Introduction

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Mannosyl-rich lysosomal hydrolases (-hexosaminidases: Hex A and B and -glucuronidase) are secreted into the extracellular space selectively by inflammatory cells such as mast cells, basophils, macrophages, eosinophils, and neutrophils (1). Increased levels of these enzymes in bronchoalveolar lavage fluid (BALF) have been reported after dust-mite allergen challenge in sensitized human subjects (4, 5) and in ozone-exposed guinea pigs (6). These mannosyl-rich lysosomal hydrolases are potent mitogens for bovine airway smooth-muscle cells (ASMC) (7), and the mitogenic action of these mannosyl-rich enzymes is mediated via airway smooth-muscle mannose-recognizing receptors (ASM-MR) (8). These ASM-MR have a molecular mass of 175 kD and belong to a family of multilectin mannose receptors, including macrophage mannose receptor, secretory phospholipase A₂ receptor, DEC 205, and mannose receptor X (9). With regard to postreceptor signaling, a transient increase in cyclic adenosine monophosphate (cAMP) (10) and activation of protein kinase C (PKC) are involved in Hex-induced mitogenesis in ASMC (11). Even though a number of growth factors, such as platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1 (12) induce sustained activation of p44/42^MAPK, the characteristics and role of p44/42^MAPK activation in Hex-induced ASMC proliferation are unknown.

Many receptor tyrosine kinases utilize p21Ras to bring Raf to the plasma membrane (13, 14). Farnesylation of p21Ras in the cysteine alanine alanine random amino acid (CAAX) motif enables the molecule to anchor to the membrane and recruit Raf-1 (15). Activation of Raf-1, which requires 14-3-3 proteins (18), is followed by phosphorylation and activation of mitogen-activated protein kinase (MAPK) kinase (MEK1) and MAPKs. In some cases, activation of MAPKs is partly dependent on PKC activation (19). In other cases, it has been proposed that PKC directly activates Raf-1, bypassing p21Ras (23). However, a recent report by Marais and coworkers (24) showed that Raf-1 activation by PKC requires Ras-guanosine triphosphate (GTP)-Raf complex formation. Whether or not p21Ras is involved in the Hex-induced activaton of p44/42^MAPK is unknown.

The objectives of this study were to examine the pattern of p44/42^MAPK activation in response to Hex in ASMC, to establish its relationship to p21Ras and PKC, and to examine the role of p44/42^MAPK activation in Hex-induced ASMC proliferation.

	Materials and Methods

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ASMC Culture

Primary cultures of bovine ASM cells were prepared from bovine trachealis muscle as described previously (7). Direct immunofluorescence staining of the cells with a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (mAb) to smooth-muscle isoactin (Sigma Chemical Co., St. Louis, MO) showed that cultures consisted of a homogeneous population of smooth-muscle cells (7). Cells at passages 1-7 were used for experiments.

Western Blot Analysis of p44/42^MAPK and Phospho-p44/42^MAPK

Confluent cells in 35-mm-well plates were starved for 48 h in medium M199 (Cellgro; Mediatech, Washington, DC) containing 0.4% (vol/vol) fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT). Cells were stimulated with Hex A (50 nM) or vehicle (phosphate buffered saline, [PBS]) for various periods at 37°C. When the effect of inhibitors was studied, the cells were treated with an inhibitor or vehicle at 37°C in a CO₂ incubator, followed by Hex A treatment for 2 h at the same temperature. The inhibitors studied were FPT III dissolved in 10% ethanol (1-10 µM, 4 h pretreatment; Calbiochem, San Diego, CA); SCH-56582 dissolved in dimethylsulfoxide (DMSO) (1-10 µM, 4 h pretreatment, a gift from Dr. Robert Bishop, Schering-Plough Research Institute, Kenilworth, NJ); PD98059 dissolved in DMSO (1-5 µM, 30 min pretreatment; Calbiochem); and Tyrphostin AG-126 dissolved in DMSO (0.1- 0.2 µM, 3 h pretreatment; Calbiochem). Purified bovine epididymal -N-acetylglucosaminidase A (-hexosaminidase A, Hex A) was purchased from Sigma Chemical Company and was dialyzed against phosphate-buffered saline (PBS) for 4 h, using a Slide-A-Lyzer dialyzer (M.W. cutoff: 10,000; Pierce, Rockford, IL). Cells were washed three times with cold PBS and lysed with radio immunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1% NP-40; 0.25% Na-deoxycholate; 1 mM phenylmethylsulfonyl fluoride [PMSF]; 1 mM ethylene glycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid [EGTA]; 1 mM Na₃VO₄; 1 mM NaF; 10 µg/ml leupeptin; and 10 µg/ ml aprotinin). Cell lysates (12 µg protein per sample) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the electrophoresed proteins were transferred onto Hybond-ECL nitrocellulose membranes (Amersham, Arlington Heights, IL). The membranes were incubated overnight at 4°C with a blocking buffer (5% nonfat dry milk in PBS/0.02% Tween-20). After three washes in PBS containing Tween-20 (PBS-T), the membranes were incubated overnight at 4°C with an antibody as follows: polyclonal anti-p44/42^MAPK (1:1,000 in PBS-T containing 5% bovine serum albumin [New England Biolabs, Inc., Beverly, MA]) or polyclonal antiphospho-p44/42^MAPK (Thr²⁰²/Tyr²⁰⁴) antibody (1:1,000 in PBS-T containing 5% nonfat dry milk [New England Biolabs]). The nitrocellulose membranes were then washed three more times in PBS-T and incubated with a 1:1,000 dilution of antirabbit, horseradish peroxidase-linked whole antibody (Amersham) in PBS-T for 1 h at room temperature. Four more extensive washes with PBS-T were performed, and the immunoreactive protein bands were visualized on X-ray film with an enhanced chemiluminescence light (ECL)-detection system (Amersham).

Ras Activation

Cells were grown to approximately 70% confluence on chamber slides (Nunc, Inc., Naperville, IL). They were rinsed with Hanks' balanced salt solution (HBSS) and arrested over a period of 24 h in 1 ml of medium M199 containing 0.1% fetal bovine serum (FBS). This starvation protocol, as compared with the usual protocol of starving cells for 48 h in 0.4% FBS-containing media, does not affect signaling-activation pathways or cell proliferation induced by Hex A in bovine ASMC. The standard cells were then stimulated with Hex A (50 nM) or vehicle (PBS) for 10 min at 37°C. When the effect of inhibitors was studied, the cells were treated with FPT III (10 µM) for 24 h at 37°C in a CO₂ incubator, followed by Hex A treatment for 10 min at the same temperature. The cells were washed for 5 min at 22°C three times in PBS to wash away the inhibitor, and fixed in 4% paraformaldehyde in PEM buffer (1,4-piperazinediethane sulfonic acid, 10 mM; EGTA, 5 mM; MgCl₂, 2 mM; pH 6.8) containing 0.2% Triton X-100, and all washings were done with PBS. The cells were rehydrated in PBS containing 0.1% BSA for 30 min, and were treated for 1 h with PBS containing 3% BSA and 0.25% gelatin. A monoclonal anti-p21Ras antibody (F235 diluted 200-fold with PBS containing BSA and gelatin; Santa Cruz Biotechnology, Santa Cruz, CA) was applied to each well. After 1 h the cells were washed three times and exposed to biotinylated antimouse IgG (10 µg/ml; Vector Laboratories, Burlingame, CA) for 30 min in the dark. The cells were washed three times and rinsed quickly with water. A drop of Fluoromount-G (Fisher Scientific, Pittsburgh, PA) was applied to the cell surface, and coverslips were mounted. Slides were viewed by confocal fluorescence microscopy (MRC-1000 Lazer Scanning Confocal Imaging system, using an argon/krypton lamp; Bio-Rad, Hercules, CA) with a ×100 objective lens.

Assessment of Proliferation

To assess cell counts and cytotoxicity, a modified, colorimetric, tetrazolium salt reduction (3-[4,5-dimethylthiozol-2-yl]-2,5-diphenyl tetrazolium bromide [MTT]) assay was used (25). After the cells reached confluence and were contact-inhibited in microtiter wells, the medium was replaced with medium M199 containing 0.4% (vol/vol) FBS. After a 48-h starvation, and without changing the medium, cells were exposed to the test inhibitors followed by Hex A (50 nM) for 48 h. MTT at 5 mg/ml, Millipore filtered (Sigma), dissolved in PBS, was added to the adherent cells in microtiter wells (20 µl/well) at the end of the experimental period. After a 4-h incubation at 37°C, media were removed from the microtiter wells, and acidified (0.04 M HCl) 2-propane/isopropanol was added to the wells (200 µl/well). The plates were left at room temperature for 30 min, and were then read on an enzyme-linked immunoassay (ELISA) plate reader (EL340 Microplate; Bio-Tek Instruments, Winooski, VT) at a wavelength of 570 nm. The values for the cell blank were obtained similarly, except for MTT, which was substituted with PBS. Cell numbers were derived from a standard curve generated from known cell numbers. It has been shown that viable cell numbers correlate with OD as determined with the MTT assay; the tetrazolium ring is cleaved in active mitochondria, and the reaction therefore occurs only in living cells (25). If the serum content is high ( 5%), however, OD is altered and the results are not reliable (26). When necessary, manual counts of viable cells were obtained with a hemocytometer, using trypan blue according to the manufacturer's description (Sigma Catalog, tissue culture media and reagents section, 1997). Experiments were performed in quadruplicate and were repeated three times.

Inhibition of MAPK Expression by Antisense DNA Oligonucleotide

The effects on p44/42^MAPK activities and cell proliferation in Hex-stimulated cells of transient transfection with antisense oligonucleotides were assessed under similar conditions to those described earlier. Antisense phosphorothioate-derivatized oligonucleotides (S-oligos, 18-mer), prepared through a polymerase chain reaction targeted to the translation initiation site (27) of human p44/42^MAPK messenger RNA (mRNA) (28), were synthesized in the Molecular Resource Center at the University of Tennessee, Memphis. As controls, we used the following sense oligonucleotides: human p44^MAPK (antisense), 5'-ATA GGC CGA GCT GAC CAT-3'; human p44^MAPK (sense), 5'-ATG GTC AGC TCG GCC TAT- 3'. Subconfluent (70-80%) ASMC in 35-mm well plates were incubated for 24 h with Optimem medium (GIBCO-BRL, Gaithersburg, MD) containing 0.1% FBS, and were then either left untreated or were treated with antisense or sense oligonucleotides (1 µM) along with 3 µl/ml of lipofectamine (GIBCO-BRL) for 6 h at 37°C in a CO₂ incubator. At the end of 6 h of incubation, FBS was added to achieve a final concentration of 10% (vol/vol), and cells were further incubated overnight for cell growth. All transfection media were removed, and fresh medium M199 containing 10% (vol/vol) FBS was added. The procedures were repeated twice (three 24-h pulse treatments). After the last pulse treatment, the medium was changed to M199 containing 0.4% (vol/vol) FBS. Cells were then stimulated with Hex A (50 nM, 2 h) for MAPK activity assays. Parallel experiments were set up for cell proliferation assays (72 h stimulation for cell counts).

p44/42^MAPK Kinase Enzyme Assay

The assays were done with the BIOTRAK MAPK assay system (Amersham) on both the membrane and cytosolic fractions of samples. This assay system is based on the p42/ 44^MAPK-catalyzed transfer of the -phosphate group of adenosine triphosphate (ATP) to a synthetic peptide that is highly selective for p42/44^MAPK. The reaction is initiated by the addition of [-³²P]ATP. After an incubation for 30 min at 30°C, the peptide is separated from unincorporated radioactivity through a binding-paper separation, according to the manufacturer's instructions. The peptide used in the assay kit (KRELVEPLTPAGEAPNQALLR) is based on the single Thr⁶⁶⁹ phosphorylation site of the EGF receptor, and although the protein is not totally specific for p44/42^MAPK, it is approximately 10 times more specific for p44/42^MAPK than the commonly used substrate, myelin basic protein, when comparing p44/42^MAPK and p34cdc2 kinase activities according to the manufacturer's data (Table 3 in the Amersham instruction manual). Enzyme activity was expressed as pmol/min/mg protein.

Data Analysis

Experiments were performed in quadruplicate for cell proliferation assays. All experiments were repeated at least three times, and data were analyzed with the StatView 4.5 and SuperANOVA software systems (Abacus Concepts, Berkeley, CA) with Dunnett's post hoc test.

	Results

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Activation of p44/42^MAPK in Response to Hex A in Bovine ASMC

To examine the pattern of p44/42^MAPK activation, quiescent cells in 35-mm well plates were stimulated with Hex A (50 nM) or vehicle (PBS) for various times (0, 5, 15, and 30 min, and 1, 2, 4, 6, and 8 h). Western blot analysis of cell lysates was done with one of the polyclonal antibodies specific for p44/42^MAPK or phospho-p44/42^MAPK. The polyclonal anti-phospho-p44/42^MAPK antibody detects only the catalytically activated forms that are phosphorylated at the Thr²⁰² and Tyr²⁰⁴ residues (29, 30). Phospho-p44/42^MAPK was increased in Hex-stimulated cells as compared with vehicle-treated cells at 30 min, 1 h, 2 h, and 4 h (Figure 1). The control blots for p44/42^MAPK showed that there were equal amounts of p44/42^MAPK proteins in the Hex-stimulated cells (Figure 1).

View larger version (35K): [in this window] [in a new window]   Figure 1.   Time course of activation of p44/42MAPK in response to Hex A in bovine ASMC. Quiescent cells in 35-well plates were treated with Hex A (H, 50 nM) or vehicle (V, PBS) for various times (0-8 h) at 37°C in a CO2 incubator. Cell lysates in RIPA buffer (12 µg protein/sample) were subjected to 10% SDS- PAGE and Western blot analysis for p44/42MAPK (left panels) and phospho-p44/42MAPK (right panels). A polyclonal anti-p44/42MAPK (1:1,000 dilution; New England Biolabs), or polyclonal antiphospho-p44/42MAPK (Thr202/Tyr204) antibody (1:1,000 dilution) was used as the primary antibody. A horseradish peroxidase-linked whole antirabbit antibody (1:1,000 dilution; Amersham) was used as the secondary antibody. The immunoreactive protein bands were visualized on X-ray film with an enhanced chemiluminescence light (ECL)-detection system.

Effect of SCH-56582, PD98059, FPT III, AG-126, and Calphostin C on Hex-Induced Activation of p44/42^MAPK

Recently, Karpova and colleagues (31) reported that MEK1 is required for platelet-derived growth factor (PDGF)-induced ERK activation and DNA synthesis in bovine tracheal smooth-muscle cells. To establish the possible dependence of Hex-induced p44/42^MAPK activation on MEK1, we tested the effect of PD98059, a specific inhibitor of MEK1 (32). Pretreatment of cells with PD98059 (5 µM) attenuated the activation of p44/42^MAPK as judged by immunoblot analysis for phospho-p44/42^MAPK (Figure 2A). Because of the known importance of sustained activation of MAPK in airway smooth-muscle proliferation (12), we examined the effect of inhibitors of MAPK activation at a later time point (2 h). To test for the possible involvement of p21Ras in Hex-induced p44/42^MAPK activation, we tested inhibitors of farnesyl protein transferases that inhibit p21Ras activity (33, 34). Quiescent cells were pretreated with SCH-56582 (10 µM), FPT III (10 µM), or vehicle for 4 h before being stimulated with Hex A (50 nM) or vehicle for 2 h. Both SCH-56582 (Figure 2A) and FPT III (Figure 2B) attenuated the activation of p44/ 42^MAPK as judged by immunoblot analysis for phospho-p44/42^MAPK. The p42^MAPK inhibitor Tyrphostin AG-126 (35) also slightly attenuated p44/42^MAPK (Figure 2B). The concentrations of inhibitors were chosen to avoid cytotoxity in the long-term culture of cells in starvation media for proliferation assays. Possible regulation of Hex-induced p44/42^MAPK activation by PKC was examined by pretreatment of cells with the PKC inhibitor calphostin C (0.2 µM, 2 h). Calphostin C completely inhibited Hex-induced p44/ 42^MAPK activation (Figure 2C), at the same concentration that has been shown to inhibit PKC activity and Hex- induced ASMC proliferation (11).

View larger version (44K): [in this window] [in a new window]   Figure 2.   Effect of SCH-56582, PD98059, FPT III, AG-126, and Calphostin C on Hex-induced activation of p44/42MAPK. Inhibitors or vehicle was added to quiescent cells in 35-mm wells for the indicated times at 37°C in a CO2 incubator, followed by Hex A (50 nM) treatment for 2 h at the same temperature. Cell lysates in RIPA buffer (12 µg protein/sample) were subjected to 10% SDS-PAGE and Western blot analysis for p44/42MAPK and phospho-p44/42MAPK. A polyclonal antiphospho-p44/42MAPK (Thr202/ Tyr204) antibody (1:1,000 dilution) was used as the primary antibody. A horseradish peroxidase-linked whole antirabbit antibody (1:1,000; Amersham) was used as the secondary antibody. The immunoreactive protein bands were visualized on X-ray film, using an ECL detection system. (A) Lane 1, PBS (2 h); lane 2, PD98059 (5 µM, 30 min pretreatment); lane 3, Hex A (50 nM, 2 h); lane 4, SCH-56582 (10 µM, 4 h pretreatment); lane 5, PD98059 + Hex A; lane 6, SCH-56582 + Hex A. (B) Lane 1, PBS (2 h); lane 2, Hex A (50 nM, 2 h); lane 3, 0.001% EtOH; lane 4, FPT III (10 µM, 4 h pretreatment); lane 5, FPT III + Hex A; lane 6, 0.025% DMSO; lane 7, AG-126 (0.2 µM, 3 h pretreatment); lane 8, AG126 + Hex A. (C) Lane 1, PBS (2 h); lane 2, Hex A (50 nM, 2 h); lane 3, DMSO; lane 4, Calphostin C (0.2 µM, photoactivated for 2 h, 2 h pretreatment); lane 5, Calphostin C + Hex A. The bar graph results are mean ± SEM of three separate experiments and directly correspond to upper A, B, and C panels (filled bar: phospho-p44MAPK; hatched bar: phospho-p42MAPK) on the relative density of protein bands (*P < 0.05, significantly higher than the value obtained with vehicle alone; P < 0.05, significantly lower than the value obtained with Hex A alone).

p21Ras Activation by Hex A in Bovine ASMC

Farnesylation of p21Ras is required for the subcellular localization of Ras to the plasma membrane, and is critical to the cell-transforming activity of Ras (34). To assess whether Hex indeed activates p21Ras, we examined fluorescent signals for p21Ras. In Hex-treated cells (50 nM, 10 min), fluorescent signals were aggregated at the plasma membrane of ASM cells (Figure 3B). The Ras activation was much more rapid (peak at 10 min) than the activation of MAPK (onset 30 min). These signals were abolished in cells that were pretreated with FPT III (10 µM, 24 h) (Figure 3D).

View larger version (81K): [in this window] [in a new window]   Figure 3.   Effect of Hex on Ras activation. Bovine ASM cells were seeded in four-well chamber slides (25,000 cells/well). Cells were starved 48 h later in 0.1% FBS containing medium M199 for 24 h. A farnesyl transferase inhibitor, FPT III (10 µM) (C), was added to some wells (C and D) at the time of starvation, and cells were stimulated 24 h later with Hex A (50 nM, 10 min) (B) or vehicle (PBS) (A). Cells were fixed in 4% paraformaldehyde in PEM buffer containing 0.2% Triton X-100. A monoclonal anti-p21Ras antibody (F235, Santa Cruz) was used as the primary antibody and biotinylated antimouse IgG was used as the secondary antibody. Fluorescent signals were obtained by confocal microscopy (×100 objective lens). Aggregation of Ras fluorescent signals at the plasma membranes (B) indicates the activation of Ras that was inhibited by FPT III pretreatment (D).

Effect of FPT III, SCH-56582, PD98059, and Tyrphostin AG-126 on Hex-Induced Bovine ASMC Proliferation

To elucidate the roles of p21Ras, MEK1, and p44/42^MAPK, we tested the effect of these agents on Hex-induced ASMC proliferation. Both farnesyl protein transferase inhibitors FPT III (1-10 µM, 34-75% inhibition) and SCH-56582 (1-10 µM, 88-100% inhibition), as well as the MEK1 inhibitor PD98059 (1-5 µM, 53-78% inhibition), significantly attenuated Hex-induced increases in cell number. Tyrphostin AG-126 (0.2 µM, 39%) had a modest inhibitory effect on Hex-induced ASMC proliferation (Figure 4).

View larger version (36K): [in this window] [in a new window]   Figure 4.   Effect of FPT III, SCH-56582, PD98059, and Tyrphostin AG-126 on Hex-induced bovine ASMC proliferation. Quiescent cells in microtiter wells (starved in 0.4% FBS-containing medium for 48 h) were pretreated with various noncytotoxic concentrations of FPT III (1-10 µM, 4 h), SCH-56582 (1-10 µM, 4 h), PD98059 (1-5 µM, 30 min), AG-126 (0.2 µM, 3 h), or vehicle prior to the addition of Hex A (50 nM) to the cells. After 48 h of incubation, viable cell counts were obtained by MTT assay as described in MATERIALS AND METHODS. Results are mean ± SEM (quadruplicate cultures in three separate experiments). *Significantly different from the value obtained with vehicle alone, P < 0.05; significantly different from the value obtained with Hex alone, P < 0.05.

Effect of p44^MAPK Antisense S-Oligonucleotides on p44/42^MAPK Activity, p44/42^MAPK Protein Expression, and Hex-Induced Bovine ASMC Proliferation

Subconfluent bovine ASMC in 35-mm-well plates were transiently transfected with either 18-mer sense or antisense S-oligonucleotides (1 µM, three 24-h pulses) according to the method described earlier. Cells were stimulated with Hex A (50 nM) or vehicle (PBS) for 2 h. Cell lysates were analyzed for MAPK activity using Biotrek (Amersham) assay kit. Antisense S-oligonucleotides effectively inhibited Hex-induced MAPK activity (Figure 5A). Antisense oligonucleotides directed to human p44^MAPK (1 µM, two or three 24-h pulses) selectively inhibited MAPK protein expression, but not that of p72^Syk in bovine ASMC (Figure 5B). Interestingly, human p44^MAPK antisense S-oligonucleotides inhibited both p44^MAPK and p42^MAPK expression. These results are consistent with the findings of Sale and colleagues (36), and are explained by a high level of sequence homology among MAPKs. Cells responded well to growth factors (FBS and Hex) after repeated treatment with lipofectamine, and antisense S-oligonucleotides directed to p44^MAPK (1 µM, three pulses) completely inhibited Hex-induced ASMC proliferation (Figure 5C).

View larger version (15K): [in this window] [in a new window]   View larger version (25K): [in this window] [in a new window]   View larger version (32K): [in this window] [in a new window]   Figure 5.   (A) Inhibition of p44/42MAPK activity by phosphorothioate (S) antisense p44MAPK oligonucleotides in bovine ASMC. Subconfluent bovine ASMC in 35-mm-well plates were transiently transfected with either 18-mer sense or antisense S-oligonucleotides (1 µM, three 24-h pulses) (see MATERIALS AND METHODS). S-oligonucleotide sequences: 5'-ATG GTC AGC TCG GCC TAT-3' (sense); 5'-ATA GGC CGA GCT GAC CAT-3' (antisense). Cells were stimulated with Hex A (50 nM) or vehicle (PBS) for 2 h. Cell lysates were analyzed for MAPK activity using the Biotrek (Amersham) assay kit. Results are mean ± SEM (n = 4). Antisense S-oligonucleotides effectively inhibited Hex-induced MAPK activity. (B) Selective inhibition of MAPK by antisense p44MAPK S-oligonucleotides. Cell lysates obtained after a treatment with either 18-mer sense or antisense S-oligonucleotides (1 µM, one, two, or three pulses) were resolved by 10% SDS-PAGE. Upper panel: Western blot analysis for p44/42MAPK using polyclonal antibody (1:1,000 dilution). In cells treated with two or three 24-h pulses, antisense S-oligonucleotides inhibited p44/42MAPK. Lipofectamine (Lf) had no effect on p44/42MAPK expression. Bottom panel: Western blot analysis for p72Syk using a monoclonal antibody (1:1,000 dilution; Santa Cruz). There was no inhibition of p72Syk protein expression by p44MAPK antisense S-oligonucleotides. (C) Effect of repeated addition of lipofectamine on FBS- and Hex-induced growth response, and inhibition by phosphorothioated p44MAPK antisense oligonucleotides of Hex-induced bovine ASMC proliferation. Subconfluent ASMC were transiently transfected with either sense or antisense S-oligonucleotides (1 µM) three times, as described earlier. Control cells underwent changes in medium without additions of lipofectamine. Cells were exposed to agonists for 72 h before cell counting with a hemocytometer. Results are mean ± SEM (n = 6). (A and C) *Significantly different from the value obtained with vehicle alone (P < 0.05); significantly different from the value obtained with Hex alone (P < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present study demonstrates that Hex-induced ASMC proliferation is mediated by activation of the p21Ras/ MEK/p44/42^MAPK pathway and PKC. -Hexosaminidase A caused activation of p44/42^MAPK, which was late in onset (30 min) and was sustained for 4 h (Figure 1). The pattern of Hex-induced p44/42^MAPK activation in bovine ASMC is consistent with the concept that sustained activation of p44/42^MAPK is critical to cell proliferation (13, 37, 38). Interestingly, the onset of Hex-induced p44/42^MAPK activation was delayed by nearly 20 min delayed after the activation of p21Ras, and coincided with the previously reported ending of cAMP increase in response to Hex in bovine ASMC (10). It has been suggested that activation of cAMP-dependent protein kinase inhibits activation of p44/42^MAPK (39, 40). It is possible that the transient increase in cAMP caused by Hex suppressed a potential early activation of p44/42^MAPK in bovine ASMC.

The finding that p44/42^MAPK antisense but not sense oligonucleotides inhibited Hex-induced expression of p44/ 42^MAPK and ASMC proliferation supports our contention that p44/42^MAPK mediates the mitogenic effect of Hex. Also supporting this view were our findings that tyrphostin AG-126 reduced MAPK activity and Hex-induced ASMC proliferation. It is known that MAPK is activated by MEK (13, 14). Since Hex-induced ASMC proliferation was inhibited by PD98059, it appears that Hex-induced p44/42^MAPK activation depends on the activation of MEK. It is well established that MEK is activated by Raf-1 that is brought to the plasma membrane for activation by p21Ras (15). Raf-1 is also known to be activated by PKC (23). The farnesyl transferase inhibitors SCH-56582 and FPT III blocked Hex-induced anchorage of p21Ras to plasma membranes (Figure 3), and inhibited Hex-induced p44/42^MAPK activation (Figures 2A and 2B) and ASMC proliferation (Figure 4), thus indicating an involvement of p21Ras in Hex-induced p44/42^MAPK activation and ASMC proliferation. Regarding the method used to assess p21Ras activation in our study, the conventional method for estimating the GTP/guanosine diphosphate exchange ratio was not feasible, owing to the high basal levels of guanosine triphosphatase activating protein in bovine ASMC (D. B. Lew, unpublished data).

Because the PKC inhibitor Calphostin C completely inhibited Hex-induced p44/42^MAPK activation (Figure 2C) and ASMC proliferation (11), it appears that PKC is involved in Hex-induced, MAPK-mediated ASMC proliferation, probably via Raf-1 activation. Limited inhibition of Hex-induced p44/42^MAPK activation by several agents was due to the lower concentrations of these agents used in our study as compared with the concentrations used in other studies (10 versus 100 µM for FPT III [33, 34], 5 versus 10- 50 µM for PD98059 [31, 32], and 0.2 versus 5 µM for Tyrphostin AG-126 [35], respectively). A necessarily noncytotoxic range of concentration of these inhibitors was chosen in the long-term culture system in starvation media used in our study, and one may therefore correlate the inhibitory effect of these agents on p44/42^MAPK activation with their effect on cell proliferation induced by Hex.

The mechanism(s) by which ligation of ASM-MR by Hex leads to the activation of p21Ras is unknown. Most recently, we have reported an interaction between ASM-MR and very late-acting antigen-5 (51), an integrin that is sensitive to pretreatment with pertussis toxin (41), which uncouples GTP-binding regulatory proteins (G-proteins: G_i and G_o) by ribosylation of adenosine diphosphate. Such findings indicate that ASM-MR is coupled to the inhibitory G-protein (G_i) that is expressed in ASM, unlike G_o (D. B. Lew, unpublished data). A few recent reports have provided insights into how G-protein-coupled receptors mediate activation of p44/42^MAPK in a p21Ras-dependent manner (42, 43). Crespo and colleagues (42) have shown that in COS-7 cells, subunits of pertussis toxin-sensitive G-protein affect the functioning of Ras and activation of p44/42^MAPK in a PKC-independent manner (42). Another report, by Della Rocca and coworkers (43), indicated that the G-protein -subunit complex is important in activation of p44/42^MAPK in HEK-293 cells, again in a PKC-independent manner. Our system seems to be unique in that Hex-induced activation of p44/42^MAPK in bovine ASMC is both p21Ras- and PKC-dependent. Studies that may establish a relationship between the G subunit complex, p21Ras, and PKC in the Hex-induced activation of p44/ 42^MAPK are underway.

We conclude that Hex stimulates a late-onset, sustained activation of p44/42^MAPK in bovine ASMC. This activation depends on the activation of p21Ras, MEK1, and PKC. Inhibition of p44/42^MAPK expression completely abolished Hex-induced proliferation in our study, suggesting that the activation of p44/42^MAPK plays an important role in Hex-induced mitogenesis in bovine ASMC. Because Hex A is a physiologically significant inflammatory mediator that is secreted into the extracellular space in asthma (4), interfering in the process of activation of p21Ras, PKC, MEK1, and p44/42^MAPK induced by Hex A may be a useful means of preventing hypertrophy/hyperplasia of ASM.