Proliferation and Signaling in Fibroblasts - Semantic Scholar

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Proliferation and Signaling in Fibroblasts Role of 5-Hydroxytryptamine2A Receptor and Transporter David J. Welsh, Margaret Harnett, Margaret MacLean, and Andrew J. Peacock Scottish Pulmonary Vascular Unit and Department of Immunology, Western Infirmary; and Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom

(Received in original form February 24, 2003; accepted in final form April 13, 2004)

crine cells was found to correlate with the extent of proliferation of myofibroblasts in the pulmonary arteries (7). On a molar basis, 5-HT is one of the most potent pulmonary vasoconstrictors identified in humans to date (8), but in the systemic vasculature, it causes vasodilation (9). We have previously shown that rat pulmonary artery fibroblast (RPAF) cells have exaggerated proliferative effects to hypoxia, whereas those from systemic arterial fibroblasts do not (10). In light of these results, we have been interested in the role of 5-HT in the proliferation of pulmonary artery fibroblasts from chronically hypoxic rats and particularly in defining which receptor subtype is of importance and in identifying a possible mechanism of this effect. At least 10 classes of 5-HT receptors have been identified: 5-HT1A–F, 5-HT2A–C, 5-HT3, and 5-HT4 (11), and different receptors have been implicated in the pathogenesis of a number of vascular disorders. 5-HT1 and 5-HT2 appear to be the principal receptors relevant to the human pulmonary arteries (12–14). Under most experimental conditions, stimulation of the 5-HT1 receptor causes vasodilation, and the 5-HT2 receptor often mediates vasospasm in the pulmonary circulation (12). There is also direct evidence that the 5-HT transporter (5-HTT) plays a key role in pulmonary vascular remodeling with the finding that mice lacking the 5-HTT developed less severe hypoxic PHT than control animals and that selective 5-HTT inhibitors attenuated hypoxic PHT (15). In this article, we have examined the effects of 5-HT on the proliferation and activation of mitogen-activated protein (MAP) kinases in rat pulmonary fibroblasts from control and chronically hypoxic animals. At the same time, the effects of specific 5-HT receptor antagonists, such as the 5-HT2A receptor antagonist, Ketanserin, as well as 5-HTT inhibition, have been used to help determine whether a specific 5-HT receptor is involved in these effects. The molecular mechanisms by which hypoxia stimulates proliferation of pulmonary artery fibroblasts, but not fibroblasts from the systemic circulation, are unknown. There is, however, considerable evidence in the literature that the MAP kinases, the classic Erk1 and Erk2 (p42/44), and the related stress-activated kinases, Jnk and p38 MAP kinase, which have been implicated as key regulators of cell proliferation, can be activated in response to hypoxic stress (16, 17). These MAP kinases, which are all activated by a common threonine-X-tyrosine regulatory motif by their distinct upstream dual-specificity (thr/tyr) MAP kinase kinases (16), are an important group of serine/threonine signaling kinases. These modulate the phosphorylation and hence the activation status of transcription factors and link transmembrane signaling with gene-induction events in the nucleus (16). Some of the results of these studies have been previously reported in the form of an abstract (18).

Supported by the British Lung Foundation and the Chest Heart and Stroke Association, Scotland.

METHODS

5-Hydroxytryptamine (5-HT) plays an important role in the remodeling of the pulmonary circulation, notably during exposure to hypoxia. Here, we have been interested in the role of 5-HT and the 5-HT transporter in the proliferation of pulmonary artery fibroblasts derived from pulmonary hypertensive animals and particularly in defining which receptor subtype is of importance and in identifying a possible mechanism of this effect. This study has examined the effects of 5-HT on the proliferation and activation of mitogenactivated protein kinases in rat pulmonary artery fibroblasts from control and chronically hypoxic animals. We have shown that 5-HT has a co-mitogenic effect with serum to produce an enhanced proliferative response in cells from chronically hypoxic rats over those from control animals. Moreover we have found that the 5-HT2A receptor is responsible for the hypoxia-associated 5-HT proliferation in these cells by using specific receptor agonist and antagonist studies and that this receptor signals via p38 mitogen-activated protein kinase. We have also shown that the 5-HT transporter is important in the mitogenic response not pertaining to hypoxic stimulation. Taken together, these data suggest that selective 5-HT2A receptor antagonists may have a role in pulmonary artery fibroblast proliferation to hypoxia. Keywords: fibroblasts; hypoxia; pulmonary; pulmonary hypertension; 5-hydroxytryptamine

Pulmonary hypertension (PHT) occurs commonly in patients with chronic hypoxic lung disease and is characterized by the remodeling of the pulmonary artery walls by processes including hypertrophy of vascular cells (1). Such structural remodeling of the pulmonary arteries together with hypoxia-induced vasoconstriction and polycythemia (2) leads to the rise of pulmonary artery pressure observed in PHT. Previous work has suggested a link with 5-hydroxytryptamine (5-HT) in the etiology of PHT. Normally, plasma levels of 5-HT are extremely low because circulating 5-HT is stored in platelets. The “5-HT hypothesis of PHT” was developed in the 1960s after an outbreak of PHT was observed in patients taking aminorex, a diet pill that increases 5-HT availability by inducing platelet release of 5-HT while inhibiting its reuptake. Since then, there has been increasing interest in the role of 5-HT in the development of PHT (3). 5-HT is released from pulmonary neuroendocrine cells and neuroepithelial bodies distributed throughout the airways. Secretion of large amounts of 5-HT from these cells occurs in response to hypoxia and thus might contribute to secondary PHT (2, 4–6). In lung transplant recipients with endstage primary PHT, the degree of hyperplasia of the neuroendo-

Correspondence and requests for reprints should be addressed to David J. Welsh, B.Sc., Ph.D., Scottish Pulmonary Vascular Unit, Level 8, Western Infirmary, Glasgow G11 6NT, UK. E-mail: [email protected] Am J Respir Crit Care Med Vol 170. pp 252–259, 2004 DOI: 10.1164/rccm.200302-264OC Internet address: www.atsjournals.org

Materials All reagents were of Analar grade and were obtained from Sigma (Poole, Dorset, UK) unless specified otherwise. [3H]Thymidine was purchased from DuPont (Stevenage, Hertfordshire, UK). All tissue culture flasks and media were obtained from Gibco (Paisley, Renfrewshire, UK). Fetal

Welsh, Harnett, MacLean, et al.: 5-HT and Hypoxia on Rat Fibroblasts Figure 1. Effect of 5-hydroxytryptamine (5-HT) and serum on [3H]thymidine uptake in rat pulmonary artery fibroblast (RPAF) cells from normoxic and chronically hypoxic rats. Uptake of [3H]thymidine from normoxic (open bar) and chronically hypoxic (closed bar) rats. RPAF cells were grown to 60% confluency in 24-well plates and then quiesced in serum-free medium. The cells were stimulated with either 3- or 10-␮M 5-HT alone or in conjunction with 0.2% serum as indicated in the graph. Cells were then allowed to grow in normoxic conditions for a period of 24 hours. The addition of either 0.2% serum, 3-␮M, or 10-␮M 5-HT alone did not significantly enhance the proliferation of fibroblast cells from either normoxic or those from chronically hypoxic animals in comparison with control untreated cells. However, 3- and 10-␮M 5-HT in the presence of 0.2% serum caused a significant increase in the [3H]thymidine uptake in cells from normoxic animals. The effect was even greater in the cells from hypoxic animals. The experiment was repeated with cells from four different animals and the results shown are typical of those obtained. * Value for hypoxia significantly greater than normoxia, p ⬍ 0.01. DPM ⫽ disintegrations per minute.

calf serum was obtained from Imperial Laboratories (Andover, Hants, UK). Rabbit polyclonal antibodies specific for the activated dualphosphorylated forms of the MAP kinase family members (Erk1/Erk2 [p42/44] and p38) and their appropriate control antibodies for total MAP kinase expression were obtained from New England Biolabs (Hertfordshire, UK).

Chronic Hypoxic Rat Model of PHT Pulmonary hypertensive rats were prepared (in the laboratory of M. MacLean) using the technique of MacLean and colleagues (19). Full details of this technique can be found in Welsh and colleagues (10).

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Figure 2. Effects of 10-␮M 5-HT and serum on the uptake of [3H]thymidine in pulmonary artery fibroblasts from control and chronically hypoxic animals. Uptake of [3H] thymidine from normoxic and chronically hypoxic rats is shown. RPAF cells were grown to 60% confluency in 24-well plates and then quiesced in serum-free medium. The cells were stimulated with a range of serum concentrations either alone or in conjunction with 10-␮M 5-HT as indicated in the graph. Cells were then allowed to grow in normoxic conditions for a period of 24 hours. The addition of 5-HT and serum gave rise to enhanced proliferation in the cells from chronically hypoxic rats, especially in serum concentrations of 1% or lower (p ⬍ 0.001). The experiment was repeated with cells from four different animals, and the results shown are typical of those obtained. *Value for hypoxia significantly greater than normoxia, p ⬍ 0.01. Open square ⫽ normoxic. Closed square ⫽ chronically hypoxic. White diamond ⫽ normoxic ⫹ 10 ␮M 5-HT. Shaded diamond ⫽ chronically hypoxic ⫹ 10 ␮M 5-HT.

Primary Culture of RPAFs Fibroblasts were prepared using the technique of Freshney (20), with some modifications (10). Cells were used between passages 3–10.

Proliferation Assay: DNA Synthesis as Measured by [3H]Thymidine Incorporation To measure DNA replication as a measure of cellular proliferation, rat pulmonary fibroblast cells were grown to approximately 60% confluence in 24-well plates at 37⬚C and then serum starved for 24 hours. After this time, the following additions were made as described below. To determine the effect of serum and 5-HT on proliferation in the normoxic and chronically hypoxic cells, 0.2% serum and 10-␮M 5-HT were added to the cells for 24 hours. Specific 5-HT agonists (10 ␮M), ␣-methyl-5-HT (5-HT2A), BW723C86 (5-HT2B), and MK212 (5-HT2C) were also studied. The incubations were also performed in the presence and absence of 5-HT receptor antagonists, 0.1-␮M ketanserin (5-HT2A), 1-␮M SDZ SER 082 (5-HT2B⫹C), RS102221 (5-HT2B), and GR55562 (5-HT1B–1D). Fluoxetine (10 ␮M) was used to block the actions of the 5-HTT. All drugs were added 2 hours before the addition of growth factors for 24 hours. In addition, the role of MAP kinases in the 5-HT replicative response was assessed with the use of a specific p38 MAP kinase inhibitor (SB203580, 1 ␮M) and a MAP kinase kinase inhibitor (U0126, 0.1 ␮M). A detailed method for this technique is available in Welsh and colleagues (10).

Western Blot Analysis RPAF cells were grown to 90% confluence in six-well plates at 37⬚C and then serum starved for 24 hours. After this time, the following additions were made as described here. To determine the effect of serum and 5-HT on p42/44 and p38 MAP kinase activity in the normoxic and chronically hypoxic cells, 0.2% serum and 10-␮M 5-HT were added to the cells for 24 hours. To determine the effects of the specific 5-HT antagonists on p42/44 and p38 MAP kinase activity in the normoxic and chronically hypoxic cells, 0.1-␮M ketanserin (5-HT2A inhibitor) and 1-␮M GR55562 (5-HT1B–1D inhibitor) were added 2 hours before the addition of growth factors for 24 hours. In addition to this, the p38 MAP kinase inhibitor (SB203580, 1 ␮M) and the p42/p44 MAP kinase inhibitor (U0126, 0.1 ␮M) were also used. A standard Western blot technique was then used as described in Welsh and colleagues (10).

Statistical Analysis Each result represents the mean of four experiments performed on cells from the same animal. All results shown were confirmed in additional experiments on four different animals. Data in Figure 2 were analyzed by two-way analysis of variance. Results were considered significant at a p value of less than 0.05. A comparison of means (Figures 1, 3, 4, and 7) was determined using Student’s t test (significant if p ⬍ 0.05). Densitometry was used to quantify results presented in Figures 5 and 6.

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Figure 3. Effects of specific 5-HT agonists and antagonists on the replication of pulmonary artery fibroblasts from control and chronically hypoxic animals. Uptake of [3H]thymidine from normoxic (open bar) and chronically hypoxic (closed bar) rats is shown. RPAF cells were grown to 60% confluence in 24-well plates and then quiesced in serum-free medium. The cells were stimulated with serum (0.2%) either alone or in conjunction with 5-HT (10 ␮M) in the presence or absence of 10-␮M ␣-methyl-5-HT (5-HT2A agonist), 10-␮M BW723C86 (5-HT2B agonist), 10-␮M MK212 (5-HT2C agonist), 0.1-␮M ketanserin (5-HT2A antagonist), 1-␮M SDZSER082 (5-HT2B⫹C antagonist), and 1-␮M RS102221 (5-HT2B antagonist) as indicated in the graph. Cells were then allowed to grow in normoxic conditions for a period of 24 hours. Of the specific 5-HT agonists studies, only ␣-methyl-5-HT in the presence of 0.2% serum enhanced the proliferation of cells from chronically hypoxic rats. Similarly, the 5-HT2A inhibitor was the only inhibitor to block the hypoxia-associated proliferation in these cells. The experiment was repeated with cells from four different animals, and the results shown are typical of those obtained. * Value for hypoxia significantly greater than normoxia, p ⬍ 0.01. 1 ⫽ control; 2 ⫽ 0.2% serum; 3 ⫽ 10-␮M 5-HT; 4 ⫽ 0.2% serum ⫹ 10-␮M 5-HT; 5 ⫽ 0.2% serum ⫹ ␣-methyl 5-HT; 6 ⫽ 0.2% serum ⫹ BW723C86; 7 ⫽ 0.2% serum ⫹ MK212; 8 ⫽ 0.2% serum ⫹ 10-␮M 5-HT ⫹ ketanserin; 9 ⫽ 0.2% serum ⫹ 10-␮M 5-HT ⫹ SDZSER082; and 10 ⫽ 0.2% serum ⫹ 10-␮M 5-HT ⫹ RS102221.

RESULTS Effects of 5-HT Agonists and Antagonists on Proliferation of RPAF Cells from Normoxic and Chronically Hypoxic Rats

The effect of 5-HT on the proliferation of pulmonary artery fibroblasts was determined by measuring its effect on the uptake of [3H]thymidine into cells. Figure 1 shows the effect of 5-HT on the replication of pulmonary artery fibroblast cells from control and chronically hypoxic animals. The addition of either 0.2% serum, 3-␮M, or 10-␮M 5-HT alone did not significantly enhance the proliferation of fibroblast cells from either normoxic or those from chronically hypoxic animals in comparison with control untreated cells (p ⬎ 0.05). However, 3-␮M and 10-␮M 5-HT in the presence of 0.2% serum caused a 1.4- and 3.5-fold increase, respectively, in the [3H]thymidine uptake in cells from normoxic animals. The effect was even greater in the cells from hypoxic animals where there was a 3.3-fold increase over control cells with the addition of 0.2% serum and 3-␮M 5-HT and a 7.2-fold increase with the addition of 10-␮M 5-HT (Figure 1). Figure 2 shows the effects of 10-␮M 5-HT on the proliferation of RPAF cells from control and chronically hypoxic animals in the presence of increasing concentrations of serum. There was an enhanced proliferative response in the presence of 5-HT and serum in the cells from chronically hypoxic animals (p ⬍ 0.001) in comparison with serum alone. This was particularly true at lower concentrations of serum (⬍ 1%). For example, the presence of 0.5% serum had no effect on the proliferation of RPAF cells from chronic hypoxic animals (p ⬎ 0.05). However, in the presence of 10-␮M 5-HT, there was an 11.7-fold increase over control levels. Normoxic cells were also affected. Figure 3 shows the effects of specific 5-HT agonists and antagonists on the replication of pulmonary artery fibroblasts from control and chronically hypoxic animals. Unstimulated RPAF

cells from control and hypoxic animals showed no difference in [3H]thymidine uptake (p ⬎ 0.05). Similarly, the addition of 0.2% serum or 5-HT (10 ␮M) alone had no significant effect. However, as described for Figure 2, the addition of 5-HT (10 ␮M) in the presence of 0.2% serum resulted in a 2.7-fold increase in proliferation in normoxic cells (p ⬍ 0.005). This response was further enhanced in cells from chronically hypoxic animals where there was a sixfold increase in comparison with control, untreated cells. To address which 5-HT receptors mediated these effects, we studied a range of more specific 5-HT agonists. In the presence of 0.2% serum, the 5-HT2A agonist ␣-methyl-5-HT (10 ␮M) enhanced the proliferation of cells from hypoxic rats to the same degree as that of 10-␮M 5-HT alone. In contrast, the 5-HT2B agonist BW723C86 (10 ␮M) did not increase proliferation in either cell type. This was also true for the 5-HT2C agonist, MK212 (10 ␮M), which did not increase proliferation in either cell type. To study this further, we also looked at the effect of a range of 5-HT antagonists on the 5-HT–induced [3H]thymidine uptake. The addition of the 5-HT2A antagonist, ketanserin (0.1 ␮M), abrogated the enhanced growth response observed in the cells from chronically hypoxic animals induced with 5-HT and 0.2% serum (926 ⫾ 115 disintegrations per minute (DPM) for the hypoxic cells, which was not significantly different from normoxic cells 849 ⫾ 310 DPM). In contrast, neither the 5-HT2B⫹C inhibitor SDZ SER 082 (1 ␮M) nor the 5-HT2B inhibitor RS102221 (1 ␮M) had any effect on reducing proliferation of cells in the presence of 5-HT and 0.2% serum (1796 ⫾ 121 and 1847 ⫾ 314 DPM for the hypoxic cells, which was significantly different, p ⬍ 0.05, from normoxic cells, 882 ⫾ 265 and 886 ⫾ 172 DPM, respectively). We have previously shown that although Erk MAP kinase activity contributes to serum-stimulated DNA synthesis in both normoxic and hypoxic fibroblasts, p38 MAP kinase activity is responsible for the enhanced levels of replication observed in

Welsh, Harnett, MacLean, et al.: 5-HT and Hypoxia on Rat Fibroblasts

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of this reagent, the increase in [3H]thymidine uptake in cells from hypoxic rats was not significantly different to that from normoxic animals (793 ⫾ 102 DPM) (p ⬎ 0.05). In contrast, the addition of 0.1 ␮M of the MAP kinase kinase inhibitor U0126 completely abolished the proliferative response of normoxic cells (312 ⫾ 28 DPM) and those from chronically hypoxic animals (331 ⫾ 38 DPM, which was not significantly different from control levels, p ⬎ 0.05). The effects of 5-HT antagonists were also further investigated (Figure 4). As shown in the previous figure, the addition of the 5-HT2A antagonist ketanserin (0.1 ␮M) abolished the enhanced 5-HT growth response seen in the cells from chronically hypoxic animals (913 ⫾ 50 DPM). Like SB203580, the enhanced response was reduced to the level of that observed for cells from normoxic animals (832 ⫾ 78 DPM) (p ⬎ 0.05). In contrast, the 5-HT1B–1D antagonist GR55562 (1 ␮M) had no effect on the proliferation of cells from either the normoxic or hypoxic animals. Effect of 5-HT and 5-HT Antagonists on p38 Phosphorylation in Fibroblast Cells from Normoxic and Chronically Hypoxic Rats Figure 4. Effects of specific 5-HT and mitogen-activate protein (MAP) kinase inhibitors on 5-HT–induced [3H]thymidine uptake of pulmonary artery fibroblasts from control and chronically hypoxic animals. Uptake of [3H]thymidine from normoxic (open bar) and chronically hypoxic (closed bar) rats is shown. RPAF cells were grown to 60% confluence in 24-well plates and then quiesced in serum-free medium. The cells were stimulated with 10-␮M 5-HT (10 ␮M) in the presence of 0.2% serum (S) in the presence and absence of 0.1-␮M SB203580 (SB; p38 MAP kinase inhibitor), 1-␮M U0126 (MAP kinase kinase inhibitor), 0.1-␮M ketanserin (Ketan; 5-HT2A antagonist), or 1-␮M GR55562 (GR; 5-HT1B-1D antagonist) as indicated in the graph. Cells were then allowed to grow in normoxic conditions for a period of 24 hours. The addition of 1-␮M SB203580 (p38 MAP kinase inhibitor) abolished the enhanced 5-HT growth response observed in the cells from chronically hypoxic rats. The MAP kinase kinase inhibitor U0126 completely abolished the proliferative response of both normoxic fibroblasts and those from chronically hypoxic rats. The 5-HT1B–1D antagonist GR55562 had no effect on proliferation in either cell type. The experiment was repeated with cells from four different animals and the results shown are typical of those obtained. * Value for hypoxia significantly greater than normoxia, p ⬍ 0.01.

Given that our data demonstrate that the p38 MAP kinase inhibitor SB203580 abolishes the enhanced response to 5-HT plus 0.2% serum observed in hypoxic fibroblasts, we were interested to see whether 5-HT could further enhance p38 phosphorylation already observed in pulmonary artery fibroblasts from hypoxic rats (10). Figure 5A shows the effect of 5-HT antagonists on pp38 RPAF cells from control and chronically hypoxic animals. The upper panel shows that 10- and 3-␮M 5-HT gave rise to a substantial increase in pp38 in the cells from chronically hypoxic animals in comparison to control untreated cell extracts. There was no increase in pp38 in extracts from normoxic cells in response to 0.2% serum plus 5-HT. The increase in pp38 in the cells from chronically hypoxic animals could be abrogated with the addition of 0.1-␮M ketanserin, a specific 5-HT2A antagonist. In contrast, addition of 1-␮M GR55562, a 5-HT1B-1D antagonist, had no effect. The apparent slight inhibition of dual phosphorylation observed reflects reduced levels of total p38 expression in these samples (Figure 5, lower panel). Figure 5B shows the percentage increase in pp38 MAP kinase activity in the relative samples shown in Figure 5A by means of densitometry. Effect of 5-HT and 5-HT Antagonists on p42/p44 MAP Kinase Phosphorylation

hypoxic fibroblasts in response to 5% serum (10). Therefore, to determine whether either of these MAP kinases play a role in the synergistic responses to 0.2% serum plus 5-HT, we tested the effect of inhibitors of p38 MAP kinase (SB203580) and Erk MAP kinase (U0126, an inhibitor of MAP kinase kinase, the Erk MAP kinase kinase) signaling on the replication of pulmonary artery fibroblast cells from control and chronically hypoxic animals treated with 5-HT plus 0.2% serum (Figure 4). The role of specific 5-HT receptor subtypes in these responses was confirmed by the use of the selective antagonists ketanserin (5-HT2A) and GR55562 (5-HTIB/D). As shown previously, basal levels of replication in normoxic cells (220 ⫾ 15 DPM) and in cells from chronically hypoxic animals (230 ⫾ 20 DPM) were not significantly different (p ⬎ 0.05); 10-␮M 5-HT with the addition of 0.2% serum gave rise to a fourfold increase in proliferation in the normoxic cells and an enhanced increase (6.2-fold) in the cells from chronically hypoxic animals (p ⬍ 0.005). The addition of 1-␮M SB203580 (a specific p38 inhibitor) abolished this enhanced 5-HT growth response observed in the cells from chronically hypoxic animals (923 ⫾ 42 DPM). In the presence

As Watts (21) has previously shown that 5-HT can lead to the phosphorylation of Erk MAP kinase, we similarly wished to see whether 5-HT could further enhance the p42/p44 MAP kinase hyperphosphorylation that we reported in pulmonary artery fibroblasts from hypoxic rats (10). Figure 6A shows the effect of 5-HT antagonists on phosphorylation of p42/44 MAP kinase in pulmonary artery fibroblast cells from control and chronically hypoxic animals. Unstimulated cells from chronically hypoxic animals displayed increased levels of p42/44 MAP kinase activity compared with normoxic cells. There was a further increase in p42/p44 MAP kinase phosphorylation with the addition of 5% serum in RPAFs from hypoxic animals. Neither 10- nor 3-␮M 5-HT gave rise to increased p42/p44 MAP kinase phosphorylation in cells from chronically hypoxic animals or in the normoxic cells over control levels. To that end, the addition of ketanserin, a specific 5-HT2A antagonist, and GR55562, a 5-HT1B–1D antagonist, had no effect on phosphorylation of p42/ p44 MAP kinase. The total amount of p42/p44 MAP kinase was examined (Figure 6, lower panel) to ensure equal loading of protein on the polyacrylamide gel. Figure 6B shows the percentage

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Figure 5. Effect of 5-HT on p38 activity in pulmonary artery fibroblast cells from control and chronically hypoxic rats. (A ) RPAF cells were grown to 90% confluence in six-well plates and then quiesced for 24 hours in serum-free medium. The cells were then preincubated with 0.1-␮M ketanserin (K; a specific 5-HT2A antagonist) or 1 ␮M GR55562 (GR; a specific 5-HT1B/1D antagonist) for 2 hours before being stimulated with 3- or 10-␮M 5-HT for 24 hours. The 5-HT increase in p38 phosphorylation in the cells from chronically hypoxic rats could be blocked by the addition of the 5-HT2A receptor antagonist ketanserin. In contrast, the 5-HT1B–1D antagonist had no effect. The experiment shown is one of four replicate experiments and is typical of those obtained. Band density is used as a measure of protein level. 1 ⫽ control; 2 ⫽ 5% serum; 3 ⫽ 10-␮M 5-HT; 4 ⫽ 3-␮M 5-HT; 5 ⫽ 10-␮M 5-HT ⫹ 0.1-␮M ketanserin; 6 ⫽ 3-␮M 5-HT ⫹ 0.1-␮M ketanserin; 7 ⫽ 10-␮M 5-HT ⫹ 1-␮M GR55562; 8 ⫽ 3-␮M 5-HT ⫹ 1-␮M GR55562. (B ) Quantitation of p38 MAP kinase phosphorylation. Densitometry was used to compare the relative amounts of p38 MAP kinase phosphorylation, and this was expressed as a percentage of the control. The values shown represent the mean of four experiments, including the one illustrated.

increase in p42/44 MAP kinase activity in the relative samples shown in Figure 6A by means of densitometry. Effects of the 5-HTT on Proliferation of RPAF Cells from Normoxic and Chronically Hypoxic Rats

The effect of the 5-HTT on the proliferation of pulmonary artery fibroblasts was determined by measuring its effect on the uptake of [3H]thymidine into cells. Figure 7 shows the effect of the 5-HTT on the replication of pulmonary artery fibroblast cells from control and chronically hypoxic animals. In the cells from chronically hypoxic animals, the addition of the 5-HTT inhibitor fluoxetine (10 ␮M) had a similar effect to that of the 5-HT2A antagonist ketanserin (0.1 ␮M), which abrogated the enhanced growth response observed in the cells from chronically hypoxic animals induced with 5-HT and 0.2% serum. However, fluoxetine and ketanserin in combination could reduce proliferation basal levels (p ⬎ 0.05). In the normoxic cells, fluoxetine could reduce the 5-HT and serum-stimulated fibroblast proliferation to basal levels (p ⬎ 0.05).

DISCUSSION This study focused on the effects of 5-HT on the proliferation and cell signaling of RPAF cells from normoxic and chronic hypoxic rats. 5-HT alone did not produce a mitogenic response in pulmonary artery fibroblast cells from either control or chronically hypoxic animals. These results are in keeping with the work of others (22, 23) who studied 5-HT–induced proliferation of bovine and rat pulmonary vascular smooth muscle cells in culture. In those studies, 5-HT had a co-mitogenic effect on the cells, requiring incubation with other growth factors such as

platelet-derived growth factor and epidermal growth factor. This agrees with our findings. The addition of a low concentration of serum (which in itself does not increase proliferation) in conjunction with 5-HT resulted in an increase of proliferation in both the normoxic and especially in the chronically hypoxic cells. The effect of 5-HT may require either binding of 5-HT to cell membrane receptors or active transport of 5-HT into the cell via the 5-HTT (15, 24, 25). Previous work has demonstrated that the proliferative effect of 5-HT on [3H]thymidine incorporation in bovine pulmonary artery smooth muscle cells was abolished after inhibition of the 5-HTT into the cell (26, 27). Our previous work (10) demonstrated that fibroblast cells from chronically hypoxic animals displayed increased basal levels of p38 and p42/p44 MAP kinase phosphorylation in comparison to those from normoxic cells and that these levels were further increased in response to 5% serum. In this study, 5-HT further increased pp38 above that of the enhanced basal levels in a dosedependent manner. This occurred in the absence of any comitogens. Other work on vascular smooth muscle cells has demonstrated that 5-HT mediates its response by phosphorylating p42/44 MAP kinase (28, 29). The mechanisms for this activation are unclear; however, signaling pathways for 5-HT have classically included activation of potential upstream regulators of Erk MAP kinase such as phosolipase C and plasma membrane calcium channels that are sensitive to inhibition by dihydropyridines (29). In our work, however, p42/p44 activation was activated by 5-HT only in the presence of co-mitogens. We believe that our study on RPAFs is the first to show that pp38 is activated by 5-HT and also that the specific p38 MAP kinase inhibitor, SB203580, can block the enhanced hypoxia-associated proliferation caused by 5-HT. We were particularly interested in investigating the effects

Welsh, Harnett, MacLean, et al.: 5-HT and Hypoxia on Rat Fibroblasts

257 Figure 6. Effect of 5-HT on p42/44 MAP kinase activity in pulmonary artery fibroblast cells from control and chronically hypoxic rats. (A ) RPAF cells were grown to 90% confluence in six-well plates and then quiesced for 24 hours in serum-free medium. The cells were then preincubated with 0.1-␮M ketanserin (a specific 5-HT2A antagonist) or 1-␮M GR55562 (a specific 5-HT1B/1D antagonist) for 2 hours before being stimulated with 3- or 10-␮M 5-HT for 24 hours. Assessment of kinase activity was by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and Western blot analysis. There was no increase in p42/ p44 MAP kinase phosphorylation observed in either the normoxic or the fibroblasts from chronically hypoxic rats with any dose of 5-HT. The specific 5-HT 1B-1D antagonist GR55562 had no effect on p42– p44 phosphorylation. The experiment shown is one of four replicate experiments and is typical of those obtained. Band density is used as a measure of protein level. 1 ⫽ control; 2 ⫽ 5% serum; 3 ⫽ 10-␮M 5-HT; 4 ⫽ 3-␮M 5-HT; 5 ⫽ 10-␮M 5-HT ⫹ 0.1-␮M ketanserin; 6 ⫽ 3-␮M 5-HT ⫹ 0.1-␮M ketanserin; 7 ⫽ 10-␮M 5-HT ⫹ 1-␮M GR55562; 8 ⫽ 3-␮M 5-HT ⫹ 1-␮M GR55562. (B ) Quantitation of p42/44 MAP kinase phosphorylation. Densitometry was used to compare the relative amounts of p42/44 MAP kinase phosphorylation, and this was expressed as a percentage of the control. The values shown represent the mean of four experiments, including the one illustrated.

of 5-HT1 and 5-HT2 receptor antagonists on MAP kinase activation and proliferation of RPAF cells, as previous work has indicated that although the 5-HT1 receptor is involved in contraction, the 5-HT2 receptor is involved in proliferation of cells (19). For example, studies by other groups in rat aortic smooth muscle cells indicated that the 5-HT2A receptor mediated 5-HT–stimulated phosphorylation and activation of the p42/p44 MAP kinases (30). This is unlikely to be the case for RPAF cells because 5-HT alone does not increase p42/p44 phosphorylation. Moreover, although we found that ketanserin blocked the increased proliferation in the pulmonary artery fibroblast cells from chronically hypoxic animals, it had no effect on either the residual response to 0.2% serum plus 5-HT in hypoxic cells or the comparable stimulation observed in normoxic cells. Inhibition of the 5-HTT

simultaneously with ketanserin was required before all 5-HT– stimulated proliferation could be inhibited in the cells from chronically hypoxic animals, whereas 5-HTT inhibition alone could block the 5-HT and serum synergistic proliferation in the normoxic cells. Furthermore, the enhanced proliferation of hypoxic cells blocked by ketanserin correlated with that blocked by the p38 MAP kinase inhibitor, SB203580. Indeed, ketanserin blocked 5-HT–mediated p38 MAP kinase activity, suggesting that the 5-HT2A receptor mediated the enhanced proliferation observed in hypoxic cells in a p38 MAP kinase-dependent manner. To investigate whether other 5-HT receptors played a role in the proliferation and MAP kinase activation in normoxic or chronically hypoxic animals with respect to 5-HT, further inhibitor studies in conjunction with specific agonist studies were

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the actions of this receptor we also blocked pp38 activation. We have previously shown pp38 (10) to be essential for hypoxic growth of pulmonary artery fibroblasts while not being so for systemic vascular cells. Taken together, our previous work and this study suggest that selective 5-HT2A receptor antagonists may be useful in reducing the pulmonary vascular remodeling associated with PHT. Conflict of Interest Statement : D.J.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.J.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Figure 7. Effects of the 5-HT transporter (5-HTT) inhibitor fluoxetine on the replication of pulmonary artery fibroblasts from control and chronically hypoxic animals. Uptake of [3H]thymidine from normoxic (open bar) and chronically hypoxic (closed bar) rats is shown. RPAF cells were grown to 60% confluence in 24-well plates and then quiesced in serum-free medium. The cells were stimulated with serum (0.2%) in conjunction with 5-HT (10 ␮M) in the presence or absence of 0.1-␮M ketanserin (5-HT2A antagonist) and 10-␮M fluoxetine (5-HTT inhibitor) as indicated in the graph. Cells were then allowed to grow in normoxic conditions for a period of 24 hours. In the cells from chronically hypoxic animals, individually, ketanserin and fluoxetine could block the apparent increase in proliferation seen in the cells from chronically hypoxic rats to 5-HT and serum. However, in combination, this inhibition could be reduced to basal levels. In the normoxic cells, fluoxetine blocked the proliferative effects of 5-HT and serum. The experiment was repeated with cells from four different animals, and the results shown are typical of those obtained. * Value for hypoxia significantly greater than normoxia, p ⬍ 0.01. 1 ⫽ control; 2 ⫽ 0.2% serum ⫹ 10-␮M 5-HT; 3 ⫽ 0.2% serum ⫹ 10-␮M 5-HT ⫹ ketanserin; 4 ⫽ 0.2% serum ⫹ 10-␮M 5-HT ⫹ fluoxetine; 5 ⫽ 0.2% serum ⫹ 10-␮M 5-HT ⫹ ketanserin ⫹ fluoxetine.

undertaken. There are three 5-HT2 receptor subtypes that have been identified: 5-HT2A, 5-HT2B, and 5-HT2C. Unfortunately, there are no specific 5-HT2A agonists commercially available; however, there are selective 5-HT2B (BW72386) and 5-HT2C (MK212) agonists and a specific 5-HT2 agonist (␣-methyl 5-HT). Use of these compounds allowed us to determine the specificity of the subtypes involved in proliferation of RPAF cells. There are also selective antagonists available namely, 5-HT2A (ketanserin), 5-HT2C (RS102221), and a specific 5-HT2B/C antagonist (SDZ SER 082), which allowed us to determine further the action of the individual receptor subtypes. Our results demonstrated that neither 5-HT2B nor 5-HT2C agonists in the presence of low serum contribute to the proliferation of normoxic or hypoxic cells. However, the selective 5-HT2 agonist gave rise to a proliferative response in both cells types, which equaled the response in terms of magnitude of 5-HT itself. This suggests that the 5-HT2A receptor may be in part responsible for proliferation of these cells. Inhibitor studies using a 5-HT2A inhibitor supported this finding. The use of specific 5-HT2B/C inhibitors could not inhibit the replicative responses of these cells to 5-HT. The hypoxic-associated growth of these cells, however, could be successfully abrogated by the use of ketanserin (5-HT2 inhibitor). These findings are consistent with the agonist studies and firmly pointed toward the 5-HT2A receptor as being the receptor responsible for hypoxicassociated enhanced growth of pulmonary artery fibroblast cells from chronically hypoxic animals, whereas the 5-HTT plays a role in the serum proliferative response. The 5-HT2A receptor also appears to signal via p38 MAP kinase because by blocking

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