Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada 89557-0046
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ABSTRACT |
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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- and PI
3-kinase-
. Muscarinic stimulation of intact muscle strips (10 µM
ACh) activated PI 3-kinase-
, ERK and p38 MAP kinases, and MAP
kinase-activated protein kinase-2, whereas PI 3-kinase-
activation
was not detected. Wortmannin (25 µM) abolished the activation of PI
3-kinase-
, ERK, and p38 MAP kinases. MAP kinase inhibition was a PI
3-kinase-
-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-
and PI 3-kinase-
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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INTRODUCTION |
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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 (p85, p85
, p101, and p46) and
catalytic (p110
, p110
, and p110
) 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-, 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-
appears
to be activated after stimulation of GPCR, which triggers dissociation
of the G
heterotrimer to G
and G
(11,
17). The mechanism of activation of PI 3-kinase-
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/p110
(referred to as PI 3-kinase-
) and p85/p110
(PI
3-kinase-
), but GTP-bound Ras in not sufficient to fully activate PI
3-kinase-
(24). It has been suggested that Ras-mediated
membrane attachment may even decrease PI 3-kinase-
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- is an open
question (3). For example, PI 3-kinase-
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-
. In vascular smooth
muscle cells, the role of PI 3-kinase-
in activation of ERK MAP
kinase is largely unexplored. There is only sketchy information to
couple PI 3-kinase-
and p38 MAP kinase activation. So far, a clear
functional role for PI 3-kinase-
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- and PI 3-kinase-
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 AND METHODS |
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Materials.
Wortmannin was purchased from Sigma (St. Louis, MO);
[-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-
/p85
antibody and recombinant activated murine
GST-p42 MAP kinase were from Upstate Biotechnology (Lake Placid, NY); rabbit polyclonal anti-PI 3-kinase-
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- 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-
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-
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-
(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- and PI 3-kinase-
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-
and PI 3-kinase-
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
[
-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
-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
-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
[
-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 -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 [
-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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RESULTS |
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PI 3-kinase- and PI 3-kinase-
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-
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 p110
subunit (29). PI
3-kinase-
does not associate with p85 subunits; hence, PI
3-kinase-
activation depends on G
and/or G
proteins
(26). We tested whether M2/M3 muscarinic receptors are
coupled to PI 3-kinase-
and PI 3-kinase-
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-
(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-
(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-
in this region. The deduced amino acid
sequence revealed 100% identity to human and bovine p110 PI
3-kinase-
and 97.8 and 96.2% identity to mouse and chicken PI
3-kinase-
, 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-
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 p85
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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ACh stimulation activates PI 3-kinase-, but not PI 3-kinase-
,
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-
, whereas PI 3-kinase-
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-
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-
in
intact canine colonic smooth muscle.
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PI 3-kinase- 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-
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-
rather than other kinases mediating
ERK MAP kinase activation. Our results thus demonstrate that PI
3-kinase-
is upstream and is required for activation of ERK and p38
MAP kinase pathways in intact canine colonic smooth muscle.
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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- and
PI 3-kinase-
in cultured colonic myocytes to more than three times
above basal activity. This activation was dependent on the inhibitor
concentration; PI 3-kinase-
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-
(Fig. 5D).
These experiments suggest that PI 3-kinase-regulated pathways are
necessary for chemotactic cell migration.
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DISCUSSION |
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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-, 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-
was not detected,
it is likely that activation of PI 3-kinase-
is an early event
mediating activation of MAP kinases. It is also possible that PI
3-kinase-
regulates both ERK and p38 MAP kinase pathways, since
inhibition of PI 3-kinase-
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-
-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- and PI
3-kinase-
. 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- 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- and PI 3-kinase-
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.
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ACKNOWLEDGEMENTS |
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The technical assistance of Michelle Deetken and Shanti Rawat is acknowledged.
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FOOTNOTES |
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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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