IGF-I increases IGFBP-5 and collagen alpha1(I) mRNAs by the MAPK pathway in rat intestinal smooth muscle cells

IGF-I is a potent fibrogenic growth factor that stimulates proliferation of intestinal smooth muscle cells and increases synthesis of collagen and IGF-I-binding proteins by the cells. These processes contribute to intestinal fibrosis that develops in patients with Crohn's disease and in Lewis-strain rats with experimental Crohn's disease. The aim of this study was to determine which early docking proteins are associated with IGF-I receptor signal transduction and which transduction pathway is involved in IGF-I-mediated gene regulation in intestinal smooth muscle cells. Primary cultures of smooth muscle cells isolated from the muscularis externa of the distal colon of Lewis rats were treated with IGF-I (100 ng/ml). Immunoprecipitation studies demonstrated that IGF-I stimulation resulted in tyrosine phosphorylation of IRS-1, IRS-2, and Shc. Coimmunoprecipitation demonstrated a close association between the IGF-I receptor and these three early docking proteins. Concurrent treatment with the MAPK inhibitor PD98059 (10 microM) resulted in an inhibition of the IGF-I-mediated increase in IGFBP-5 and collagen alpha(1)(I) mRNAs, while concurrent treatment with the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin (100 nM) had no effect. In additional experiments, cells were transiently transfected with adenoviral vectors dominantly expressing inactive mutant Akt or constitutively expressing wild-type Akt. In both cases, the IGF-I-mediated increase in collagen I protein did not differ from that observed in control cultures that had been transfected with an adenoviral vector carrying the LacZ reporter gene. These results suggest that the MAPK pathway is key to IGF-I-mediated gene regulation in intestinal smooth muscle cells, whereas data do not suggest a role for the Akt-dependent pathway in our system.     

Increased IRS-2 content and activation of IGF-I pathway contribute to enhance beta-cell mass in fetuses from undernourished pregnant rats

We have previously shown that fetuses from undernourished (U) pregnant rats exhibited an increased beta-cell mass probably related to an enhanced IGF-I replicative response. Because IGF-I signaling pathways have been implicated in regulating beta-cell growth, we investigated in this study the IGF-I transduction system in U fetuses. To this end, an in vitro model of primary fetal islets was developed to characterize glucose/IGF-I-mediated signaling that specially influences beta-cell proliferation. We found that U fetal islets showed a greater replicative response to glucose and IGF-I than controls. Furthermore, insulin receptor substrate (IRS)-2 protein and its association with p85 were also increased. In the complete absence of IGF-I or stimulatory glucose, U islets presented an increased basal phosphorylation of downstream signals of the phosphatidylinositol 3-kinase (PI3K) pathway such as PKB, glycogen synthase kinase (GSK)3alpha/beta, PKCzeta, and mammalian target of rapamycin (mTOR). Similarly, phosphorylation of these proteins (except GSK3alpha/beta) by glucose and IGF-I was augmented even though total protein content remained unchanged. Downstream of PKB, direct glucose activation of mTOR was increased as well. In contrast, ERK1/2 phosphorylation was unaffected by undernutrition, but ERK activation seemed to be required to induce a higher proliferative response in U islets. In conclusion, we have demonstrated that fetal U islets show increased IRS-2 content and an enhancement in both basal and glucose/IGF-I activations of the IRS-2/PI3K/PKB pathway. These molecular changes may be responsible for the greater glucose/IGF-I islet replication and contribute to the increased beta-cell mass found in these fetuses.     

Stimulatory effects of insulin-like growth factor-I on growth plate chondrogenesis are mediated by nuclear factor-kappaB p65

Insulin-like growth factor-I (IGF-I) is an important regulator of endochondral ossification. However, little is known about the signaling pathways activated by IGF-I in growth plate chondrocytes. We have previously shown that NF-kappaB-p65 facilitates growth plate chondrogenesis. In this study, we first cultured rat metatarsal bones with IGF-I and/or pyrrolidine dithiocarbamate (PDTC), a known NF-kappaB inhibitor. The IGF-I-mediated stimulation of metatarsal growth and growth plate chondrogenesis was neutralized by PDTC. In rat growth plate chondrocytes, IGF-I induced NF-kappaB-p65 nuclear translocation. The inhibition of NF-kappaB-p65 expression and activity (by p65 short interfering RNA and PDTC, respectively) in chondrocytes reversed the IGF-I-mediated induction of cell proliferation and differentiation and the IGF-I-mediated prevention of cell apoptosis. Moreover, the inhibition of the phosphatidylinositol 3-kinase and Akt abolished the effects of IGF-I on NF-kappaB activation. In conclusion, our findings indicate that IGF-I stimulates growth plate chondrogenesis by activating NF-kappaB-p65 in chondrocytes.     

Insulin-like growth factor-I stimulates H4II rat hepatoma cell proliferation: dominant role of PI-3'K/Akt signaling

Although hepatocytes are the primary source of endocrine IGF-I and -II in mammals, their autocrine/paracrine role in the dysregulation of proliferation and apoptosis during hepatocarcinogenesis and in hepatocarcinomas (HCC) remains to be elucidated. Indeed, IGF-II and type-I IGF receptors are overexpressed in HCC cells, and IGF-I is synthesized in adjacent non-tumoral liver tissue. In the present study, we have investigated the effects of type-I IGF receptor signaling on H4II rat hepatoma cell proliferation, as estimated by 3H-thymidine incorporation into DNA. IGF-I stimulated the rate of DNA synthesis of serum-deprived H4II cells, stimulation being maximal 3 h after the onset of IGF-I treatment and remaining elevated until at least 6 h. The IGF-I-induced increase in DNA replication was abolished by LY294002 and only partially inhibited by PD98059, suggesting that phosphoinositol-3' kinase (PI-3'K) and to a lesser extent MEK/Erk signaling were involved. Furthermore, the 3- to 19-fold activation of the Erks in the presence of LY294002 suggested a down-regulation of the MEK/Erk cascade by PI-3'K signaling. Finally, the effect of IGF-I on DNA replication was almost completely abolished in clones of H4II cells expressing a dominant-negative form of Akt but was unaltered by rapamycin treatment of wild-type H4II cells. Altogether, these data support the notion that the stimulation of H4II rat hepatoma cell proliferation by IGF-I is especially dependent on Akt activation but independent on the Akt/mTOR signaling.     

Inhibition of Src-like kinases reveals Akt-dependent and -independent pathways in insulin-like growth factor I-mediated oligodendrocyte progenitor survival

Insulin-like growth factor I (IGF-I) has been previously shown to promote survival of oligodendrocyte progenitors; however, the underlying mechanisms are not fully understood. Our aim was to investigate the involvement of phosphatidylinositol 3-kinase (PI3K), MEK1, and Src family tyrosine kinases in IGF-I-mediated oligodendrocyte progenitor survival. In agreement with previous studies, IGF-I promoted cell survival. We show that IGF-I prevented apoptosis induced by growth factor deprivation in a PI3K-dependent and MEK/ERK-independent manner. In addition, IGF-I activated Akt while inhibiting caspase-3 activation, and these effects were reversed by the PI3K inhibitors LY 294002 and wortmannin, but not by the MEK1 inhibitor PD 98059. Interestingly, PP2, a specific Src-like kinase inhibitor, blocked the tyrosine phosphorylation of Src, Fyn, and Lyn and IGF-I-stimulated Akt activation, yet had no significant effects on caspase-3 activation or progenitor survival. To further determine whether Akt is required for IGF-I-mediated survival, oligodendrocyte progenitors were transduced with defective Akt mutants or treated with an Akt inhibitor. Although the Akt mutants and inhibitor decreased Akt activity and reduced basal cell survival, IGF-I could partially rescue oligodendrocyte progenitors by decreasing caspase-3 activation. These results suggest that 1) PI3K is essential for IGF-I-promoted cell survival, 2) downstream activation of Akt-dependent and -independent pathways is involved, and 3) Src-like tyrosine kinases participate in IGF-I-induced Akt activation. Therefore, an unidentified effector(s) of PI3K appears to be involved in conferring complete IGF-I-mediated protection of oligodendrocyte progenitors.     

Dual regulation of MMP-2 expression by the type 1 insulin-like growth factor receptor: the phosphatidylinositol 3-kinase/Akt and Raf/ERK pathways transmit opposing signals

The matrix metalloproteinase (MMP)-2 has been recognized as a major mediator of basement membrane degradation, angiogenesis, tumor invasion, and metastasis. The factors that regulate its expression have not, however, been fully elucidated. We previously identified the type I insulin-like growth factor (IGF-I) receptor as a regulator of MMP-2 synthesis. The objective of the present study was to investigate the signal transduction pathway(s) mediating this regulation. We show here that in Lewis lung carcinoma subline H-59 cells treated with IGF-I (10 ng/ml), the PI 3-kinase (phosphatidylinositol 3'-kinase) /protein kinase B (Akt) and C-Raf/ERK pathways were activated, and MMP-2 promoter activity, mRNA, and protein synthesis were induced. MMP-2 induction was blocked by the PI 3-kinase inhibitors LY294002 and wortmannin, by overexpression of a dominant-negative Akt or wild-type PTEN (phosphatase and tensin homologue deleted on chromosome 10), and by rapamycin. In contrast, a MEK inhibitor PD98059 failed to reduce MMP-2 promoter activation and actually increased MMP-2 mRNA and protein synthesis by up to 30%. Interestingly, suppression of PI 3-kinase signaling by a dominant-negative Akt enhanced ERK activity in cells stimulated with 10 ng/ml but not with 100 ng/ml IGF-I. Furthermore, at the higher (100 ng/ml) IGF-I concentration, C-Raf and ERK, but not PI 3-kinase activation, was enhanced, and this resulted in down-regulation of MMP-2 synthesis. This effect was reversed in cells expressing a dominant-negative ERK mutant. The results suggest that IGF-I can up-regulate MMP-2 synthesis via PI 3-kinase/Akt/mTOR (the mammalian target of rapamycin) signaling while concomitantly transmitting a negative regulatory signal via the Raf/ERK pathway. The outcome of IGF-IR (the receptor for IGF-I) activation may ultimately depend on factors, such as ligand bioavailability, that can shift the balance preferentially toward one pathway or the other.     

IL-1beta suppresses prolonged Akt activation and expression of E2F-1 and cyclin A in breast cancer cells

Cell cycle aberrations occurring at the G(1)/S checkpoint often lead to uncontrolled cell proliferation and tumor growth. We recently demonstrated that IL-1beta inhibits insulin-like growth factor (IGF)-I-induced cell proliferation by preventing cells from entering the S phase of the cell cycle, leading to G(0)/G(1) arrest. Notably, IL-1beta suppresses the ability of the IGF-I receptor tyrosine kinase to phosphorylate its major docking protein, insulin receptor substrate-1, in MCF-7 breast carcinoma cells. In this study, we extend this juxtamembrane cross-talk between cytokine and growth factor receptors to downstream cell cycle machinery. IL-1beta reduces the ability of IGF-I to activate Cdk2 and to induce E2F-1, cyclin A, and cyclin A-dependent phosphorylation of a retinoblastoma tumor suppressor substrate. Long-term activation of the phosphatidylinositol 3-kinase/Akt signaling pathway, but not the mammalian target of rapamycin or mitogen-activated protein kinase pathways, is required for IGF-I to hyperphosphorylate retinoblastoma and to cause accumulation of E2F-1 and cyclin A. In the absence of IGF-I to induce Akt activation and cell cycle progression, IL-1beta has no effect. IL-1beta induces p21(Cip1/Waf1), which may contribute to its inhibition of IGF-I-activated Cdk2. Collectively, these data establish a novel mechanism by which prolonged Akt phosphorylation serves as a convergent target for both IGF-I and IL-1beta; stimulation by growth factors such as IGF-I promotes G(1)-S phase progression, whereas IL-1beta antagonizes IGF-I-induced Akt phosphorylation to induce cytostasis. In this manner, Akt serves as a critical bridge that links proximal receptor signaling events to more distal cell cycle machinery.     

Cross-talk between IGF-I and TGF-beta signaling pathways

Insulin-like growth factor-I (IGF-I) has gained broad recognition as an important survival factor for epithelial cells in numerous tissues. The IGF-I receptor signaling pathway is deregulated in the majority of carcinomas, and such deregulation has also been reported to be tightly associated with enhanced tumor progression and metastasis. One of the key proteins that transduces IGF-I signals and is phospho-activated downstream of the IGF-I receptor, is the non-receptor serine/threonine kinase proto-oncogene protein kinase B (PKB, also known as Akt). This kinase serves as a major molecular node to control the function of many cell survival and death proteins through phosphorylation-mediated protein modification. The end result of the activation of Akt is enhanced cell survival and proliferation, pre-requisites for malignant transformation. Recent studies show that IGF-I signals cross-talk at multiple levels with various components of the TGF-beta signaling pathway, which depending on context may function either as tumor suppressor or as tumor promoter. Thus, a better understanding of how the IGF-I and TGF-beta signaling pathways are mutually interconnected is likely to unveil novel targets for the therapeutic intervention of many cancers.     

Dual control of muscle cell survival by distinct growth factor-regulated signaling pathways

In addition to their ability to stimulate cell proliferation, polypeptide growth factors are able to maintain cell survival under conditions that otherwise lead to apoptotic death. Growth factors control cell viability through regulation of critical intracellular signal transduction pathways. We previously characterized C2 muscle cell lines that lacked endogenous expression of insulin-like growth factor II (IGF-II). These cells did not differentiate but underwent apoptotic death in low-serum differentiation medium. Death could be prevented by IGF analogues that activated the IGF-I receptor or by unrelated growth factors such as platelet-derived growth factor BB (PDGF-BB). Here we analyze the signaling pathways involved in growth factor-mediated myoblast survival. PDGF treatment caused sustained activation of extracellular-regulated kinases 1 and 2 (ERK1 and -2), while IGF-I only transiently induced these enzymes. Transient transfection of a constitutively active Mek1, a specific upstream activator of ERKs, maintained myoblast viability in the absence of growth factors, while inhibition of Mek1 by the drug UO126 blocked PDGF-mediated but not IGF-stimulated survival. Although both growth factors activated phosphatidylinositol 3-kinase (PI3-kinase) to similar extents, only IGF-I treatment led to sustained stimulation of its downstream kinase, Akt. Transient transfection of a constitutively active PI3-kinase or an inducible Akt promoted myoblast viability in the absence of growth factors, while inhibition of PI3-kinase activity by the drug LY294002 selectively blocked IGF- but not PDGF-mediated muscle cell survival. In aggregate, these observations demonstrate that distinct growth factor-regulated signaling pathways independently control myoblast survival. Since IGF action also stimulates muscle differentiation, these results suggest a means to regulate myogenesis through selective manipulation of different signal transduction pathways.     

High-frequency electrically stimulated skeletal muscle contractions increase p70 phosphorylation independent of known IGF-I sensitive signaling pathways

Insulin-like growth factor (IGF-I) is hypothesized to be a critical upstream regulator of mammalian target of rapamycin (mTOR)-regulated protein synthesis with muscle contraction. We utilized a mouse model that expresses a skeletal muscle specific dominant-negative IGF-I receptor to investigate the role of IGF-I signaling of protein synthesis in response to unilateral lengthening contractions (10 sets, 6 repetitions, 100 Hz) at 0 and 3 h following the stimulus. Our results indicate that one session of high frequency muscle contractions can activate mTOR signaling independent of signaling components directly downstream of the receptor.     

IGF-I/PI3K/Akt and IGF-I/MAPK/ERK pathways in vivo in skeletal muscle are regulated by nutrition and contribute to somatic growth in the fine flounder

The insulin-like growth factor-I (IGF-I) is a key regulator of skeletal muscle growth in vertebrates, promoting mitogenic and anabolic effects through the activation of the MAPK/ERK and the PI3K/Akt signaling pathways. Nutrition also affects skeletal muscle growth, activating intracellular pathways and inducing protein synthesis and accretion. Thus, both hormonal and nutritional signaling regulate muscle mass. In this context, plasma IGF-I levels and the activation of both pathways in response to food were evaluated in the fine flounder using fasting and refeeding trials. The present study describes for the first time in a nonmammalian species that the MAPK/ERK and PI3K/Akt are activated by exogenous circulating IGF-I, as well as showing that the MAPK/ERK pathway activation is modulated by the nutritional status. Also, these results show that there is a time-dependent regulation of IGF-I plasma levels and its signaling pathways in muscle. Together, these results suggest that the nutritionally managed IGF-I could be regulating the activation of the MAPK/ERK and the PI3K/Akt signaling pathways differentially according to the nutritional status, triggering different effects in growth parameters and therefore contributing to somatic growth in fish. This study contributes to the understanding of the nutrient regulation of IGF-I and its signaling pathways in skeletal muscle growth in nonmammalian species, therefore providing insight concerning the events controlling somatic growth in vertebrates.     

Rapamycin promotes vascular smooth muscle cell differentiation through insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt2 feedback signaling

The phenotypic plasticity of mature vascular smooth muscle cells (VSMCs) facilitates angiogenesis and wound healing, but VSCM dedifferentiation also contributes to vascular pathologies such as intimal hyperplasia. Insulin/insulin-like growth factor I (IGF-I) is unique among growth factors in promoting VSMC differentiation via preferential activation of phosphatidylinositol 3-kinase (PI3K) and Akt. We have previously reported that rapamycin promotes VSMC differentiation by inhibiting the mammalian target of rapamycin (mTOR) target S6K1. Here, we show that rapamycin activates Akt and induces contractile protein expression in human VSMC in an insulin-like growth factor I-dependent manner, by relieving S6K1-dependent negative regulation of insulin receptor substrate-1 (IRS-1). In skeletal muscle and adipocytes, rapamycin relieves mTOR/S6K1-dependent inhibitory phosphorylation of IRS-1, thus preventing IRS-1 degradation and enhancing PI3K activation. We report that this mechanism is functional in VSMCs and crucial for rapamycin-induced differentiation. Rapamycin inhibits S6K1-dependent IRS-1 serine phosphorylation, increases IRS-1 protein levels, and promotes association of tyrosine-phosphorylated IRS-1 with PI3K. A rapamycin-resistant S6K1 mutant prevents rapamycin-induced Akt activation and VSMC differentiation. Notably, we find that rapamycin selectively activates only the Akt2 isoform and that Akt2, but not Akt1, is sufficient to induce contractile protein expression. Akt2 is required for rapamycin-induced VSMC differentiation, whereas Akt1 appears to oppose contractile protein expression. The anti-restenotic effect of rapamycin in patients may be attributable to this unique pattern of PI3K effector regulation wherein anti-differentiation signals from S6K1 are inhibited, but pro-differentiation Akt2 activity is promoted through an IRS-1 feedback signaling mechanism.     

Insulin-like growth factor (IGF)-I regulates IGF-binding protein-5 gene expression through the phosphatidylinositol 3-kinase, protein kinase B/Akt, and p70 S6 kinase signaling pathway

Expression of the insulin-like growth factor-binding protein 5 (IGFBP-5) gene in vascular smooth muscle cells is up-regulated by IGF-I through an IGF-I receptor-mediated mechanism. In this study, we studied the possible involvement of the mitogen-activated protein kinase (MAPK) and PI 3-kinase signaling pathways in mediating IGF-I-regulated IGFBP-5 gene expression. The addition of Des(1-3)IGF-I, an IGF analog with reduced affinity to IGFBPs, resulted in a transient activation of p44 and p42 MAPK. Inhibition of the MAPK activation by PD98059, however, did not affect IGF-I-stimulated IGFBP-5 expression. Des(1-3)IGF-I treatment also strongly activated PI 3-kinase. This activation was probably mediated through IRS-1, because IGF-I stimulation resulted in a significant increase in IRS-1- but not IRS-2-associated PI 3-kinase activity. This activation occurred within 5 min and was sustained at high levels for over 6 h. Likewise, Des(1-3)IGF-I caused a long lasting activation of PKB/Akt and p70(s6k). When LY294002 and wortmannin, two specific inhibitors of PI 3-kinase, were added with Des(1-3)IGF-I, the IGF-I-regulated IGFBP-5 expression was negated. The addition of rapamycin, which inhibits IGF-I-induced p70(s6k) activation, significantly inhibited IGF-I-regulated IGFBP-5 gene expression. These results suggest that the action of IGF-I on IGFBP-5 gene expression requires the activation of the PI 3-kinase-PKB/Akt-p70(s6k) pathway but not the MAPK pathway in vascular smooth muscle cells.     

Insulin-like growth factor-I induces renal cell hypertrophy via a calcineurin-dependent mechanism

Insulin-like growth factor-I (IGF-I) may play an important role in the development of renal hypertrophy. In this study we determined the effect of IGF-I on cultured mesangial cells (MCs) and examined activation of key signaling pathways. IGF-I induced hypertrophy as determined by an increase in cell size and an increase in protein to DNA ratio and increased accumulation of extracellular matrix (ECM) proteins. IGF-I also activated both Erk1/Erk2 MAPK and phosphatidylinositol 3-kinase (PI3K) in MCs. Inhibition of either MAPK or PI3K, however, had no effect on IGF-I-induced hypertrophy or ECM production. Next, we examined the effect of IGF-I on activation of the calcium-dependent phosphatase calcineurin. IGF-I treatment stimulated calcineurin activity and increased the protein levels of calcineurin and the calcineurin binding protein, calmodulin. Cyclosporin A, an inhibitor of calcineurin, blocked both IGF-I-mediated hypertrophy and up-regulation of ECM. In addition, calcineurin resulted in sustained Akt activation, indicating possible cross-talk with other signaling pathways. Finally, IGF-I treatment resulted in the calcineurindependent nuclear localization of NFATc1. Therefore, IGF-I induces hypertrophy and increases ECM accumulation in MCs. IGF-I-mediated hypertrophy is associated with activation of Erk1/Erk2 MAPK and PI3K but does not require either of these pathways. Instead, IGF-I mediates hypertrophy via a calcineurin-dependent pathway.