Pulmonary and Critical Care Division/Tupper Research Institute, Department of Medicine, New England Medical Center/Tufts University School of Medicine, Boston, Massachusetts 02111
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ABSTRACT |
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Our previous
studies have shown that 5-hydroxytryptamine (5-HT) induces cellular
hyperplasia/hypertrophy through protein tyrosine phosphorylation, rapid
formation of superoxide (O
5-hydroxytryptamine; serotonin; reactive oxygen species; extracellular signal-regulated kinase; mitogen-activated protein kinase; mitogenesis
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INTRODUCTION |
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THE CELLULAR REDOX
STATE has been increasingly recognized as playing an important
role in mediating cellular growth in response to growth factors and
cytokines. We have previously reported that 5-hydroxytryptamine (5-HT)
produces hyperplasia/hypertrophy of bovine pulmonary artery
smooth muscle cells (BPASMCs) via its active transporter and signaling
through tyrosine phosphorylation of GTPase-activating protein (GAP),
Ras activation, activation of NAD(P)H oxidase producing superoxide
(O
In addition, we have explored the possible role of p38 MAP kinase
in the proliferative responses of BPASMCs to 5-HT. Although this
kinase has been previously shown to be responsive to angiotensin II and
other stimuli (29, 31, 32, 40, 41), 5-HT failed to
activate p38 MAP kinase in the BPASMCs, whereas SB-203580 and SB-202190, reported inhibitors of p38 MAP kinase, inhibited
5-HT-induced growth of the BPASMCs. These agents also blocked cellular
production of O
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MATERIALS AND METHODS |
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Reagents.
Phospho-specific p44/42 MAP kinase
(Thr202/Tyr204) and phospho-specific p38 MAP
kinase (Thr180/Tyr182) antibodies were from New
England BioLabs (Beverly, MA). Anti-catalase antibody, SB-203580, and
SB-202190 were from Calbiochem (San Diego, CA). WB-4101 HCl,
(±)-8-hydroxy-dipropylaminotetralin hydrobromide (DPAT), BW-723C86,
-methyl-5-hydroxytryptamine maleate (m-5-HT), R-(
)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI),
clorgyline HCl, R-(
)-deprenyl HCl, Ro-41-1049, and
Ro-16-6491 were from Research Biochemicals International (Natick, MA).
N,N'-diethyldithiocarbamate (DETC), catechol,
tetraethylenepentamine (TEPA), triethylenetetramine (TRIEN), and all
other reagents were from Sigma Chemical (St. Louis, MO).
Cell culture. SMCs from bovine pulmonary artery were isolated and cultured by a modification of the method of Ross as previously described (24). Third- to fifth-passage SMCs were used.
Incorporation of [3H]thymidine. In brief, plated cells were cultured for 72 h in RPMI medium containing 10% FBS, followed by growth arrest in medium without FBS for 72 h. Cells were then incubated with and without 5-HT in the same medium for 20 h before being labeled with [methyl-3H]thymidine (0.1 mCi/ml, specific activity 20 Ci/mmol, New England Nuclear, Boston, MA) for 4 h. Four-hour preincubation was used for copper chelators to inhibit SOD and for buthionine sulfoximine (BSO) to inhibit biosynthesis of glutathione (2). Other inhibitors were added 30 min before the 5-HT. These agents alone, at the concentrations reported, did not alter the incorporation of [3H]thymidine into these cells. After labeling, experiments were terminated by aspiration of medium and washing of the cellular monolayer, first with ice-cold phosphate-buffered saline (PBS) and then with cold 6% trichloroacetic acid. Cells were then dissolved in 0.2 N NaOH, and radioactivity was counted.
Catalase infection. BPASMCs were infected with adenovirus containing catalase (AdSCMOCat) for 16 h using 20,000 and 40,000 particles/cell. Cells were growth arrested in medium without FBS for 72 h before exposure to 1 µM 5-HT and were then processed for [3H]thymidine incorporation or for electrophoresis. Infection with an adenovirus vector without the cDNA insert was done as a control. Adenovirus containing catalase was provided by Dr. John Englehardt, University of Iowa.
Measurement of O
Preparation of whole cell extracts for electrophoresis. Cells were grown in 100-mm petri dishes and were growth arrested in medium as described above. The cells were preincubated with inhibitors for 1-2 h before the addition of 1 µM 5-HT for the periods indicated in RESULTS. Cellular monolayers were then washed twice with ice-cold PBS. Cell lysates were obtained by incubating the cellular monolayer in 1 ml of cell lysis buffer for 10 min at 4°C. The insoluble material was removed by centrifugation (14,000 g, 2 min), and the supernatant fraction was used for analysis. Twenty micrograms of protein of the whole cell lysate were subjected to SDS-PAGE on a Novex 12% precast gel (Novex, San Diego, CA).
Immunoblot. After electrophoresis, gel proteins were electrophorectically transferred to a polyvinylidene difluoride (PVDF) membrane (Tropifluor; Tropix, Bedford, MA). After transfer, nonspecific PVDF binding sites were blocked with 5% HiPure liquid gelatin (Norland, New Brunswick, NJ) in buffer, pH 7.4. Blocking was done for 1 h at ambient temperature. The membrane was then treated overnight with a 1:1,000 dilution of phospho-specific p44/p42 or p38 MAP kinase antibody in blocking buffer at 4°C. The membrane was then washed with washing buffer for 30 min, the wash buffer being changed every 10 min before incubation for 1 h at ambient temperature with a 1:25,000 dilution of secondary antibodies (horseradish peroxidase-conjugated anti-rabbit antibody from Santa Cruz Biotechnology, Santa Cruz, CA) in HiPure buffer. Protein phosphorylation was then detected with SuperSignal chemiluminescent substrate (Pierce, Rockford, IL). To confirm catalase infection, the blot was then stripped and reprobed with anti-catalase antibody at 1:3,000 dilution. Densitometry was performed with a Millipore densitometer with Visage version 4.6p software (Millipore, Bedford, MA). Data are expressed as degree of stimulation compared with control sample.
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RESULTS |
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We have previously reported that O
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5-HT activated ERK1/ERK2 MAP kinase three- to fourfold at 10 min of incubation (Fig. 3B, top), and this activation was inhibited when cells were infected with adenovirus containing catalase at 20,000 and 40,000 particles/cell (Fig. 3B, lanes 3 and 4). The adenoviral vector alone did not affect the 5-HT-induced activation of ERK MAP kinase (Fig. 3B, lane 5 vs. 2).
Although activation of ERK MAP kinase was induced by 5-HT, we failed to
observe any activation of p38 MAP kinase (data not shown).
Nevertheless, the p38 MAP kinase inhibitors SB-202190 and SB-203580
dose dependently inhibited 5-HT-induced [3H]thymidine
incorporation, as shown in Fig. 4,
A and B. These inhibitors were also found to
block O
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Various pathways for induction of cellular mitogenesis by 5-HT have
been proposed via both the 5-HT transporter and 5-HT receptors (7). However, receptor agonists of 5-HT1A
(1-10 µM of WB-4101 and DPAT), 5-HT2B (10 µM
BW-723C86), and 5-HT2 (10 µM of m-5HT and DOI) all failed
to show any enhancement in O
Exogenous application of H2O2 to BPASMCs
activated ERK1/ERK2 MAP kinase dose and time dependently as shown in
Fig. 6A but failed to induce
DNA synthesis in this cell type (Fig. 6B).
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DISCUSSION |
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O
ERK MAP kinase activation is now well recognized to be associated with
cellular proliferation induced by growth factors and cytokines through
the formation of ROS. Aside from the studies with 5-HT, angiotensin II
is the most widely examined vasoactive agent that signals through ROS
and MAP kinase. However, activation of specific pathways for
angiotensin II-induced SMC hyperplasia/hypertrophy remains
controversial. Some laboratories have reported that ROS participate in
angiotensin II-induced protein tyrosine phosphorylation of receptor
tyrosine kinases and mediate the downstream signaling events including
activation of ERK MAP kinase (1, 9, 34, 43, 44). Others
(40, 41) have reported that c-Jun amino-terminal kinase
(JNK) and p38 MAP kinase, but not ERK, are the critical redox-sensitive
signaling pathways activated by angiotensin II. p38 MAP kinase is a
Ser/Thr protein kinase activated by various inflammatory cytokines and
a variety of stress stimuli (29, 31, 32, 34, 35), but it
also acts as a negative regulator of cell proliferation (3,
22). Our studies have shown that p38 MAP kinase activation is
not in the intermediate signaling pathway for 5-HT (data not shown);
rather, the participation of SB-202190 and SB-203580 (inhibitors of p38
MAP kinase) in the blockade of 5-HT-induced DNA synthesis is more
likely through O
Recently, production of ROS (O
Recently Jin et al. (19) reported that exogenously added H2O2-induced egr-1, fra-1 and c-jun gene expression is mediated through platelet-derived growth factor receptor tyrosine kinase activation. Our data show that the extracellular application of 0.01-50 µM H2O2 activated ERK MAP kinase but failed to induce mitogenesis. This is consistent with observations by Rao (35), who found that H2O2 induced eukaryotic translation initation factor 4E phosphorylation and expression of c-fos, c-jun, and Hsp70 mRNA but failed to stimulate hypertrophy in SMCs. Because of this observation, Rao questioned the role of intracellular H2O2 in angiotensin II-induced hypertrophy as proposed by Zafari et al. (44) and concluded that these activations were associated with cell survival. Application of H2O2 extracellularly may differ considerably in cellular response from that generated intracellularly at specific locations in response to an applied signaling agent such as 5-HT or angiotensin II.
The mitochondrial enzyme monoamine oxidase B has been recently reported
to generate H2O2, which may lead to
ERK-dependent cellular mitogenesis (42). This enzyme is
unlikely to be involved in 5-HT-induced mitogenesis because we failed
to observe any inhibition by monoamine oxidase type A (10 µM of
clorgyline and Ro-41-10490) or type B (10 µM of deprenyl and
Ro-16-6491) inhibitors (data not shown). Our previous data showing that
O
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ACKNOWLEDGEMENTS |
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This study was supported by Research Grant R01-HL-32723 from the National Heart, Lung, and Blood Institute of the National Institutes of Health.
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. L. Fanburg, New England Medical Center, Pulmonary and Critical Care Division, 750 Washington St., NEMC#257, Boston, MA 02111 (E-mail: bfanburg{at}lifespan.org).
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.
Received 8 February 2001; accepted in final form 23 April 2001.
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