INTRODUCTION

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Several pulmonary diseases are associated with chronic hypoxia and a consequence of this is pulmonary hypertension. This is characterized by structural changes in the pulmonary artery walls, including marked hypertrophy and hyperplasia (1). In addition to blood-borne and locally derived mitogens, such as platelet-derived growth factor (PDGF) and endothelin (ET-1), chronic hypoxia is a major stimulus for cells of the pulmonary vasculature. Fibroblast, smooth muscle, and endothelial cells derived from the pulmonary artery and maintained under hypoxic conditions (35 mm Hg) proliferate at a higher rate than cells under normoxic conditions (2, 3). Thus, an understanding of the mechanisms regulating this hyperactive cell division may lead to the development of novel therapies for treatment of patients with pulmonary hypertension.

The intracellular signaling mechanisms by which hypoxia stimulates cell proliferation in pulmonary artery fibroblasts have not been characterized. Of particular importance in growth factor signaling is the activation of a series of kinase cascades involving multiple serine/threonine and tyrosine phosphorylation events (4). One such pathway is the mitogen-activated protein (MAP) kinase cascade (5), which stimulates the phosphorylation of several intracellular substrates, such as p90^rsk and Elk-1, believed to play a role in initiating mitogenesis (6, 7). The MAP kinase protein itself is also regulated in a cell cycle-dependent fashion (8). In rat cardiac myocytes, components of the MAP kinase cascade, including Raf-1, MAP kinase kinase, and MAP kinase, were shown to be activated in response to hypoxia (9). However, these responses are very small when compared with mitogen stimulation of these pathways. Recently, protein serine/threonine kinases related to the "classic" forms of MAP kinase have been identified, and are referred to as the stress-activated protein (SAP) kinases (10). These kinases, consisting of at least two homologues, c-Jun NH₂-terminal kinase (JNK) (11) and p38 MAP kinase, are strongly activated by environmental stress, including heat shock and ultraviolet (UV) irradiation, cytokines such as tumor necrosis factor (TNF), and interleukin-1 (IL-1) and toxins such as lipopolysaccharide (LPS) (11).

In this study we show that hypoxia stimulates strongly the SAP kinases JNK and p38 MAP kinase in the absence of significant stimulation of the classic p42/44 MAP kinase pathway. In particular, we observe a second peak of p38 MAP kinase activity associated with G1/S phase transition. Thus, both SAP kinase pathways may play a role in the regulation of cell growth and division stimulated by hypoxia.

	METHODS

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Materials

Antibodies to p38 MAP kinase and JNK were purchased from (Santa Cruz Biotechnology, Santa Cruz, CA). Second antibodies, ECL detection reagents, and the BIOTRAK MAP kinase assay kit were purchased from Amersham International (Bucks, UK). [³H]thymidine (38 Ci/mmol) and [-³²P]-ATP (3,000 Ci/mmol) were from New England Nuclear (Hertfordshire, UK). All general reagents were of the highest commercial purity available. Plasmid vectors encoding glutathione-S-transferase-mitogen activated-protein kinase-activated protein kinase-2 (GST-MAPKAP kinase-2) and GST-c-Jun (5-89) were generous gifts from C. J. Marshall (Institute of Cancer Research, Chester Beatty Laboratories, London, UK) and J. R. Woodgett (Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Canada).

Cell Culture

Bovine pulmonary arteries from adult cows were obtained from the local abbatoir. Fibroblast cultures were isolated from these by a primary explant procedure previously described (16, 17). Cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS), 2 mM L-glutamine, and 250 U/ml and 100 mg/ml penicillin/streptomycin, respectively, and used between passages 4-8. Cells were rendered quiescent in serum-free media for 48 h prior to experimentation. Hypoxic conditions were attained in an Haerus hypoxic incubator (Philip Harris, Glasgow, Scotland, UK) at 37° C and stabilized to an oxygen tension of 1% (PO₂ = 30 to 35 mm Hg). Cells remained in hypoxic conditions for up to 30 h.

Solid-phase JNK Assay

JNK activity was measured by a solid-state kinase assay with GST-c-Jun as the substrate (18). After hypoxic incubation for the indicated times, cells (3-cm culture dishes) were washed twice in phosphate-buffered saline (PBS), then were lysed in 300 ml of buffer A (20 mM HEPES, pH 7.7, 50 mM NaCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 0.1 mM Na₃VO₄, 2 mg/ml leupeptin, and 100 mg/ml phenylmethylsulfonyl fluoride [PMSF]). Extracts were incubated on ice for 30 min then centrifuged at 10,000 × g for 10 min at 4° C. Supernatants were added to 20 mg of GST-c-Jun immobilized on glutathione (GSH)-sepharose. After 3 h at 4° C, the beads were washed extensively with buffer A and bound JNK was resuspended in kinase buffer (25 mM HEPES, pH 7.6, 5 mM -glycerophosphate, 0.1 mM Na₃VO₄, 2 mM dithiothreitol, 2 mg/ml leupeptin, and 100 mg/ml PMSF). The reaction was initiated by the addition of 20 mM ATP and 1 mCi [-³²P]ATP. After 20 min at 30° C, the reaction was terminated by the addition of 4× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and the samples boiled for 10 min. Phosphorylated proteins were separated by SDS-PAGE and detected by autoradiography.

Solid-phase p38 MAP Kinase Assay

p38 MAP kinase activity was measured by a solid-state kinase assay utilizing GST-MAPKAP kinase-2 as the substrate (19). After stimulation of cells as previously described, they were washed twice in PBS, then were lysed in 300 ml of buffer B (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 5 mM -glycerophosphate, 20 mM NaF, 0.1 mM Na₃VO₄, 2 mg/ml leupeptin, and 100 mM PMSF). Extracts were incubated on ice and centrifuged as before. Supernatants were added to 20 mg of GST-MAPKAP kinase-2 immobilized on GSH-sepharose. After 3 h at 4° C, the beads were washed extensively with buffer B and resuspended in the same kinase buffer as for JNK but at pH 7.4. The reaction was initiated by the addition of 50 mM ATP and 2 mCi [-³²P]ATP. After 30 min at 30° C, the reaction was terminated and phosphorylated proteins were separated as above. Preliminary immunoblotting experiments identified specific association of JNK or p38 with c-Jun and MAPKAP kinase-2, respectively, and linearity of the kinase assays with time and protein concentration.

p42/44 MAP Kinase Activity

MAP kinase activity was initially assessed by Western blotting using retardation on SDS-PAGE gels as a marker of activation (20, 21). Activity was also assayed in solubilized lysates using the epidermal growth factor (EGF) receptor peptide EGF^660-681 as substrate. The phosphorylated peptide was separated from other products by ion- exchange chromatography on Whatman p81 paper and quantified by liquid scintillation counting.

MAPKAP Kinase-2 Activity

MAPKAP kinase-2 was measured as outlined previously (12). Briefly, cellular MAPKAP kinase-2 was immunoprecipitated from the cells using 4 mg/ml of a specific antibody and immunoprecipitates incubated with the MAPKAP kinase-2 substrate peptide (KKLNRTLSVA) in an in vitro kinase assay. Peptide phosphorylation was assessed by ion-exchange chromatography on Whatman p81 paper and quantified by liquid scintiallation counting.

	RESULTS

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Effect of Hypoxia on c-Jun NH₂-terminal and p38 MAP Kinases

Figure 1 shows the effect of chronic hypoxia upon both JNK and p38 MAP kinase activity over a 30-h period. After a lag period of 1 to 2 h a large increase in JNK activity was observed in response to hypoxia, which peaked at approximately 6 h before decreasing slowly toward basal values for the remainder of the time course (Figure 1A). Stimulation of JNK activity under hypoxic conditions at the 6-h time point was 10- to 12-fold increased over basal activity, and was at least as great as that induced by 0.5 M sorbitol, a known activator of the SAP kinases.

View larger version (30K): [in this window] [in a new window]   Figure 1.   Stimulation of JNK and p38 MAP kinase activity by chronic hypoxia in bovine pulmonary artery fibroblast cells. Cells were exposed to sorbitol (0.5 M, 30 min) or incubated under hypoxic conditions (1% O2) for the indicated times. Cell lysates were assayed for JNK (panel A) or p38 MAP kinase activity (panels B and C ) as described in the experimental procedures. C = control; S = sorbitol. Numbers represent time in hours. The blot is a representative example from at least four independent experiments. Hypoxia caused stimulation of JNK and p38 MAP kinase activity at 3 to 6 h and a second peak of p38 MAP kinase activity at 24 h.

Using a similar approach, we also found that low oxygen stimulated p38 MAP kinase activity. This activity coincided with the rise in JNK, reaching a maximum after 6 h before returning to basal between 6 and 12 h (Figure 1B). However, in contrast to JNK activity, a second peak of p38 MAP kinase activity was observed, which was maximal between 18 to 24 h before returning to basal levels by 30 h (Figure 1C).

Effect of Hypoxia on p42/44 MAP Kinase Activity

To determine whether the observed increases in SAP kinase activity were components of a general response to hypoxic conditions, we analyzed the effect of lowered O₂ on other signaling enzymes. Figure 2 shows the effect of chronic hypoxia upon the activity of p42/44 MAP kinase. Cells incubated over a time course of up to 24 h in 1% O₂ showed no change in electrophoretic mobility shift of p42 or p44 MAP kinase isoforms (Figure 2A). In addition there was only a weak activation of MAP kinase activity assessed in vitro (Figure 2B) in response to lowered O₂ with a maximum 1.8-fold increase in activity after 3 h of hypoxic challenge. In contrast, both sorbitol and 10% FCS stimulated a substantial retardation of MAP kinase and a 6- to 8-fold increase in activity. In preliminary experiments we found that there was no hypoxic-mediated activation of p70 ribosomal S6 kinase or phosphatidylinositol 3-kinase (3), indicating that there was no initiation of these mitogen-responsive signaling events.

View larger version (21K): [in this window] [in a new window]   Figure 2.   Time course of hypoxic stimulation of p42 and p44 MAP kinase phosphorylation and activation in bovine pulmonary artery fibroblast cells. Confluent and quiescent cells were incubated in hypoxic conditions (1% O2) for the times indicated or serum (10%, 10 min, n). MAP kinase phosphorylation (A) and activity (B) were assessed as outlined in the experimental procedures. Hypoxia for 2, 4, and 6 h, unlike serum and sorbitol, had no effect on electrophoretic mobility shift of p42/44 MAP kinase isoforms (A) or activity (B). Numbers represent time in hours. C = control; H = hypoxia; Ser = serum; S = sorbitol. Each blot or value is representative of at least three independent experiments.

Identity of p38-like MAP Kinase Activity Stimulated in Response to Hypoxia

We sought to correlate the effects of hypoxia upon p38 MAP kinase with the ability to activate the downstream target of p38 MAP kinase, MAPKAP kinase-2. After 6 h of hypoxic challenge, a 9.9 ± 3.8 (n = 3)-fold activation of MAPKAP kinase-2 was observed (Figure 3A). However, this response was approximately 50% of the stimulation recorded by sorbitol (fold stimulation: 18.2 ± 4.5, n = 3). At the 24-h time point, a 4- to 5-fold increase in activity was observed in response to hypoxia (4.9 ± 2.8, n = 3). SB 203580, a specific inhibitor of p38 MAP kinase, prevented the rise in p38 MAP kinase activity caused by hypoxia and sorbitol (Figure 3A).

View larger version (25K): [in this window] [in a new window]   Figure 3.   Effect of SB 203580, a specific p38 MAP kinase inhibitor, upon p38 MAP kinase and MAPKAP kinase-2 activity in bovine pulmonary artery fibroblast cells. In panel A cells were incubated in chronic hypoxic conditions (H) or sorbitol (0.5 M, 30 min) for 6 (H6) or 24 (H24) hours and then assayed for MAPKAP kinase-2 activity as outlined in experimental procedures. The experiments were repeated after preincubation with 20 mM SB 203580 for 60 min (S + SB and H6 + SB). Each value is the mean ± SEM of three independent experiments. In panel B cells were preincubated with 20 mM SB 203580 for 60 min before hypoxic challenge and then assayed for p38 MAP kinase activity. SB 203580 decreased the activity of both p38 MAP kinase (panel A) and MAPKAP kinase-2. C = control; H = hypoxia; S = sorbitol; SB = SB 203580. Each experiment is representative of at least two others.

Figure 3B shows the effect of SB 203580, a specific inhibitor of p38 MAP kinase (22), upon hypoxia-induced solid-phase p38 MAP kinase activity. Concentrations of SB 203580 (20 mM), which almost totally abolished both sorbitol and hypoxia-stimulated MAPKAP kinase-2 activity (Figure 3A), caused a large reduction in hypoxic-mediated p38 MAP kinase activity.

Reversal of the Late Phase of Hypoxic-mediated p38 MAP Kinase Activity by Reoxygenation

In order to determine if continuous hypoxia was required for the late phase of p38 MAP kinase activity, cells were reoxygenated at different times after the initial stimulation period. Reoxygenation after 6 h resulted in almost complete abolition of the p38 MAP kinase activity peak at 24 h (Figure 4), suggesting the requirement of a continued hypoxic environment for the initiation of the second phase of the response.

View larger version (12K): [in this window] [in a new window]   Figure 4.   Effect of reoxygenation upon hypoxic-stimulated p38 MAP kinase activity in bovine pulmonary artery fibroblast cells. Cells were exposed to 1% O2 for 6 h before reoxygenation in 20% O2 at the times indicated. After 24 h samples were assayed for p38 MAP kinase activity as outlined in experimental procedures. Reoxygenation prevented hypoxia-stimulated p38 MAP kinase activity at 6 and 12 h (H6, H12) but not at 18 and 24 h (H18, H24). C = control; H = hypoxia; S = sorbitol. Each value is representative of at least three experiments.

	DISCUSSION

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Our earlier observations showed that low O₂ conditions stimulated a significant increase in incorporation of [³H]thymidine into DNA in fibroblast cells derived from the pulmonary artery, implying increased replication in these cells (3). A recent study implicated a role for the classic MAP kinase isoforms in the initiation of hypoxia-mediated increases in cell growth and division (9). However, in this study only a small stimulation of p42/44 MAP kinase activity was observed (9). Whereas some studies have implicated SAP kinase in the responses of cardiac myocytes to ischemic damage and reperfusion (23), this is the first study that implicates a role for SAP kinase activation in the response of cells derived from the pulmonary vasculature to hypoxia.

An initial peak of JNK and p38 MAP kinase activity was observed between 3 and 6 h of hypoxic challenge, which was comparable in magnitude to sorbitol stimulation, a well-recognized activator of the SAP kinases. This is unlike SAP kinase responses to other environmental stress agents and cytokines where peak activation occurs between 30 and 60 min (11). One possible reason for this may be the time required to equilibrate the media in low oxygen conditions. However, this time course of activation is similar to that observed in PC12 cells (a neuronal-derived cell line) following serum deprivation (26) and it is more likely that this is a physiologically relevant pattern of cellular activation of SAP kinases following certain stimuli, including hypoxia. We also found that the classic p42/44 MAP kinases were stimulated in response to hypoxia, however, this was a weak stimulation in comparison to that seen with the stress-activated forms.

A second peak of p38 MAP kinase activity was observed under hypoxic conditions in pulmonary artery fibroblasts, occurring between 18 and 24 h at a time when JNK activity was low in these cells. Significantly, this second phase of p38 MAP kinase activity coincided with the time required for the initiation of DNA synthesis in this cell type (18). This second phase also required the continued presence of a hypoxic environment, suggesting a clear specificity in the signals required to initiate this response that are unrelated to mitogen stimulation. The effect was blocked by the specific p38 MAP kinase inhibitor SB 203580. To date, only one other study has indicated activation of p38 MAP kinase in the absence of JNK, and this is following ischemic challenge of cardiac myocytes (24). Thus, under certain conditions related to the oxygen environment, p38 MAP kinase may be activated specifically.

These results indicate the potential for p38 MAP kinase to be associated with signaling events late in G1 in response to hypoxic challenge. One possible mechanism involves the induction of cyclin D1, which has been shown to be regulated by p38 MAP kinase in fibroblasts (27) although in that study p38 MAP kinase activity was associated with an inhibition of CCL39 hamster lung fibroblast cell division and prolongation of the quiescent state. However, since pulmonary artery cells are stimulated to contract and proliferate in response to hypoxia, it is still possible that p38 MAP kinase plays a positive role in cell division by regulating cell cycle function.