IGF-I stimulates intestinal muscle cell growth by activating distinct PI 3-kinase and MAP kinase pathways

John F. Kuemmerle and Toni L. Bushman

Departments of Medicine and Physiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298-0711

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -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 [gamma -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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -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% beta -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 [gamma -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 beta -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 [gamma -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 beta -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, [gamma -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).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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Fig. 1.   Insulin-like growth factor I (IGF-I) induces growth of human intestinal muscle cells. IGF-I induced concentration-dependent growth in human muscle cells (control, bullet ). IGF-I-induced growth was significantly inhibited in the presence of either the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY-294002 (50 µM; open circle ) or the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor PD-98059 (50 µM; black-square). IGF-I-induced growth was abolished in presence of the 2 inhibitors combined (square ). Growth was measured by incorporation of [3H]thymidine as described in MATERIALS AND METHODS. Results are expressed as percentage of basal thymidine incorporation in control cells [129 ± 32 counts per minute (cpm)/µg protein]. Values are means ± SE of 6 experiments. * P < 0.05 compared with basal; ** P < 0.05 vs. control; + P < 0.01 vs. control; ++ P < 0.001 vs. control.

The involvement of PI 3-kinase in the signaling cascade initiated by IGF-I, leading to growth of the muscle cells, was investigated using the selective PI 3-kinase inhibitor LY-294002 (38). The results of preliminary studies showed that the inhibition of IGF-I-stimulated [3H]thymidine incorporation by LY-294002 was maximal at a 50 µM concentration (data not shown). At the 50 µM concentration used in the current study LY-294002 has no inhibitory effects on protein serine/threonine kinases, including MAP kinase or other protein tyrosine kinases (38). The selective PI 3-kinase inhibitor LY-294002 (50 µM) significantly inhibited IGF-I-induced [3H]thymidine incorporation (86 ± 7% inhibition of 100 nM IGF-I; P < 0.01) (Fig. 1).

The involvement of MAP kinase was similarly investigated using PD-98059, a selective inhibitor of MEK1 that blocks phosphorylation and activation of MAP kinase (1). The results of preliminary studies showed that the inhibition of IGF-I-stimulated [3H]thymidine incorporation by PD-98059 was maximal at a 50 µM concentration (data not shown). At the 50 µM concentration used in the current study, PD-98059 had negligible effects on PI 3-kinase activity (1). The MEK inhibitor PD-98059 significantly inhibited IGF-I-induced [3H]thymidine incorporation (35 ± 6% inhibition of 100 nM IGF-I; P < 0.05) (Fig. 1).

In the presence of both the PI 3-kinase inhibitor LY-294002 and the MEK inhibitor PD-98059, IGF-I-induced [3H]thymidine incorporation was completely abolished (114 ± 18% inhibition of 100 nM IGF-I; P < 0.001) (Fig. 1). The ability of the PI 3-kinase and MEK inhibitors individually to inhibit [3H]thymidine incorporation suggested that both pathways were involved in IGF-I-stimulated growth. That the combination of the two inhibitors abolished [3H]thymidine incorporation implied that the two pathways accounted fully for the growth stimulation in response to IGF-I.

IGF-I-induced activation of PI 3-kinase. PI 3-kinase activity was measured by the incorporation of [gamma -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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Fig. 2.   IGF-I induces time-dependent increase in PI 3-kinase activity. A: representative autoradiogram of time-dependent increase in PI 3-kinase activity elicited by 10 nM IGF-I. B: densitometric analysis of time-dependent increase in PI 3-kinase activity. IGF-I (10 nM) elicited a prompt increase in PI 3-kinase activity that was significant within 2 min, maximal by 10 min, and declined to low suprabasal levels by 240 min. PI 3-kinase activity was measured by immune complex in vitro kinase assay as described in MATERIALS AND METHODS. Results are expressed as percentage increase in 32P incorporation into phosphatidylinositol 3,4,5-triphosphate (PIP3) above basal unstimulated values (basal 212 ± 55 cpm). Values are means ± SE of 7 experiments. * P < 0.05 compared with basal values.


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Fig. 3.   IGF-I induces concentration-dependent increase in PI 3-kinase activity. A: representative autoradiogram of concentration-dependent increase in PI 3-kinase activity elicited by IGF-I (10 nM) and effects of LY-294002 (50 µM) and PD-98059 (50 µM). B: densitometric analysis of concentration-dependent increase in PI 3-kinase activity. IGF-I elicited a concentration-dependent increase in PI 3-kinase activity (bullet ). Increase in PI 3-kinase activity in response to 10-100 nM IGF-I, measured at the 10-min maximum, was inhibited by LY-294002 (50 µM; open circle ) but was not affected by PD-98059 (50 µM; black-square). PI 3-kinase activity was measured by immune complex in vitro kinase assay as described in MATERIALS AND METHODS. Results are expressed as percentage increase in 32P incorporation into PIP3 above basal unstimulated values (basal 212 ± 55 cpm). Values are means ± SE of 3-7 experiments. * P < 0.05 compared with basal values; ** P < 0.05 for inhibition of response.

Inhibition of IGF-I-induced growth in the presence of PD-98059 suggested that MAP kinase activation was also involved in the proliferative response of the muscle cells to IGF-I. However, the finding that the IGF-I-stimulated increase in PI 3-kinase activity was abolished by the selective PI 3-kinase inhibitor LY-294002 but was not affected by the MEK inhibitor PD-98059 implied that IGF-I-induced activation of the PI 3-kinase occurred either upstream of MAP kinase activation or independently of the MAP kinase pathway. This possibility was explored further by measuring IGF-I-induced activation of MAP kinase.

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 [gamma -32P]ATP incorporation into a synthetic MAP kinase substrate.

IGF-I caused a rapid, time-dependent phosphorylation of both the p44 and p42 isoforms of MAP kinase, as measured by Western blot analysis of pp44/pp42 MAP kinase. Maximum MAP kinase phosphorylation (1,300 ± 200% increase over basal; P < 0.05) was observed within 10 min and declined to low suprabasal levels within 240 min (Fig. 4). Phosphorylation of both the p44 and p42 isoforms was strongly inhibited at the 10-min maximum by the MEK inhibitor PD-98059 (78 ± 9% inhibition of control; P < 0.05) but was not affected by the PI 3-kinase inhibitor LY-264002 [6.2 ± 8.8% inhibition; not significant (NS)] (Fig. 4).


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Fig. 4.   IGF-I induces time-dependent phosphorylation of p44/p42 MAP kinase. A: representative Western blot of time-dependent phosphorylation of p44/p42 MAP kinase induced by IGF-I (100 nM). B: densitometric analysis of pp44/pp42 MAP kinase. IGF-I (100 nM; bullet ) induced prompt, within 2 min, phosphorylation of p44/p42 MAP kinase that was maximal within 10 min, sustained for up to 60 min, and declined to low suprabasal levels within 240 min. IGF-I induced phosphorylation of p44/p42 MAP kinase, measured at the 10-min maximum, was inhibited by PD-98059 (50 µM; black-square) but not affected by LY-294002 (50 µM; open circle ). p44/p42 MAP kinase phosphorylation was measured by Western blot analysis using an antibody specific for phosphorylated (pp44/pp42) MAP kinase as described in MATERIALS AND METHODS. Results are expressed as percentage increase in protein band density above basal levels in arbitrary units. Values are means ± SE of 5 separate experiments. * P < 0.05 compared with basal; ** P < 0.05 for inhibition of response.

A similar time-dependent increase in MAP kinase activity in response to IGF-I was also observed. IGF-I (100 nM) elicited a prompt increase in MAP kinase activity that was maximal within 10 min (8.8 ± 1.3 pmol Pi · min-1 · mg protein-1 above basal; P < 0.05) and declined to low suprabasal levels by 240 min (Fig. 5). The increase in MAP kinase activity induced by IGF-I was concentration dependent (Fig. 6). The MEK inhibitor PD-98059 abolished the increase in maximal MAP kinase activity in response to MAP kinase by 98 ± 7% (P < 0.05). LY-294002 had no effect on the IGF-I-induced increase in MAP kinase activity (5.7 ± 12% inhibition of control; NS) (Fig. 6).


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Fig. 5.   IGF-I induces time-dependent activation of MAP kinase. IGF-I (100 nM) induced prompt, within 2 min, activation of MAP kinase that was maximal within 10 min and remained elevated for up to 60 min before declining to basal levels within 240 min. MAP kinase activation was measured by an in vitro kinase assay as the incorporation of [32P]ATP into a synthetic MAP kinase substrate as described in MATERIALS AND METHODS. Results are expressed as the increase in 32P incorporation (pmol Pi · min-1 · mg protein-1) above basal values (basal 0.87 ± 0.22 pmol Pi · min-1 · mg protein-1). Values are means ± SE of 4 separate experiments. * P < 0.05 compared with basal values.


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Fig. 6.   IGF-I induces concentration-dependent activation of MAP kinase. IGF-I (bullet ) induced concentration-dependent activation of MAP kinase measured at the 10-min maximum. MAP kinase activation elicited by IGF-I was inhibited by PD-98059 (50 µM; black-square) but was not affected by LY-294002 (50 µM; open circle ). MAP kinase activation was measured at the 10-min maximum by in vitro kinase assay as described in MATERIALS AND METHODS. Results are expressed as the increase in 32P incorporation (pmol Pi · min-1 · mg protein-1) above basal values (basal 0.87 ± 0.22 pmol Pi · min-1 · mg protein-1). Values are means ± SE of 4 separate experiments. * P < 0.05 compared with basal values; ** P < 0.05 for inhibition of response from control values.

The finding that the phosphorylation of both the p44 and p42 MAP kinase isoforms and the increase in MAP kinase activity induced by IGF-I were inhibited by the MEK inhibitor PD-98059 but were not affected by the PI 3-kinase inhibitor LY-294002 implied that the activation of the MAP kinase pathway by IGF-I did not occur downstream from PI 3-kinase activation but rather that the two pathways were activated independently by IGF-I in these cells.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -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-gamma 1 (PLCgamma 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-delta , PKC-epsilon , and PKC-zeta (10, 20, 35). In vascular smooth muscle cells, activation of PKC-epsilon 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.

    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49691.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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