Involvement of ligand occupancy in Insulin-like growth factor-I (IGF-I) induced cell growth in osteoblast like MC3T3-E1 cells

Growth factors and matrix proteins regulate the proliferation and differentiation of osteoblasts. The insulin-like growth factor (IGF) system comprises IGF-I, IGF-II, and six high-affinity IGF-binding proteins (IGFBPs). IGFs stimulate cell growth in many types of tissue; IGF-binding proteins regulate cellular actions and can affect cell growth. IGF-I is involved in differentiation, proliferation, and matrix formation in osteoblasts; IGFBP-5 is associated with the extracellular matrix (ECM) and can potentiate the actions of IGF-I. We investigated the effect of ECM proteins on the responses of MC3T3-E1 osteoblast cells to IGF-I and IGFBP-5. In addition, because extracellular signal-regulated kinases 1 and 2 (Erk 1/2) affect cell growth, we evaluated the effects of IGFBP-5 on Erk 1/2 phosphorylation in MC3T3-E1 cells. IGF-I caused an increase in IGFBP-5 expression in cultured MC3T3-E1 cells, and IGF-I plus IGFBP-5 significantly increased cell growth. Likewise, the addition of IGF-I and IGFBP-5 to cultured MC3T3-E1 cells increased the synthesis of the ECM proteins osteopontin (OPN) and thrombospondin-1 (TSP-1), which can bind to alphaVbeta3 integrin receptors on the cell surface. By contrast, the addition of an antibody against ECM proteins inhibited the effects of OPN and TSP-1 on IGFBP-5 expression. The stimulatory effect of IGFBP-5 was mediated via Erk 1/2 activation. These data suggest that IGFBP-5 regulates Erk 1/2 phosphorylation in cultured MC3T3-E1 cells via ECM proteins that may ultimately stimulate the growth of osteoblasts. We determined whether occupation of the alphaVbeta3 integrin receptor affects IGF-I receptor (IGF-IR)-mediated signaling and function in MC3T3-E1 osteoblast cells. Occupation of the alphaVbeta3 integrin receptor with ECM proteins induced IGF-I-stimulated IGF-IR phosphorylation. Conversely, in the presence of the alphaVbeta3-specific disintegrin echistatin, IGF-I-stimulated IGF-IR activation was inhibited. IGF-I-stimulated IGF-IR phosphorylation was accompanied by IRS-1 phosphorylation and MAPK activation. However, these effects were attenuated by echistatin. Thus, occupancy of the alphaVbeta3 disintegrin receptor modulates IGF-I-induced IGF-IR activation and IGF-IR-mediated function in MC 3T3-E1 osteoblasts.     

Phosphatidylinositol 3-Kinase (PI3K) Activity Bound to Insulin-like Growth Factor-I (IGF-I) Receptor, which Is Continuously Sustained by IGF-I Stimulation, Is Required for IGF-I-induced Cell Proliferation

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G(1) phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G(1) to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G(1) phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr(1316)-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR(-/-) fibroblasts expressing exogenous mutant IGF-IR in which Tyr(1316) was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation.     

Insulin-Like Growth Factor-I E-Peptide Activity Is Dependent on the IGF-I Receptor

Insulin-like growth factor-I (IGF-I) is an essential growth factor that regulates the processes necessary for cell proliferation, differentiation, and survival. The Igf1 gene encodes mature IGF-I and a carboxy-terminal extension called the E-peptide. In rodents, alternative splicing and post-translational processing produce two E-peptides (EA and EB). EB has been studied extensively and has been reported to promote cell proliferation and migration independently of IGF-I and its receptor (IGF-IR), but the mechanism by which EB causes these actions has not been identified. Further, the properties of EA have not been evaluated. Therefore, the goals of this study were to determine if EA and EB possessed similar activity and if these actions were IGF-IR independent. We utilized synthetic peptides for EA, EB, and a scrambled control to examine cellular responses. Both E-peptides increased MAPK signaling, which was blocked by pharmacologic IGF-IR inhibition. Although the E-peptides did not directly induce IGF-IR phosphorylation, the presence of either E-peptide increased IGF-IR activation by IGF-I, and this was achieved through enhanced cell surface bioavailability of the receptor. To determine if E-peptide biological actions required the IGF-IR, we took advantage of the murine C2C12 cell line as a platform to examine the key steps of skeletal muscle proliferation, migration and differentiation. EB increased myoblast proliferation and migration while EA delayed differentiation. The proliferation and migration effects were inhibited by MAPK or IGF-IR signaling blockade. Thus, in contrast to previous studies, we find that E-peptide signaling, mitogenic, and motogenic effects are dependent upon IGF-IR. We propose that the E-peptides have little independent activity, but instead affect growth via modulating IGF-I signaling, thereby increasing the complexity of IGF-I biological activity.     

IGF-I stimulates cooperative interaction between the IGF-I receptor and CSK homologous kinase that regulates SHPS-1 phosphorylation in vascular smooth muscle cells

IGF-I plays an important role in smooth muscle cell proliferation and migration. In vascular smooth muscle cells cultured in 25 mm glucose, IGF-I stimulated a significant increase in Src homology 2 domain containing protein tyrosine phosphatase substrate-1 (SHPS-1) phosphorylation compared with 5 mm glucose and this increase was required for smooth muscle cell proliferation. A proteome-wide screen revealed that carboxyl-terminal SRC kinase homologous kinase (CTK) bound directly to phosphotyrosines in the SHPS-1 cytoplasmic domain. Because the kinase(s) that phosphorylates these tyrosines in response to IGF-I is unknown, we determined the roles of IGF-I receptor (IGF-IR) and CTK in mediating SHPS-1 phosphorylation. After IGF-I stimulation, CTK was recruited to IGF-IR and subsequently to phospho-SHPS-1. Expression of an IGF-IR mutant that eliminated CTK binding reduced CTK transfer to SHPS-1, SHPS-1 phosphorylation, and cell proliferation. IGF-IR phosphorylated SHPS-1, which provided a binding site for CTK. CTK recruitment to SHPS-1 resulted in a further enhancement of SHPS-1 phosphorylation. CTK knockdown also impaired IGF-I-stimulated SHPS-1 phosphorylation and downstream signaling. Analysis of specific tyrosines showed that mutation of tyrosines 428/452 in SHPS-1 to phenylalanine reduced SHPS-1 phosphorylation but allowed CTK binding. In contrast, the mutation of tyrosines 469/495 inhibited IGF-IR-mediated the phosphorylation of SHPS-1 and CTK binding, suggesting that IGF-IR phosphorylated Y469/495, allowing CTK binding, and that CTK subsequently phosphorylated Y428/452. Based on the above findings, we conclude that after IGF-I stimulation, CTK is recruited to IGF-IR and its recruitment facilitates CTK's subsequent association with phospho-SHPS-1. This results in the enhanced CTK transfer to SHPS-1, and the two kinases then fully phosphorylate SHPS-1, which is necessary for IGF-I stimulated cellular proliferation.     

Tyrosine phosphorylation of the beta3-subunit of the alphaVbeta3 integrin is required for embrane association of the tyrosine phosphatase SHP-2 and its further recruitment to the insulin-like growth factor I receptor

Ligand occupancy of the alphaVbeta3 integrin is required for IGF-I receptor (IGF-IR) phosphorylation of an appropriate duration and for stimulation of IGF-I actions. In vascular smooth muscle cells (SMCs), the tyrosine phosphatase SHP-2 regulates the duration of IGF-IR phosphorylation and biological actions. We determined the role of ligand occupancy of the alphaVbeta3 integrin on beta3 phosphorylation and studied the role of beta3 phosphorylation in regulating both SHP-2 recruitment to the cell membrane and IGF-I-dependent biological responses. Vitronectin binding to alphaVbeta3 induced tyrosine phosphorylation of the beta3-subunit in subconfluent SMCs and was accompanied by increased association of SHP-2 with beta3. In confluent SMCs, the beta3-subunit was constitutively phosphorylated leading to basal binding of SHP-2. The Src kinase inhibitor PP2 caused a concentration-dependent decrease in beta3 phosphorylation and resulted in decreased SHP-2 association with beta3 and with the cell membrane. In contrast to control cells, SMCs expressing a mutant beta3 that had two tyrosines changed to phenylalanines showed a 89.9 +/- 1.2% decrease in beta3 phosphorylation. This decrease was associated with reduced SHP-2 binding to nonphosphorylated beta3 and a corresponding decrease in the membrane association of SHP-2. When IGF-I was added to cells expressing mutant beta3, SHP-2 was not recruited to the Src homology 2 domain-containing tyrosine phosphatase substrate-1 or to IGF-IR. This was associated with prolonged IGF-IR phosphorylation and an impaired cellular DNA synthesis response to IGF-I. These results define a mechanism by which ligand occupancy of alphaVbeta3 regulates the SMC response to IGF-I.     

Regulation of insulin-like growth factor I receptor dephosphorylation by SHPS-1 and the tyrosine phosphatase SHP-2.

Activation of insulin-like growth factor I receptor (IGF-IR) kinase is an important site of control of IGF-I-linked intracellular signaling pathways. One potentially important regulatory variable is IGF-IR dephosphorylation. It has been shown that SHP-2, a tyrosine phosphatase, can bind to the activated IGF-IR in vitro; however, its role in IGF-IR dephosphorylation in whole cells is unknown. These studies were undertaken to determine whether SHP-2 was a candidate for mediating IGF-IR dephosphorylation. The IGF-IR in smooth muscle cells was dephosphorylated rapidly beginning 10 min after ligand addition, and this was temporally associated with SHP-2 binding to the receptor. IGF-I stimulated SHPS-1 phosphorylation and the subsequent recruitment of SHP-2. In cells expressing a SHPS-1 mutant that did not bind SHP-2 there was no recruitment of SHP-2 to the IGF-IR. Cells expressing a catalytically inactive form of SHP-2 showed SHP-2 recruitment to SHPS-1, but this did not result in SHPS-1 dephosphorylation, and there was a prolonged IGF-IR phosphorylation response after IGF-I stimulation. These studies indicate that IGF-IR stimulates phosphorylation of SHPS-1 which is critical for SHP-2 recruitment to the plasma membrane and for its recruitment to the IGF-IR. Recruitment of SHP-2 to the receptor then results in receptor dephosphorylation. The regulation of this process may be an important determinant of IGF-IR-mediated signaling.     

Occupation of alphavbeta3-integrin by endogenous ligands modulates IGF-I receptor activation and proliferation of human intestinal smooth muscle

We have previously shown that endogenous IGF-I regulates growth of human intestinal smooth muscle cells by stimulating proliferation and inhibiting apoptosis. In active Crohn's disease, expression of IGF-I and the alpha(v)beta(3)-integrin receptor ligands fibronectin and vitronectin is increased. The aim of the present study was to determine whether occupation of the alpha(v)beta(3)-receptor influences IGF-I receptor tyrosine kinase activation and function in human intestinal smooth muscle cells. In untreated cells, IGF-I elicited time-dependent tyrosine phosphorylation of its cognate receptor that was maximal within 2 min and sustained for 30 min. In the presence of the alpha(v)beta(3)-ligand fibronectin, IGF-I-stimulated IGF-I receptor activation was augmented. Conversely, in the presence of the alpha(v)beta(3)-specific disintegrin echistatin, IGF-I-stimulated IGF-I receptor tyrosine kinase phosphorylation was inhibited. IGF-I-stimulated IGF-I receptor activation was accompanied by recruitment of the adapter protein IRS-1, activation of Erk1/2, p70S6 kinase, and proliferation. These effects were augmented by fibronectin and attenuated by echistatin. IGF-I also elicited time-dependent recruitment of protein tyrosine phosphatase SHP-2 that coincided with dephosphorylation of the tyrosine phosphorylated IGF-I receptor tyrosine kinase. The alpha(v)beta(3)-disintegrin echistatin accelerated the rate of SHP-2 recruitment and deactivation of the IGF-I receptor tyrosine kinase. The results show that occupancy of the alpha(v)beta(3)-integrin receptor modulates IGF-I-induced IGF-I receptor activation and function in human intestinal muscle cells. We hypothesize that the concomitant increases in the expression of alpha(v)beta(3)-ligands and of IGF-I in active Crohn's disease may contribute to muscle hyperplasia and stricture formation by acting in concert to augment IGF-I-stimulated IGF-I receptor tyrosine kinase activity and IGF-I-mediated muscle cell growth.     

Role of SHPS-1 in the regulation of insulin-like growth factor I-stimulated Shc and mitogen-activated protein kinase activation in vascular smooth muscle cells

Insulin-like growth factor I (IGF-I) stimulates smooth muscle cell (SMC) proliferation, and the mitogen-activated protein kinase (MAPK) pathway plays an important role in mediating IGF-I-induced mitogenic signaling. Our prior studies have shown that recruitment of Src homology 2 domain tyrosine phosphatase (SHP-2) to the membrane scaffolding protein Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) is required for IGF-I-dependent MAPK activation. The current studies were undertaken to define the upstream signaling components that are required for IGF-I-stimulated MAPK activation and the role of SHPS-1 in regulating this process. The results show that IGF-I-induced Shc phosphorylation and its subsequent binding to Grb2 is required for sustained phosphorylation of MAPK and increased cell proliferation in SMCs. Furthermore, for Shc to be phosphorylated in response to IGF-I requires that Shc must associate with SHPS-1 and this association is mediated in part by SHP-2. Preincubation of cells with a peptide that contains a phospho-tyrosine binding motif sequence derived from SHPS-1 inhibited IGF-I-stimulated SHP-2 transfer to SHPS-1, the association of Shc with SHPS-1, and IGF-I-dependent Shc phosphorylation. Expression of an SHPS-1 mutant that did not bind to Shc or SHP-2 resulted in decreased Shc and MAPK phosphorylation in response to IGF-I. In addition, SMCs expressing a mutant form of the beta3 subunit of the alphaVbeta3, which results in impairment of SHP-2 transfer to SHPS-1, also showed attenuated IGF-I-dependent Shc and MAPK phosphorylation. Further analysis showed that Shc and SHP-2 can be coimmunoprecipitated after IGF-I stimulation. A cell-permeable peptide that contained a polyproline sequence from Shc selectively inhibited Shc/SHP-2 association and impaired Shc but not SHP-2 binding to SHPS-1. Exposure to this peptide also inhibited IGF-I-stimulated Shc and MAPK phosphorylation. Cells expressing a mutant form of Shc with the four prolines substituted with alanines showed no Shc/SHPS-1 association in response to IGF-I. We conclude that SHPS-1 functions as an anchor protein that recruits both Shc and SHP-2 and that their recruitment is necessary for IGF-I-dependent Shc phosphorylation, which is required for an optimal mitogenic response in SMCs.     

Mitogenic effect of the insulin analogue glargine in malignant cells in comparison with insulin and IGF-I.

The aim of the study was to investigate if the insulin analogue glargine, with an increased affinity for the IGF-I receptor (IGF-IR), affects the cell growth to a larger extent than human insulin in malignant cells expressing IGF-IRs. The breast cancer cell lines MCF-7 and SKBR-3, and the osteosarcoma cell line SaOS-2 were used. Gene expression was determined by real-time RT-PCR and receptor protein quantified by ELISAs. Receptor phosphorylation was assessed by immunoprecipitation and Western blot. Mitogenic effect was determined as (3)H-thymidine incorporation into DNA. The gene expression of insulin receptor (IR) varied between 4.3-7.5 x 10(-3) and the expression of IGF-IR between 7.7-147.7 x 10(-3) in relation to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Insulin receptor and IGF-IR protein varied between 2.0-4.1 ng/mg protein and 2.0-40.4 ng/mg protein, respectively. The IGF-IR was phosphorylated by IGF-I at a concentration of 10(-10)-10(-9) M. All three polypeptides stimulated DNA synthesis in MCF-7, SKBR-3, and SaOS-2 cells. SaOS-2 cells were more sensitive to IGF-I than to insulin and glargine. MCF-7 cells were more sensitive to des(1-3)IGF-I than to IGF-I. In SKBR-3 and SaOS-2 cells, glargine tended to be more potent than human insulin to stimulate DNA synthesis. Our results suggest that glargine, compared to human insulin, has little or no increased mitogenic effect in malignant cells expressing IGF-IRs.     

Insulin and IGF-I action on insulin receptors, IGF-I receptors, and hybrid insulin/IGF-I receptors in vascular smooth muscle cells

Insulin and insulin-like growth factor I (IGF-I) are known to affect cardiovascular disease. We have investigated ligand binding and the dose-response relationship for insulin and IGF-I on vascular smooth muscle cells (VSMCs) at the receptor level. VSMCs from rat thoracic aorta were serum starved, stimulated with IGF-I or insulin, lysed, immunoprecipitated, and analyzed by Western blot. d-[U-(14)C]Glucose accumulation and [6-(3)H]thymidine incorporation into DNA were also measured. Specific binding of both insulin and IGF-I was demonstrated, being higher for IGF-I. Both IGF-I receptor (IGF-IR) and insulin receptor (IR) beta-subunits were detected and coprecipitated after immunoprecipitation (IP) against either of the two. No coprecipitation was found after reduction of disulphide bonds with dithiotreitol before IP. After stimulation with 10(-10)-10(-9) M IGF-I, IP of the IGF-IR, or IR beta-subunit and immunoblot with anti-phosphotyrosine antibody, we found two distinct bands indicating phosphorylation of both the IGF-IR and the IR beta-subunit. Stimulation with 10(-10)-10(-9) M insulin and IP against the IGF-IR did not show phosphorylation of either beta-subunit, whereas after IP of the IR we found phosphorylation of the IR beta-subunit. [(14)C]Glucose accumulation and [(3)H]thymidine incorporation were elevated in cells stimulated with IGF-I at 10(-10)-10(-7) M, reaching maximum by 10(-9) M. Insulin stimulation showed measurable effects only at supraphysiological concentrations, 10(-8)-10(-7) M. In conclusion, coprecipitation of both the IGF-IR and the IR beta-subunit indicates the presence of hybrid insulin/IGF-I receptors in VSMC. At a physiological concentration, insulin activates the IR but does not affect either glucose metabolism or DNA synthesis, whereas IGF-I both activates the receptor and elicits biological effect.     

The role of Src kinase in insulin-like growth factor-dependent mitogenic signaling in vascular smooth muscle cells

Activation of the MAPK pathway mediates insulin-like growth factor-I (IGF-I)-dependent proliferation in vascular smooth muscle cells (SMC). Our previous studies have shown that IGF-I-induced Shc phosphorylation is necessary for sustained activation of MAPK and increased cell proliferation of SMCs, and both Shc and the tyrosine phosphatase SHP-2 must be recruited to the membrane protein SHPS-1 in order for Shc to be phosphorylated. These studies were undertaken to determine whether Src kinase activity is required to phosphorylate Shc in response to IGF-I in SMC and because SHP-2 binds to Src whether their interaction was also required for IGF-I-stimulated mitogenesis. Our results show that IGF-I induces activation of Src kinase and that is required for Shc phosphorylation and for optimal MAPK activation. We tested whether Shc is a substrate of c-Src in SMC by disrupting Src/Shc association using a peptide containing a YXXL (Tyr328) motif sequence derived from Src. The peptide blocked the binding of Src and Shc in vitro and in vivo. Cells expressing a mutant Src (Src-FF) that had Tyr328/Tyr358 substituted with phenylalanines (Src-FF) showed defective Src/Shc binding, impaired IGF-I-dependent Shc phorylation, and impaired mitogenesis. This supports the conclusion that Src phosphorylates Shc. IGF-I induced both Src/SHP-2 and Src/SHPS-1 association. SMCs expressing an SHP-2 mutant that had the polyproline-rich region of SH2 deleted (SHP-2Delta10) had disrupted SHP-2/Src association, impaired IGF-I-dependent Shc phosphorylation, and an attenuated mitogenic response. IGF-I-induced association of Src and SHPS-1 was also impaired in SHP-2Delata10-expressing cells, although SHP-2/SHPS-1 association was unaffected. Upon IGF-I stimulation, a complex assembles on SHPS-1 that contains SHP-2, c-Src, and Shc wherein Src phosphorylates Shc, a signaling step that is necessary for an optimal mitogenic response.     

Effects of RACK1 on cell migration and IGF-I signalling in cardiomyoctes are not dependent on an association with the IGF-IR

RACK1 can act as a scaffolding protein to integrate IGF-IR and integrin signalling in transformed cells but its actions in regulating IGF-IR signalling in non-transformed cells are less well understood. Here, we investigated the function of RACK1 in the non-transformed cardiomyocyte cell line H9c2. Overexpression of RACK1 in H9c2 cells was sufficient to increase cell size, increase adhesion to collagen 1, enhance protection from hydrogen peroxide-induced cell death, and increase cell migration. However, cell proliferation was decreased in these cells. Small interfering RNA (siRNA)-mediated suppression of RACK1 in H9c2 cells resulted in decreased cell adhesion and migration, but had no effect on cell proliferation or size. Increased basal and IGF-I-mediated Erk phosphorylation was observed in RACK1-overexpressing H9c2 cells. Interestingly, contrary to observations in transformed cells, RACK1 was not observed to interact with the IGF-IR in H9c2 cells. Also in contrast to observations in transformed cells, IGF-I promoted recruitment of Src to RACK1 as well as recruitment of PKC, and PKC to RACK1. Overall, the data indicate that in H9c2 cells RACK1 can influence cell size, cell survival, adhesion, migration, but its responses to IGF-I are independent of an association with the IGF-IR. Thus, the composition of the RACK1 scaffolding complex and its effects on IGF-I signalling may be different in transformed and non-transformed cells.     

Aflatoxin B1 Up-Regulates Insulin Receptor Substrate 2 and Stimulates Hepatoma Cell Migration

Aflatoxin B1 (AFB1) is a potent carcinogen that can induce hepatocellular carcinoma. AFB1-8,9-exo-epoxide, one of AFB1 metabolites, acts as a mutagen to react with DNA and induce gene mutations, including the tumor suppressor p53. In addition, AFB1 reportedly stimulates IGF receptor activation. Aberrant activation of IGF-I receptor (IGF-IR) signaling is tightly associated with various types of human tumors. In the current study, we investigated the effects of AFB1 on key elements in IGF-IR signaling pathway, and the effects of AFB1 on hepatoma cell migration. The results demonstrated that AFB1 induced IGF-IR, Akt, and Erk1/2 phosphorylation in hepatoma cell lines HepG2 and SMMC-7721, and an immortalized human liver cell line Chang liver. AFB1 also down-regulated insulin receptor substrate (IRS) 1 but paradoxically up-regulated IRS2 through preventing proteasomal degradation. Treatment of hepatoma cells and Chang liver cells with IGF-IR inhibitor abrogated AFB1-induced Akt and Erk1/2 phosphorylation. In addition, IRS2 knockdown suppressed AFB1-induced Akt and Erk1/2 phosphorylation. Finally, AFB1 stimulated hepatoma cell migration. IGF-IR inhibitor or IRS2 knockdown suppressed AFB1-induced hepatoma cell migration. These data demonstrate that AFB1 stimulates hepatoma cell migration through IGF-IR/IRS2 axis.     

Synergistic Effect of Glucagon-Like Peptide 2 (GLP-2) and of Key Growth Factors on the Proliferation of Cultured Rat Astrocytes. Evidence for Reciprocal Upregulation of the mRNAs for GLP-2 and IGF-I Receptors

The aim of this work was to determine whether the stimulating effect of glucagon-like peptide (GLP)-2 on astrocyte proliferation could be reinforced by proliferating substances, including growth factors such as EGF, platelet-derived growth factor, insulin-like growth factor type I (IGF-I) or a hormone such as insulin. Both DNA synthesis and astrocyte density, as well as the expression of c-Fos, Ki-67, proliferating cell nuclear antigen and glial fibrillary acidic proteins, were found to be higher in the presence of GLP-2 than in its absence. In an attempt to get a better understanding of this process, intracellular cyclic adenosine monophosphate (cAMP) production, extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and the expression of GLP-2R and IGF-I receptor (IGF-IR) mRNAs were studied in response to growth factors. Our results indicate that, in the presence of different growth factors, GLP-2 does not increase cAMP production but raises ERK 1/2 phosphorylation. In addition, GLP-2R mRNA expression was increased by IGF-I, whilst mRNA expression of IGF-IR was higher in cells incubated with GLP-2 than in control cells. These results suggest for the first time that GLP-2 and several growth factors show synergistic effects on the proliferation of rat astrocytes, a process in which an enhanced expression of GLP-2R and IGF-IR may be involved, providing additional insights into the physiological role of this novel neuropeptide, specially during astroglial regeneration.     

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

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-3beta 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.     

IGF-I induces caveolin 1 tyrosine phosphorylation and translocation in the lipid rafts

Caveolin 1, a component of caveolae, regulates signalling pathways compartmentalization interacting with tyrosine kinase receptors and their substrates. The role of caveolin 1 in the Insulin Receptor (IR) signalling has been well investigated. On the contrary, the functional link between caveolin 1 and IGF-I Receptor (IGF-IR) remains largely unknown. Here we show that (1) IGF-IR colocalizes with caveolin 1 in the lipid rafts enriched fractions on plasmamembrane in R-IGF-IR(WT) cells, (2) IGF-I induces caveolin 1 phosphorylation at the level of tyrosine 14, (3) this effect is rapid and results in the translocation of caveolin 1 and in the formation of membrane patches on cell surface. These actions are IGF-I specific since we did not detect caveolin 1 redistribution in insulin stimulated R(-) cells overexpressing IRs.