Endogenous IGF-I protects human intestinal smooth muscle cells from apoptosis by regulation of GSK-3{beta} activity

John F. Kuemmerle

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

Submitted 20 January 2004 ; accepted in final form 28 July 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have previously shown that endogenous IGF-I regulates human intestinal smooth muscle cell proliferation by activation of phosphatidylinositol 3 (PI3)-kinase- and Erk1/2-dependent pathways that jointly regulate cell cycle progression and cell division. Whereas insulin-like growth factor-I (IGF-I) stimulates PI3-kinase-dependent activation of Akt, expression of a kinase-inactive Akt did not alter IGF-I-stimulated proliferation. In other cell types, Akt-dependent phosphorylation of glycogen synthase kinase-3{beta} (GSK-3{beta}) inhibits its activity and its ability to stimulate apoptosis. The aim of the present study was to determine whether endogenous IGF-I regulates Akt-dependent GSK-3{beta} phosphorylation and activity and whether it regulates apoptosis in human intestinal muscle cells. IGF-I elicited time- and concentration-dependent GSK-3{beta} phosphorylation (inactivation) that was measured by Western blot analysis using a phospho-specific GSK-3{beta} antibody. Endogenous IGF-I stimulated GSK-3{beta} phosphorylation and inhibited GSK-3{beta} activity (measured by in vitro kinase assay) in these cells. IGF-I-dependent GSK-3{beta} phosphorylation and the resulting GSK-3{beta} inactivation were mediated by activation of a PI3-kinase-dependent, phosphoinositide-dependent kinase-1 (PDK-1)-dependent, and Akt-dependent mechanism. Deprivation of serum induced {beta}-catenin phosphorylation, increased in caspase 3 activity, and induced apoptosis of muscle cells, which was inhibited by either IGF-I or a GSK-3{beta} inhibitor. Endogenous IGF-I inhibited {beta}-catenin phosphorylation, caspase 3 activation, and apoptosis induced by serum deprivation. IGF-I-dependent inhibition of apoptosis, similar to GSK-3{beta} activity, was mediated by a PI3-kinase-, PDK-1-, and Akt-dependent mechanism. We conclude that endogenous IGF-I exerts two distinct but complementary effects on intestinal smooth muscle cell growth: it stimulates proliferation and inhibits apoptosis. The growth of intestinal smooth muscle cells is regulated jointly by the net effect of these two processes.

phosphoinositidol 3-kinase; phosphoinositide-dependent kinase-1; Akt; proliferation


THE SURVIVAL OF MAMMALIAN cells is regulated by growth factors and hormones that actively suppress apoptosis. Survival factors such as insulin and insulin-like growth factor-I (IGF-I) prevent apoptosis through activation of phosphatidylinositol 3-kinase (PI3-kinase) and the subsequent activation of the downstream protein serine/threonine kinase, Akt (9, 13, 22). Glycogen synthase kinase-3 (GSK-3), also a serine/threonine kinase, is a principle substrate of Akt and exists as two highly homologous isoforms: {alpha} and {beta} (1). Both GSK-3{alpha} and GSK-3{beta} are inhibited by Akt-dependent phosphorylation. In addition to glycogen synthase, from which it takes its name, the GSK-3{beta} isoform phosphorylates a number of substrates including metabolic and signaling proteins, structural proteins, and transcription factors that regulate cell survival (6, 7, 39).

IGF-I can regulate the growth of normal cells through two complementary mechanisms. IGF-I stimulates proliferation and inhibits apoptosis (12, 22, 23, 29). Although both effects are mediated by the interaction of IGF-I with the cognate IGF-I receptor tyrosine kinase, the particular effects are both species and cell-type specific. IGF-I stimulates proliferation of human intestinal smooth muscle cells by activation of the IGF-I receptor tyrosine kinase, which is coupled to distinct Erk1/2- and PI3-kinase-dependent pathways that stimulate the cells to enter the cell cycle and divide as well as provide the necessary increase in cell size required for cell division to occur (17, 16, 21). IGF-I also activates Akt downstream of PI3-kinase via PDK-1. Unlike PI3-kinase and PDK-1, which, when activated by IGF-I, stimulate cellular proliferation, Akt activation is not required for cell division to occur because transfection of a kinase-inactive Akt did not alter IGF-I-induced proliferation (16).

GSK-3{beta} has been identified as a target of Akt that plays a role in the regulation of apoptosis in a variety of normal and transformed cell types including muscle cells, hepatocytes, neurons, and colon cancer cells (5, 27, 31, 38). Akt-dependent phosphorylation of GSK-3{beta} causes its inactivation, blocking downstream proapoptotic pathways, e.g., caspase and {beta}-catenin signaling (1, 10). Akt-dependent regulation of GSK-3{beta} activity contributes to the growth and hypertrophy of skeletal and cardiac muscle and inhibits apoptosis in vascular smooth muscle (11, 36).

A role for endogenous IGF-I in regulating proliferation of human intestinal smooth muscle has been identified; however, its role in regulating apoptosis has not previously been investigated. A number of clinical conditions are associated with increased expression of IGF-I in the muscularis propria including pyloric stenosis and Crohn's disease. We hypothesize that the increased IGF-I expression by intestinal muscle cells plays a role in the muscle hyperplasia by both stimulating proliferation and inhibiting apoptosis of smooth muscle.

The present study shows that in human intestinal smooth muscle, IGF-I regulates the phosphorylation and activity of GSK-3{beta} and thereby inhibits apoptosis. GSK-3{beta} activity and apoptosis are regulated via IGF-I-dependent sequential activation of PI3-kinase, phosphoinositide-dependent kinase-1 (PDK-1), and Akt. Endogenous IGF-I inhibits apoptosis in these cells by regulating not only GSK-3{beta}, but also {beta}-catenin phosphorylation and caspase 3 activity. In summary, IGF-I regulates two complementary processes in human intestinal smooth muscle: stimulation of proliferation and inhibition of apoptosis that jointly regulate the growth of muscle cells.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Culture of smooth muscle cells isolated from normal human jejunum. Muscle cells were isolated and cultured from the circular muscle layer of human jejunum as described previously (1417, 20). 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 Virginia Commonwealth University institutional review board. After the segments were opened along the mesenteric border, the mucosa was dissected away and the remaining muscle layer was cut into 2 x 2-cm strips. Slices were obtained separately from the circular layer using a Stadie-Riggs tissue slicer. The slices were incubated overnight at 37°C in 20 ml of DMEM plus 10% fetal bovine serum (DMEM-10) containing 200 U/ml penicillin, 200 µg/ml streptomycin, 100 µg/ml gentamycin, and 2 µg/ml amphotericin B to which was added 0.0375% collagenase (CLS type II) and 0.1% soybean trypsin inhibitor. 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. After resuspension in DMEM-10 containing the same antibiotics, the cells were plated at a concentration of 5 x 105 cells/ml as determined by counting in a hemocytometer. 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 on reaching confluence and were used in first passage. We have previously shown that these cells express a phenotype characteristic of intestinal smooth muscle as determined by immunostaining for smooth muscle markers and expression of {gamma}-enteric actin. Epithelial cells, endothelial cells, neurons, and interstitial cells of Cajal are not detected in these cultures (15, 33).

Nucleosome apoptosis assay. Apoptosis of smooth muscle cells in culture was quantitated by measurement of cytoplasmic histone-associated DNA fragments (nucleosomes) using a quantitative sandwich-enzyme immunoassay ELISA (Roche Applied Science, Indianapolis, IN) according to the manufacturer's directions. Briefly, muscle cells growing in 48-well culture plates were washed free of serum and incubated for 0–72 h in serum-free DMEM in the presence and absence of various test agents. The medium was removed and replaced with 100 µl of lysis buffer. The cells were incubated at room temperature for 20 min and centrifuged for 10 min at 200 g. Aliquots of supernatant (20 µl) containing cytoplasmic DNA fragments were transferred to each well of a 96-well ELISA plate precoated with streptavidin. To each well was added 80 µl of a mixture containing mouse monoclonal antibody to histone-biotin (clone H11–4) and mouse monoclonal antibody to anti-DNA-POD (clone MCA-33). The microtiter plates were incubated at room temperature on a microplate shaker at 250 rpm for 2 h. The unbound components were removed by washing each well three times with 250 µl. After substrate (2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] diammonium salt) solution was added to each well, the microtiter plates were incubated with shaking (200 rpm) for 10 min. Absorbance at 405 nm was measured using a Victor2 1420 multichannel counter (Perkin-Elmer, Boston, MA). All samples were run in duplicate and corrected for substrate blanks. Results were reported as the percent change in A405.

Caspase 3 activity assay. Apoptosis of muscle cells was also measured by quantification of caspase 3 activity using a fluorimetric immunosorbent assay (Roche Applied Science) according to manufacturer's directions. Briefly, muscle cells growing in 60-mm dishes were washed free of serum and incubated for 24 h in serum-free DMEM in the presence and absence of various test agents. The reaction was terminated by washing cells in PBS. The cells were incubated for 1 min at 4°C in lysis buffer, and the lysates were transferred to Microfuge tubes before centrifugation for 1 min at 14,000 rpm in a Microfuge. The supernatant was removed, and 100-µl aliquots were added in duplicate to a 96-well microtiter plate precoated with antibody recognizing human caspase 3. The plate was incubated at 37°C for 1 h and washed three times with washing buffer and then with substrate solution for 1 h at 37°C in a light-tight container. Caspase 3 activity was measured with Ex405/Em500 using a Victor2 1420 multichannel counter (Perkin-Elmer). All samples were run in duplicate and corrected for substrate blanks. Results were reported as the change in relative fluorescence, Ex405/Em500.

Assay of GSK-3{beta} activity. The activity of GSK-3{beta} was assayed using an immune complex in vitro kinase assay by the methods of Fang et al. (8). Briefly, confluent muscle cells were rendered quiescent by incubation in serum-free DMEM for 24 h. Cells were stimulated with IGF-I (100 nM) for the indicated periods of time (0–24 h) in the presence or absence of various inhibitors. The reaction was terminated by washing the cells in ice-cold PBS. The cells were lysed by addition of a lysis buffer consisting of (in mM): 50 HEPES (pH 7.4), 150 NaCl, 1.5 MgCl2, 1 EGTA, 100 NaF, 1 sodium orthovanadate, 10 sodium pyrophosphate, 25 {beta}-glycerol phosphate, 1 DTT, and 1 benzamidine, containing added 10% glycerol, 1% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.1 µM okadaic acid. Cell lysates were clarified by centrifugation, and aliquots of the supernatant containing equal amounts of total cellular protein (75 µg) were incubated with 0.75 µg of anti-GSK-3{beta}. After rotation for 2 h at 4°C, protein G-sepharose was added for an additional 1.5 h of incubation. Immunoprecipitates were washed twice with lysis buffer and twice with kinase assay buffer. Kinase assay buffer consisted of (in mM): 10 4-3-(N-morpholino)propanesulfonic acid (pH 7.4), 1 EDTA, 10 Mg acetate, 50 {beta}-glycerol phosphate, 1 Na orthovanadate, 0.5 NaF, 1 benzamidine, and 1 DTT, with added 0.1 µM okadaic acid and 1 µg/ml aprotinin. Kinase activity of immunoprecipitated GSK-3{beta} was assayed in a total reaction volume of 40 µl containing 20 mM MgCl2 and 125 µM cold ATP using 62.5 µM phosphoglycogen synthase peptide-2 as substrate or 62.5 µM glycogen synthase peptide-2 [Ala21] as a negative control peptide. The reaction was initiated by the addition of 10 µCi [{gamma}-32P]ATP and incubated for 20 min at 30°C. The reaction was terminated by centrifugation. Duplicate 15-µl aliquots of supernatant were spotted on phosphocellulose paper. The phosphocellulose paper was washed three times with 0.75% phosphoric acid and one time with acetone, transferred to scintillation vials, and counted on a {beta}-scintillation counter. The results were corrected for endogenous substrates by subtraction of values obtained in the presence of the negative control peptide and for background obtained by immunoprecipitation with nonimmune serum. Values were expressed in picomoles of phosphate incorporated into peptide substrate per minute per milligram of protein.

Western blot analysis. The phosphorylation of GSK-3{beta}(Ser9) and {beta}-catenin(Ser33/37/Thr41) was measured by Western blot analysis using standard methods (4, 1620). Briefly, confluent muscle cells were rendered quiescent by incubation for 24 h in serum-free medium. The cells were stimulated with recombinant human IGF-I for periods of time from 0 to 72 h. The cells were rapidly washed with ice-cold PBS and lysed in sample buffer. Lysates were boiled for 5 min, and equal amounts of total cell protein were separated with SDS-PAGE under denaturing conditions. After the proteins were electrotransferred to nitrocellulose, the membranes were incubated overnight with a 1:1,000 dilution of antibody recognizing phosphorylated (inactive) GSK-3{beta}(Ser9) or {beta}-catenin(Ser33/37/Thr41). Nitrocellulose membranes were stripped and reblotted to determine levels of total (phosphorylated + nonphosphorylated) GSK-3{beta} or {beta}-catenin. Bands of interest were visualized with enhanced chemiluminescence using a FluorChem 8800 (Alpha Innotech, San Leandro, CA), and the resulting digital images were quantified using AlphaEaseFC version 3.1.2 software.

Expression of kinase-inactive Akt and constitutively active Akt. Substitution of methionine for lysine at residue 179 (K179M) of Akt results in a kinase-inactive Akt (32). Conversely, myristoylation of Akt (Myr-Akt) confers membrane targeting to Akt resulting in constitutively active Akt (3). cDNA for Myc-tagged, kinase-inactive Akt(K179M) in the pUSEamp(+) expression vector or Myc-tagged, Myr-Akt in the pUSEamp(+) expression vector was purified, and muscle cells growing in six-well plates transiently transfected with 1 µg of pUSEamp(+)-Akt(K179M), 2 µg of pUSEamp(+)-Myr-Akt cDNA, or with pUSEamp(+) vector alone as a control using a LipofectAMINE PLUS Reagent kit (Life Technologies). Cells were incubated for 3 h at 37°C with the transfection reagent-DNA complexes. The DNA-containing medium was replaced with DMEM + 10% FCS. After 48 h incubation, the expression of kinase-inactive Akt(K179M) or Myr-Akt was confirmed by Western blot analysis using anti-Myc tag antibody. The dominant negative effect of Akt(K179M) on IGF-I-activated signaling was previously confirmed by analysis of this mutation's effect on IGF-I-induced Akt(Ser473) phosphorylation (16).

Measurement of protein content. The protein content of cell lysates was measured using the Bio-Rad DC protein assay kit according to manufacturer's directions. Samples were adjusted to provide aliquots of equal protein content before in vitro kinase assay or Western blot analysis.

Statistical analysis. Values given represent the 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 appropriate. Densitometric values for protein bands of phosphorylated signaling intermediates were reported in arbitrary units above background values after normalization to total signaling intermediate protein levels. Bands of interest were visualized with enhanced chemiluminescence using a FluorChem 8800 (Alpha Innotech, San Leandro, CA), and the resulting digital images were quantified using AlphaEaseFC version 3.1.2 software.

Materials. Recombinant human IGF-I was obtained from Austral Biologicals (San Ramon, CA); collagenase and soybean trypsin inhibitor were obtained from Worthington Biochemical (Freehold, NJ); HEPES was obtained from Research Organics (Cleveland, OH); DMEM and Hank's balanced salt solution were obtained from Mediatech (Herndon, VA); fetal bovine serum was obtained from Summit Biotechnologies (Fort Collins, CO); [{gamma}-32P]ATP (specific activity 3,000 Ci/mmol) was obtained from New England Nuclear (Boston, MA); rabbit polyclonal antibodies to phospho-GSK-3{beta}(Ser9), phospho-{beta}-catenin(Ser33/37/Thr41), total {beta}-catenin, anti-mouse IgG-HRP, and anti-rabbit IgG-HRP were obtained from Cell Signaling Technology (Beverly, MA); mouse monoclonal antibody to GSK-3{beta} (clone 7) was obtained from Transduction Laboratories (Lexington, KY); mouse monoclonal antibody to the Myc tag on Akt(K179M) and Myr-Akt, phosphoglycogen synthase peptide-2, and glycogen synthase peptide-2 [Ala21] were obtained from Upstate Biotechnology (Lake Placid, NY); the kinase inhibitors LY294002, N-{alpha}-tosyl-Phe chloromethyl ketone (TPCK), and 3-(3-carboxy-4-chloroanilinio)-4-(3-nitrophenyl)maleimide were obtained from Calbiochem (San Diego, CA); Western blot analysis materials and DC protein assay kit were obtained from Bio-Rad Laboratories (Hercules, CA); and plastic cultureware was obtained from Corning (Corning, NY). All other chemicals were obtained from Sigma (St. Louis, MO).


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
IGF-I regulates GSK-3{beta} phosphorylation and activity. The effects of IGF-I on GSK-3{beta} and the signaling mechanisms regulating IGF-I-induced GSK-3{beta} activation were investigated in two complementary ways. The first method examined the phosphorylation (inactivation) of GSK-3{beta}(Ser9) in Western blot analysis using a phosphorylation state-specific antibody. The second method examined the activity of GSK-3{beta} by in vitro kinase assay.

The effect of IGF-I on GSK-3{beta} phosphorylation was measured by Western blot analysis using an antibody specific for Ser9 phosphorylated (inactive) GSK-3{beta}. Quiescent smooth muscle cells were incubated for increasing periods of time (0–72 h) with IGF-I. IGF (100 nM) caused phosphorylation of GSK-3{beta}(Ser9) that was prompt, occurring within 30 min, that was maximal within 6 h (180 ± 19% above basal), and that was sustained for up to 72 h (Fig. 1A). Incubation of muscle cells for 6 h with increasing concentrations of IGF-I (0–100 nM) elicited concentration-dependent GSK-3{beta} phosphorylation (Fig. 1B).



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Fig. 1. Insulin-like growth factor-I (IGF-I) stimulates glycogen synthase kinase-3{beta} (GSK-3{beta}) phosphorylation. A: IGF-I causes time-dependent GSK-3{beta}(Ser9) phosphorylation. Quiescent muscle cells were incubated for increasing periods of time with 100 nM IGF-I and phosphorylation of GSK-3{beta} on Ser9 measured by Western blot analysis using a phosphospecific antibody. Inset: representative Western blot of IGF-I-stimulated GSK-3{beta} phosphorylation and total GSK-3{beta} levels. B: IGF-I causes concentration-dependent phosphorylation of GSK-3{beta}(Ser9). Quiescent muscle cells were incubated for 6 h with increasing concentrations of IGF-I. Inset: representative Western blot analysis of IGF-I-stimulated GSK-3{beta} phosphorylation. Results are expressed as a percent above basal values in relative densitometric units and normalized to total GSK-3{beta} values. Values represent the means ± SE of 3–4 experiments. *P < 0.05 vs. basal.

 
The effect of IGF-I on GSK-3{beta} activity was determined by in vitro kinase assay using phosphoglycogen synthase peptide-2 as substrate as described in METHODS. In muscle cells deprived of serum for 24 h, control GSK-3{beta} activity was 20.8 ± 0.5 pmol phosphate·min–1·mg protein–1. In cells incubated for the final 6 h of the 24-h period in the presence of 100 nM IGF-I, GSK-3{beta} activity was inhibited by 68 ± 15% (Fig. 2). GSK-3{beta} activity measured after 24 h of serum deprivation was also inhibited 60 ± 8% by the GSK inhibitor 3-(3-carboxy-4-chloroanilinio)-4-(3-nitrophenyl)maleimide (10 µM; Fig. 2).



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Fig. 2. IGF-I regulates GSK-3{beta} activity. GSK-3{beta} activity was inhibited by IGF-I and augmented in the presence of the IGF-I receptor antagonist. GSK-3{beta} activity was also inhibited in the presence of the GSK-3{beta} inhibitor 3-(3-carboxy-4-chloroanilino)-4-(3-nitrophenyl)maleimide. Quiescent muscle cells were incubated for 6 h with 100 nM IGF-I, with 100 nM IGF-I receptor antagonist (IGF analog), or with 10 µM GSK-3{beta} inhibitor. The activity of GSK-3{beta} was measured by in vitro kinase assay from the incorporation of [{gamma}-32P]ATP into phosphoglycogen synthase peptide-2 as substrate. Basal GSK-3{beta} activity was 20.8 ± 0.5 pmol phosphate·min–1·mg protein–1. Results represent the means ± SE of 3–4 experiments. *Decrease with P < 0.05 vs. control; **increase with P < 0.05 vs. control.

 
Endogenous IGF-I regulates GSK-3{beta} phosphorylation and activity. We have previously shown that human intestinal smooth muscle cells secrete IGF-I that regulates muscle cell growth (15). The role of endogenous IGF-I in the regulation of GSK-3{beta} phosphorylation and activity was examined in cells treated with the selective IGF-I receptor antagonist IGF analog (28). In muscle cells deprived of serum (incubated in serum-free DMEM) for periods of time from 0 to 72 h, dephosphorylation (activation) of GSK-3{beta}(Ser9) was significant after 12 h and progressed for up to 72 h (Fig. 3A). In the presence of the IGF-I receptor antagonist IGF analog GSK-3{beta}, dephosphorylation induced by serum deprivation was significantly enhanced at all time periods between 24 and 72 h.



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Fig. 3. Endogenous IGF-I inhibits GSK-3{beta} phosphorylation. Levels of GSK-3{beta} phosphorylation were decreased in time-dependent fashion in response to serum deprivation. Serum deprivation-induced GSK-3{beta} dephosphorylation was augmented in the presence of the IGF-I receptor antagonist (Antag). Intestinal muscle cells were incubated for increasing periods of time with serum-free DMEM alone (DMEM-0) or with added IGF-I receptor antagonist (100 nM). GSK-3{beta} phosphorylation was measured by Western blot analysis using a phosphospecific antibody for GSK-3{beta}(Ser9). Results were expressed in relative densitometric units normalized to total GSK-3{beta} levels. Values represent the means ± SE of 4 separate experiments. *P < 0.05 vs. DMEM-0 alone.

 
These results were confirmed by measurement of GSK-3{beta} activity. Incubation of quiescent muscle cells for 6 h with the IGF-I receptor antagonist IGF analog (100 nM) augmented basal GSK-3{beta} activity by 70 ± 12% (basal: 20.8 ± 0.5 pmol phosphate·min–1·mg protein–1; Fig. 2). The results implied that endogenous IGF-I regulates GSK-3{beta} phosphorylation and activity.

Pathways mediating the effects of IGF-I on GSK-3{beta} activity. We have previously shown that IGF-I activates distinct PI3-kinase and Erk1/2 pathways in human intestinal smooth muscle cells. The involvement of PI3-kinase and the PI3-kinase-dependent signaling intermediates PDK-1 and Akt and of Erk1/2 in regulating GSK-3{beta} activity were examined using two techniques. The first technique used selective inhibitors of PI3-kinase, PDK-1, GSK-3{beta}, and Erk1/2. Initial experiments had confirmed the ability of 3-(3-carboxy-4-chloroanilinio)-4-(3-nitrophenyl)maleimide to inhibit GSK-3{beta} activity (see above and Fig. 2). In the second, the role of Akt was identified by transient transfection of a kinase-inactive Akt gene, Akt(K179M), or a constitutively active Akt gene, Myr-Akt. We have previously shown that kinase-inactive Akt(K179M) exerts a dominant negative effect in these cells (16). Akt is activated by its recruitment to the plasma membrane in a PI3-kinase- and PDK-1-dependent fashion. Expression of a Myr-Akt gene has been shown to result in Akt that is constitutively associated with the plasma membrane and constitutively active (3).

Muscle cells were incubated for 24 h in serum-free DMEM and with 100 nM IGF-I for the final 6 h. IGF-I increased GSK-3{beta} phosphorylation by 220 ± 13% above basal levels. IGF-I-induced GSK-3{beta} phosphorylation was inhibited 87 ± 15% in the presence of the PI3-kinase inhibitor (35) LY294002 (10 µM) and was inhibited 96 ± 5% in the presence of the PDK-1 inhibitor TPCK (50 µM) (Fig. 4A) (2). GSK-3{beta} phosphorylation was unaffected (0 ± 8% inhibition) by the Erk1/2 inhibitor U1026 (1 µM) and was also unaffected (–3 ± 5% inhibition) by the p70S6 kinase inhibitor rapamycin (10 nM).



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Fig. 4. IGF-I regulates GSK-3{beta} phosphorylation and activity via phosphatidylinositol (PI3)-kinase-, phosphoinositide-dependent kinase-1 (PDK-1)-, and Akt-dependent pathways. A: GSK-3{beta}(Ser9) phosphorylation elicited by IGF-I was blocked by the PI3-kinase inhibitor LY294002 (10 µM) or by the PDK-1 inhibitor N-{alpha}-tosyl-Phe chloromethyl ketone (TPCK; 1 µM). Inset: representative Western blot analysis of GSK-3{beta}(Ser9) phosphorylation. B: IGF-I-elicited GSK-3{beta}(Ser9) phosphorylation was abolished in cells expressing kinase-inactive Akt(K179M). Inset: representative Western blot analysis demonstrating the expression of myc-tagged Akt(K179M) in transfected human intestinal muscle cells. C: the increase in GSK-3{beta}(Ser9) phosphorylation in the presence of the IGF-I receptor antagonist was abolished in cells expressing constitutively active myristolated Akt (Myr-Akt). Inset: representative Western blot analysis demonstrating the expression of myc-tagged Myr-Akt in transfected human intestinal muscle cells. D: IGF-I inhibits GSK-3{beta} activity. Inhibition of GSK-3{beta} activity by IGF-I was abolished by the PI3-kinase inhibitor or by the PDK-1 inhibitor. E: IGF-I-induced inhibition of GSK-3{beta} activity was abolished in cells expressing a kinase-inactive Akt(K179M) gene. E: quiescent muscle cells were incubated for 24 h in serum-free DMEM and for the final 6 h with 100 nM IGF-I in the presence or absence of various inhibitors. Naïve or transfected cells were incubated for 24 h in serum-free DMEM in the presence of various test agents. GSK-3{beta}(Ser9) phosphorylation was measured by Western blot analysis using a phosphorylation state-specific antibody, and the results were normalized to total GSK-3{beta} levels. GSK-3{beta} activity was measured by in vitro kinase assay. Basal GSK-3{beta} activity was 20.8 ± 0.5 pmol phosphate·min–1·mg protein–1. Results represent the means ± SE of 3–5 separate experiments. *P < 0.05 vs. control or basal values as appropriate, **P < 0.05 vs IGF-I alone.

 
Transfection of muscle cells with vector alone did not block the ability of IGF-I (100 nM for 6 h) to elicit GSK-3{beta} phosphorylation (naïve cells: 180 ± 19% above basal, vector transfected: 130 ± 9% above basal). Transfection of muscle cells with kinase-inactive Akt(K179M) inhibited IGF-I-induced GSK-3{beta} phosphorylation by 91 ± 3% (kinase-inactive Akt1: 14 ± 1% above basal; Fig. 4B). In muscle cells transfected with a Myr-Akt gene, basal GSK-3{beta} phosphorylation was increased to 200 ± 20% of basal. The ability of the IGF-I antagonist to inhibit basal GSK-3{beta} phosphorylation in vector-transfected cells (40 ± 8% inhibition) was abolished in the presence of constitutively active Akt (Fig. 4C).

In complementary experiments, the effect of various inhibitors on IGF-I-induced inhibition of GSK-3{beta} activity was examined. In the presence of the PI3-kinase inhibitor, the ability of IGF-I to inhibit GSK-3{beta} activity was blocked (IGF-I alone: 68 ± 5% inhibition; IGF-I + LY294002: 13 ± 6% inhibition, P < 0.05). Similarly, the effects of IGF-I on GSK-3{beta} activity were also blocked in the presence of the PDK-1 inhibitor (IGF-I + TPCK: 15 ± 4% inhibition, P < 0.05 vs. IGF-I alone; Fig. 4D). Transfection of muscle cells with the kinase-inactive Akt(K179M) mutant also blocked the ability of IGF-I to inhibit GSK-3{beta} activity (vector: 64 ± 5% inhibition, kinase-inactive Akt1: –6 ± 12% inhibition; Fig. 4E). These results implied that IGF-I regulates the phosphorylation and activity of GSK-3{beta} via a PI3-kinase-, PDK-1-, and Akt-dependent mechanism.

IGF-I protects muscle cells from apoptosis. Initial experiments were performed to characterize the time course of apoptosis induced by serum deprivation in human intestinal muscle cells using cytoplasmic, histone-associated DNA (nucleosomes) as a marker of apoptosis as described in METHODS. This technique allows quantification of apoptosis and differentiates apoptosis from necrosis. Incubation of muscle cells in serum-free DMEM elicited an increase in nucleosomes within 12 h of serum deprivation that increased progressively for up to 72 h (Fig. 5A). The role of GSK-3{beta} in the development of muscle cell apoptosis was confirmed by incubation of muscle cells in serum-free DMEM for 0–72 h in the presence of the GSK-3{beta} inhibitor. The GSK-3{beta} inhibitor significantly inhibited the apoptosis induced by serum deprivation for periods up to 72 h (Fig. 5). In muscle cells incubated in serum-free DMEM, the addition of exogenous IGF-I (100 nM) also significantly inhibited the apoptosis at all time periods up to 72 h (Fig. 5).



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Fig. 5. Endogenous IGF-I protects intestinal smooth muscle cells from apoptosis. A: serum deprivation of intestinal muscle cells for 0–72 h caused a time-dependent increase in apoptosis measured as nucleosomes ({bullet}). Serum deprivation of muscle cells in the presence of 100 nM IGF-I ({circ}) or the GSK-3{beta} inhibitor ({square}) 3-(3-carboxy-4-chloroanilino)-4-(3-nitrophenyl)maleimide (10 µM) inhibited apoptosis, whereas in the presence of the 100 nM IGF-I receptor antagonist ({blacksquare}) apoptosis was augmented, implying that endogenous IGF-I inhibits apoptosis. Apoptosis was measured as the increase in nucleosomes using an ELISA-based assay as described in METHODS. Results are expressed as %basal absorbance (A405–490). Values represent the means ± SE of 4 experiments. *P < 0.05 vs. control. B: serum deprivation of intestinal muscle cells for 24 h elicited an increase in caspase 3 activity. Incubation of serum-deprived muscle cells with either 100 nM IGF-I or 10 µM GSK-3{beta} inhibitor (Inh.) decreased caspase 3 activity, whereas incubation of muscle cells with 100 nM IGF antagonist (Ant.) augmented caspase 3 activity. Caspase 3 activity was measured by fluorometric immunosorbent assay as described in METHODS. Results are expressed in relative fluorescence values using Ex405/Em500. Values represent the means ± SE of 3–4 experiments. *P < 0.05 vs. serum-free DMEM alone (DMEM-0).

 
The role of endogenous IGF-I in regulation of muscle cell apoptosis was therefore examined using the IGF-I receptor antagonist (28) IGF analog (100 nM). In muscle cells deprived of serum for up to 72 h, the addition of the IGF-I receptor antagonist significantly increased the appearance of nucleosomes after 12–72 h (Fig. 5). The results implied that endogenous IGF-I protects cells from apoptosis via a GSK-3{beta}-dependent pathway.

The ability of IGF-I to protect cells from apoptosis induced by serum deprivation was confirmed by measurement of caspase 3 activity. Caspase 3 activity induced by serum deprivation for 24 h (control: 4,700 ± 200) was decreased by 2,980 ± 84 in relative fluorescence (Fig. 5B). In contrast, when cells were incubated in the presence of the IGF-I receptor antagonist, caspase 3 activity was increased by 5,300 ± 94 in relative fluorescence. In the presence of the GSK-3{beta} inhibitor, caspase 3 activity induced by serum deprivation was also decreased by 1,840 ± 160. The results confirmed the ability of endogenous IGF-I to protect cells from apoptosis and suggested that caspase 3 activity was regulated by IGF-I via a GSK-3{beta}-dependent mechanism.

Mechanisms mediating the inhibitory effects of IGF-I on apoptosis. The pathway that was shown to mediate the effects of IGF-I on GSK-3{beta} activity (PI3-kinase -> PDK-1 -> Akt -> GSK-3{beta}) was examined for its role in regulating IGF-I-dependent inhibition of apoptosis using specific inhibitors of PI3-kinase and PDK-1. We have previously shown that IGF-I regulates Akt activity by sequential activation of PI3-kinase, PDK-1, and Akt (16).

The ability of IGF-I to inhibit apoptosis in intestinal smooth muscle cells induced by serum deprivation was examined in cells treated with the PI3-kinase inhibitor LY294002 (10 µM) and the PDK-1 inhibitor TPCK (10 µM). Muscle cells were incubated in serum-free DMEM for 0–72 h with 100 nM IGF-I. The ability of IGF-I to block apoptosis induced by serum deprivation was inhibited at each time point in the presence of the PI3-kinase inhibitor LY294002 or by the PDK-1 inhibitor TPCK (Fig. 6). The results implied that IGF-I inhibited apoptosis via PI3-kinase- and PDK-1-dependent mechanisms.



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Fig. 6. IGF-I-dependent inhibition of apoptosis is mediated via PI3-kinase and PDK-1. IGF-I-dependent inhibition of apoptosis induced by serum deprivation in human intestinal muscle cells was blocked by either the PI3-kinase inhibitor LY294002 (10 µM) or the PDK-1 inhibitor TPCK (1 µM). Muscle cells were incubated in serum-free DMEM for increasing periods of time (0–72 h) in the presence of 100 nM IGF-I alone or with IGF-I and either the PI3-kinase inhibitor or the PDK-1 inhibitor. Apoptosis was measured as the increase in nucleosomes using an ELISA-based assay as described in METHODS. Results are expressed as %basal absorbance (A405–490). Values represent the means ± SE of 3–4 experiments.

 
Akt-dependent regulation of IGF-I-induced GSK-3{beta} phosphorylation and protection from apoptosis. The role of Akt in mediating IGF-I-stimulated inhibition of apoptosis was examined in cells transfected with the constitutively active Myr-Akt gene and with the kinase-inactive Akt(K179M) gene. Transfection of muscle cells with vector alone did not affect apoptosis induced by a serum deprivation (A405–490: 1.52 ± 0.09) for 24 h compared with naïve cells (A405–490: 1.55 ± 0.19).

In vector-transfected cells deprived of serum for 24 h, IGF-I (100 nM) inhibited apoptosis by 82 ± 6%. In cells expressing kinase-inactive Akt(K179M), the ability of 100 nM IGF-I to inhibit apoptosis was lost (Fig. 7A).



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Fig. 7. IGF-I-dependent inhibition of apoptosis is mediated via Akt. A: IGF-I-dependent inhibition of apoptosis was abolished in cells expressing kinase-inactive Akt(K179M). B: in cells expressing constitutively active Myr-Akt, basal apoptosis was inhibited and the ability of the IGF-I receptor antagonist to increase apoptosis was abolished. The results implied that endogenous IGF-I inhibited apoptosis in an Akt-dependent manner. Muscle cells were transfected with either kinase-inactive Akt(K179M) or constitutively active Myr-Akt as detailed in METHODS. After 24 h, cells were deprived of serum for an additional 24 h in the presence or absence of 100 nM IGF-I or 100 nM IGF-I receptor antagonist. Apoptosis was measured as the increase in nucleosomes using an ELISA-based assay as described in METHODS. Results are expressed as %basal absorbance (A405–490). Values represent the means ± SE of 3–4 experiments. *P < 0.05 vs. basal levels (in vector-transfected cells); **P < 0.05 vs. vector transfected control cells.

 
Conversely, in cell vector-transfected cells deprived of serum for 24 h, the IGF-I receptor antagonist (100 nM) augmented apoptosis by 87 ± 16% above basal levels. Transfection of cells with Myr-Akt inhibited basal rates of apoptosis by 65 ± 15%. In these cells expressing constitutively active Akt, the ability of the IGF-I receptor antagonist to increase apoptosis was lost (Fig. 7B).

Endogenous IGF-I regulates {beta}-catenin phosphorylation. One potential target of GSK-3{beta} through which IGF-I might regulate apoptosis is {beta}-catenin. GSK-3{beta} induces phosphorylation of {beta}-catenin that leads to {beta}-catenin instability. Phosphorylated {beta}-catenin undergoes degradation, which, in turn, allows apoptosis to proceed. The possibility that IGF-I regulates the levels of {beta}-catenin phosphorylation was therefore investigated in experiments in which the levels of phospho-{beta}-catenin(Ser33/37/Thr41) were measured by Western blot analysis. Quiescent muscle cells were incubated for 24 h in either serum-free DMEM (DMEM-0) or with DMEM-0 to which was added 100 nM IGF-I, 100 nM IGF antagonist, or 10 µM GSK-3{beta} inhibitor. The levels of phospho-{beta}-catenin(Ser33/37/Thr41) were decreased in the presence of either 100 nM IGF-I (27 ± 6% decrease from control) or in the presence of 10 µM GSK-3{beta} inhibitor (46 ± 7% decrease from control; Fig. 8). In contrast, in the presence of the IGF antagonist (100 nM), the levels of phospho-{beta}-catenin(Ser33/37/Thr41) was 136 ± 11% of control levels (Fig. 8).



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Fig. 8. Endogenous IGF-I inhibits and GSK-3{beta} augments {beta}-catenin phosphorylation. Levels of phospho-{beta}-catenin(Ser33/36/Thr41) were decreased by either IGF-I or the GSK-3{beta} inhibitor and were increased in the presence of the 100 nM IGF antagonist. Inset: representative Western blot analyses of phospho-{beta}-catenin(Ser33/36/Thr41) and total {beta}-catenin levels. Quiescent muscle cells incubated for 24 h in DMEM-0 or in DMEM-0 with added 100 nM IGF-I, 100 nM IGF antagonist, or 10 µM GSK-3{beta} inhibitor. Levels of phospho-{beta}-catenin(Ser33/36/Thr41) were measured by Western blot analysis. Results are expressed as the relative levels of phospho-{beta}-catenin normalized to total {beta}-catenin levels. Values represent the means ± SE of 3 experiments. *P < 0.05 vs. control.

 

    DISCUSSION
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 ABSTRACT
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IGF-I mediates three distinct effects on the growth of intestinal smooth muscle cells. IGF-I regulates muscle development, proliferation, and survival (1618, 25, 40). Each of these effects is mediated through the cognate IGF-I receptor tyrosine kinase. In human intestinal smooth muscle cells, the IGF-I receptor tyrosine kinase is coupled to Erk1/2- and PI3-kinase-dependent signaling pathways that jointly convey mitogenic signals to the nucleus, which stimulates cell cycle progression (17, 21). PI3-kinase also regulates p70S6 kinase activity, which, via the S6 protein, mediates the protein synthesis needed for cell division to occur (16). In human intestinal muscle cells, IGF-I-activated PI3-kinase also causes activation of Akt. This pathway seems to mediate cellular functions other than proliferation, because transfection of a kinase-inactive Akt did not affect IGF-I-stimulated proliferation (16). In other muscle cell types, such as cardiac and vascular muscle, Akt regulates GSK-3{beta}(Ser9) phosphorylation (24, 36). Phosphorylation on Ser9 induced by Akt inactivates GSK-3{beta} and results in an inhibition of the proapoptotic signaling pathways to which GSK-3{beta} is coupled.

The evidence that IGF-I regulates the activity of GSK-3{beta} via an Akt-dependent mechanism can be summarized as follows: 1) exogenous IGF-I increased GSK-3{beta} phosphorylation in a time- and concentration-dependent fashion and decreased GSK-3{beta} activity. 2) An IGF-I receptor antagonist augmented GSK-3{beta} dephosphorylation induced by serum deprivation and increased GSK-3{beta} activity. 3) IGF-I-induced GSK-3{beta} phosphorylation and inhibition of GSK-3{beta} activity were blocked by the PI3-kinase inhibitor, the PDK-1 inhibitor, and expression of kinase-inactive Akt, but they were not affected by the Erk1/2 or the p70S6 kinase inhibitors.

GSK-3{beta} activity is both stimulated and inhibited by phosphorylation. Although GSK-3{beta} activity and its ability to promote apoptosis are reduced by phosphorylation of Ser9, its activity and proapoptotic effects are also stimulated by phosphorylation on Tyr216 (1, 6, 7). Several kinases are capable of mediating Ser9 phosphorylation including Akt, p90Rsk, PKA, and certain isoforms of PKC ({alpha}, {beta}I, {beta}II, {gamma}, but not {epsilon}) (1, 68). p90Rsk appears to be an important mediator of GSK-3{beta}(Ser9) phosphorylation in response to EGF. IGF-I and insulin inactivate GSK-3{beta} through Ser9 phosphorylation, but rather than via p90Rsk, these effects are mediated via a PI3-kinase-dependent pathway involving PDK-1 and Akt activation. Substantial evidence supports the notion that the interaction between Akt and GSK-3{beta} is a direct one (34).

Acting in opposition to the inhibition of GSK-3{beta} activity via Ser9 phosphorylation is GSK-3{beta} activation by phosphorylation on Tyr216. The intracellular mechanisms that have been shown to participate in GSK-3{beta}(Tyr216) phosphorylation include changes in intracellular Ca2+, the src tyrosine kinase family member Fyn, and the novel tyrosine kinase ZAK1. ZAK1, for example, interacts directly with and phosphorylates GSK-3{beta}(Tyr216) (1, 6, 7).

The effects of GSK-3{beta} are also regulated by protein complex formation, a process involved in modulating {beta}-catenin levels. In this paradigm, GSK-3{beta}, when bound to GSK-3{beta} binding protein and disheveled, is held in an inactive state that leads to stabilization of the {beta}-catenin protein (5). Once GSK-3{beta} is activated, it binds to axin, and a tetrameric complex forms among GSK-3{beta}, axin, the adenomatous polyposis coli gene product, and {beta}-catenin, which facilitates the ability of GSK-3{beta} to phosphorylate {beta}-catenin and results in enhanced {beta}-catenin degradation (10). These regulatory mechanisms are not necessarily mutually exclusive, because the binding of GSK-3{beta} to GSK-3{beta} binding protein also inhibits its catalytic activity toward others, but not all, of its peptide substrates.

The evidence that IGF-I-dependent inactivation of GSK-3{beta} protects cells from apoptosis, measured by either nucleosome accumulation or caspase 3 activity, can be summarized as follows: 1) IGF-I blocked apoptosis induced by serum deprivation; 2) the GSK-3{beta} inhibitor blocked apoptosis; 3) the IGF-I receptor antagonist augmented apoptosis; 4) the augmented apoptosis that occurred in the presence of the IGF-I receptor antagonist was reversed by the GSK-3{beta} inhibitor or by expression of a constitutively active Akt.

GSK-3{beta} phosphorylates a diverse group of substrates: metabolic and signaling proteins, structural proteins, and transcription factors through which cellular survival and apoptosis are influenced. In most cases, GSK-3{beta} inhibits transcription factor activation. GSK-3{beta} reduces the levels and transcriptional activity of NF-{kappa}B, CREB, AP-1, and {beta}-catenin (10). The effects of these latter transcription factors on cell survival are mediated by their ability to sequester CREB binding protein (CBP). GSK-3{beta} activation reduces transcription factor levels and allows for the downstream activation of p53 and stimulation of apoptosis by releasing CBP from CREB (30). This is a dynamic process, because inhibition of GSK-3{beta} activity (by phosphorylation on Ser9) increases transcription factor levels and their association with CBP, limiting p53 activity and inhibiting apoptosis.

Altered levels of IGF-I expression have also been observed in several disease states. In regions of active Crohn's disease and muscle stricture and in animal models of enterocolitis, the expression of IGF-I in the muscularis propria is increased (41, 42). This suggests that the increase in growth-stimulatory IGF-I could be responsible, in part, for the hypertrophy of smooth muscle that results in stricturing of the intestine. This notion is further supported by the selective hypertrophy of smooth muscle tissues, including intestinal smooth muscle, that occurs in animals overexpressing an IGF-I cDNA (25, 37). Children with hypertrophic pyloric stenosis also have increased IGF-I expression in the pyloric muscle compared with pyloric muscle from unaffected children (26). Taken together, these observations suggest that the increase in local IGF-I production and intestinal smooth muscle hyperplasia are linked. We previously provided evidence that endogenous IGF-I regulates the proliferation of human intestinal smooth muscle (15). The findings of the current study suggest that intestinal muscle hyperplasia and stricture formation may be due not only to the stimulation of proliferation by IGF-I, but also by IGF-I-dependent inhibition of apoptosis. Normal intestinal smooth muscle cells turn over at a slow rate, i.e., both proliferation and apoptosis occur at low rates. Whereas the short-term effects of altering either of these processes might be negligible on morphology of the muscularis propria, the long-term effects of a concomitant increase in proliferation and a decrease in apoptosis induced by IGF-I may be significant, i.e., muscle hyperplasia and/or stricture formation whether during development of the pyloric sphincter or during the chronic intestinal inflammation of Crohn's disease.

In summary, the present paper shows that IGF-I, acting via PI3-kinase-dependent activation of PDK-1 and Akt, causes GSK-3{beta}(Ser9) phosphorylation and GSK-3{beta} inactivation that lead to inhibition of apoptosis, in part via regulation of {beta}-catenin phosphorylation. The effects of IGF-I are both concentration and time dependent. Endogenous IGF-I promotes cell survival and inhibits apoptosis in human intestinal smooth muscle. Thus IGF-I exerts two distinct yet complementary effects in intestinal smooth muscle cells: it stimulates proliferation and inhibits apoptosis, two complementary processes that jointly regulate muscle cell growth.


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


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. F. Kuemmerle, Division of Gastroenterology, Medical College of Virginia Campus, Virginia Commonwealth Univ., PO Box 980711, Richmond, VA 23298–0711 (E-mail: jkuemmerle{at}hsc.vcu.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. Section 1734 solely to indicate this fact.


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