lysis buffer composed of 20 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (pH 7.4), 80 mM -glycerophosphate, 1 mM ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid, 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM dithiothreitol (DTT), 0.2 mM Na₃VO₄, and 0.1 mM phenylmethylsulfonyl fluoride. The homogenate was subjected to sonication (10 s, three times), followed by centrifugation for 30 min at 12,000 × g to remove cellular debris, and the protein concentration of each supernatant was determined. Equal amounts of protein (10 µg) from each sample were resolved on a 10% polyacrylamide gel by electrophoresis. Proteins were transferred to a nitrocellulose membrane. The membranes were blocked for 1 h at room temperature with 2% bovine serum albumin (BSA) in 20 mM Tris, 500 mM NaCl, and 0.05% Tween 20. The blots were incubated with 1:1,000 primary antibodies (anti-phospho-JNK, ERK, and p38) at 4°C overnight, followed by incubation for 1 to 2 h with the secondary antibody (HRP-conjugated antirabbit IgG, 1:5,000). Tyrosine phosphorylated proteins were visualized by ECL.

To verify that equal amount of proteins were loaded on the gel, the same blots were subsequently stripped and reprobed with JNK₁, ERK₁, or p38 antibodies as internal controls. The results were quantified by densitometry using a GS-670 Imaging Densitometer (Bio-Rad) and calculated as a ratio of phosphorylated kinase versus total kinase.

Measurement of JNK, ERK, and p38 Kinase Activity

MAPK activities were determined by measuring the amount of phosphorylation of MAPK substrates (c-Jun, ATF-2, and MBP) in the presence of ³²P--ATP as described by Duff and colleagues (5) and Modur and associates (21). The homogenate (10 µg protein) of pulmonary arteries from control and hypoxic rats were immunoprecipitated with 1 to 2 µg of anti-JNK, ERK₁, or p38 kinase antibodies, respectively, for 2 h. The immunocomplexes were added to protein A/G agarose macro beads, rocked for an additional 2 h, and washed twice with lysis buffer. Immunocomplex was resuspended in a total 25 µl of reaction buffer (20 mM Tris [pH 7.4], 5 mM -glycerophosphate, 10 mM MgCl₂, 2 mM DTT, 0.02% Triton X-100, 100 µM Na₃VO₄, and 50 µM ATP) containing 1 µCi of [³²P]--ATP and relevant substrate, and incubated at 30°C for 30 min. Substrates used in kinase assays were 500 ng (each sample) of human recombinant c-Jun for JNK, 250 ng ATF-2 for p38, and 50 µg MBP for ERK. Reactions were terminated by adding 250 µl of ice-cold 20% trichloroacetic acid containing 60 mM sodium pyrophosphate. Precipitates were collected on nitrocellulose filters and washed twice with 5% trichloroacetic acid. Filters were dried and the radioactivity was counted by liquid scintillation spectrophotometry. Background counts assayed without immunokinases were subtracted from the counts assayed in the presence of immunokinases to determine MAPK activities. Phosphorylation of MBP by whole tissue lysate was also measured to compare with the specific kinase activities.

Total RNA Isolation and Northern Blot Analysis

Individual pulmonary artery isolated from control and hypoxic rats was ground to powder in liquid nitrogen with a diethyl pyrocarbonate H₂O-treated mortar and pestle. Total RNA was extracted using TRI reagent following manufacturer's protocol. Ground tissue was placed in a test tube and 1 ml of TRI reagent was added. Chloroform (0.2 ml for each sample) was added and vortexed vigorously. After centrifugation, the top layer was removed to a new microcentrifuge tube and the total RNA was precipitated with isopropanol. The total RNA from each sample was determined with a spectrophotometer (Beckman DU64).

For Northern analysis, equivalent amounts of total RNA (10 µg/sample) were fractionated on a 1.1% agarose, 2.2 mol/liter formaldehyde gel and transferred to a nylon membrane. The membrane was hybridized overnight at 42°C in a solution (250 mmol/ liter Na₂HPO₄ [pH 7.2], 50% formamide, 5% sodium dodecyl sulfate (SDS), 1% BSA, 4 mmol/liter EDTA, and 0.1 mg/ml transfer RNA containing 2 × 10⁷ counts per min/ml of [³²P]-labeled cDNA probes. The cDNA probes VEGF, PDGF receptor B, egr-1, c-jun, and c-fos were labeled with [³²P]--dCTP by random priming. After hybridization, blots were washed in 2× saline sodium citrate (SSC), 0.1% SDS at room temperature for 15 min and washed twice in 0.2× SSC, 0.1% SDS at 55°C for 15 min. The hybridized blots were subjected to autoradiography at 70°C for 1 to 2 d. Signals of growth factor messenger RNA (mRNA) and the immediate early gene mRNA were measured by densitometry. Blots were then dehybridized and reprobed with CHOB that was used as an internal marker. The results were expressed as ratios of target mRNA versus CHOB mRNA.

Measurement of c-Jun Protein Levels

Pulmonary arteries from control and hypoxic rats were homogenized and the homogenate (10 µg) was immunoprecipitated with anti-c-Jun/AP-1 antibody. Western blots were prepared and probed with anti-c-Jun/AP-1 antibody (1:1,000) and followed by ECL. Protein levels were quantified by densitometry.

Statistics

Data are expressed as means ± standard error (SE) from three to seven individual experiments. Student's t test was used for comparing two mean values. When three or more groups were being compared, the one-way analysis of variance was used. Values were considered significantly different when P < 0.05.

	Results

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Hypoxia-Induced Pulmonary Arterial Remodeling

Chronic hypoxia caused a significant remodeling in rat pulmonary arteries. Previous studies showed that pulmonary arterial remodeling occurs at both large and small pulmonary arteries (15). In the present study we found that remodeling also occurs in extrapulmonary vessels, including the trunk and right and left branches (Figure 1). Pulmonary arteries isolated from hypoxic rats (exposed to 10% O₂ for 14 d) were enlarged and thicker (Figure 1A, right panel) than control pulmonary arteries (Figure 1A, left panel). Pulmonary arterial tissue mass increased 2-fold after 7 d of exposure to hypoxia and remained at that level through 14 d of exposure (Table 1). Microscopic studies showed a significant thickness of tunica media and adventitia in the pulmonary arterial walls from hypoxic rats (Figure 1B). Our previous work showed that the cross-section area of pulmonary artery increased from 0.566 ± 0.039 mm² in control to 1.255 ± 0.367 mm² in 14 d of hypoxia (16). The average total RNA in each control pulmonary artery was 8.1 ± 0.9 µg (Table 1), and increased approximately 4-fold after 7 d of hypoxia. After normalization, the ratio of RNA versus tissue mass also increased significantly at 7 d of exposure to hypoxia (Table 1), suggesting an increased gene activity in hypoxic vessels. However, these data do not provide information about what specific gene expression increased. Phosphorylation of MBP, a substrate for MAPK and other serine/threonine kinases, was 2.6 ± 0.1 pmol/µg/min in control arteries (Table 1). Hypoxia (7 and 14 d) caused a nearly 2-fold increase in MBP phosphorylation (4.1 ± 0.5 and 4.1 ± 0.9 pmol/µg/min, respectively; Table 1). These data show that chronic hypoxia caused significant structural and functional changes in pulmonary artery.

Hypoxia-Induced Activation of JNK

Because MBP can be phosphorylated by many kinases, including MAPK, protein kinase C, and other serine/threonine kinases, experiments were further carried out to determine the effects of hypoxia on specific MAPK isoforms. JNK activation was investigated first. Activation of JNK requires a dual phosphorylation of threonine and tyrosine residues which are on a Thr-Pro-Tyr motif. Using a specific anti-phospho-JNK antibody, our data showed a significant tyrosine phosphorylation of JNK (54 KD) in the hypoxic pulmonary artery (Figure 2A). Tyrosine phosphorylation of JNK increased more than 2-fold at the early exposure time (Day 1, n = 4; Figure 2B), and then declined with longer periods of hypoxia (determined by densitometry and normalized to total JNK). After 14 d of hypoxia, JNK tyrosine phosphorylation returned to control level. JNK activity was assayed with JNK-immunocomplex and the recombinant c-Jun was used as a substrate. As shown in Figure 2C, hypoxia significantly increased JNK activities in pulmonary arteries as measured by c-Jun phosphorylation. Hypoxia stimulated JNK activities nearly 2-fold (n = 3, * P < 0.05). JNK activities then declined with prolonged exposure, which is similar to the kinetics of JNK tyrosine phosphorylation.

View larger version (29K): [in this window] [in a new window]   Figure 2.   Hypoxia-induced JNK activation. (A) A typical Western blot probed with anti-phospho-JNK antibody (top row of bands). The lower row of bands is the same blot stripped and reprobed with anti-JNK1 antibody. (B) Mean data of Western analysis from four individual experiments (n = 4, *P < 0.05). (C ) Effects of hypoxia on JNK activity in the pulmonary arteries. JNK activities were assayed by recombinant c-Jun phosphorylation from JNK immunocomplexes (n = 3, *P < 0.05).

Hypoxia-Induced Activation of ERK₁ and ERK₂

Activation of p44 ERK₁ and p42 ERK₂ is characterized by a dual phosphorylation of motif Thr-Glu-Tyr. An anti-phospho-ERK antibody recognizes tyrosine-phosphorylated p44 and p42 ERKs. Figure 3A is a typical immunoblot showing that hypoxia induced a remarkable tyrosine phosphorylation of ERK₁ and ERK₂ in the pulmonary artery. Exposure of rats to hypoxia for 7 d caused a 3.6-fold increase of tyrosine phosphorylation of p44 ERK₁ (n = 4; Figure 3B) and 4.9-fold increase of p42 ERK₂ (n = 4; data not shown). Hypoxia also increased about 2-fold of ERK's activities in the pulmonary artery after 7 d exposure (n = 3; Figure 3C).

View larger version (28K): [in this window] [in a new window]   Figure 3.   Hypoxia-induced activation of ERK1 and ERK2. (A) A typical immunoblot showing the tyrosine phosphorylation of ERKs. The Western blot was probed with a specific anti-phospho-MAPK antibody. The same blot was stripped and reprobed with anti-ERK1 antibody. (B) Mean data of Western analysis from four individual experiments (n = 4, *P < 0.05). (C) Effects of hypoxia on ERK activities that were determined by MBP phosphorylation from ERK-immunocomplexes (n = 3, *P < 0.05).

Hypoxia-Induced Activation of p38 Kinase

Activation of p38 kinase requires a dual threonine and tyrosine phosphorylation on a Thr-Gly-Tyr motif. Because p38 kinase shares about 40% amino acid sequence identity with ERK₁ and ERK₂ (10), anti-phospho-MAPK (New England Biolabs) recognizes phosphorylated p38 kinase. By using this antibody, we found that hypoxia caused a significant increase in tyrosine phosphorylation of p38 kinase, as shown in Figure 4A. In the control pulmonary arteries the amount of activated p38 kinase was very low. Hypoxia caused 3.5-fold increase in p38 kinase phosphorylation by 7 d exposure (n = 4; Figure 4B). A similar result was found when the blots were probed with anti-phospho-p38 kinase antibody (data not shown). The activity of p38 kinase, assayed in the immunocomplex of p38 kinase using ATF-2 as a substrate, showed a significant increase at 3 and 7 d of exposure to hypoxia (n = 3; Figure 4C). The kinetics of p38 kinase activity were well correlated with the tyrosine phosphorylation levels of p38 kinase.

View larger version (27K): [in this window] [in a new window]   Figure 4.   Hypoxia-induced activation of p38 kinase. (A) A typical immunoblot MAPK showing hypoxia-induced p38 phosphorylation in pulmonary arteries. (B) Mean data of Western analysis from four individual experiments (n = 4, *P < 0.05). (C ) Hypoxia-induced increase in p38 kinase activity. The kinase activity was determined by ATF-2 phosphorylation by p38 kinase-immunocomplex (n = 3). *P < 0.05.

Hypoxia-Induced Immunostaining of Phospho-MAPK and ERK in the Intrapulmonary Artery

Lung sections (10 µm) from control and hypoxic rats were immunostained with anti-ERK₁ antibody or anti-phospho- MAPK antibody, respectively. Exposure of the rat to hypoxia caused a significant remodeling in the intrapulmonary conductive artery (~ 800 µm) and small resistant vessels (~ 200 µm), characterized by the proliferation of endothelium and thickening of the media and adventitia layers (Figures 5 and 6). Hypoxia-induced arterial remodeling was associated with an increase in phospho-MAPK immunostaining in those arteries (Figure 5). Increased phospho-MAPK staining was more profound in endothelium and subendothelium areas than in media in large arteries (Figure 5). In the small arteries, phospho-MAPK staining increased in both endothelial and medial layers (Figure 5). Hypoxia did not significantly alter the immunostaining of unphosphorylated ERK, which was consistent with our Western analysis. The levels of ERK₁ staining in media and adventitia layers from both hypoxic large and small vessels were similar to the controls (Figure 6). The only notable increase in ERK₁ immunostaining was seen in a part of the endothelium of the large artery (Figure 6). This increase in ERK₁ expression may contribute to the increased ERK activity seen in Figure 3C.

View larger version (132K): [in this window] [in a new window]   Figure 5.   Immunostaining of phospho-MAPK in large conductive and small resistant intrapulmonary arteries. Control (0 d) and hypoxia (7 d) lungs were stained with anti-phospho-MAPK antibodies. Hypoxia increased phospho-MAPK staining mainly in endothelium and subendothelium areas (red color). A, a pulmonary artery; B, a bronchiole. Bar: 200 microns.

View larger version (118K): [in this window] [in a new window]   Figure 6.   Immunostaining of ERK1 in large and small intrapulmonary arteries. Control (0 d) and hypoxia (7 d) lungs were stained with anti-ERK1 antibodies. A, a pulmonary artery; B, a bronchiole. Bar: 100 microns.

Hypoxia-Induced Upregulation of VEGF, PDGF, and PDGF Receptor B mRNA Levels

Because ERK₁ and ERK₂ have been reported to respond mainly to growth factor or mitogen stimulation, these experiments were designed to measure the VEGF and PDGF mRNA expression to determine the possible mechanism of hypoxia-induced activation of ERKs. VEGF and PDGF were chosen because VEGF is a potent growth factor for vascular endothelium, and PDGF not only is a most potent growth factor for vascular smooth muscle but also stimulates matrix protein synthesis (19). As shown in Figure 7, exposure to hypoxia for 7 d caused a 5-fold upregulation of VEGF mRNA in pulmonary arteries (n = 3). Hypoxia did not alter the PDGF mRNA in pulmonary arteries but did increase this mRNA in rat lungs (data not shown). However, hypoxia dramatically increased PDGF receptor B mRNA in pulmonary arteries. Increased PDGF receptor B mRNA was about 4- and 7-fold, respectively, by 7 and 14 d exposure (n = 3; Figure 8). These results suggest that pulmonary artery is a major target for PDGF. Although pulmonary artery itself did not express the PDGF gene during the hypoxic stimulation, increased PDGF receptor B mRNA indicates a upregulated response to PDGF signals that was released from other sources.

View larger version (30K): [in this window] [in a new window]   Figure 7.   Hypoxia-induced increase in VEGF mRNA levels in the pulmonary arteries. (A) A typical Northern blot of VEGF mRNA expression in response to hypoxia. (B) An average of VEGF mRNA from three individual experiments (n = 3). *P < 0.05.

View larger version (33K): [in this window] [in a new window]   Figure 8.   Hypoxia-induced increase in PDGF receptor B mRNA levels in the pulmonary arteries. (A) A typical Northern blot shows PDGF receptor B mRNA expression in response to hypoxia. (B) An average of PDGF receptor B mRNA from three individual experiments (n = 3).

Effects of Hypoxia on the Immediate Early Gene egr-1, c-jun, and c-fos Expression

Activation of MAPKs leads to increased expression of a group of proto-oncogenes, including c-jun, c-fos, and egr-1 genes. Additional experiments were carried out to determine whether hypoxia-induced activation of MAPKs is associated with increases of transcription factor gene expression. Figure 9A shows a typical Northern blot demonstrating the amount of egr-1, c-jun, and c-fos mRNA in pulmonary arteries in response to hypoxia. The levels of egr-1 mRNA markedly increased about 3-fold at the early hypoxic exposure time. However, exposure to hypoxia did not stimulate the c-jun mRNA level. Actually, c-jun mRNA was downregulated by hypoxia. Hypoxia also inhibited c-fos mRNA expression (Figure 9A). To confirm that downregulation of c-jun gene occurred during hypoxia, c-jun mRNA was measured from seven individual experiments. After exposure to hypoxia for 3 d, the average of c-jun mRNA levels was only 40% of the control and returned to 90% of the control by 14 d exposure (n = 7; Figure 9B).

View larger version (35K): [in this window] [in a new window]   Figure 9.   Effect of hypoxia on transcription of the immediate early genes egr-1, c-jun, and c-fos in pulmonary arteries. (A) A typical Northern blot probed separately with egr-1, c-jun, c-fos, and CHOB cDNA. (B) Average of c-jun mRNA in pulmonary arteries during hypoxic stimulation (n = 7, *P < 0.05).

Proto-oncogene products c-Jun and c-Fos, which are intrinsically unstable nuclear proteins, are subject to ubiquitin-dependent degradation. Because our data show that c-jun mRNA levels were downregulated by hypoxia at the early exposure time, the next set of experiments was designed to measure c-Jun protein content in hypoxic pulmonary arteries to determine whether there is hypoxia-induced degradation of c-Jun protein. Corresponding to the c-jun mRNA levels, c-Jun protein levels showed similar decreases during hypoxic stimulation by Western analysis (Figure 10A). At 1 d of hypoxia the c-Jun protein level was 68% of the control and returned to normal (105%) after 14 d (n = 3; Figure 10B).

View larger version (33K): [in this window] [in a new window]   Figure 10.   Effects of hypoxia on c-Jun/AP-1 protein levels in the pulmonary arteries. (A) A typical Western blot probed with anti- c-Jun/AP-1 antibody. (B) Mean level of c-Jun/AP-1 protein in the pulmonary arteries during hypoxic stimulation (n = 3, *P < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the MAPK superfamily, activation of p42/p44 ERK is a key step in the kinase cascade leading to cell proliferation in response to growth factors, agonists, and hormones (5- 7). In contrast, JNK/SAPK mediates signals in response to cytokines and environmental stress, such as tumor necrosis factor, ultraviolet light, oxidative stress, and heat shock (8, 10, 11, 21, 22). p38 kinase, also called HOG1 kinase, has been found to be involved in apoptosis, endotoxic response, and osmotic stress (2, 12, 13, 23). However, it is not known whether hypoxia activates MAPKs in an in vivo model and whether activation of MAPKs is involved in the signaling of hypoxia-induced pulmonary remodeling. The present study provides direct evidence that these isoforms of MAPK are activated in hypoxic pulmonary arteries. Hypoxia caused a time-dependent increase in kinase activity and kinase tyrosine phosphorylation. Increased phospho-MAPK immunostaining was observed in both large and small intrapulmonary arteries in association with significant arterial remodeling in response to hypoxia. Increases in phospho-MAPK immunostaining in hypoxic lungs (Figure 5) were relatively small compared with the Western analysis. The lack of signals was probably due to dephosphorylation that occurred during the procedures of paraffin slicing and immunostaining.

Increases in JNK tyrosine phosphorylation and JNK activity occurred at an early hypoxic exposure time. This time dependency suggests that JNK activation is an immediate response to low oxygen stress. In contrast, activation of ERK₁/ERK₂ measured by both phosphorylation and activity assay peaked at 7 d of hypoxia. This later activation of ERKs suggests that activation of ERK may be a secondary response to hypoxia-induced release of growth factors or mediators. Results from our laboratory and from others are consistent with this hypothesis (24, 25). Tuder and coworkers reported that hypoxia for 14 d increased VEGF mRNA in the lung (25). Oparil and colleagues found that endothelin production in the blood and endothelin mRNA in the pulmonary arteries were increased after 14 d of hypoxia (24). In the present study, we found VEGF and PDGF receptor mRNA upregulated in hypoxic pulmonary arteries, indicating the important role of these growth factors in the pulmonary arterial remodeling. Our results also show that hypoxia activates p38 kinase, as demonstrated by increased phosphorylation of p38 kinase and increased ATF-2 phosphorylation by immunoprecipitated p38 kinase. The mechanism for the hypoxia-induced activation of p38 kinase is unclear. Previous studies have shown that p38 kinase responds to the same extracellular stimuli as JNK/SAPK (21, 26). However, our data show that hypoxia-induced activation of JNK had a significantly different time course compared with that of p38 kinase, suggesting that different mechanism(s) are involved.

Activation of MAPK causes phosphorylation of transcription factors c-Jun, ATF-2, and c-Fos, and results in activation of the AP-1 complex (27). The transcription of c-Jun is regulated post-translationally by phosphorylation of c-Jun. Both JNK and ERK families can phosphorylate c-Jun; however, JNK phosphorylates c-Jun at a rate 10 times faster than do ERK₁ and ERK₂ (9). Activation of c-Jun has been reported to be involved in responses to a variety of stimuli. Ausserer and coworkers reported that hypoxia (6 h) increases proto-oncogene c-jun mRNA in human carcinoma cells (28). Actually, our study shows that c-jun mRNA and its protein levels were downregulated by hypoxia in rat pulmonary arteries. The mechanism of hypoxia-induced inhibition of c-jun and c-fos is not clear. One possible reason is that the proto-oncogene products c-Jun and c-Fos undergo degradation during hypoxia. Recently, an important observation has been reported that c-jun and c-fos, which are nuclear proteins with a very short half-life, are subjected to ubiquitin-dependent degradation (29, 30). The breakdown of c-Jun and c-Fos can be either accelerated or reduced by activation of JNK and other protein kinases (29). In our studies, we found that short-term hypoxia exposure caused a downregulation of c-jun and c-fos mRNA and the degradation of c-Jun proteins in pulmonary arteries. The activation of JNK preceded the degradation of c-Jun, whereas ERKs were activated concomitantly with recovery of c-Jun degradation. However, at this stage we do not know why hypoxia induces degradation of Jun and Fos and what mechanisms and what significance are involved in this degradation.

Although hypoxia downregulates c-jun and c-fos mRNA expression, the other growth-related gene, egr-1, was upregulated by hypoxia. The opposite responses of the early genes to hypoxia indicate that downregulation is specific only for c-jun and c-fos. The egr-1 transcription factor, which stands for "early growth response," has been shown to be upregulated by a variety of stimuli, including stress, growth factors, and hypoxia in cultured cells (32). Upregulation of egr-1 can occur with or without a dependency on JNK, p38, or ERK (33, 34). Our data show that hypoxia-stimulated egr-1 mRNA expression in an in vivo model is similar to that found in the in vitro study (35). However, it is not clear whether hypoxia-induced egr-1 expression is a direct downstream effector of MAPK activation.

The results from the present study show that chronic hypoxia stimulates JNK, ERK, and p38 kinase activity and increases their tyrosine phosphorylation. Hypoxia also causes pulmonary arterial remodeling in both large and small pulmonary arteries, and is associated with increases in phospho-MAPK immunostaining. Hypoxia-induced upregulation of growth factor and egr-1 mRNA expression suggests the involvement of these factors in hypoxia-induced pulmonary arterial remodeling. However, our in vivo model has limitations and the data presented here provide only a correlation, not a direct relationship, between these signaling molecules. Activation of MAPK isoforms in the hypoxic pulmonary artery may serve as a subsequent response to hypoxia-induced release/generation of growth factors and/ or vasomediators instead of a direct response to hypoxia.