Phosphatidylinositol 3-kinases regulate ERK and p38 MAP kinases in canine colonic smooth muscle

Ilia A. Yamboliev, Kevin M. Wiesmann, Cherie A. Singer, Jason C. Hedges, and William T. Gerthoffer

Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada 89557-0046


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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In canine colon, M2/M3 muscarinic receptors are coupled to extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases. We tested the hypothesis that this coupling is mediated by enzymes of the phosphatidylinositol (PI) 3-kinase family. RT-PCR and Western blotting demonstrated expression of two isoforms, PI 3-kinase-alpha and PI 3-kinase-gamma . Muscarinic stimulation of intact muscle strips (10 µM ACh) activated PI 3-kinase-gamma , ERK and p38 MAP kinases, and MAP kinase-activated protein kinase-2, whereas PI 3-kinase-alpha activation was not detected. Wortmannin (25 µM) abolished the activation of PI 3-kinase-gamma , ERK, and p38 MAP kinases. MAP kinase inhibition was a PI 3-kinase-gamma -specific effect, since wortmannin did not inhibit recombinant activated murine ERK2 MAP kinase, protein kinase C, Raf-1, or MAP kinase kinase. In cultured muscle cells, newborn calf serum (3%) activated PI 3-kinase-alpha and PI 3-kinase-gamma isoforms, ERK and p38 MAP kinases, and stimulated chemotactic cell migration. Using wortmannin and LY-294002 to inhibit PI 3-kinase activity and PD-098059 and SB-203580 to inhibit ERK and p38 MAP kinases, we established that these enzymes are functionally important for regulation of chemotactic migration of colonic myocytes.

muscarinic receptors; cell migration; mitogen-activated protein kinases; reverse transcriptase-polymerase chain reaction; phosphatidylinositol 3-kinases


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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PHOSPHATIDYLINOSITOL (PI) 3-kinases are a family of multifunctional enzymes that have been implicated in regulation of DNA synthesis and cell proliferation, cell survival, regulation of actin assembly, and cell motility (3). PI 3-kinases phosphorylate PI lipids at position D3 of the inositol ring and fall into three classes based on their lipid substrate specificity. In vitro, class I PI 3-kinases phosphorylate both PI 4-phosphate and PI 4,5-bisphosphate, although in vivo the preferred substrate is PI 4-phosphate (28). Class II PI 3-kinases phosphorylate only PI 4-phosphate in vitro, whereas the substrate specificity of class III PI 3-kinases is limited to PI (28). Most of class I PI 3-kinases are heterodimers composed of different adaptor (p85alpha , p85beta , p101, and p46) and catalytic (p110alpha , p110beta , and p110delta ) subunits (29). The heterodimeric PI 3-kinases are activated after stimulation of receptor tyrosine kinases (RTKs), although there is evidence to suggest that some isoforms can be activated by G protein-coupled receptors (GPCRs; see Ref. 11). The Src-homology 2 (SH2) domains of the regulatory p85 subunits are involved in initial binding to phosphorylated tyrosine residues in the COOH-terminal cytosolic tail of RTKs, followed by activation of the catalytic p110 subunit.

The catalytic activity of some class I PI 3-kinases does not depend on association with a p85 subunit. One PI 3-kinase isoform of this group, referred to as PI 3-kinase-gamma , lacks the NH2-terminal sequence necessary for association with regulatory p85 subunits (26). Instead, its NH2-terminal region has been found to be associated with a p101 adapter protein with a currently unknown function (14). PI 3-kinase-gamma appears to be activated after stimulation of GPCR, which triggers dissociation of the Galpha beta gamma heterotrimer to Galpha and Gbeta gamma (11, 17). The mechanism of activation of PI 3-kinase-gamma and its downstream effectors is not completely understood. Association with GTP-bound Ras promotes activation of other class I PI 3-kinases, such as p85/p110alpha (referred to as PI 3-kinase-alpha ) and p85/p110beta (PI 3-kinase-beta ), but GTP-bound Ras in not sufficient to fully activate PI 3-kinase-gamma (24). It has been suggested that Ras-mediated membrane attachment may even decrease PI 3-kinase-gamma activity (2).

A variety of stimuli from the extracellular environment that activate GPCRs has been shown to activate mitogen-activated protein (MAP) kinases, but whether this is mediated by PI 3-kinase-gamma is an open question (3). For example, PI 3-kinase-gamma was not sufficient to fully activate extracellular signal-regulated kinase (ERK) MAP kinases in bovine aortic endothelial cells although it may be sufficient for activation of Jun NH2-terminal kinase MAP kinases (5). In contrast, in Chinese hamster ovary (27) and COS-7 cells (2), ERK MAP kinase activation strongly depends on PI 3-kinase-gamma . In vascular smooth muscle cells, the role of PI 3-kinase-gamma in activation of ERK MAP kinase is largely unexplored. There is only sketchy information to couple PI 3-kinase-gamma and p38 MAP kinase activation. So far, a clear functional role for PI 3-kinase-gamma has not been proposed.

In previous studies, we established that stimulation of smooth muscle with excitatory neurotransmitters such as ACh leads to activation of ERK and p38 MAP kinases (4, 7, 16). In the present study, we hypothesize that enzymes of the PI 3-kinase family mediate coupling between the M2/M3 muscarinic receptors and MAP kinases of canine colonic smooth muscle. We demonstrate expression of PI 3-kinase-alpha and PI 3-kinase-gamma isoforms and reveal that PI 3-kinases are required for activation of ERK and p38 MAP kinases. We also show that PI 3-kinases and MAP kinases may be necessary for regulation of colonic smooth muscle cell chemotactic migration.


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Materials. Wortmannin was purchased from Sigma (St. Louis, MO); [gamma -32P]ATP was from ICN Biomedicals (Costa Mesa, CA); phosphospecific p38 MAP kinase antibody (PhosphoPlus, catalog no. 9210) was purchased from New England Biolabs (Beverly, MA); rabbit polyclonal anti-PI 3-kinase-alpha /p85alpha antibody and recombinant activated murine GST-p42 MAP kinase were from Upstate Biotechnology (Lake Placid, NY); rabbit polyclonal anti-PI 3-kinase-gamma antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rabbit and anti-mouse IgG alkaline phosphatase conjugate antibodies and RNase H were from Promega (Madison, WI). LY-294002, PD-098059, and SB-203580 were purchased from Calbiochem (La Jolla, CA). Oligonucleotides were from Bio-Synthesis (Lewisville, TX). Trizol reagent, dATP, dTTP, dGTP, cCTP, and SuperScript II RT were from GIBCO-BRL (Gaithersburg, MD). P81 phosphocellulose filter paper was from Whatman (Maidstone, UK); Transwell cell migration plates were from Corning Costar (Corning, NY); type I collagen (Vitrogen 100) was from Collagen Biomaterials (Palo Alto, CA); Diff-Quik staining solutions I and II were from Baxter Diagnostics (McGaw Park, IL). All other reagents came from commercial sources.

Smooth muscle stimulation and homogenization. A circular layer of the proximal colon was isolated from adult mongrel dogs of either sex killed with pentobarbital sodium (62 mg/kg iv). Muscle strips were cut parallel to the long axis of circular muscle fibers. The longitudinal muscle layer and mucosa were removed, and the strips were mounted on stainless steel hooks and incubated at 37°C in oxygenated physiological saline solution (PSS) composed of (in mM) 2 MOPS (pH 7.4), 140 NaCl, 4.7 KCl, 1.2 MgSO4, 2.5 CaCl2, 1.2 Na2HPO4, 0.02 EDTA, and 5.6 D-glucose. Muscle strips were stimulated three times for 5 min with 70 mM K+ to produce stable, reproducible contractions. The muscles were then stimulated with 10 µM ACh to obtain control contractile responses. ACh treatment was repeated after 1 h of incubation with 25 µM wortmannin. Muscle strips were frozen 5 min after ACh stimulation by immersion in ice-cold acetone containing 5 mM NaF (-80°C). The frozen strips were homogenized in extraction buffers as specified below. Tissue extracts were centrifuged at 10,000 g for 20 min, and the protein content of the clear supernatant was assayed by the bicinchoninic acid method.

RT-PCR and sequencing of PI 3-kinases. Total RNA was extracted from canine colonic circular smooth muscle, canine heart, or human K-562 cells with Trizol reagent (1 ml/100 mg wet muscle). First-strand cDNA synthesis was performed at 42°C from 2 µg of RNA using 500 ng of oligo(dT), 50 mM Tris·HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT), 0.125 mM dATP, dTTP, dGTP, and cCTP, and 1 unit of SuperScript II RT. Twenty units of RNase H were added, and the reaction was further incubated at 37°C for 20 min. Amplification of the cDNA encoding the p110 subunit of PI 3-kinase-alpha took place at 94°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min for 35 cycles in a thermal cycler (GeneAmp PCR System 2400; Perkin-Elmer). The following oligonucleotides designed complementary to the bovine PI 3-kinase-alpha sequence (M93252) were used: 5'-TGCCTCCAAGACCATCATCA-3' (sense) and 5'-CCACTATTATTTGCCCTTTATCCA-3' (anti-sense). The reaction mixture contained 60 mM Tris·HCl (pH 8.5), 15 mM (NH4)2SO4, 1.5 mM MgCl2, 0.25 mM dATP, dCTP, dGTP, and dTTP, 0.2 ng/ml of each primer, template cDNA, and 2.5 units of thermus aquaticus polymerase (Taq). Amplication of the p110 subunit of PI 3-kinase-gamma was carried out at 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min using the primers 5'-GGAGCAGATGAAGGCCCAGGTGT-3' (sense) and 5'-GGCGCCGGGGGTGTCGTC-3' (antisense) obtained from the human sequence of p110 PI 3-kinase-gamma (X83368) in the same reaction mixture described above with the addition of 10% DMSO. The reaction products (10 µl) were examined by electrophoresis through a 1% agarose-Tris-acetate-EDTA gel and were visualized with ethidium bromide. PCR products were sequenced by dye terminator cycle sequencing on an ABI Prism 310 Genetic Analyzer (Perkin-Elmer) according to the manufacturer's instructions.

Western immunoblotting. Colonic smooth muscle strips or cultured cells were homogenized with SDS extraction buffer [25 mM Tris (pH 7.4), 2% SDS, 10% glycerol, 1 mM DTT, 1 µM leupeptin, 10 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF)] or RIPA extraction buffer [50 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM Na2EDTA, 0.5% (vol/vol) Nonidet P-40, 0.5% (vol/vol) Triton X-100, 10 mM NaF, 1 µM leupeptin, 100 µM 4-(2-aminoethyl)benzenesulfonl fluoride, 1 mM sodium orthovanadate, and 10% glycerol]. Equal amounts of total sample protein (15 µg) were resolved by SDS-PAGE and were transferred to nitrocellulose membranes in transfer buffer (25 mM Tris·HCl-192 mM glycine-10% methanol) at 24 V and 4°C for 1 h (Genie blotter; Idea Scientific, Minneapolis, MN). Membranes were blocked for 2 h with 0.5% gelatin solution in TNT buffer [100 mM Tris (pH 7.5), 0.1% Tween 20, and 150 mM NaCl] at room temperature. Labeling with primary antibodies took place in 0.1% gelatin/TNT for 2 h at room temperature, with antibody dilution as specified below. Membranes were then washed with TNT buffer three times for 5 min each and were incubated for 1 h with alkaline phosphatase-conjugated secondary goat anti-rabbit or goat anti-mouse antibodies (Promega) diluted at 1:10,000 in 0.1% gelatin/TNT. Membranes were washed as before, color was developed as appropriate, and blots were scanned with a UMAX Powerlook flatbed scanner (Bio-Rad, Hercules, CA) to obtain band densities. In preliminary experiments, we have established that, with the alkaline phosphatase assay, band densities are linearly dependent on the total protein amount per lane in the range of 3-25 µg. Unless otherwise specified, band densities of different samples within a treatment group were presented relative to the control group.

PI 3-kinase activity assay. Colonic smooth muscle strips stimulated with 10 µM ACh or 1 µM methacholine (MCh) before or 1 h after incubation with wortmannin or LY-294002 were frozen in liquid N2 and homogenized in RIPA extraction buffer (20 µl buffer/1 mg wet tissue wt). Smooth muscle cells, cultured in 100-mm petri dishes were stimulated with 3% newborn calf serum (NCS) and then lysed and scraped with 200 µl RIPA buffer. Muscle tissue and cell homogenates were then sonicated for 3 min and clarified by centrifugation at 10,000 g for 15 min, and PI 3-kinase-alpha and PI 3-kinase-gamma were immunoprecipitated with Protein A/G agarose plus beads and polyclonal antibodies purchased from UBI and Santa Cruz Biotechnology, respectively. Immune complexes were washed two times with 0.5 ml of RIPA buffer and two times with 0.5 ml of PI 3-kinase assay buffer [50 mM Tris·HCl (pH 7.8), 2 mM MgCl2, 0.5 mM EDTA, and 50 mM NaCl]. Before the second PI 3-kinase assay, buffer wash immunoprecipitates were split into two tubes, which were then centrifuged, and the clear supernatants were removed. One tube was used in a Western blotting protocol to verify successful immunoprecipitation of PI 3-kinase. The immunoprecipitate in the second tube was used for PI 3-kinase activity assay. Both PI 3-kinase-alpha and PI 3-kinase-gamma activities were assayed by in vitro phosphorylation of PI (Avanti, Alabaster, AL) in a final volume of 40 µl containing PI 3-kinase immunoprecipitate, 5 µg of PI, and 0.25 mM ATP/10 µCi [gamma -32P]ATP. Phosphorylation was carried out at 30°C for 30 min and was stopped by adding 200 µl of 1 N HCl and cooling on ice. Lipids were extracted with 0.5 ml of a 1:1 mix of CHCl3 and MeOH, and extracts were transferred to clean tubes and evaporated under a stream of N2. Dry residues were recovered in 10 µl of CHCl3, and the whole volume was spotted on TLC plates (Silica gel HL; Analtech, Newark, DE) and developed with a mobile phase composed of CHCl3, MeOH, 50% ammonium hydroxide, and water at a volume ratio of 130:80:8:10. The radioactive bands of phosphorylation product, PI 3-phosphate, were identified by iodine staining using PI 4-phosphate as the TLC standard (Avanti). The TLC plates were then exposed to a phosphorimaging screen, and radioactive spot densities were assayed using a Bio-Rad model 525 Molecular Imager (Bio-Rad). Radioactive signals were normalized to the respective band densities derived from the Western blots, and kinase activities were expressed relative to controls.

ERK MAP kinase activity assay. An in-gel kinase assay was used as previously described (4). Colonic muscle strips were stimulated according to the experimental protocol, frozen in acetone, and homogenized in MAP kinase extraction buffer containing the following (50 µl buffer/1 mg wet tissue wt): 2% SDS, 10% glycerol, 5 mM NaF, 1 µM leupeptin, 10 mM EDTA, and 1 mM PMSF. DTT (1 mM) was added, and samples were incubated for 20 min at 37°C. The reduced protein (15 µg total sample protein/lane) was resolved by SDS-PAGE using 10% acrylamide gels containing 0.5 mg/ml MBP incorporated in the separating gel. The gels were washed in Tris·HCl buffer (pH 8.0) with 20% isopropanol, and the latter was removed by washing in 50 mM Tris-5 mM beta -mercaptoethanol. The proteins were denatured with 6 M guanidine, which was removed by a wash in 50 mM Tris (pH 8.0)-0.04% Tween 20-5 mM beta -mercaptoethanol, and then the gels were equilibrated in 40 mM HEPES (pH 8.0)-10 mM MgCl2-2 mM DTT. The kinase reaction was carried out in 40 mM HEPES (pH 8.0)-10 mM MgCl2-0.5 mM EGTA and 0.1 mM ATP (5 µCi [gamma -32P]ATP/ml) for 2 h at room temperature. Excess radioactivity was removed from the gels by extensive washing with 5% TCA/10 mM sodium pyrophosphate for 12-16 h followed by a 1-h wash in 3% glycerol. The gels were air-dried between cellophane sheets and subjected to phosphorimaging and densitometry (model 525 Molecular Imager; Bio-Rad).

p38 MAP kinase phosphorylation assay. Canine colonic strips were treated and homogenized as described above. Protein was separated by SDS-PAGE (10% acrylamide) and transferred to nitrocellulose membranes. Phosphorylated p38 MAP kinase was immunodetected by PhosphoPlus p38 MAP kinase antibody (dilution 1:1,000). Immunoreactive proteins were visualized by alkaline phosphatase detection using goat anti-rabbit alkaline phosphatase conjugate. The immunoblots were scanned with a UMAX Powerlook flatbed scanner, the bands were analyzed by Molecular Analyst software (Bio-Rad), and the changes in p38 MAP kinase phosphorylation were expressed relative to the control muscle strips.

MAP kinase-activated protein kinase-2 activity assay. Colonic muscle strips were mounted in an isometric muscle bath, stimulated with agonist, frozen in acetone-5 mM NaF (-80°C), and homogenized in extraction buffer (20 µl/mg wet tissue) containing 25 mM HEPES, 150 mM NaCl, 1 mM PMSF, 1 µM leupeptin, 1 mM Na3VO4, and 10 mM NaF. MAP kinase-activated protein (MAPKAP) kinase-2 activity was assayed in vitro using bacterially expressed full-length canine Hsp-27 (rHsp-27; see Ref. 16). The kinase reaction (40 µl) contained 25 mM Tris·HCl (pH 7.0), 0.1 mM EGTA, 0.2 mM Na3VO4, 10 mM magnesium acetate, 1 mM DTT, 3.3 mM rHsp-27, and clarified tissue homogenate. After incubation at 37°C for 2 h, the reaction was stopped by adding 13.3 µl of 4× SDS sample buffer to produce final concentrations of 0.06 M Tris·HCl (pH 6.8), 2% SDS, 10% glycerol, 1 mM DTT, and 0.03% bromphenol blue and boiling for 5 min. Protein was resolved by SDS-PAGE (12% acrylamide), and the gels were stained with Coomassie brilliant blue and destained with four to five changes of solution. The phosphorylation of rHsp-27 was detected with a Bio-Rad model 525 Molecular Imager. Background signal due to the phosphorylation of endogenous Hsp-27 comprised <9% of the total kinase activity. The total Hsp-27 kinase activity was corrected by subtracting the signal of reactions lacking rHsp-27 from reactions containing rHsp-27.

In vitro MAP kinase activity assay. A model peptide (APRTPGGRR) that includes the MAP kinase phosphorylation sequence of MBP was used as substrate for recombinant activated murine GST-p42 MAP kinase in an in vitro phosphorylation assay. The kinase reaction contained 25 mM MOPS (pH 7.2), 25 mM beta -glycerophosphate, 15 mM MgCl2, 1 mM EGTA, 0.1 mM NaF, 1 mM Na3VO4, 4 mM DTT, 10 mM substrate peptide, and 0.033 units (0.1 µg protein) of recombinant activated murine GST-p42 MAP kinase without (control) or with different concentrations of wortamnnin (1 nM to 100 µM). The reaction was started with the addition of 250 µM ATP-2 µCi [gamma -32P]ATP in a final volume of 40 µl. After 2 h of incubation at 37°C, 20-µl aliquots were spotted on P81 phosphocellulose filter squares that were washed five times for 10 min each with ice-cold 0.75% H3PO4. After a final 95% ethanol wash for 1 min, the filters were air-dried and counted on a LKB 1217 Rackback scintillation counter (Wallac, Gaithersburg, MD).

Cell migration. First-passage canine colonic smooth muscle cells grown to 80% confluence and starved for 24 h were used for the migration assays. Membrane inserts of Transwell culture plates (diameter 6.5 mm, 8-µm pore size) were coated by immersion in 1 mg/ml type I collagen (Vitrogen 100) and were sterilized under ultraviolet (UV) light for 30 min. The wells of migration plates were filled with 0.6 ml cell culture medium 199 (M199) containing 0.3 mg/ml BSA, with or without 3% NCS as chemoattractant (bottom solution). The insert was filled with 100 µl of cell suspension (8 × 104 cells) in 0.3 mg/ml BSA/M199 (top solution). Plates were then incubated in a humidified CO2 incubator at 37°C. After 5 h, the cells on the top membrane surface were gently scraped with a cotton swab, and the cells on the bottom surface were fixed and stained in Diff-Quik Solutions I and II as recommended by the manufacturer. To evaluate the role of PI 3-kinases and MAP kinases in the migration, cells were preincubated for 15 min with different concentrations of wortmannin, LY-294002, PD-098059, or SB-203580. These inhibitors were also present in both bottom and top solutions throughout the experiment. The degree of change of cell migration was determined by dividing the total number of cells in five contiguous fields of polycarbonate membrane (0.113 mm2/field) in the presence of attractant by the total number of spontaneously migrated cells (without attractant). In all treatment groups, the spontaneous cell migration (without chemoattractant, chemokinesis) was significantly lower compared with cell migration stimulated by NCS present only in the bottom solution (chemotaxis). Because NCS could also stimulate cell division and hence lead to misinterpretation of migration data, in preliminary experiments we tested the cell growth-stimulating effect of NCS. Colonic myocytes were trypsinized, plated on a collagen-coated culture plate, and incubated in M199-0.3 mg/ml BSA-3% NCS at 37°C for 1 h. Unattached cells were washed out with M199-0.3 mg/ml BSA, and fresh medium containing 3% NCS was added. Cells in one marked microscope field were counted, plates were incubated at 37°C for another 4 h, and cell number in the same microscope field was obtained again. The presence of NCS during this protocol resulted in a total cell number increase of 1.013 times the number of plated cells (average of 4 parallel assays). Because in our routine migration experiments the number of NCS-attracted cells on the bottom membrane surface increases four to five times the number of spontaneously migrated cells, <1% percent of the total cell number increase is due to cell division.

Statistical methods. Results are presented as means ± SE of five to eight experiments for each experimental point. Student's t-test for paired and unpaired data was applied to compare the differences between treatment means, as appropriate. P < 0.05 was accepted as a statistically significant difference.


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PI 3-kinase-alpha and PI 3-kinase-gamma are expressed in canine colonic smooth muscle. Several PI 3-kinase isoforms have been identified based on p85 and p110 subunit diversity. Activation of PI 3-kinase-alpha is initiated by binding of the SH2 domain of the regulatory p85 subunits to the tyrosine-phosphorylated cytosolic tail of RTKs, which triggers activation of the catalytic p110alpha subunit (29). PI 3-kinase-gamma does not associate with p85 subunits; hence, PI 3-kinase-gamma activation depends on Galpha and/or Gbeta gamma proteins (26). We tested whether M2/M3 muscarinic receptors are coupled to PI 3-kinase-alpha and PI 3-kinase-gamma isoforms in intact canine colonic smooth muscle. We used RT-PCR with isoform-specific primer sets to first test PI 3-kinase expression in this tissue. A 576-bp portion of PI 3-kinase-alpha (Fig. 1A) was amplified from canine colon, and nucleotide sequence analysis of this PCR product demonstrated that it is 98% identical to nucleotide fragment 51-626 of human PI 3-kinase-alpha (U79143). The canine sequence was also highly homologous to other vertebrate sequences as follows: 97% to bovine (M93252), 90.4% to mouse (U03279), and 86% to chicken (AF001076) PI 3-kinase-alpha in this region. The deduced amino acid sequence revealed 100% identity to human and bovine p110 PI 3-kinase-alpha and 97.8 and 96.2% identity to mouse and chicken PI 3-kinase-alpha , respectively. PCR products with the same size were amplified from human myelogenous leukemia K-562 cells (positive control), canine heart, tracheal, and vascular smooth muscles (Fig. 1A), and lung, pancreas, skeletal muscle, and liver (not shown). To further examine expression of the PI 3-kinase-alpha isoform, we resolved total protein from K-562 cell and tissue extracts by SDS-PAGE, transferred the protein to a nitrocellulose membrane, and probed with a rabbit polyclonal antibody against the p85alpha subunit of PI 3-kinase. Immunoreactive bands were detected in K-562 cells, colonic, tracheal, and vascular smooth muscle (Fig. 1B), and in other tissue extracts of the dog.


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Fig. 1.   Expression of phosphatidylinositol (PI) 3-kinase isoforms in K-562 cells (positive controls) and in canine heart (H), colonic (C), tracheal (T), and pulmonary artery (PA) smooth muscle. RT-PCR of PI 3-kinase-alpha (A) and PI 3-kinase-gamma (C) was carried out with primers designed complementary to sequences of bovine PI 3-kinase-alpha (M93252, predicted product size 576 bp) and human PI 3-kinase-gamma (X83368, predicted product size 495 bp), respectively. Western blots depicted immunoreactive bands of the p85alpha subunit of PI 3-kinase-alpha (B) and p110gamma subunit of PI 3-kinase-gamma (D), which were visualized by alkaline phosphatase-conjugated secondary antibodies.

A 495-bp portion of p110 PI 3-kinase-gamma was amplified from K-562 cells and canine heart, colon, tracheal, and vascular smooth muscles (Fig. 1C). Nucleotide sequence analysis of the PCR product demonstrated that it is 83% identical to human PI 3-kinase-gamma (nucleotides 201-727, X83368) and 84% identical to pig PI 3-kinase-gamma (Y10743). The deduced amino acid sequences revealed 82 and 86% identity to human and pig PI 3-kinase-gamma , respectively. Amplification of PI 3-kinase-gamma also produced smaller PCR products whose sequence analysis did not show homology with p110gamma . Western immunoblotting of PI 3-kinase-gamma immunoprecipitated from total tissue homogenates with a polyclonal antibody detected light immunoreactive bands, suggesting low abundance of the enzyme in colonic (Fig. 1D), tracheal, and pulmonary artery smooth muscle. These results indicate that both isoforms, PI 3-kinase-alpha and PI 3-kinase-gamma , are expressed in various canine tissues.

ACh stimulation activates PI 3-kinase-gamma , but not PI 3-kinase-alpha , in intact colonic smooth muscle. ACh triggers intracellular effects by stimulation of cell surface M2/M3 receptors, which belong to the family of heptahelical transmembrane GPCR. To test which PI 3-kinase isoform is coupled to muscarinic receptors, we stimulated smooth muscle strips with 1 µM MCh and, after immunoprecipitation from total tissue homogenate, assayed activation of both PI 3-kinase isoforms by phosphorylation of PI in vitro. In a 10-min time course of MCh stimulation, we did not detect activation of PI 3-kinase-alpha , whereas PI 3-kinase-gamma was transiently activated with a peak at 3 min (Fig. 2A). In tissue strips incubated for 1 h with 10 and 25 µM wortmannin, MCh activation of PI 3-kinase-gamma was abolished; kinase activation was reduced 85% by 2.5 µM wortmannin (Fig. 2B). These experiments establish coupling between M2/M3 muscarinic receptors and PI 3-kinase-gamma in intact canine colonic smooth muscle.


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Fig. 2.   Methacholine (MCh; 1 µM) transiently activates PI 3-kinase-gamma in intact smooth muscle. Colonic muscle strips were frozen at different times after MCh stimulation and homogenized in RIPA buffer. PI 3-kinase-gamma was immunoprecipitated, and the activity was assayed by phosphorylation of PI in vitro. Phosphorylation products were separated by TLC, and the radioactivity incorporated in PI 3-phosphate (PI3P) was assayed by phosphorimaging (A, top). Kinase activation is presented relative to basal activity (A, bottom, n = 3). The catalytic activity of PI 3-kinase-gamma was abolished by 10 µM and significantly reduced by 2.5 µM wortmannin (Wm) added to tissue baths 1 h before MCh stimulation (B, n = 3). * P < 0.05 compared with basal. ** P < 0.05 compared with maximum PI 3-kinase-gamma activity without wortmannin, Student's t-test.

PI 3-kinase-gamma is required for ACh-stimulated activation of ERK and p38 MAP kinases. In our previous work, we have shown that stimulation of canine colonic smooth muscle with ACh leads to activation of ERK (4) and p38 MAP kinases (7). Several signaling pathways could mediate activation of MAP kinases. Therefore, in this study, we set out to determine whether PI 3-kinase-gamma is a component of pathways coupling muscarinic receptors to activation of ERK and p38 MAP kinases. To address this issue, we incubated colonic smooth muscle strips in tissue bath with 0.1% DMSO (vehicle) or 25 µM wortmannin for 1 h before a 5-min stimulation with 10 µM ACh. DMSO had no effect on the contractile response to ACh or on ACh-stimulated activation of ERK1 (3.05 ± 0.86 times basal), ERK2 (2.74 ± 0.48), or p38 MAP kinases (4.15 ± 0.47) or MAPKAP kinase-2 (2.52 ± 0.43; Fig. 3). Wortmannin abolished ACh-induced activation of ERK MAP kinases, p38 MAP kinases, and MAPKAP kinase-2. Wortmannin also blocked smooth muscle contraction, which is due to inhibition of myosin light chain kinase (MLCK; see Ref. 19). Wortmannin has not been reported to inhibit enzymes downstream of PI 3-kinases that mediate activation of ERK MAP kinases. We conducted two experiments in an attempt to validate this notion. First, we used a model synthetic peptide substrate (APRTPGGRR) containing the MAP kinase phosphorylation sequence of MBP. We used recombinant activated GST-p42 MAP kinase to phosphorylate this peptide substrate in vitro without or after a 10-min incubation with wortmannin. GST-p42 MAP kinase potently phosphorylated the peptide in a control reaction (without wortmannin). This phosphorylation remained unchanged in the presence of wortmannin (1 nM to 100 µM, not shown), suggesting lack of a direct inhibitory effect on ERK MAP kinase activity in vitro. Second, we assayed the activity of ERK1 and ERK2 MAP kinases after a 1-h incubation of colonic smooth muscle strips with DMSO or wortmannin, followed by a 20-min stimulation with 1 µM phorbol dibutyrate (PDBu). We reasoned that phorbol ester should activate protein kinase C (PKC), Raf-1, and mitogen/extracellular signal-regulated kinase (MEK) and hence the ERK MAP kinases. PI 3-kinase is thought to be more proximal to the muscarinic receptors than Raf-1. If wortmannin nonselectively inhibits activation of Raf-1 and MAP kinase kinases (MEKs/MKKs), then PDBu-stimulated ERK MAP kinase activation should be blocked by wortmannin. We found a significant PDBu-stimulated activation of both ERK1 and ERK2 MAP kinases to 3.39 ± 0.67 and 2.07 ± 0.35 times above basal, which remained unaffected by wortmannin (Fig. 4). These results suggest that wortmannin does not directly inhibit PKC, Raf-1, MEK, or ERK MAP kinases. Therefore, wortmannin interrupts muscarinic activation of MAP kinases via inhibition of PI 3-kinase-gamma rather than other kinases mediating ERK MAP kinase activation. Our results thus demonstrate that PI 3-kinase-gamma is upstream and is required for activation of ERK and p38 MAP kinase pathways in intact canine colonic smooth muscle.


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Fig. 3.   M2/M3 receptor agonist ACh activates extracellular signal-regulated kinase (ERK) 1 and ERK2 MAP kinases, p38 mitogen-activated protein (MAP) kinase, and MAP kinase-activated protein (MAPKAP) kinase-2; kinase activation is attenuated by the PI 3-kinase inhibitor wortmannin. Canine muscle strips were incubated with 25 µM wortmannin for 1 h followed by a 5-min stimulation with 10 µM ACh. Strips were frozen and homogenized, and kinase activities were assayed as described in MATERIALS AND METHODS. Traces in A show a typical ACh-induced muscle contraction (left) that is inhibited by wortmannin (right). Bar graph in B shows the relative activation of ERK1, ERK2, p38 MAP kinases, and MAPKAP kinase-2 without and with wortmannin. * P < 0.05 compared with basal (open bar). ** P < 0.05 compared with samples without wortmannin, Student's t-test.



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Fig. 4.   Phorbol dibutyrate (PDBu) induces a PI 3-kinase-independent activation of ERK1 and ERK2 MAP kinases. Muscle strips were treated with 1 µM PDBu for 20 min before or after 1 h of incubation with 25 µM wortmannin. Strips were frozen and homogenized, and kinase activities were assayed as described. A: radioactive bands from an in-gel ERK MAP kinase activity assay. B: bar graphs representing degree of activation of MAP kinases. * P < 0.05 compared with basal (open bar), Student's t-test.

PI 3-kinase, ERK, and p38 MAP kinases regulate colon smooth muscle cell migration. It has been suggested that, in addition to eliciting nuclear responses, activation of MAP kinase pathways may be involved in other cellular responses such as smooth muscle contraction (4). In the present study, we tested the hypothesis that PI 3-kinases and MAP kinases modulate chemotactic migration of canine colonic smooth muscle cells. First-passage canine colonic myocytes grown to 70-80% confluence and starved for 24 h were stimulated to migrate with 3% NCS. After 5 h at 37°C, the number of cells that migrated as a result of NCS stimulation increased to 4.29 ± 0.82 times the number of spontaneously migrated cells (without NCS, Fig. 5). NCS-stimulated migration was inhibited by wortmannin (range 0.3-25 µM) present in bottom and top solutions throughout the assay (Fig. 5A). Another PI 3-kinase inhibitor, LY-294002 (1-50 µM), also produced a dose-dependent decrease of cell migration (Fig. 5C). To verify that this is a PI 3-kinase-mediated event, in another group of experiments, 3% NCS significantly activated both PI 3-kinase-alpha and PI 3-kinase-gamma in cultured colonic myocytes to more than three times above basal activity. This activation was dependent on the inhibitor concentration; PI 3-kinase-alpha activation was abolished by a 15-min incubation with 25 µM wortmannin and was reduced by 1 µM (Fig. 5B). At a concentration of 50 µM, LY-294002 eliminated the NCS-stimulated activation of PI 3-kinase-alpha (Fig. 5D). These experiments suggest that PI 3-kinase-regulated pathways are necessary for chemotactic cell migration.


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Fig. 5.   PI 3-kinase inhibitors wortmannin (A) and LY-294002 (LY; C) dose dependently decrease chemotactic migration of canine colonic smooth muscle cells (n = 6). First-passage cultured cells were growth arrested for 24 h and then stimulated to migrate with 3% normal calf serum (NCS). After 5 h at 37°C, cells in 5 contiguous fields on the bottom membrane surface were counted. The degree of change in cell migration was determined by dividing the total number of NCS-attracted cells (A and C, filled bars) by the total number of spontaneously migrated cells (open bars). B and D: NCS activates PI 3-kinase-alpha activity in control colonic myocytes, and this activation is inhibited by wortmannin and LY-294002 (n = 3). * P < 0.05 compared with basal (open bars). ** P < 0.05 compared with control NCS activation (filled bars), Student's t-test.

Cell migration was also reduced when PD-098059 (10 and 50 µM) and SB-203580 (10 and 25 µM) were used to block ERK and p38 MAP kinases (Fig. 6A). Although 50 µM PD-098059 and 25 µM SB-203580 completely inhibited NCS-stimulated activation of ERK and p38 MAP kinases, full inhibition of cell migration was not achieved (Fig. 6B). These results imply that MAP kinases play a minor role in modulation of chemotactic migration of canine colonic smooth muscle cells.


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Fig. 6.   ERK MAP kinase inhibitor PD-098059 (PD; A) and p38 MAP kinase inhibitor SB-203580 (SB; C) reduce chemotactic migration of canine colonic smooth muscle cells (n = 6). First-passage cultured cells were growth arrested for 24 h and then stimulated to migrate with 3% NCS. After 5 h at 37°C, cells in 5 contiguous fields on the bottom membrane surface were counted. The degree of change in cell migration was determined by dividing the total number of NCS-attracted cells (A and C; filled bars) by the total number of spontaneously migrated cells (open bars). B and D: NCS activates ERK and p38 MAP kinase pathways in control colonic myocytes, and their activation is inhibited by PD-098059 and SB-203580 (n = 3). * P < 0.05 compared with basal (open bars). ** P < 0.05 compared with control NCS activation (filled bars), Student's t-test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In our previous results, as well in the present study, we have demonstrated that muscarinic receptor stimulation of colonic smooth muscle activates ERK and p38 MAP kinase pathways (4, 7). ACh stimulates both most abundantly expressed muscarinic receptor types in canine gastrointestinal tract, M2 and M3 receptors. M3 receptors comprise ~18% of the total muscarinic receptor population, and they stimulate production of inositol trisphosphate via coupling to G proteins of the Gq subtype and hence may be largely responsible for contractile muscle responses. The dominating M2 muscarinic receptor subtype (82%) is coupled to Gi protein thus inhibiting adenylate cyclase activity (30). Here we show that, in addition to ERK MAP kinases, these two receptor subclasses are coupled to intracellular signal transduction pathways that were presumed to be activated by oxidative or thermal stress, cytokines, or UV light, such as the p38 MAP kinases. The enzymes that mediate activation of ERK MAP kinases have been more extensively studied over the years, although less is known about the kinases involved in activation of p38 MAP kinase by GPCRs. Recent evidence has indicated a role for PI 3-kinases in regulation of MAP kinases in various cell systems such as COS-7 cells (18), neutrophils (13), and human vascular smooth muscle cells (9). At least two pathways were involved in activation of ERK MAP kinases (one dependent on PI 3-kinases or other wortmannin-sensitive enzymes and another wortmannin-insensitive pathway; see Refs. 10 and 25). It is presently unclear whether there is a role for PI 3-kinases in regulation of the p38 MAP kinase pathway. Studies in neutrophils demonstrate either partially dependent (15) or entirely PI 3-kinase-independent p38 MAP kinase activation (13). From our results it appears that in canine colon, M2/M3 receptor-mediated activation of both ERK and p38 MAP kinases requires PI 3-kinase activity. PI 3-kinase-gamma , an isoform that is activated by GPCR in other cell systems (26), is expressed in a number of canine tissues, including colonic smooth muscle (Fig. 1), and is activated upon ACh receptor stimulation. Because ACh-stimulated activation of PI 3-kinase-alpha was not detected, it is likely that activation of PI 3-kinase-gamma is an early event mediating activation of MAP kinases. It is also possible that PI 3-kinase-gamma regulates both ERK and p38 MAP kinase pathways, since inhibition of PI 3-kinase-gamma with wortmannin abolished ACh-stimulated activation of both MAP kinases. Although wortmannin has been shown to inhibit enzymes other than PI 3-kinases such as the DNA-dependent protein kinase PI 4-kinase, cytosolic phospholipase A2, and MLCK (21), in our tissue wortmannin did not inhibit the PDBu-sensitive PKC isoforms Raf-1, MEK, or ERK MAP kinases. We believe, therefore, that the observed inhibition of MAP kinases in intact smooth muscle in vivo is a specific PI 3-kinase-gamma -dependent effect.

The signaling mechanisms to these kinases in migrating smooth muscle cells may be different. The NCS used as chemoattractant in our migration experiments activates multiple receptors of the RTK and GPCR types and should be expected to activate both PI 3-kinase-alpha and PI 3-kinase-gamma . Because activation of these two isoforms was established in our migration experiments (Fig. 5), it was unclear what is the functional role of PI 3-kinases in chemotactic cell migration. PI 3-kinases have been found to regulate migration of several cell types, including neutrophils (13), fibroblasts, and epithelial cells (1). In contrast, from the work of Higaki and collaborators (8) it appears that PI 3-kinase may be unnecessary for migration of vascular smooth muscle and Swiss 3T3 cells. ERK and p38 MAP kinases have been shown to modulate migration of vascular smooth muscle cells (20), but it is possible that activation of MAP kinases is either not sufficient or is not required for migration of other cell types (1).

In the gastrointestinal tract, cell migration takes place in some basic processes such as healing of intestinal ulcer or in the pathogenesis of gut tumors. Therefore, we further investigated the role of PI 3-kinases and MAP kinases in chemotactic migration of cultured colonic myocytes. We provided new evidence that in colonic smooth muscle cells, PI 3-kinases are critical components, whereas ERK and p38 MAP kinases may participate in regulation of chemotactic migration. Even a complete inhibition of ERK MAP kinases by PD-098059 produced only a modest effect on migration, which may be mediated by phosphorylation of caldesmon or other currently unrecognized actin-binding proteins. Full inhibition of the p38 MAP kinase pathway by SB-203580 more vigorously reduced cell migration, but a complete inhibition of cell migration was not achieved in this cell type. The p38 MAP kinase pathway has been shown to mediate phosphorylation of proteins coupled to regulation of actin assembly, such as the small heat-shock protein Hsp-27 (23). A recent study in our laboratory reported that migration of cultured canine tracheal smooth muscle cells is modulated by p38 MAP kinases probably via phosphorylation of Hsp-27 (6). Overexpression of a constitutively active mutant of an upstream activator of p38 MAP kinases, MKK6, increased basal activity of p38 MAP kinase and cell migration compared with uninfected cells. In contrast, overexpression of a dominant negative p38 MAP kinase-alpha isoform almost completely suppressed cell migration as did an Hsp-27 mutant whose phosphorylation sites Ser-15, Ser-78, and Ser-82 were mutated to alanine (6). In studies of other laboratories, expression of an active MEK1 mutant caused activation of ERK MAP kinases and enhanced cell migration (12). Mutations of threonine and tyrosine residues within the activation lip of ERK MAP kinases caused rearrangement of actin assembly and increased cell spreading and chemotactic cell migration (22). Together these results indicate that ERK and p38 MAP kinases may be important to modulate chemotactic migration of smooth muscle cells.

In conclusion, in this study, we present evidence that both PI 3-kinase-alpha and PI 3-kinase-gamma isoforms are expressed in colonic smooth muscle of the dog and are required to couple M2/M3 muscarinic receptors to ERK and p38 MAP kinases. We demonstrate a functional role of PI 3-kinase/MAP kinase pathways in the chemotactic migration of colonic smooth muscle cells.


    ACKNOWLEDGEMENTS

The technical assistance of Michelle Deetken and Shanti Rawat is acknowledged.


    FOOTNOTES

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41315.

Address for reprint requests and other correspondence: I. A. Yamboliev, Dept. of Pharmacology, MS 318, Univ. of Nevada School of Medicine, Reno, NV 89557-0046 (E-mail: yambo{at}med.unr.edu).

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. §1734 solely to indicate this fact.

Received 8 February 1999; accepted in final form 8 February 2000.


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DISCUSSION
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