Departments of Medicine and Physiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298-0711
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ABSTRACT |
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Insulin-like growth factor I (IGF-I), acting via its cognate receptor, plays an autocrine role in the regulation of growth of intestinal muscle cells. In the present study the signaling pathways mediating the growth effects of IGF-I were characterized in cultured human intestinal smooth muscle cells. Growth induced by a maximally effective concentration of IGF-I (100 nM), measured as [3H]thymidine incorporation, was only partially inhibited by LY-294002 [phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor] or PD-98059 [mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor] (86 ± 7% and 35 ± 6% inhibition, respectively) alone but was abolished by the two combined (114 ± 18% inhibition), implying the participation of both pathways. IGF-I elicited time- and concentration-dependent increases in PI 3-kinase activity. This effect was inhibited only by LY-294002 (89 ± 12%). IGF-I elicited time- and concentration-dependent phosphorylation of p44/p42 MAP kinase and increased MAP kinase activity. These effects were inhibited only by PD-98059 (78 ± 9% and 98 ± 7%, respectively). We conclude that in human intestinal muscle cells IGF-I activates distinct PI 3-kinase and MAP kinase signaling pathways, which act in conjunction to mediate growth.
proliferation; p85; phosphatidylinositol 3,4,5-triphosphate; LY-294002; PD-98059
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INTRODUCTION |
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INSULIN-LIKE GROWTH FACTOR I (IGF-I) mediates three distinct regulatory effects on cell growth through activation of its cognate IGF-I receptor. Acting either alone or in concert with other growth factors, IGF-I is required for optimal proliferation of many mammalian cells, including adipocytes, epithelial cells, fibroblasts, and muscle cells (2, 3, 14, 26, 29, 36, 40). Transformation and maintenance of the transformed state also requires IGF-I receptor activation in some cells (2, 31). IGF-I-mediated activation of the IGF-I receptor can protect PC-12 and BALB/3T3 cells from apoptosis (24, 39). In addition to its effects on growth, IGF-I has also been observed in skeletal myoblasts to mediate the transition from proliferation to differentiation (6, 9, 28).
Mutational analysis of the IGF-I receptor has identified specific
phosphorylation sites within the COOH-terminal intracellular domain of
the IGF-I receptor -subunit that confer specificity for the
proliferative, transforming, or antiapoptotic effects elicited in
response to stimulation by IGF-I (13, 18, 22). The results of studies
on the intracellular signaling cascades activated in response to IGF-I
receptor activation have identified two primary pathways by which these
signals are transmitted. Similar to other receptor tyrosine kinases,
after IGF-I-induced receptor autophosphorylation, the
phosphatidylinositol 3-kinase (PI 3-kinase) pathway and the
mitogen-activated protein (MAP) kinase pathway are activated (6, 11,
24, 29, 32, 34). These pathways can act either in conjunction, in
opposition, or individually to mediate the IGF-I response, whether
proliferative, transforming, antiapoptotic, or differentiating (6, 11,
24, 28).
IGF-I causes proliferation of vascular smooth muscle cells, including aortic (26), arterial smooth muscle (40), pulmonary artery (8), and portal vein (5), as well as visceral smooth muscle cells, including myometrial (37), airway (21), and genitourinary smooth muscle cells (4). The importance of MAP kinase pathway activation in the proliferative response to IGF-I has been demonstrated for a number of smooth muscle cell types (25, 34). More recently, the potential role for PI 3-kinase in the proliferative response to growth factors has been appreciated, since PI 3-kinase may either function as an upstream activator of MAP kinase or exert MAP kinase-independent actions on cellular events (35). In skeletal myoblasts the MAP kinase and PI 3-kinase pathways act in opposition to mediate proliferation and differentiation, respectively. In contrast, in smooth muscle cells the interrelationship of MAP kinase and PI 3-kinase in the IGF-I-mediated effects on growth is less clearly understood (6, 11, 28).
Within the gastrointestinal tract, IGF-I receptors have been localized to both the mucosal and muscle layers (16, 33, 44, 46). The effect of exogenous IGF-I on the mucosal layer, acting via IGF-I receptors, is to increase mucosal growth (17, 42). These observations were confirmed by studies of transgenic mice, in which overexpression of the IGF-I gene was associated with increased small bowel length and mucosal mass and proliferation of crypt cells (23). Intestinal smooth muscle cells also express IGF-I and IGF-I receptors under normal conditions in vivo; their expression is upregulated in models of experimental colitis (44-46). The growth effect of IGF-I in intestinal smooth muscle cells depends on the species and the time in culture. IGF-I is an autocrine regulator of growth in muscle cells cultured from the human intestine. It acts most potently in proliferating cells and with lesser potency in postconfluent cells (15). Although IGF-I receptors were expressed in sparse cultures of neonatal rabbit gastric muscle cells, no effects on proliferation were observed (43). The intracellular mechanisms mediating the growth effects of IGF-I in intestinal smooth muscle cells are unknown.
In the present study, growth of smooth muscle cells cultured from the
circular layer of normal human intestine in response to IGF-I was
measured by
[3H]thymidine
incorporation, activation of MAP kinase and PI 3-kinase were measured
by in vitro kinase assay as the incorporation of [-32P]ATP into
selective substrates, and phosphorylation of MAP kinase was measured by
Western immunoblot analysis. The selective MAP kinase kinase (MEK)
inhibitor, PD-98059 (1), and the selective PI 3-kinase inhibitor,
LY-294002 (38), were used to identify the contribution of each
signaling pathway to the overall effects elicited by IGF-I. The results
show that IGF-I-induced activation of the IGF-I receptor is coupled to
activation of two distinct pathways, a PI 3-kinase-dependent, MAP
kinase-independent pathway and a MAP kinase-dependent, PI
3-kinase-independent pathway. The two signaling cascades act in
conjunction to mediate the IGF-I-stimulated growth of human intestinal
smooth muscle cells.
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MATERIALS AND METHODS |
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Preparation of isolated muscle cells from human jejunum. Muscle cells were isolated from the circular muscle layer of human jejunum as described previously (15). Briefly, 4- to 5-cm segments of normal jejunum were obtained from patients undergoing surgery for morbid obesity, according to a protocol approved by the Institutional Committee on the Conduct of Human Research. After the segments were opened along the mesenteric border, the mucosa was dissected away and the remaining muscle layer was cut into 2 × 2 cm strips. Slices were obtained separately from the circular layer using a Stadie-Riggs tissue slicer. The slices were incubated for two successive 60-min periods at 37°C in 25 ml of medium containing 0.2% collagenase (CLS type II) and 0.1% soybean trypsin inhibitor. The medium consisted of (in mM) 120 NaCl, 4 KCl, 2.6 KH2PO4, 2 CaCl2, 0.6 MgCl2, 25 HEPES, 14 glucose, and 2.1% Eagle's essential amino acid mixture. After the second incubation, the partially digested muscle strips were washed and incubated in enzyme-free medium for 30 min to allow the cells to disperse spontaneously.
Cell culture of human intestinal muscle cells. Primary cultures of human intestinal muscle cells were initiated and maintained according to methods described previously (15). Briefly, muscle cells dispersed from the circular layer were harvested by filtration through 500-µm Nitex mesh and centrifugation at 150 g for 5 min. Cells were resuspended and washed twice by centrifugation at 150 g for 5 min and resuspension in Ca2+- and Mg2+-free Hank's balanced salt solution (HBSS) containing 200 U/ml penicillin, 200 µg/ml streptomycin, 100 µg/ml gentamycin, and 2 µg/ml amphotericin B. The muscle cells were washed and then resuspended in DMEM containing 10% FBS (DMEM-10) and the same antibiotics. The cells were plated at a concentration of 5 × 105 cells/ml as determined by counting in a hemacytometer. Cultures were incubated in a 10% CO2 environment at 37°C. DMEM-10 medium was replaced every 3 days until the cells reached confluence.
Primary cultures of muscle cells were passaged after reaching confluence by first washing three times with PBS. After PBS was removed, cells were treated for 2 min with 0.05% trypsin and 0.53 mM EDTA. The trypsin activity was neutralized by addition of a fourfold excess of DMEM-10. The resulting cell suspension was centrifuged at 350 g for 10 min at 4°C. The pellet was washed twice by centrifugation at 350 g and resuspension in HBSS. The final cell pellet was resuspended in DMEM-10 at a concentration of 2.5 × 105 cells/ml and plated in the appropriate cultureware. The medium was changed after 24 h. All subsequent studies were performed in first-passage cultured cells after 7 days, at which time the cells were confluent.[3H]thymidine proliferation
assay.
Proliferation of smooth muscle cells in culture was measured by the
incorporation of
[3H]thymidine, as
described previously (15). Briefly, the cells were washed free of serum
and incubated for 24 h in DMEM-0. After 24-h incubation in the absence
of serum, the cells were incubated for an additional 24 h with 0.1 nM
to 1 µM IGF-I. During the final 4 h of this incubation period, 1 µCi/ml [3H]thymidine
was added to the medium. The incubation was terminated by washing cells
twice with 1 ml HBSS at 4°C and then incubating for two 10-min
periods with 1 ml of 10% TCA at 4°C. TCA was removed and 0.5 ml of
2 N perchloric acid was added to each well, and the cells were
incubated for 30 min at 60°C. After cooling, 400-µl aliquots of
the supernatant were removed to scintillation vials and counted on a
-scintillation counter. The cell residue was dissolved in 0.3 N NaOH
and neutralized with 0.3 N HCl, and protein was measured
spectrophotometrically using the Bio-Rad assay system. [3H]thymidine
incorporation was expressed as counts per minute (cpm) per microgram
protein.
Measurement of MAP kinase phosphorylation by Western blot.
MAP kinase phosphorylation in response to IGF-I was measured by Western
blotting with an antibody specific for the phosphorylated MAP kinase
isoforms, pp44 and pp42 MAP kinase. Briefly, confluent muscle cells
grown in six-well plates were incubated with IGF-I for various time
periods. The reaction was terminated by aspirating the medium and
rapidly washing two times with 2 ml PBS at 4°C. The cells were
lysed by addition of a 100 µl sample buffer containing 62.5 mM Tris
(pH 6.8) with 2% SDS, 25% glycerol, 0.01% bromophenol blue, and 5%
-mercaptoethanol. Samples were sonicated to shear DNA and heated to
boiling for 5 min. After centrifugation, 20-µl samples (containing 30 µg protein) were separated by SDS-PAGE on 10% polyacrylamide gels.
The separated proteins were electrotransferred to 0.2-µm
polyvinylidine difluoride membranes overnight at 4°C, using a buffer containing 25 mM Tris (pH 8.3) and 192 mM glycine, with
20% methanol and 0.1% SDS. After transfer the membranes were incubated in a Tris-buffered saline (TBS) blocking buffer containing 0.1% Tween 20 and 5% milk protein for 1 h at room temperature. The
membranes were incubated overnight with a 1:1,000 dilution of an
antibody specific for the phosphorylated (pp44/pp42) MAP kinase species
in TBS with 0.1% Tween 20 and 5% BSA. After incubation the membranes
were washed three times in TBS with 0.1% Tween 20 at room temperature.
The membranes were incubated in a 1:2,000 dilution of horseradish
peroxidase-conjugated secondary antibody for 1 h at room temperature
and then washed three times with washing buffer. The resulting protein
bands were visualized with enhanced chemiluminescence and quantitated
by image scanning densitometry.
Measurement of MAP kinase activity by in vitro kinase assay.
Confluent muscle cells growing in six-well plates were incubated in
serum-free DMEM for 72 h. IGF-I was then added for various time
periods, and the reaction was terminated by washing cells rapidly with
PBS at 4°C. The cells were lysed in a buffer containing (in mM) 10 Tris (pH 7.4), 150 NaCl, 2 EGTA, 2 dithiothreitol, 1 orthovanadate, and
1 phenylmethylsulfonyl fluoride, with 10 µg/ml leupeptin and 10 µg/ml aprotinin. Cellular debris in the lysates was precipitated by
centrifugation at 12,000 g for 20 min
at 4°C. The supernatant containing cytosolic MAP kinase activity was stored at 80°C until assayed. MAP kinase activity was
measured as the incorporation of phosphate from
[
-32P]ATP (1 µCi/30-µl reaction volume) into a synthetic MAP kinase substrate
(Amersham Life Science, Arlington Heights, IL) during a 30-min
incubation at 30°C. The reaction was terminated, and phosphorylated
peptide substrate was separated using phosphocellulose microcentrifuge
spin tubes (Pierce, Rockford, IL). The phosphocellulose units were
washed twice with 500-µl aliquots of 75 mM phosphoric acid. After
washing, the phosphocellulose units were counted using
-scintillation. The results were expressed in picomoles of phosphate incorporated per minute per milligram of protein.
Measurement of PI 3-kinase activity by in vitro kinase assay.
PI 3-kinase activity was measured by a modification of the method of
Higaki et al. (12). Briefly, muscle cells grown to confluence in 100-mm
dishes were incubated in serum-free DMEM for 72 h. Cells were
stimulated for various periods of time from 0 to 24 h and with various
concentrations of IGF-I from 10 pM to 100 nM. The reaction was
terminated by rapidly washing cells with 4°C PBS and then lysing
them. Lysis buffer consisted of (in mM) 50 Tris · HCl
(pH 7.4), 150 NaCl, 1 Na2VO3,
2 EDTA, 1 MgCl2, 1 CaCl2, and 30 nM leupeptin, with
1% Trasylol (wt/vol) and 1% Nonidet P-40 (vol/vol). DNA in the lysate
was sheared by passage through a 22-gauge needle, and the lysate was
incubated for 20 min at 4°C. After this incubation the lysate was
centrifuged at 12,000 g for 20 min at
4°C. The supernatant was removed and stored at 80°C
until assayed. Aliquots of the thawed lysate containing equal amounts
of protein were incubated with 25 µl of antiphosphotyrosine antibody
(PY20) coupled to agarose beads with gentle mixing for 2 h at 4°C.
The beads were collected by centrifugation at 12,000 g for 5 min at 4°C and washed
three times with lysis buffer and two times with kinase assay buffer.
Kinase assay buffer consisted of (in mM) 50 Tris · HCl (pH 7.8), 50 NaCl, 2 MgCl2, and 0.5 EDTA. After the
final washing the beads were resuspended in 30 µl of kinase assay
buffer, to which 10 µl of sonicated 1 mg/ml phosphatidylinositol were
added. The reaction was initiated by addition of 5 µl of 50 mM ATP
containing 0.5 µCi
[
-32P]ATP and
continued for 10 min at 30°C. The reaction was terminated by
addition of 0.5 ml of 1 N HCl and 2 ml of chloroform-methanol (2:1,
vol/vol). Phospholipids were recovered from the lower organic phase and
dried under N2 gas. The dried
phospholipids were dissolved in chloroform and spotted on Silica H gel
thin-layer chromatography (TLC) plates impregnated with 1% potassium
oxalate. Chromatograms were developed in a mixture of chloroform,
methanol, 28% NH3, and water
(70:100:15:25, vol/vol). The plates were air-dried, and phospholipids
were visualized with autoradiography. The spots corresponding to
authentic phosphatidylinositol 3,4,5-triphosphate (PIP3) were scraped
off the plates, and incorporated
32P was quantified by
-scintillation counting. Results are expressed as the increase in
32P incorporation into
PIP3 in counts per minute above
basal values.
Statistical analysis. Values are means ± SE of n experiments, where n represents the number of experiments on cells derived from separate primary cultures. Statistical significance was tested by Student's t-test for either paired or unpaired data as was appropriate. Densitometric analysis was performed using computerized densitometry and ImageQuant NT software (Molecular Dynamics). Densitometric values for protein bands were determined in areas of equal size and are reported in arbitrary units above background values.
Materials.
Recombinant human IGF-I was obtained from Collaborative Biomedical
Products (Bedford, MA), collagenase and soybean trypsin inhibitor were
obtained from Worthington Biochemical (Freehold, NJ), HEPES was from
Research Organics (Cleveland, OH), DMEM and HBSS from Mediatech
(Herndon, VA), and FBS from BioWhittaker (Walkersville, MD). The MAP
kinase assay kit,
[-32P]ATP (sp act
3,000 Ci/mmol), and
[3H]thymidine (sp act
6 Ci/mmol) were obtained from Amersham. Antibody to pp44/pp42 MAP
kinase was obtained from New England Biolabs (Beverly, MA), Western
blotting materials and the protein assay kit were obtained from Bio-Rad
(Hercules, CA), antiphosphotyrosine-PY20 agarose beads were obtained
from Transduction Laboratories (Lexington, KY), phosphocellulose spin
columns were obtained from Pierce, TLC plates were obtained from
Analtech (Newark, DE), and plastic cultureware was obtained from
Corning (Corning, NY). Phosphatidylinositol and all other chemicals
were obtained from Sigma (St. Louis, MO).
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RESULTS |
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Effect of IGF-I on smooth muscle cell growth. Addition of IGF-I to serum-deprived confluent human intestinal muscle cells caused an increase in [3H]thymidine incorporation. This increase was concentration dependent with an EC50 of 2.7 ± 0.4 nM and a maximal increase (at 100 nM) of 178 ± 28% above basal (basal 129 ± 32 cpm/µg protein) (Fig. 1).
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IGF-I-induced activation of PI 3-kinase.
PI 3-kinase activity was measured by the incorporation of
[-32P]ATP into
PIP3, the specific product of
phosphatidylinositol hydrolysis by PI 3-kinase. IGF-I (10 nM) caused a
rapid, time-dependent increase in the activity of PI 3-kinase, with a
maximal increase of 124 ± 31% above basal values after 10 min
(P < 0.01). The increase was
sustained at submaximal levels for periods of up to 60 min and fell to
basal levels after 240 and 1,440 min (Fig.
2). The maximal increase in PI 3-kinase
activity induced by IGF-I measured at 10 min was also concentration
dependent (Fig. 3). The selective PI
3-kinase inhibitor LY-294002 (50 µM) significantly inhibited IGF-I-induced PI 3-kinase activity (89 ± 12% inhibition of 100 nM
IGF-I; P < 0.05). The MEK inhibitor
PD-98059 (50 µM) had no effect on PI 3-kinase activity induced by 10 nM IGF-I (Fig. 3).
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Effect of IGF-I on MAP kinase activation.
Two methods were used to examine the involvement of the MAP kinase
pathway in IGF-I-stimulated growth and its relationship to the PI
3-kinase pathway. The first method measured IGF-I-induced MAP kinase
phosphorylation by Western blot analysis using an antibody specific for
the phosphorylated (pp44/pp42) MAP kinase isoforms. The second method
measured IGF-I-induced MAP kinase activity by an in vitro kinase assay,
by measuring
[-32P]ATP
incorporation into a synthetic MAP kinase substrate.
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DISCUSSION |
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This study shows that in human intestinal smooth muscle cells, growth mediated by IGF-I occurs as a result of the activation of two distinct signaling cascades. Stimulation of these cells by IGF-I results in activation of both a PI 3-kinase-dependent, MAP kinase-independent pathway and a MAP kinase-dependent, PI 3-kinase-independent pathway.
The results of studies on the intracellular signaling by IGF-I in
various cell types have revealed two primary second messenger cascades
activated by this growth factor: MAP kinase and PI 3-kinase (6, 11, 24,
29, 32, 34). The binding of IGF-I to its cognate heterotetrameric
receptor results in receptor -chain autophosphorylation, which then
leads to tyrosine phosphorylation of several intracellular substrates.
The two primary initial substrates of the activated IGF-I receptor are
insulin receptor substrate-1 (IRS-1) and Shc (19, 30). IRS-1 is an
adaptor protein with multiple sites undergoing tyrosine phosphorylation
after IGF-I activation. These regions act as docking sites for
interaction with proteins having SH2 domains, including the p85 subunit
of PI 3-kinase, Grb2, and phospholipase C-
1 (PLC
1) (6). The role
of this latter protein in the growth effects of IGF-I has as yet not
been well established (11). The Grb2 protein associates with mSos, a
guanine nucleotide exchange factor, which in turn activates p21
Ras and initiates activation of the Raf-MAP kinase pathway. Phosphorylated Shc can also associate with Grb2 and activate the Raf-MAP kinase pathway. Although either IRS-1 or Shc can link the
IGF-I receptor and Ras, evidence suggests that the Shc-Grb2-mSos pathway is most important for Ras activation in the context of IGF-I-stimulated growth (30). A second signaling pathway, the PI
3-kinase pathway, can also be activated by the interaction of
phosphorylated IRS-1 with the SH2 domains of the p85 regulatory subunit
of PI 3-kinase (7). p85 contains both
NH2-terminal and COOH-terminal SH2
domains. There is mounting evidence that after IGF-I receptor
activation, the receptor itself can directly associate with the
COOH-terminal SH2 domain of the p85 subunit and can activate PI
3-kinase (32). This observation would provide one mechanism by which
the MAP kinase and PI 3-kinase signaling pathways could be activated
separately. Alternatively, because it contains a binding site for Ras,
the p110 subunit of PI 3-kinase may be a direct effector of Ras and may
thus provide a mechanism for coactivation of the PI 3-kinase and
Raf-MAP kinase pathways (27). In some systems, however, PI 3-kinase
lies upstream of Ras activation after stimulation with IGF-I or insulin
(30, 41). This would provide a possible mechanism for activation of the
Raf-MAP kinase pathway downstream of PI 3-kinase and independently of
IRS-1 or Shc acting via Grb2-mSos.
In a number of cells, PI 3-kinase has been associated with activation
of various novel and atypical protein kinase C (PKC) isoforms,
including PKC-, PKC-
, and PKC-
(10, 20, 35). In vascular
smooth muscle cells, activation of PKC-
by IGF-I provides synergy
with second messengers activated by platelet-derived growth factor
(34). Neither the ability of IGF-I nor a potential role for PKC
activation and its ability to activate the Raf-MAP kinase pathway in
intestinal muscle cells were explored in the current study.
In the human intestinal smooth muscle cells used in the current study, the evidence supporting the involvement of a PI 3-kinase-dependent, MAP kinase-independent pathway stimulated by IGF-I can be summarized as follows. 1) IGF-I causes time-dependent and concentration-dependent activation of PI 3-kinase, which is abolished by the PI 3-kinase inhibitor but is not affected by the MEK inhibitor PD-98059. 2) IGF-I causes concentration-dependent growth, which is only partially inhibited by the PI 3-kinase inhibitor.
The evidence supporting the involvement of a MAP kinase-dependent, PI 3-kinase-independent pathway stimulated by IGF-I can be summarized as follows. 1) IGF-I causes time-dependent and concentration-dependent phosphorylation of p44/p42 MAP kinase, which is abolished by the MEK inhibitor PD-98059 but is not affected by the PI 3-kinase inhibitor LY-294002. 2) IGF-I causes time-dependent and concentration-dependent activation of MAP kinase, which is also abolished by the MEK inhibitor PD-98059 but is not affected by the PI 3-kinase inhibitor LY-294002. 3) IGF-I causes concentration-dependent stimulation of growth, which is only partially inhibited by the MEK inhibitorPD-98059. That both the PI 3-kinase and the MAP kinase pathways contribute independently to IGF-I-mediated growth is supported by the finding that although the PI 3-kinase inhibitor and the MEK inhibitor were singularly capable of partially inhibiting IGF-I-stimulated growth, in combination they abolished IGF-I-mediated growth.
The importance of activating two distinct signaling pathways, i.e., a MAP kinase pathway and a PI 3-kinase pathway, depends on the type of muscle cell examined. In skeletal muscle myoblasts, the two pathways are coupled to two distinct actions of IGF-I and act to oppose the actions of the other pathway: the Raf-MAP kinase pathway is coupled to the mitogenic actions of IGF-I and inhibits its myogenic, differentiating actions, whereas the PI 3-kinase pathway is essential for activating the myogenic, differentiating actions of IGF-I (6). In rat cardiac myocytes, IGF-I also activates both the Raf-MAP kinase and PI 3-kinase pathways (11). These pathways appear to play an important role in the hypertrophic response of cardiac muscle cells. It is not clear in these cells how the two pathways might interact, but IGF-I appears to mediate only a hypertrophic response in these cells, without a change in proliferation. In contrast, IGF-I elicits a proliferative response in a number of vascular smooth muscle cell types, including aortic, arterial, pulmonary artery, and portal vein (5, 8, 26, 40). Proliferation in response to IGF-I also occurs in visceral smooth muscle cells from the human myometrium (37) and airway (21). A feature common to many of these smooth muscle cells is the ability of IGF-I to act synergistically with other growth factors to stimulate growth. In these cases, the ability of IGF-I to activate two distinct signaling cascades may be crucial for it to produce its effects.
IGF-I, IGF-I receptors, and several IGF-binding proteins (IGFBP) have been identified within the muscular layers of the rat colon in vivo (44-46). IGF-I is also secreted by human intestinal muscle in culture, where it acts in an autocrine fashion to stimulate growth (15). Induction of experimental colitis in rats, either by peptidoglycan polysaccharide (46) or by trinitrobenzene sulfonate (44), has been shown to increase IGF-I mRNA and to result in an upregulation of IGFBP-4 and IGFBP-5, the effect of which is to increase IGF-I binding to the cells (44, 45). These findings, that the IGF-I system plays a role in the growth of normal intestinal muscle and in its response to experimental inflammation or injury, suggest that the regulation of IGF-I actions is an important component not only of the normal growth of human intestinal smooth muscle but also in the regulation of the muscle's response to injury and inflammation.
In summary, the present study shows that IGF-I-stimulated growth of human intestinal smooth muscle cells occurs as a result of the activation of two distinct signaling pathways: a MAP kinase-dependent, PI 3-kinase-independent pathway and a PI 3-kinase-dependent, MAP kinase-independent pathway.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49691.
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FOOTNOTES |
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Address for reprint requests: J. F. Kuemmerle, Division of Gastroenterology, Medical College of Virginia Campus, Virginia Commonwealth Univ., PO Box 980711, Richmond, VA 23298-0711.
Received 4 November 1997; accepted in final form 30 March 1998.
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