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Am J Physiol Lung Cell Mol Physiol 287: L919-L927, 2004. First published June 25, 2004; doi:10.1152/ajplung.00012.2004
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Differential effects of TGF-{beta}1 and BMP-4 on the hypoxic induction of cyclooxygenase-2 in human pulmonary artery smooth muscle cells

Karen K. K. Sheares, Trina K. Jeffery, Lu Long, Xudong Yang, and Nicholas W. Morrell

Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Papworth Hospitals, Cambridge CB2 2QQ, United Kingdom

Submitted 15 January 2004 ; accepted in final form 23 June 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chronic hypoxia-induced pulmonary hypertension results partly from proliferation of smooth muscle cells in small peripheral pulmonary arteries. Previously, we demonstrated that hypoxia modulates the proliferation of human peripheral pulmonary artery smooth muscle cells (PASMCs) by induction of cyclooxygenase-2 (COX-2) and production of antiproliferative prostaglandins (55). The transforming growth factor (TGF)-{beta} superfamily plays a critical role in the regulation of pulmonary vascular remodeling, although to date an interaction with hypoxia has not been examined. We therefore investigated the pathways involved in the hypoxic induction of COX-2 in peripheral PASMCs and the contribution of TGF-{beta}1 and bone morphogenetic protein (BMP)-4 in this response. In the present study, we demonstrate that hypoxia induces activation of p38MAPK, ERK1/2, and Akt in PASMCs and that these pathways are involved in the hypoxic regulation of COX-2. Whereas inhibition of p38MAPK or ERK1/2 activity suppressed hypoxic induction of COX-2, inhibition of the phosphoinositide 3-kinase pathway enhanced hypoxic induction of COX-2. Furthermore, exogenous TGF-{beta}1 induced COX-2 mRNA and protein expression, and our findings demonstrate that release of TGF-{beta}1 by PASMCs during hypoxia contributes to the hypoxic induction of COX-2 via the p38MAPK pathway. In contrast, BMP-4 inhibited the hypoxic induction of COX-2 by an MAPK-independent pathway. Together, these findings suggest that the TGF-{beta} superfamily is part of an autocrine/paracrine system involved in the regulation of COX-2 expression in the distal pulmonary circulation, and this modulates hypoxia-induced pulmonary vascular cell proliferation.

hypoxia; prostaglandins; bone morphogenetic protein; transforming growth factor-{beta}1; pulmonary hypertension

PROLONGED EXPOSURE TO ALVEOLAR HYPOXIA leads to structural changes in the walls of small pulmonary arteries, known as pulmonary vascular remodeling, and a sustained elevation of pulmonary vascular resistance (40). The characteristic pathological findings in the hypoxic hypertensive pulmonary circulation are increased wall thickness of small muscular arteries and muscularization of normally nonmuscular arteries at the level of the alveolar ducts (21, 52). These processes occur as a result of smooth muscle cell hypertrophy, hyperplasia, and migration (30, 39). Regulation of pulmonary vascular growth under hypoxic conditions may involve the interaction between locally derived growth factors, circulating hormones, and differences in genetic susceptibility (24). In addition, the pulmonary vascular smooth muscle cell responds directly to hypoxia by changes in ion channel activity, activation of protein kinases, for example protein kinase C and p38MAPK (7), and expression of transcription factors such as hypoxia-inducible factor-1 (46), all of which may impact on growth signals.

We have previously demonstrated that smooth muscle cells from human peripheral pulmonary arteries comprise at least two distinct cell types that may be either inhibited or stimulated to proliferate under hypoxic conditions. In both cell types, hypoxic modulation of cell proliferation occurs via induction of cyclooxygenase-2 (COX-2) and production of antiproliferative prostaglandins. In addition, inhibition of COX-2 activity was associated with a marked reduction in prostaglandin release and enhanced proliferation of cells under hypoxic conditions (55). COX, also known as prostaglandin H synthase, is the rate-limiting enzyme for the conversion of arachidonic acid to prostaglandins and thromboxanes and comprises two isoforms. COX-1 is constitutively expressed and is responsible for the basal production of prostanoids, whereas COX-2 is highly inducible by a number of cytokines and mitogens (20). Vasodilating prostaglandins such as prostacyclin (PGI2) and prostaglandin E2 (PGE2) are critical in the regulation of pulmonary vascular remodeling. COX-2 induction by cytokines and growth factors has been shown to regulate the growth of vascular smooth muscle cells (37). In vivo, mice overexpressing prostaglandin synthase are protected from the development of hypoxia-induced pulmonary hypertension (12).

The transforming growth factor (TGF)-{beta} superfamily, which includes the bone morphogenetic proteins (BMPs), has recently been shown to be a critical regulator of pulmonary vascular remodeling. Familial primary pulmonary hypertension is associated with the inheritance of heterozygous germline mutations in the gene encoding the BMP type II receptor (BMPRII) (8, 26, 28). We have recently demonstrated that BMPs suppress proliferation of pulmonary artery smooth muscle cells (PASMCs) derived from the proximal pulmonary arteries of normal subjects and patients with secondary forms of pulmonary hypertension but fail to suppress proliferation of cells from patients with primary pulmonary hypertension (32).

Given the critical role of the TGF-{beta} superfamily in pulmonary vascular remodeling, we hypothesized that the TGF-{beta}/BMPs may be involved in the hypoxic regulation of growth of human peripheral PASMCs. Simultaneous treatment with TGF-{beta} and hypoxia has a synergistic effect on endothelial cell apoptosis (22). In human vascular endothelial cells, hypoxia and TGF-{beta} cooperate in the activation of the vascular endothelial growth factor (VEGF) promoter (42). A TGF-{beta} response element has been identified in the human COX-2 promoter (53), and exogenous TGF-{beta}1 has also been shown to induce COX-2 and PGE2 release in cultured human airway smooth muscle cells (10) and proximal PASMCs (4). However, there is little information on the interaction between the TGF-{beta} superfamily and hypoxia on the hypoxic induction of COX-2 in peripheral PASMCs or the contribution of BMPs to this response.

The MAPKs play important roles in the cellular response to hypoxia and TGF-{beta}/BMPs. For example, p38MAPK has been shown to be activated by BMP (36) and other TGF-{beta} superfamily receptors (6, 14). All three MAPKs have been implicated in the regulation of COX-2 expression in many different cell types and signaling settings (17, 18, 41). Interleukin-1 stimulation of rat renal mesangial cells mediates COX-2 expression and PGE2 production with concomitant activation of the p38MAPK and c-jun NH2-terminal kinase (JNK) signaling pathways (16). Activation of p38MAPK and JNK has also been shown to be activated during exposure to hypoxia (7, 43, 45). Recently, another signaling pathway, mediated by the phosphoinositide 3-kinase family of enzymes, has been shown to be a key regulator of human PAMSC proliferation and migration via activation of serine/threonine kinase Akt/protein kinase B and its substrate the ribosomal protein kinase p70s6k (15). Selective inhibition of phosphoinositide 3-kinase by LY-294002 completely inhibits platelet-derived growth factor and thrombin-stimulated [3H]methyl-thymidine incorporation in PASMCs. Hypoxia has been shown to activate the phosphoinositide 3-kinase/Akt pathway in rat pheochromocytoma (PC12) cells where it protects against apoptosis (1), and phosphoinositide 3-kinase activation regulates hypoxia-induced gene expression of VEGF in human breast cancer cell lines (2).

In this study, we sought to determine the main signaling pathways involved in the hypoxic regulation of COX-2 in PASMCs. In addition, we investigated the possible interaction between hypoxia and TGF-{beta}/BMP on COX-2 expression and cell proliferation. Our findings demonstrate that p38MAPK and ERK1/2 positively regulate the hypoxic induction of COX-2, whereas phosphoinositide 3-kinase is a negative regulator. TGF-{beta}1 and hypoxia-exerted additive effects on COX-2 induction and autocrine/paracrine release of TGF-{beta}1 during hypoxic exposure may contribute to the hypoxic induction of COX-2. In contrast, BMP-4 inhibited the hypoxic induction of COX-2 in a MAPK-independent fashion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Distal PASMCs were isolated from peripheral segments of artery (0.3–1.0 mm external diameter) by microdissection of human lung tissue, as previously described (50). The use of human lung tissue was approved by the Huntington Local Research Ethics Committee (approval no. H00/531/T). For experiments, PASMCs were plated in DMEM containing 10% FBS and used between passages 3 and 10. The smooth muscle lineage of cells was confirmed by positive immunofluorescence staining using antibodies to {alpha}-smooth muscle actin and smooth muscle myosin (Sigma-Aldrich, Poole, UK) (50).

Exposure of cells to hypoxia. Peripheral PASMCs were grown under standard normoxic conditions in a CO2 incubator before exposure to hypoxic conditions. Hypoxia was induced by pregassing cell culture medium (DMEM + 25 mM HEPES) with a gas mixture containing 95% N2-5% CO2 for 30 min inside a gas-tight isolator. The hypoxic culture medium was then added to cells plated in 24- or 48-well plates. Plates were then maintained inside specially designed Perspex chambers (Bellco) gassed with 95% N2-5% CO2. Chambers were regassed daily. The pH, PO2, and PCO2 in the medium were checked at the beginning and end of each experiment using a blood gas analyzer (ABL5, Radiometer). Hypoxic cells were not reoxygenated at any stage of the experimental procedure.

Measurement of TGF-{beta}1. TGF-{beta}1 in the cell supernatant was measured by ELISA according to the manufacturer’s instructions (R&D Systems). Cells were grown to 80% confluence in six-well plates and quiesced for 48 h in 0.1% FBS/DMEM. Each hypoxic well was treated with 2 ml of pregassed DMEM and exposed to hypoxia for 24 h. The control normoxic plates were also treated with 2 ml of DMEM and left in a normoxic incubator for 24 h. At the end of each experiment, the cell supernatant was removed and immediately stored at –80°C until assay. The total cell protein in each well was also measured, and the results are expressed as picograms per milliliter per microgram of protein.

Western blotting. Expression of COX isoforms in PASMCs was assessed by Western blotting, following exposure to normoxia or hypoxia, for up to 24 h. Quiescent cells were exposed to experimental conditions in 0.1% FBS/DMEM in six-well plates. Cells were lysed and extracts were boiled at a 1:1 ratio with 2x protein Tris/SDS loading buffer for 5 min. Samples (10–15 µg) were loaded onto 7.5% SDS-PAGE gels and separated by electrophoresis for 1–2 h. Protein was then transferred to nitrocellulose membrane and incubated with blocking buffer, followed by incubation with specific antibodies to COX-1 or COX-2 (1:2,000; Santa Cruz Biotechnology). The blots were then incubated with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody in blocking buffer (1:4,000) overnight at 4°C. Blots were developed using ECL reagent (Amersham Biosciences), and rainbow markers were used for molecular weight determinations. In further experiments, the effect of inhibition of p38MAPK, ERK1/2, or JNK activity on COX-2 expression was studied using the selective inhibitors SB-203580 (1–5 µM), PD-98059 (1–5 µM), or JNKi (1–5 µM), respectively (Calbiochem). To examine the effect of exogenous cytokines on COX-2 expression, cells were incubated with TGF-{beta}1 (0.01–10 ng/ml) or BMP-4 (10–50 ng/ml). Anti-TGF-{beta} antibody (10 µg/ml; R&D Systems) was used to neutralize endogenous TGF-{beta}1 activity. Cells were pretreated for 1 h with the inhibitors, cytokines, or neutralizing antibody and then exposed to hypoxia or normoxia for 24 h.

To determine whether hypoxia activated the MAPKs in PASMCs, cells were exposed to pregassed hypoxic medium for up to 2 h. Protein was harvested by washing cells in cold PBS and immediately freezing them in an ethanol/dry ice bath. Cells were scraped into 200 µl of protein loading buffer containing protease inhibitors and subjected to SDS-PAGE electrophoresis, as previously described (7, 48). After being washed, blots were incubated with anti-phospho-p38MAPK, anti-phospho-ERK1/2, or anti-phospho-JNK antibodies (1:1,000; Cell Signaling Technology) overnight at 4°C. Blots were next washed and incubated with an anti-rabbit HRP-conjugated secondary antibody (1:2,500; Dako) for 1 h at room temperature. Blots were then stripped and reprobed using an antibody to total p38MAPK, total ERK1/2, or total JNK (1:1,000; Cell Signaling Technology).

Cell proliferation. The effect of hypoxia in the presence or absence of MAPK inhibitors on proliferation of PASMCs was determined by examining [3H]methyl-thymidine incorporation, an index of DNA synthesis. Cells were plated at 104 cells/well in 10% FBS/DMEM in 48-well plates and left to adhere overnight under normoxic conditions. At the beginning of the experimental period, fresh DMEM was added either alone or containing the MAPK inhibitors. [3H]methyl-thymidine (0.25 µCi/well; Amersham) was added for the final 6 h. Cells were exposed to 24 h of normoxia or hypoxia.

RT-PCR amplication of RNA. Total RNA was extracted from PASMCs using TRIzol reagent (Life Technologies). Temporal expression during hypoxic exposure was compared by semiquantitative RT-PCR performed with the Access RT-PCR System according to the manufacturer’s instructions (Promega). Primers were taken from previously published sequences (38), where possible, or designed with the computer program Prime(+). All primers were synthesized by Sigma-Genosys. The sequences were: Smad6 (forward, 5'-CCCCCG-GCTACTCCATCAAGGTGT-3'; reverse, 5'-GTCCGTGGGGGCTGTGTCTCTGG-3'); Smad7 (forward, 5'-gtggggaggctctactgtgtc-3'; reverse, 5'-GTCGAAAGCCTTGATGGAGAAACC-3'); COX-1 (forward, 5'-TGCCCAGCTCCTGGCCCGCCGCTT-3'; reverse, 5'-GCAGCTCTGGGTCAAATTTCAG-3'); COX-2 (forward, AGTCAAAGATACTCAGGCAGA-3'; reverse, 5'-GTAGTTCTGGGTCAAATTTCAG-3'). A human {beta}-actin primer set was used to normalize the amount of cDNA in each sample. The {beta}-actin sequences were as follows: forward, 5'-ATGAAGTGT-GACGTTGACATCCG-3'; and reverse, 5'-GCTTGCTGATCCACATCTGCTG-3'. PCR products were separated by electrophoresis in 2% agarose gel and visualized with ethidium bromide. Control reactions were run without the addition of reverse transcriptase.

Statistics. Data were expressed as means ± SE and analyzed with GraphPad Prism version 3.0 (GraphPad Software). Comparisons were made by Student’s t-test (2-tailed), ANOVA with Tukey’s post hoc test where appropriate, or two-way ANOVA and then either a one-way ANOVA with Tukey’s post hoc test or paired t-test where appropriate. A value of P < 0.05 indicated statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Induction of COX-2 by hypoxia is partly dependent on activation of p38MAPK and ERK1/2. We have previously reported that COX-2 is induced in response to hypoxia in peripheral PASMCs (55). In the present study, immunoblotting showed that p38MAPK is rapidly phosphorylated during hypoxia without a change in total p38MAPK (Fig. 1A). This effect lasted for at least 60 min. In addition, incubation of hypoxic PASMCs with the p38MAPK inhibitor SB-203580 (1–5 µM) inhibited the induction of COX-2 in a concentration-dependent manner (Fig. 1, B and C). In contrast, COX-1 protein levels were unchanged by hypoxia or SB-203580. Hypoxia also caused a temporal induction of phospho-ERK1/2 (Fig. 2A). The specific ERK inhibitor, PD-98059 (1–5 µM), partially inhibited the hypoxic induction of COX-2 without any change in COX-1 protein expression (Fig. 2, B and C). Cell viability studies using trypan blue exclusion demonstrated that concentrations of SB-203580 from 1–5 µM and PD-98059 from 1–5 µM had no effect on cell viability. Hypoxia did not activate phospho-JNK in peripheral PASMCs (data not shown).



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  		Fig. 1. A: time course of the hypoxic induction in phospho-p38MAPK without a change in total p38MAPK. B refers to baseline and C refers to positive control with heat shock-treated NIH/3T3 whole cell lysates. B: incubation of hypoxic cells with the p38MAPK inhibitor SB-203580 resulted in a concentration-dependent inhibition of cyclooxygenase (COX)-2 protein expression. Immunoblots are representative of at least 3 separate experiments. C: densitometric analysis of 3 separate experiments shows a significant inhibition in the hypoxic induction of COX-2 by pretreatment with 1 and 5 µM SB-203580. Each lane was normalized against its {beta}-actin lane to check loading (data not shown). Each hypoxic response was compared with its corresponding normoxic control by one-way ANOVA (*P < 0.05 vs. untreated control). In each figure, N represents normoxia and H hypoxia.

 


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  		Fig. 2. A: time course of the hypoxic induction of phospho-ERK1/2. Equal loading of lanes was confirmed by stripping and reprobing for total ERK1/2. B: ERK inhibitor, PD-98059, caused partial inhibition of COX-2 induction by hypoxia. Immunoblots are representative of at least 3 separate experiments. C: Densitometric analysis comparing the hypoxic response with its corresponding normoxic response showed inhibition of the hypoxic induction of COX-2 by increasing concentrations of PD-98059 (*P < 0.05 vs. untreated control by 1-way ANOVA).

 
Inhibition of p38MAPK or ERK1/2 modulates growth responses to hypoxia. We have previously shown that DNA synthesis is inhibited by hypoxia in this population of peripheral PASMCs (55). Because COX-2 induction was found to be regulated by p38MAPK and ERK1/2, we studied the effect of selective inhibitors on the growth response to hypoxia. The [3H]methyl-thymidine incorporation responses of PASMCs to 24 h of hypoxia in the presence of SB-203580 (1 µM) or PD-98059 (1 µM) are shown in Fig. 3, A and B, respectively. The inhibition of DNA synthesis by hypoxia was partly reversed by pretreatment of cells for 1 h with SB-203580 and PD-98059. The results suggest that in these cells, hypoxic induction of p38MAPK and ERK1/2 are coupled to COX-2 induction and growth inhibition.



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  		Fig. 3. A and B: [3H]methyl-thymidine incorporation studies demonstrate that hypoxic inhibition of DNA synthesis was partly reversed by the p38MAPK inhibitor SB-203580 or the ERK1/2 inhibitor PD-98059. Cells in 48-well plates were pretreated with 1 µM SB-203580 or 1 µM PD-98059 for 1 h and then exposed to hypoxia or normoxia for 24 h (*P < 0.05). Results are representative of at least 3 separate experiments in quadruplicate.

 
COX-2 induction is modulated by phosphoinositide 3-kinase activity. Western blot analysis of whole cell lysates showed that hypoxic stimulation of PASMCs induced a time-dependent phosphorylation of Akt, one of the major downstream targets of phosphoinositide 3-kinase signaling (Fig. 4A). LY-294002, a competitive inhibitor of phosphoinositide 3-kinase, induced expression of COX-2 in normoxic cells and markedly increased the hypoxic induction of COX-2, suggesting that the phosphoinositide 3-kinase pathway negatively regulates hypoxia-induced COX-2 production (Fig. 4B). In contrast, phosphoinositide 3-kinase inhibition did not affect COX-1 protein expression.



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  		Fig. 4. A: temporal induction of phospho-Akt by hypoxia, without any change in total Akt. B represents baseline and PDGF was used as a positive control. B: phosphoinositide 3-kinase inhibitor LY-294002 caused a concentration-dependent increase in COX-2 induction during normoxia and hypoxia. Pulmonary artery smooth muscle cells were pretreated with LY-294002 for 1 h and then exposed to normoxia or hypoxia. Both immunoblots are representative of at least 3 separate experiments.

 
TGF-{beta}1 induces COX-2 and enhances hypoxic induction of COX-2. PASMCs were incubated in hypoxic or normoxic conditions, with or without TGF-{beta}, and Western analyses for COX-2 protein were performed. Incubation with exogenous TGF-{beta}1 for 24 h (0.1–10 ng/ml) caused a concentration-dependent increase in COX-2 expression without a change in COX-1 protein levels (Fig. 5A). At the mRNA level, TGF-{beta}1 (10 ng/ml) caused an early increase in COX-2 expression with later increases in COX-1 expression (Fig. 5B). Hypoxia andTGF-{beta}1 increased COX-2 protein compared with untreated cells under normoxic conditions. The simultaneous stimulation with both TGF-{beta} and hypoxia had an additive effect on COX-2 protein expression (Fig. 5C).



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  		Fig. 5. A: incubation with exogenous transforming growth factor (TGF)-{beta}1 for 24 h caused a concentration-dependent increase in the induction of COX-2 protein expression without a change in COX-1 expression. B: TGF-{beta}1 caused an early increase in COX-2 mRNA expression. C: hypoxia and TGF-{beta}1 increased COX-2 expression compared with untreated cells under normoxic conditions. Results are representative of at least 3 separate experiments. Stimulation with hypoxia and TGF-{beta}1 had an additive effect on COX-2 protein expression as assessed by densitometry. #P < 0.05 vs. corresponding normoxic control; *P < 0.05 vs. corresponding control without TGF-{beta}1 by 2-way ANOVA. OD, optical density.

 
Hypoxia induces TGF-{beta}1 release and expression of inhibitory Smads. We questioned whether the effect of hypoxia on COX-2 induction could be contributed to by increased autocrine/paracrine release of TGF-{beta}1 from hypoxic cells. Exposure of PASMCs to hypoxia for 24 h significantly increased TGF-{beta}1 release into the supernatant (105.5 ± 6.1 {rho}g·ml–1·µg–1 of protein) compared with normoxic exposure (44.3 ± 7.9 {rho}g·ml–1·µg–1 of protein; Fig. 6A). In addition, semiquantitative RT-PCR showed hypoxia-induced increases in mRNA expression for inhibitory Smad6 and 7, genes known to be induced by TGF-{beta}, over 24 h. Smad6 mRNA increased by 1 h of hypoxic exposure, whereas there was a later increase in Smad7 that persisted for at least 24 h (Fig. 6B).



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  		Fig. 6. A: hypoxia caused a significant increase in TGF-{beta}1 release into cell supernatant as measured by ELISA. Results were standardized to protein concentration per well, and data are means ± SE of 3 independent experiments run in duplicate (*P < 0.01 vs. normoxic control by Student’s t-test). B: RT-PCR analysis showed a temporal induction of mRNA for inhibitory Smad6 and Smad7 by hypoxia over 24 h. Gels are representative of at least 3 separate experiments.

 
Anti-TGF-{beta} antibody modulates the hypoxic induction of COX-2. Neutralizing antibodies to TGF-{beta}1 were employed to determine whether endogenous TGF-{beta}1 induction contributed to COX-2 induction by hypoxia. PASMCs were incubated in hypoxic or normoxic conditions, with or without anti-TGF-{beta} (10 µg/ml), and Western blots for COX proteins were performed. Anti-TGF-{beta} antibody partially inhibited the hypoxic induction of COX-2 and inhibited the additive effect of exogenous TGF-{beta}1 on COX-2 protein expression during hypoxia (Fig. 7). There was no effect on COX-1 protein levels (Fig. 7) or {beta}-actin expression (data not shown).



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  		Fig. 7. Anti-TGF-{beta} antibody (10 µg/ml) inhibits the hypoxic induction of COX-2 and modulates the additive effect of exogenous TGF-{beta}1 (5 ng/ml) on the induction of COX-2 during hypoxia. Immunoblots are representative of at least 3 separate experiments.

 
TGF-{beta}1 also activates p38MAPK and ERK1/2. Because hypoxia and TGF-{beta} exerted additive effects on COX-2 expression, we questioned whether these stimuli activated similar signaling pathways in PASMCs. Incubation of PASMCs with exogenous TGF-{beta}1 (10 ng/ml) led to the activation of both phospho-p38MAPK and phospho-ERK1/2 with no change in total p38MAPK or ERK1/2 (Fig. 8). TGF-{beta}1 did not activate phospho-JNK (data not shown).



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  		Fig. 8. TGF-{beta}1 (10 ng/ml) resulted in the induction of phospho-p38MAPK and phospho-ERK1/2. Anisomycin or 10% FCS was used as a positive control (PC). Lane U represents the untreated control. Immunoblots are representative of at least 3 separate experiments.

 
BMP-4 inhibited the hypoxic induction of COX-2. Incubation of PASMCs with increasing concentrations of exogenous BMP-4 (10 and 50 ng/ml) had no effect on COX-2 mRNA and protein expression during normoxic conditions (Fig. 9, A and B) but inhibited the induction of COX-2 protein during exposure to hypoxia for 24 h (Fig. 9B). In further experiments, cells were incubated with 5 ng/ml of TGF-{beta}1, 50 ng/ml of BMP-4, or a combination of TGF-{beta}1 and BMP-4. Pretreatment with BMP-4 partially inhibited the induction of COX-2 by TGF-{beta}1 during exposure to hypoxia (Fig. 9C). Exogenous BMP-4 (50 ng/ml), similar to the finding with TGF-{beta}1, resulted in the activation of both p38MAPK and ERK1/2 (Fig. 10A). To elucidate whether the MAPKs were involved in the differential effects of TGF-{beta}1 and BMP-4 on the induction of COX-2, PASMCs were preincubated with SB-203580, PD-98059, and a JNK inhibitor. In contrast to the effect of SB-203580 on TGF-{beta}-induced COX-2 induction, none of the MAPK inhibitors modulated the effect of BMP-4 (Fig. 10B).



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  		Fig. 9. A and B: exogenous bone morphogenetic protein (BMP)-4 does not alter COX-2 and COX-1 mRNA or protein expression. BMP-4 inhibits the hypoxic induction of COX-2 in a concentration-dependent manner. C: cells were incubated with 5 ng/ml of TGF-{beta}1, 50 ng/ml of BMP-4, or a combination of TGF-{beta}1 and BMP-4. Preincubation with BMP-4 inhibited the induction of COX-2 by TGF-{beta}1 in the presence of hypoxia. Experiments are representative of at least 3 separate experiments.

 


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  		Fig. 10. A: exogenous BMP-4 (50 ng/ml) stimulates the phosphorylation of p38MAPK and ERK1/2 without a change in total p38MAPK or total ERK. B: effect of MAPK inhibitors on the modulation of COX-2 expression by exogenous TGF-{beta}1 (10 ng/ml). Of the 3 MAPK inhibitors, only the p38MAPK inhibitor SB-203580 inhibited TGF-{beta}1 (T)-induced COX-2 expression, but none of the inhibitors modulated the effect of BMP-4 (B). Lane U represents untreated control. Immunoblots are representative of at least 3 separate experiments. JNKi, c-jun NH2-terminal kinase inhibitor.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study has demonstrated that hypoxia induces activation of p38MAPK, ERK1/2, and Akt and that these pathways are involved in the hypoxic regulation of COX-2 expression in human PASMCs derived from the peripheral pulmonary circulation. We have previously shown that in normoxia-derived peripheral PASMCs, hypoxia inhibits cell proliferation by induction of COX-2 and production of antiproliferative prostaglandins (55). Here, we show that inhibition of p38MAPK or ERK1/2 suppressed the hypoxic induction of COX-2 and the antiproliferative effect of hypoxia in these distal PASMCs. In addition, the phosphoinositide 3-kinase pathway negatively regulated hypoxia-induced COX-2 production. Furthermore, exogenous TGF-{beta}1 induced COX-2 protein expression, and our findings suggest that release of TGF-{beta}1 by PASMCs during hypoxia contributes to the hypoxic induction of COX-2 via the p38MAPK pathway. In contrast, BMP-4 inhibited the hypoxic induction of COX-2 by an MAPK-independent pathway. Together, these findings suggest that the TGF-{beta} superfamily may be part of an autocrine/paracrine system involved in the regulation of COX-2 expression and prostaglandin production, and this modulates hypoxia-induced pulmonary vascular cell proliferation.

Previous studies have demonstrated variable patterns of activation of p38MAPK, ERK1/2, and JNK activity in response to hypoxia depending on the species, cell type, and experimental conditions. In an in vivo rat study, activation of all three MAPK pathways has been demonstrated in both large and small intrapulmonary arteries in association with significant arterial remodeling in response to hypoxic exposure (25). In pulmonary artery fibroblasts, hypoxic activation of p38MAPK, ERK1/2, and JNK have been described with the hypoxic proliferative response reported to be dependent on p38MAPK in rat fibroblasts (48) and ERK1/2, JNK1, and p38MAPK in neonatal bovine fibroblasts (7). In adult bovine pulmonary artery fibroblasts in which hypoxia stimulated proliferation, JNK and p38MAPK, but not ERK1/2, were also activated (43). Interestingly, the growth of peripheral PASMCs used in the present study was inhibited under hypoxic conditions, and in these cells p38MAPK and ERK1/2 pathways appear to be coupled to growth inhibition via induction of COX-2. These observations suggest a complex integration of signaling pathways in the regulation of cellular responses to hypoxic exposure that is not only species and cell type specific, but also developmentally regulated.

Phosphoinositide 3-kinase is a heterodimeric dual-function lipid and protein kinase that has been linked to cell survival, transcription factor activation, and multiple signaling pathways (11, 47). Recently, hypoxia has been shown to activate the phosphoinositide 3-kinase/Akt pathway in HepG2 cells (34). Our data show that hypoxia increases Akt activity in human PASMCs, and phosphoinositide 3-kinase inhibition increases COX-2 expression during normoxia and hypoxia. In LPS-stimulated human alveolar macrophages, phosphoinositide 3-kinase inhibition also results in an increase in COX-2 protein by increasing COX-2 mRNA stability (31). Thus it is possible that phosphoinositide 3-kinase/Akt contributes to cell proliferation in PASMCs partly via suppression of COX-2.

TGF-{beta} regulates cell growth and differentiation, extracellular matrix synthesis, cytokine production, and angiogenesis. The COX-2 promoter has been shown to have a TGF-{beta} responsive element (53), and our results demonstrate that TGF-{beta} increased COX-2 mRNA and protein expression in human peripheral PASMCs. Similarly, exogenous TGF-{beta}1 has also been shown to induce COX-2 expression and PGE2 release in cultured human airway smooth muscle cells (10) and proximal PASMCs (4). To examine whether TGF-{beta} may have a role in the hypoxic regulation of growth in PASMCs, we measured TGF-{beta} release and found that TGF-{beta} release is increased during exposure to hypoxia. Furthermore, neutralizing antibodies to TGF-{beta} inhibited the hypoxic induction of COX-2 in peripheral PASMCs. COX-2 induction by cytokines and growth factors has been shown to regulate the growth of vascular smooth muscle cells (37). We have also shown that hypoxia modulates peripheral PASMC proliferation by induction of COX-2 and production of antiproliferative prostaglandins (55). Vasodilating prostaglandins such as PGI2 and PGE2 are critical in the regulation of pulmonary vascular remodeling. Mice overexpressing prostaglandin synthase are protected from the development of hypoxia-induced pulmonary hypertension (12). Conversely, PGI2 receptor-deficient mice develop a greater degree of pulmonary hypertension and pulmonary arterial media thickening than wild-type mice after exposure to chronic hypoxia for 3 wk (23). Studies have shown a reduction in smooth muscle cell TGF-{beta}1 protein and mRNA expression in hypertensive remodeling pulmonary arteries of neonatal calves with hypoxia-induced pulmonary hypertension (3). Thus impaired TGF-{beta}1 release and/or PGE2 generation may contribute to pulmonary vascular remodeling in hypoxic-induced pulmonary hypertension.

Signaling by TGF-{beta} is mediated by Smad proteins that regulate gene transcription through functional cooperations and association with other DNA-binding proteins (29, 35). Although Smad proteins are the classic mediators of TGF-{beta} signal transduction, other signaling pathways, including the MAPKs, can also be activated in various cell systems (19, 56). Here, we show that hypoxia and TGF-{beta} can induce both phospho-p38MAPK and ERK1/2 in peripheral PASMCs. Furthermore, the hypoxic induction of COX-2 was prevented by inhibitors of p38MAPK and ERK1/2. TGF-{beta}-induced activation of COX-2, however, was only abrogated by the p38MAPK inhibitor and not the ERK1/2 inhibitor. Thus induction of phospho-p38MAPK may be partly responsible for the additive effect of TGF-{beta}1 on the hypoxic induction of COX-2.

Recent studies have demonstrated that primary pulmonary hypertension is associated with germline mutations in the gene encoding the BMPRII, a receptor member of the TGF-{beta} superfamily (8, 26). We have recently reported that BMPs suppress proliferation of PASMCs from normal subjects and patients with secondary forms of pulmonary hypertension but fail to suppress proliferation of cells from patients with primary pulmonary hypertension (32). Thus we examined the potential role of BMP-4 in the regulation of COX-2. In contrast to the effect of TGF-{beta}1 on COX-2 expression, exogenous BMP-4 had no effect on COX-2 expression under normoxic conditions and inhibited the hypoxic induction of COX-2 in peripheral PASMCs. The mechanisms underlying these antagonistic interactions remain unknown. In murine cell lines, BMP-7 reverses TGF-{beta}1-induced epithelial to mesenchymal transition by reinduction of the adhesion molecule, E-cadherin, through a Smad-dependent pathway (57). Our studies appear to exclude MAPK-dependent pathways as the mechanism for the differential effects of TGF-{beta} and BMP-4. However, in this study, we have not determined whether the mechanism involves differences in the pattern of Smad activation by TGF-{beta}/BMPs. The BMP family signals via a BMP-restricted set of Smads 1, 5, and 8, whereas TGF-{beta} signals via Smads 2 and 3 (5). In view of the signaling specificity in the TGF-{beta} superfamily, the inhibitory effect of BMP-4 on hypoxic induction of COX-2 in PASMCs may be mediated by the different pattern of Smad activation. It is also possible that antagonism between BMP and TGF-{beta} pathways occurs further downstream of Smad proteins at the level of DNA binding partners. BMP and TGF-{beta} are potent inducers of the inhibitory Smads 6 and 7, which feed back to inhibit TGF-{beta} and BMP receptor signaling. Here, we have shown that mRNAs for Smads 6 and 7 are induced by hypoxia in PASMCs. This induction may be secondary to the increased autocrine release of TGF-{beta} and BMPs under hypoxic conditions. An alternative explanation is that the promoter regions of Smads 6 and 7 may be directly regulated by hypoxia, a possibility that warrants further investigation.

In summary, this study has shown that hypoxia induces COX-2 expression via the MAPK pathway in human peripheral PASMCs. In addition, TGF-{beta}1 is released by PASMCs during hypoxia, and TGF-{beta}1 and hypoxia exert additive effects on COX-2 induction via activation of MAPKs. Our data indicate that activation of p38MAPK and ERK1/2 by hypoxia in human PASMCs is coupled to growth inhibitory pathways via induction of COX-2 in these cells. In contrast, BMP-4 inhibits the hypoxic induction of COX-2, probably via a MAPK-independent system. We speculate that induction of COX-2 and the enhanced release of TGF-{beta}1 may prevent excessive PASMC proliferation during the development of hypoxia-induced proliferation. Disruption of TGF-{beta} superfamily signaling and/or COX-2 pathway may result in an imbalance between critical antiproliferative and proproliferative programs in the pulmonary vasculature during hypoxia, thus contributing to the development of hypoxia-induced pulmonary hypertension.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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This work was supported by funding from the British Heart Foundation. K. K. K. Sheares is supported by a British Heart Foundation PhD Fellowship.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. W. Morrell, Dept. of Medicine, Univ. of Cambridge School of Clinical Medicine, Addenbrooke’s and Papworth Hospitals, Cambridge CB2 2QQ, United Kingdom (E-mail: nwm23{at}cam.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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  1. Alvarez-TejadoM, Naranjo-Suarez S, Jimenez C, Carrera AC, Landazuri MO, and del Peso L. Hypoxia induces the activation of the phosphatidylinositol 3-kinase/Akt cell survival pathway in PC12 cells: protective role in apoptosis. J Biol Chem 276: 22368–22374, 2001.[Abstract/Free Full Text]
  2. BlancherC, Moore JW, Robertson N, and Harris AL. Effects of ras and von Hippel-Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1{alpha}, HIF-2{alpha}, and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3-kinase/Akt signaling pathway. Cancer Res 61: 7349–7355, 2001.[Abstract/Free Full Text]
  3. BotneyMD, Parks WC, Crouch EC, Stenmark K, and Mecham RP. Transforming growth factor-{beta}1 is decreased in remodeling hypertensive bovine pulmonary arteries. J Clin Invest 89: 1629–1635, 1992.[Web of Science][Medline]
  4. BradburyDA, Newton R, Zhu YM, Stocks J, Corbett L, Holland ED, Pang LH, and Knox AJ. Effect of bradykinin, TGF-{beta}1, IL-1{beta}, and hypoxia on COX-2 expression in pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 283: L717–L725, 2002.[Abstract/Free Full Text]
  5. ChenYG, Hata A, Lo RS, Wotton D, Shi Y, Pavletich N, and Massague J. Determinants of specificity in TGF-{beta} signal transduction. Genes Dev 12: 2144–2152, 1998.[Abstract/Free Full Text]
  6. CocolakisE, Lemay S, Ali S, and Lebrun JJ. The p38 MAPK pathway is required for cell growth inhibition of human breast cancer cells in response to activin. J Biol Chem 276: 18430–18436, 2001.[Abstract/Free Full Text]
  7. DasM, Bouchey DM, Moore MJ, Hopkins DC, Nemenoff RA, and Stenmark KR. Hypoxia-induced proliferative response of vascular adventitial fibroblasts is dependent on G protein-mediated activation of mitogen-activated protein kinases. J Biol Chem 276: 15631–15640, 2001.[Abstract/Free Full Text]
  8. DengZ, Morse JH, Slager SL, Cuervo N, Moore KJ, Venetos G, Kalachikov S, Cayanis E, Fischer SG, Barst RJ, Hodge SE, and Knowles JA. Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene. Am J Hum Genet 67: 737–744, 2000.[CrossRef][Web of Science][Medline]
  9. FongCY, Pang L, Holland E, and Knox AJ. TGF-{beta}1 stimulates IL-8 release, COX-2 expression, and PGE2 release in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 279: L201–L207, 2000.[Abstract/Free Full Text]
  10. FrumanDA and Cantley LC. Phosphoinositide 3-kinase in immunological systems. Semin Immunol 14: 7–18, 2002.[CrossRef][Web of Science][Medline]
  11. GeraciMW, Gao B, Shepherd DC, Moore MD, Westcott JY, Fagan KA, Alger LA, Tuder RM, and Voelkel NF. Pulmonary prostacyclin synthase overexpression in transgenic mice protects against development of hypoxic pulmonary hypertension. J Clin Invest 103: 1509–1515, 1999.[Web of Science][Medline]
  12. GoldbergPL, MacNaughton DE, Clements RT, Minnear FL, and Vincent PA. p38 MAPK activation by TGF-{beta}1 increases MLC phosphorylation and endothelial monolayer permeability. Am J Physiol Lung Cell Mol Physiol 282: L146–L154, 2002.[Abstract/Free Full Text]
  13. GoncharovaEA, Ammit AJ, Irani C, Carroll RG, Eszterhas AJ, Panettieri RA, and Krymskaya VP. PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 283: L354–L363, 2002.[Abstract/Free Full Text]
  14. GuanZ, Baier LD, and Morrison AR. p38 Mitogen-activated protein kinase down-regulates nitric oxide and up-regulates prostaglandin E2 biosynthesis stimulated by interleukin-1{beta}. J Biol Chem 272: 8083–8089, 1997.[Abstract/Free Full Text]
  15. GuanZ, Buckman SY, Miller BW, Springer LD, and Morrison AR. Interleukin-1{beta}-induced cyclooxygenase-2 expression requires activation of both c-Jun NH2-terminal kinase and p38 MAPK signal pathways in rat renal mesangial cells. J Biol Chem 273: 28670–28676, 1998.[Abstract/Free Full Text]
  16. GuanZ, Buckman SY, Pentland AP, Templeton DJ, and Morrison AR. Induction of cyclooxygenase-2 by the activated MEKK1 –-> SEK1/MKK4 –-> p38 mitogen-activated protein kinase pathway. J Biol Chem 273: 12901–12908, 1998.[Abstract/Free Full Text]
  17. HartsoughMT and Mulder KM. Transforming growth factor {beta} activation of p44mapk in proliferating cultures of epithelial cells. J Biol Chem 270: 7117–7124, 1995.[Abstract/Free Full Text]
  18. HerschmanHR. Prostaglandin synthase 2. Biochim Biophys Acta 1299: 125–140, 1996.[Medline]
  19. HislopA and Reid L. New findings in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension. Br J Exp Pathol 57: 542–554, 1976.[Web of Science][Medline]
  20. HoggN, Browning J, Howard T, Winterford C, Fitzpatrick D, and Gobe G. Apoptosis in vascular endothelial cells caused by serum deprivation, oxidative stress and transforming growth factor-{beta}. Endothelium 7: 35–49, 1999.[Web of Science][Medline]
  21. HoshikawaY, Voelkel NF, Gesell TL, Moore MD, Morris KG, Alger LA, Narumiya S, and Geraci MW. Prostacyclin receptor-dependent modulation of pulmonary vascular remodeling. Am J Respir Crit Care Med 164: 314–318, 2001.[Abstract/Free Full Text]
  22. JefferyTK and Morrell NW. Molecular and cellular basis of pulmonary vascular remodeling in pulmonary hypertension. Prog Cardiovasc Dis 45: 173–202, 2002.[CrossRef][Web of Science][Medline]
  23. JinN, Hatton N, Swartz DR, Xia X, Harrington MA, Larsen SH, and Rhoades RA. Hypoxia activates jun-N-terminal kinase, extracellular signal-regulated protein kinase, and p38 kinase in pulmonary arteries. Am J Respir Cell Mol Biol 23: 593–601, 2000.[Abstract/Free Full Text]
  24. LaneKB, Machado RD, Pauciulo MW, Thomson JR, Phillips JA III, Loyd JE, Nichols WC, and Trembath RC. Heterozygous germline mutations in BMPR2, encoding a TGF-{beta} receptor, cause familial primary pulmonary hypertension. The International PPH Consortium. Nat Genet 26: 81–84, 2000.[CrossRef][Web of Science][Medline]
  25. MachadoRD, Pauciulo MW, Thomson JR, Lane KB, Morgan NV, Wheeler L, Phillips JA III, Newman J, Williams D, Galie N, Manes A, McNeil K, Yacoub M, Mikhail G, Rogers P, Corris P, Humbert M, Donnai D, Martensson G, Tranebjaerg L, Loyd JE, Trembath RC, and Nichols WC. BMPR2 haploinsufficiency as the inherited molecular mechanism for primary pulmonary hypertension. Am J Hum Genet 68: 92–102, 2001.[CrossRef][Web of Science][Medline]
  26. MassagueJ and Chen YG. Controlling TGF-{beta} signaling. Genes Dev 14: 627–644, 2000.[Free Full Text]
  27. MeyrickB and Reid L. The effect of continued hypoxia on rat pulmonary arterial circulation. An ultrastructural study. Lab Invest 38: 188–200, 1978.[Web of Science][Medline]
  28. MonickMM, Robeff PK, Butler NS, Flaherty DM, Carter AB, Peterson MW, and Hunninghake GW. Phosphatidylinositol 3-kinase activity negatively regulates stability of cyclooxygenase 2 mRNA. J Biol Chem 277: 32992–33000, 2002.[Abstract/Free Full Text]
  29. MorrellNW, Yang X, Upton PD, Jourdan KB, Morgan N, Sheares KK, and Trembath RC. Altered growth responses of pulmonary artery smooth muscle cells from patients with primary pulmonary hypertension to transforming growth factor-{beta}1 and bone morphogenetic proteins. Circulation 104: 790–795, 2001.[Abstract/Free Full Text]
  30. MottetD, Dumont V, Deccache Y, Demazy C, Ninane N, Raes M, and Michiels C. Regulation of hypoxia-inducible factor-1{alpha} protein level during hypoxic conditions by the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3{beta} pathway in HepG2 cells. J Biol Chem 278: 31277–31285, 2003.[Abstract/Free Full Text]
  31. MoustakasA, Souchelnytskyi S, and Heldin CH. Smad regulation in TGF-{beta} signal transduction. J Cell Sci 114: 4359–4369, 2001.[Web of Science][Medline]
  32. NoheA, Hassel S, Ehrlich M, Neubauer F, Sebald W, Henis YI, and Knaus P. The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J Biol Chem 277: 5330–5338, 2002.[Abstract/Free Full Text]
  33. OhnakaK, Numaguchi K, Yamakawa T, and Inagami T. Induction of cyclooxygenase-2 by angiotensin II in cultured rat vascular smooth muscle cells. Hypertension 35: 68–75, 2000.[Abstract/Free Full Text]
  34. OhnoR, Yoshinaga K, Fujita T, Hasegawa K, Iseki H, Tsunozaki H, Ichikawa W, Nihei Z, and Sugihara K. Depth of invasion parallels increased cyclooxygenase-2 levels in patients with gastric carcinoma. Cancer 91: 1876–1881, 2001.[CrossRef][Web of Science][Medline]
  35. OrtonEC, LaRue SM, Ensley B, and Stenmark K. Bromodeoxyuridine labeling and DNA content of pulmonary arterial medial cells from hypoxia-exposed and nonexposed healthy calves. Am J Vet Res 53: 1925–1930, 1992.[Web of Science][Medline]
  36. RabinovitchM, Gamble W, Nadas AS, Miettinen OS, and Reid L. Rat pulmonary circulation after chronic hypoxia: hemodynamic and structural features. Am J Physiol Heart Circ Physiol 236: H818–H827, 1979.[Abstract/Free Full Text]
  37. ReddyST, Wadleigh DJ, and Herschman HR. Transcriptional regulation of the cyclooxygenase-2 gene in activated mast cells. J Biol Chem 275: 3107–3113, 2000.[Abstract/Free Full Text]
  38. Sanchez-ElsnerT, Botella LM, Velasco B, Langa C, and Bernabeu C. Endoglin expression is regulated by transcriptional cooperation between the hypoxia and transforming growth factor-{beta} pathways. J Biol Chem 277: 43799–43808, 2002.[Abstract/Free Full Text]
  39. ScottPH, Paul A, Belham CM, Peacock AJ, Wadsworth RM, Gould GW, Welsh D, and Plevin R. Hypoxic stimulation of the stress-activated protein kinases in pulmonary artery fibroblasts. Am J Respir Crit Care Med 158: 958–962, 1998.[Abstract/Free Full Text]
  40. SekoY, Takahashi N, Tobe K, Kadowaki T, and Yazaki Y. Hypoxia and hypoxia/reoxygenation activate p65PAK, p38 mitogen-activated protein kinase (MAPK), and stress-activated protein kinase (SAPK) in cultured rat cardiac myocytes. Biochem Biophys Res Commun 239: 840–844, 1997.[CrossRef][Web of Science][Medline]
  41. SemenzaGL. HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol 88: 1474–1480, 2000.[Abstract/Free Full Text]
  42. TokerA and Cantley LC. Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature 387: 673–676, 1997.[CrossRef][Medline]
  43. WelshDJ, Peacock AJ, MacLean M, and Harnett M. Chronic hypoxia induces constitutive p38 mitogen-activated protein kinase activity that correlates with enhanced cellular proliferation in fibroblasts from rat pulmonary but not systemic arteries. Am J Respir Crit Care Med 164: 282–289, 2001.[Abstract/Free Full Text]
  44. WhartonJ, Davie N, Upton PD, Yacoub MH, Polak JM, and Morrell NW. Prostacyclin analogues differentially inhibit growth of distal and proximal human pulmonary artery smooth muscle cells. Circulation 102: 3130–3136, 2000.[Abstract/Free Full Text]
  45. WilkinsonM, Langhorne CA, Heath D, Barer GR, and Howard P. A pathophysiological study of 10 cases of hypoxic cor pulmonale. QJM 66: 65–85, 1988.[Abstract/Free Full Text]
  46. YangX, Hou F, Taylor L, and Polgar P. Characterization of human cyclooxygenase 2 gene promoter localization of a TGF-{beta} response element. Biochim Biophys Acta 1350: 287–292, 1997.[Medline]
  47. YangX, Sheares KK, Davie N, Upton PD, Taylor GW, Horsley J, Wharton J, and Morrell NW. Hypoxic induction of cox-2 regulates proliferation of human pulmonary artery smooth muscle cells. Am J Respir Cell Mol Biol 27: 688–696, 2002.[Abstract/Free Full Text]
  48. YuL, Hebert MC, and Zhang YE. TGF-{beta} receptor-activated p38 MAP kinase mediates Smad-independent TGF-{beta} responses. EMBO J 21: 3749–3759, 2002.[CrossRef][Web of Science][Medline]
  49. ZeisbergM, Hanai J, Sugimoto H, Mammoto T, Charytan D, Strutz F, and Kalluri R. BMP-7 counteracts TGF-{beta}1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med 9: 964–968, 2003.[CrossRef][Web of Science][Medline]



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