Departments of 1Internal Medicine, 4Neurology, 3Obstetrics and Gynecology, and 5Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan 48109; and 2Shanghai Institute of Materia Medica, Shanghai, China 201203
Submitted 11 July 2003 ; accepted in final form 18 December 2003
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ABSTRACT |
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insulin-like growth factor I; signal transduction; Crohn's disease; inflammatory bowel disease
IGF-I is a potent fibrogenic peptide that stimulates proliferation of intestinal smooth muscle cells and an increase in their rate of collagen synthesis. The actions of IGF-I are modulated by unique binding proteins (IGFBP-1 through -6) that are expressed in a tissue-specific manner. One of the binding proteins, IGFBP-5, is highly expressed in intestinal smooth muscle and, similar to IGF-I, is increased in intestinal tissue in Crohn's disease and in Lewis-strain rats with peptidoglycan-induced experimental Crohn's disease (33, 34). Unlike most of the other binding proteins, which act as competitive inhibitors of the IGF-I receptor (IGF-IR), IGFBP-5 acts to enhance IGF-I actions (1, 3, 11). IGF-I increases the synthesis of both IGFBP-5 and collagen in cultured rat intestinal smooth muscle cells (RISM). A positive feedback loop may thus be created in which IGF-I, synthesized in the inflamed intestinal wall, acts on smooth muscle cells to cause proliferation and increased synthesis of collagen and IGFBP-5, and IGFBP-5 then enhances the actions of IGF-I on the smooth muscle cells.
The IGF-IR is an 2
2-heterotetrameric protein with intrinsic ligand-stimulated tyrosine kinase activity (21). Binding of IGF-I to the IGF-IR
-subunit induces receptor autophosphorylation of the intracellular tyrosine kinase domain of the
-subunit, resulting in its activation. There are several substrates for the IGF-IR including insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), Shc, an adaptor molecule with Src homology and collagen homology domains, and probably other proteins with Src-homology 2 (SH2) domains. The substrates act as docking proteins and have multiple tyrosine-rich motifs that are potential phosphorylation sites and that serve as binding sites for IGF-IR
-subunit (upstream) as well as downstream pathways including the Akt-dependent pathway [downstream of phosphatidylinositol 3-kinase (PI3-K)] and the MAPK pathways. The substrates for the IGF-IR vary among cell types and probably depend on the cellular repertoire (27). Studies using inhibitors, transfection studies, and knockout models underlie the classic teaching that IGF-I signaling through the PI3-K pathway mediates metabolic functions of IGF-I and effects of IGF-I on the membrane cytoskeleton, whereas the MAPK pathway generally mediates effects on cell growth, mitogenesis, and differentiation. However, newly emerging data do not support separate roles for the PI3-K and the MAPK pathways and strongly suggest that the biological effects of each pathway vary from cell type to cell type (21). In gastrointestinal smooth muscle, for example, both pathways appear to be involved in mitogenic effects of IGF-I (14, 16). The pathway(s) by which IGF-I regulates gene expression have been less well studied.
The aim of this study was to determine which cellular proteins undergo tyrosine phosphorylation after IGF-I stimulation and to determine which early docking proteins are associated with the IGF-IR in RISM. We used RISM derived from Lewis-strain rats that are genetically predisposed to develop intense intestinal fibrosis in response to intramural injection of peptidoglycan in a rat model of Crohn's disease (32). These studies show that the RISM repertoire of early docking proteins includes IRS-1, IRS-2, and Shc. Studies using the MAPK inhibitor PD98059 demonstrate that the MAPK pathway is linked to collagen and IGFBP-5 synthesis in RISM. Studies using the PI3-K inhibitor wortmannin, as well as Akt (protein kinase B) transfection studies, do not suggest a role for the Akt-dependent pathway in the IGF-I-mediated regulation of these genes in our system.
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METHODS |
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RISM were washed three times in serum-free DMEM and then cultured in serum-free medium for 24 h before experiments to decrease the effect of binding proteins present in the serum. Cells were treated in triplicate with IGF-I (0100 ng/ml; Upstate Biotechnology, Lake Placid, NY) for up to 30 min. In experiments in which RISM were pretreated with the MAPK pathway inhibitor, cells were incubated in serum-free medium for 24 h, then treated with PD98059 (10 µM; Calbiochem, La Jolla, CA) for 1 h before treatment with IGF-I. In experiments in which RISM were exposed concurrently to IGF-I and the MAPK or PI3-K pathway inhibitors, cells were incubated in serum-free medium for 24 h, then treated with PD98059 (10 µM) or wortmannin (100 nM; Calbiochem) with or without IGF-I (100 ng/ml) for 24 h.
At the end of each experiment, the medium was removed, and cells were washed twice with cold Ca2+/Mg2+-free PBS. Cells were then scraped and processed for analysis of protein or mRNA.
Western blot analyses. After aspiration of the medium, 150 µl of cold lysis buffer were placed on the cells. The protein concentrations of the cell lysates were determined by the Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA). Lysates were subjected to SDS-PAGE on 8 or 9% gels. Specific proteins were detected on Western immunoblots (18) by incubation with antiphosphotyrosine [monoclonal antibodies 4G10 (Upstate Biotechnology) and PY20 (BD Transduction Laboratories, San Diego, CA) in combination], anti-IRS-1, anti-IRS-2 (gifts from Dr. A. Saltiel, Pfizer, Ann Arbor, MI), or anti-Shc (BD Transduction Laboratories). After being washed, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), then washed again and incubated with enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL) reagents. For some studies, the membranes were stripped and reprobed with an antibody to IGF-IR -subunit (Santa Cruz Biotechnology) or to IRS-1. Densitometry was performed on scanned films using National Institutes of Health (NIH) Image software (NIH, available on-line at http://rsb.info.nih.gov/nih-image/).
Immunoprecipitation. Cell lysates were prepared as described above. Immunoprecipitation was performed as described previously (13, 18). Cell lysates (500 µg protein) were incubated overnight at 4°C with one of the following antibodies: anti-IRS-1 (2 µl/ml), anti-IRS-2 (2 µl/ml), or anti-Shc (4 µl/ml). Then, 20 µl of Protein A/G Plus-agarose (Santa Cruz Biotechnology) was added, and samples were incubated with gentle rocking for 2 h at 4°C. The precipitates were pelleted by centrifugation, washed three times, and size-separated on 9% SDS-PAGE gels. Resolved proteins were transferred to nitrocellulose membranes. Immunodetection of specific proteins was performed as above.
ERK activity assay. The kinase activity of the ERKs was measured using the p44/42 MAP kinase assay kit (Cell Signaling Technology, Beverly, MA) according to the manufacturer's protocol. RISM were incubated in serum-free medium for 24 h, and then treated with IGF-I for varying periods with or without pretreatment with PD98059 (10 µM) for 1 h. Cell lysates were prepared as above. Lysate (500 µg protein) was incubated with an immobilized phosphospecific p44/42 MAP kinase (Thr202/Try204) monoclonal antibody. The pellets obtained by subsequent centrifugation were washed three times and then incubated with 200 µM ATP and 2 µg Elk-1 fusion protein for 30 min at 30°C. The reaction was terminated, and supernatants were loaded onto 12% SDS-PAGE gels. The samples were analyzed by Western immunoblotting using a phosphospecific Elk-1 antibody.
Northern analyses. RNA was isolated as described previously (33). Briefly, cells were lysed with guanidine isothiocyanate (500 µl). RNA was extracted with phenol and cholorform-isoamyl alcohol (49:1) and precipitated with isopropanol. The pellet was dissolved in guanidine isothiocyanate and reprecipitated in ethanol. The pellet was washed with 70% ethanol, air-dried, and dissolved in DEPC-treated H2O. The absorbance at 260 nm was used to determine the RNA concentration of each sample. For Northern analysis, RNA was electrophoresed on 1% agarose/6% formaldehyde gels and then transferred overnight to Nytran (Schleicher & Schuell, Keene, NH). Blots were baked, prehybridized in buffer, then hybridized overnight with the labeled probes. The IGFBP-5 cDNA probe (33) was radiolabeled with 32P (Amersham) using a random priming kit (Roche Molecular Biochemicals, Indianapolis, IN). An antisense oligonucleotide probe for rat procollagen 1(I) (31, 33) was synthesized by the University of Michigan Biomedical Research DNA Synthesis Core Facility using an automated synthesizer (Applied Biosystems, Foster City, CA). The oligonucleotide was purified by HPLC and the 5'-end was 32P-labeled by the kinase reaction (Roche Molecular Biochemicals). After hybridization, membranes were washed, dried, and then exposed to radiographic film with intensifying screens overnight at -80°C. The autoradiogram was digitized by flatbed scanning and imported for densitometric analysis into NIH Image (NIH, Bethesda, MD). The relative densitometric value for each band was adjusted for minor variations in loading by using the corresponding signal for blots probed with a 32P-labeled cDNA probe for GAPDH (American Type Culture Collection, Rockville, MD).
Transfection studies. Replication-defective recombinant adenoviral vectors (6, 24, 25) dominantly expressing an inactive mutant Akt or constitutively expressing wild-type Akt under the control of the cytomegalovirus (CMV) promoter were gifts from Dr. K. Walsh (Tufts University, Boston, MA). The mouse gene for Akt was mutated to a dominant-negative form by sequence changes resulting in the substitution of critical amino acids (T308A, S473A) in the protein or to a constitutively active form by fusing the c-src myristoylation sequence in-frame to the NH2 terminus of the Akt coding sequence. The recombinant adenoviral vector carrying the LacZ reporter gene under the control of the CMV promoter was obtained from the University of Michigan Vector Core.
Medium was aspirated from 80% confluent RISM cultures. Viruses (4 X 109 particles/ml) were applied for 24 h. The cells were then washed twice with medium, and incubation was continued for an additional 3 days. The cells were then incubated in serum-free medium for 24 h, followed by application of IGF-I (100 ng/ml) or vehicle for 24 h. Lysates were prepared and subjected to SDS-PAGE on 8% gels. Duplicate Western immunoblots were probed sequentially with antibodies to collagen I (Rockland, Gilbertsville, PA), Akt (Santa Cruz Biotechnology), or phospho-Akt (Ser473; Cell Signaling Technology), and smooth muscle -actin (DAKO, Carpinteria, CA). HRP-conjugated secondary antibodies and an ECL system were used to visualize the specific proteins.
Statistical analysis. Comparisons were made between groups of paired samples by Wilcoxon's signed-rank test. Results from unpaired samples were analyzed using a t-test or Mann-Whitney U-test. Data were considered significant if P < 0.05.
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RESULTS |
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IGF-I increased RISM protein tyrosine phosphorylation in a time-dependent manner. RISM were exposed to IGF-I (100 ng/ml) in serum-free medium for 010 min, and protein tyrosine phosphorylation was determined by Western immunoblots of cell lysates. IGF-I increased the phosphotyrosine content of several proteins, with the strongest bands having apparent molecular masses of 200, 175, 100, and 66 kDa, consistent with IRS-2, IRS-1, IGF-IR, and an Shc isoform (Fig. 1B). All four bands were strongest following 510 min of IGF-I treatment. At 5 min, the band densities measured 6.3-, 3.9-, 4.6-, and 2.4-fold over baseline for the 200-, 175-, 100-, and 66-kDa bands, respectively.
IGF-I receptor is linked to several docking proteins. IRS-1, IRS-2, and Shc are upstream docking proteins that are known to mediate early events in the IGF-I signal-transduction pathway. Their relative roles vary from cell system to cell system, and their immediate downstream effectors in RISM are unknown. Data above suggest that IRS-1, IRS-2, and Shc are phosphorylated by interaction of IGF-I with its receptor. Whole cell lysates from IGF-I-stimulated RISM were immunoprecipitated with antibodies specific for IRS-1, IRS-2, or Shc. Immunoprecipitates were size separated on polyacrylamide gels alongside whole cell lysates from identically treated cultures from the same experiment. Western immunoblotting was performed using the same antibody as was used for immunoprecipitation. Our results show that immunoprecipitation with the IRS-1 antibody (identified as the 175-kDa band on whole cell lysate) coimmunoprecipitated a 100-kDa band consistent with the IGF-IR and a 66-kDa band consistent with an Shc isoform (Fig. 2A). This suggests the association of IRS-1 with Shc and the IGF-IR. Immunoprecipitation with IRS-2 antibody (identified as a 200-kDa band on whole cell lysate) coimmunoprecipitated a 100-kDa band consistent with the IGF-IR and a 66-kDa band consistent with an Shc isoform (Fig. 2B). This suggests association of IRS-2 with Shc and the IGF-IR. Minor known cross-reactivity of the IRS-2 antibody with IRS-1 can be seen at 175 kDa on the whole cell lysate portion of the gel. Immunoprecipitation with Shc antibody (identified as the 66-kDa predominant band in whole cell lysates) coimmunoprecipitated a faint 100-kDa band consistent with the IGF-IR (Fig. 2C). Taken together, these data are consistent with a close association between the IGF-IR and the three known upstream docking proteins IRS-1, IRS-2, and Shc.
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PD98059 blocked IGF-I-stimulated tyrosine phosphorylation of IGF-IR. A specific inhibitor of the MAPK pathway, PD98059, was studied (Fig. 3). RISM were incubated in serum-free medium for 24 h, then treated with PD98059 (10 µM) for 1 h before treatment with varying concentrations of IGF-I for 5 min. Protein tyrosine phosphorylation was determined by Western immunoblot. PD98059 resulted in decreased phosphorylation of several proteins and abolished phosphorylation of the IGF-IR (100 kDa; Fig. 3A). This effect was associated with the appearance of two phosphorylated proteins with molecular mass of 30 kDa, the identities of which are unknown. To confirm that IGF-IR was present in the samples from cells treated with PD98059, the blot was stripped and reprobed with an antibody to the IGF-IR
-subunit, demonstrating the presence of the 100-kDa IGF-IR band in all samples (Fig. 3B).
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PD98059 inhibited ERK1/2 kinase activity. To demonstrate that PD98059 decreased kinase activity in our system, we measured the ability of ERK1/2 from IGF-I-treated RISM, with or without PD98059 pretreatment, to phosphorylate Elk-1 fusion protein. RISM were incubated in serum-free medium for 24 h, and then some were pretreated with PD98059 (10 µM) for 1 h before treatment with IGF-I (100 ng/ml) for 1, 5, or 30 min. To assay ERK1/2 activity, phosphorylated ERK1/2 was immunoprecipitated from the cell lysates and then incubated in a kinase reaction with the substrate Elk-1. Phosphorylation of Elk-1 was then examined on immunoblots developed with a phosphospecific Elk-1 antibody. IGF-I treatment of RISM induced ERK1/2 tyrosine phosphorylase activity as seen by increased levels of phosphorylated Elk-1 (Fig. 4; 1.82 ± 0.31, 1.93 ± 0.31 and 2.45 ± 0.35 fold at 1, 5 and 30 min, respectively; mean of 2 experiments). The level of induction was diminished by PD98059 (Fig. 4; 5%, 12% and 38% decrease at 2, 5 and 30 min, respectively; mean of 2 experiments). The data show that PD98059 prevents IGF-I-mediated activation of ERK1/2 kinase activity in RISM.
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PD98059 blocked IGF-I-induced IGFBP-5 and collagen 1(I) mRNA expression, whereas wortmannin had no effect. It was previously shown (33) that IGF-I increases IGFBP-5 and collagen
1(I) mRNA abundance in RISM. PD98059 was used to determine the role of the MAPK pathway in mediating this effect of IGF-I; wortmannin was used to determine the role of the PI3-K pathway. RISM were exposed to IGF-I (100 ng/ml) in the presence or absence of PD98059 (10 µM) or wortmannin (100 nM) for 24 h. RNA was isolated from the cells, and IGFBP-5 and collagen
1(I) mRNAs were measured by Northern blot analysis (Fig. 5, A and B, respectively). As expected, IGF-I increased IGFBP-5 and collagen
1(I) mRNA abundance (lane 1 vs. 2). In the presence of PD98059, there were 31.4 ± 0.8 and a 20.9 ± 0.7% reductions in IGF-I-induced IGFBP-5 and collagen mRNAs, respectively (lane 2 vs. 6; P < 0.001 for each; n = 3 experiments). PD98059 alone had no effect on either mRNA (lane 1 vs. 4). Wortmannin did not decrease IGF-I-induced IGFBP-5 or collagen
1(I) mRNA abundance (lane 2 vs. 5; 8.6 ± 7.4% increased collagen mRNA and 3.4 ± 13% increased collagen mRNA; P > 0.05 for each; n = 3 experiments).
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Transfection of RISM with a dominant negative Akt did not affect the IGF-I-induced increase in collagen I protein. To further study the role of the Akt-dependent pathway in mediating this effect, transfection studies were performed. RISM were transiently transfected with adenoviral vectors carrying a dominantly expressed inactive mutant Akt gene, a constitutively expressed wild-type Akt gene, or the LacZ reporter gene, each under the control of the CMV promoter. The transfected cells were exposed to IGF-I (100 ng/ml) for 24 h. Cell lysates were examined on immunoblots. Successful transfection was confirmed on the immunoblots using an antibody to Akt (Fig. 6) or by X-Gal staining (not shown) of intact cells in additional parallel LacZ-transfected cultures. Compared with cells transfected with LacZ, transfection of RISM with a dominant negative Akt did not alter the level of induction of collagen I protein following IGF-I treatment (Fig. 6; 1.86 ± 0.26 vs. 1.47 ± 0.19-fold; n = 3 experiments; P > 0.05). Similarly, transfection of RISM with a constitutively expressed wild-type Akt had no effect on the level of IGF-I-mediated increase in collagen I protein (Fig. 6).
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DISCUSSION |
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Our studies demonstrate IGF-I-induced tyrosine phosphorylation of several intracellular proteins. The sizes of the most abundant phosphorylated proteins are consistent with IRS-1, IRS-2, the IGF-IR, and Shc (12, 13). This suggests that RISM have a cellular repertoire similar to that observed in other systems and supports activation of multiple signal-transduction pathways by IGF-I in these cells. The observation that all of the docking proteins are phosphorylated by IGF-I in our system suggests that all may participate in IGF-I signal transduction.
Coimmunoprecipitation studies suggest that IRS-1, IRS-2, and Shc are all associated with the IGF-IR (100 kDa) in RISM. In addition, the IRS-1 and -2 antibodies immunoprecipitate a protein consistent with the an Shc isoform, suggesting that a complex may exist between the IRS proteins, the IGF-IR, and Shc.
Shc appears to be an important mediator in the MAPK pathway. Shc exists in three isoforms [46, 52, and 66 kDa; (20)]. The significance of the different isoforms is incompletely understood. Certain cell systems appear to express all three isoforms, whereas others express a predominance of one or another isoform. RISM appear to express predominately the 66-kDa isoform.
The ERK1/2 inhibitor PD98059 resulted in decreased phosphorylation of several RISM proteins. The most dramatic decrease in phosphorylation was a decrease in phosphorylation of the IGF-IR. To our knowledge, this effect of PD98059 has not been previously reported in other systems. The mechanism of decreased receptor phosphorylation with inhibition of ERK1/2 is unknown, but the finding may be evidence of complexity in the system and may represent cross-talk between the receptor and downstream effectors. There is evidence in certain systems that protein complexes in the pathway can be bypassed, such as IRS-1/Shc-independent Ras activation (2), and even evidence of Ras-independent activation of Raf (17, 23). This phenomenon awaits further study.
The decrease in IGF-IR phosphorylation was associated with phosphorylation of two 30-kDa proteins of unknown identity. The 30-kDa proteins may represent proteins in the MAPK cascade that are phosphorylated as an alternative to ERK1/2 whose phosphorylation is blocked by PD98059. This effect has not been described in other systems to our knowledge and awaits further study.
PD98059 decreased the IGF-I-induced increase in IGFBP-5 and collagen 1(I) mRNA abundance in RISM. This suggests that IGF-I increases mRNA abundance through the MAPK pathway. Wortmannin, the inhibitor of PI3-K, did not affect the IGF-I-induced increase in IGFBP-5 or collagen
1(I) mRNA abundance in our system. Transfection of RISM with a dominant negative Akt did not affect the IGF-I-induced increase in collagen I protein. These two findings do not suggest a role for the Akt-dependent pathway in IGF-I-mediated regulation of gene expression in our system. In addition, because the inhibition of IGF-I effects by PD98059 was only partial, other pathways in addition to the MAPK pathway may be involved. Possible involvement of p70(s6k) must be considered because it has been implicated in studies of porcine vascular smooth muscle (4).
Whereas the Akt-dependent pathway has been implicated in the regulation of IGFBP-5 by IGF-I in other systems such as cultured human intestinal smooth muscle cells (15) and porcine vascular smooth muscle cells (4), evidence does not suggest a role in our system. This significant difference between our studies and prior published work may reflect differences among species and/or differences among smooth muscle cells from different organs or tissues. The Lewis strain of inbred rats used in our experiments are highly susceptible to intestinal inflammation and fibrosis induced by intramural injection of peptidoglycan, whereas other inbred rat strains are resistant to PG-induced inflammation (19, 32). IGF-I and IGFBP-5 are important to the development of intestinal inflammation after peptidoglycan injection in this model (10, 33). It is possible that differences in signal transduction in response to IGF-I underlie the marked strain-dependent differences in response to peptidoglycan injection.
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ACKNOWLEDGMENTS |
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This research was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants RO-1 DK-56750 (to E. M. Zimmermann) and 5-P30-DK-34933 (to the Michigan Gastrointestinal Peptide Research Center, which provided a pilot grant to Y. T. Hou and support to the University of Michigan Vector Core).
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FOOTNOTES |
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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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REFERENCES |
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