Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells

The insulin-like growth factor type 1 receptor (IGF-1R) is part of the receptor tyrosine kinase superfamily. The activation of IGF-1R regulates several key signaling pathways responsible for maintaining cellular homeostasis, including survival, growth, and proliferation. In addition to mediating signal transduction at the plasma membrane, in serum-based models, IGF-1R undergoes SUMOylation by SUMO 1 and translocates to the nucleus in response to IGF-1. In corneal epithelial cells grown in serum-free culture, however, IGF-1R has been shown to accumulate in the nucleus independent of IGF-1. In this study, we report that the insulin-like growth factor binding protein-3 (IGFBP-3) mediates nuclear translocation of IGF-1R in response to growth factor withdrawal. This occurs via SUMOylation by SUMO 2/3. Further, IGF-1R and IGFBP-3 undergo reciprocal regulation independent of PI3k/Akt signaling. Thus, under healthy growth conditions, IGFBP-3 functions as a gatekeeper to arrest the cell cycle in G0/G1, but does not alter mitochondrial respiration in cultured cells. When stressed, IGFBP-3 functions as a caretaker to maintain levels of IGF-1R in the nucleus. These results demonstrate mutual regulation between IGF-1R and IGFBP-3 to maintain cell survival under stress. This is the first study to show a direct relationship between IGF-1R and IGFBP-3 in the maintenance of corneal epithelial homeostasis.     

AMPK activation inhibits the expression of HIF-1alpha induced by insulin and IGF-1

Insulin, insulin like growth factor (IGF)-1, and AMP-activated protein kinase (AMPK) signaling regulate independently angiogenesis through vascular endothelial growth factor (VEGF) expression. In the present study, we investigated a potential cross-talk between these signaling pathways on hypoxia-inducible factor (HIF)-1alpha and VEGF expression. Retinal epithelial ARPE-19 cells were treated with AICAR, an AMPK activator, alone or in combination with insulin and IGF-1. AICAR stimulated VEGF mRNA expression, but did not modify the insulin- and IGF-1-induced VEGF expression. We have investigated the effect of AICAR on insulin and IGF-1 signaling pathways. We observed that AICAR increased insulin- and IGF-1-induced phosphorylation of PKB, whereas phosphorylation of S6K-1 was decreased. Moreover, AICAR and metformin inhibited the ability of insulin and IGF-1 to induce HIF-1alpha expression. These results show that AICAR and insulin/IGF-1 regulate VEGF expression through different mechanisms.     

Differences in survival-promoting effects and intracellular signaling properties of BDNF and IGF-1 in cultured cerebral cortical neurons

Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) act on various neurons of the CNS as neurotrophic factors promoting neuronal differentiation and survival. We examined the survival-promoting effects of BDNF and IGF-1 on serum deprivation-induced death in cultured cerebral cortical neurons, and compared the intracellular signaling pathways stimulated by BDNF and IGF-1 in the neurons. We found that the survival-promoting effect of BDNF was much weaker than that of IGF-1 in serum deprivation-induced death of cultured cortical neurons. We found no differences in the levels of phosphatidylinositol 3-kinase (PtdIns3-K) activity or Akt (also called PKB) phosphorylation induced by BDNF and IGF-1 in the cultured cortical neurons, although many reports suggest that PtdIns3-K and Akt are involved in survival promotion. In addition, phosphorylation signals of mitogen-activated protein kinase (MAPK) and cAMP responsive element-binding protein (CREB), which have also been reported to be involved in survival promotion, were stimulated by BDNF much more potently than by IGF-1. These results show that there may be, as yet unidentified, intracellular signaling pathways other than the PtdIns3-K-Akt, MAPK and CREB signaling, to regulate survival promotion. These unidentified signaling pathways may be responsible for the distinct strengths of the survival-promoting effects of BDNF and IGF-1.     

Simvastatin inhibits the proliferation of human prostate cancer PC-3 cells via down-regulation of the insulin-like growth factor 1 receptor

Recently, statins have been being studied for their proapoptic and antimetastatic effects. However, the exact mechanisms of their anticancer action are still unclear. Dolichyl phosphate is a nonsterol isoprenoid derivative in the mevalonate pathway that affects the expression of the Insulin-like growth factor 1 receptor (IGF-1R). IGF-1R activation is required for prostate cell proliferation; therefore, IGF-1R inhibitory agents may be of preventive and/or therapeutic value. In this study, the effects of simvastatin on IGF-1R signaling in prostate cancer PC-3 cells were examined. Simvastatin suppressed proliferation and induced apoptosis of PC-3, and the expression of IGF-1R was suppressed by simvastatin. Knockdown of IGF-1R by siRNA led to inhibition of proliferation of PC-3. Simvastatin also inhibited IGF-1-induced activation of both ERK and Akt signaling and IGF-1-induced PC-3 cell proliferation. Our results suggest statins are potent inhibitors of the IGF-1/IGF-1R system in prostate cancer cells and may be beneficial in prostate cancer treatment.     

Comparative signaling pathways of insulin-like growth factor-1 and brain-derived neurotrophic factor in hippocampal neurons and the role of the PI3 kinase pathway in cell survival

Insulin-like growth factor-1 (IGF-1) and brain-derived neurotrophic factor (BDNF) are trophic factors required for the viability and normal functions of various neuronal cells. However, the detailed intracellular mechanism(s) involved in these effects in neuronal cells remains to be fully elucidated. In present study, the respective intracellular signaling pathway induced by IGF-1 and BDNF and their possible role in neuronal survival were investigated. Both IGF-1 and BDNF protected hippocampal neurons from serum deprivation-induced death with IGF-1 apparently being more potent. Western blot analyses showed that both IGF-1 and BDNF induced the activation of the phosphatidylinositide 3 kinase (PI3)/Akt (protein kinase B) kinase and the mitogen-activated protein kinase (MAPK) pathways. The phosphorylation of Akt and its downstream target, FKHRL1, induced by IGF-1 was rapid and sustained while that of MAPK was transient. The reverse situation was observed for BDNF. Moreover, IGF-1 potently induced the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and its association with PI3 kinase while BDNF was weak in these assays. In contrast, the tyrosine phosphorylation of Shc proteins was dramatically stimulated by BDNF, with IGF-1 having only a minimal effect. Most interestingly, only the inhibitor of the PI3K/Akt pathway, LY294002, was able to block the survival effects of both IGF-1 and BDNF; an inhibitor of the MAPK pathway inhibitor, PD98059, being ineffective. Taken together, these data reveal that the survival properties of both IGF-1 and BDNF against serum deprivation are mediated by the activation of the PI3K/Akt, but not the MAPK, pathway in hippocampal neurons.     

Enhanced levels of endogenous endothelin-1 contribute to the over expression of Giα protein in vascular smooth muscle cells from SHR: Role of growth factor receptor activation

We earlier showed that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit increased expression of Gi proteins. Since the levels of endothelin-1 (ET-1) are enhanced in VSMC from SHR, we undertook the present study to examine the implication of endogenous ET-1 and the underlying mechanisms in the enhanced expression of Giα proteins in VSMC from SHR. The enhanced expression of Giα-2 and Giα-3 proteins in VSMC from SHR was inhibited by ET_A and ET_B receptor antagonists, BQ123 and BQ788 respectively. In addition, these antagonists also attenuated the enhanced inhibition of forskolin-stimulated adenylyl cyclase activity by low concentrations of GTPγS and by inhibitory hormones in VSMC from SHR compared to WKY. Furthermore, AG1295, AG1024 and PP2, inhibitors of platelet derived growth factor receptor (PDGFR), insulin-like growth factor 1 receptor (IGF-1R) and c-Src respectively, inhibited the enhanced expression of Giα protein and the enhanced phosphorylation of PDGFR and IGF-1R in VSMC from SHR to WKY levels. In addition, NAD(P)H oxidase inhibitor DPI and N-acetylcysteine (NAC), a scavenger of superoxide anion (O₂⁻) also inhibited the enhanced phosphorylation of PDGFR and IGF-1R and c-Src in VSMC from SHR to control levels. Furthermore, the augmented phosphorylation of ERK1/2 in VSMC from SHR was attenuated by BQ123 and BQ788, growth factor receptors inhibitors and PP2. These results suggest that the enhanced levels of endogenous ET-1 in VSMC from SHR increase oxidative stress, which through c-Src-mediated activation of growth factor receptors and associated MAP kinase signaling, contribute to the enhanced expression of Giα proteins.     

Sustained Akt/PKB activation and transient attenuation of c-jun N-Terminal kinase in the inhibition of apoptosis by IGF-1 in vascular smooth muscle cells

Characteristics of hVSMC apoptosis and its inhibition by insulin-like growth factor-1 (IGF-1) remain unclear. Also unclear is whether a balance in hVSMCs exists whereby c-Jun N-terminal stress kinases (JNK) promote apoptosis while extracellular signal-regulated (ERK1/2) MAP kinases inhibit cell death. In this study, we examined the involvement of Akt/PKB and its upstream kinase, PDK1 and whether JNK activation correlated with human and rat VSMC apoptosis induced by staurosporine and by c-myc, respectively. We observed a strong, sustained JNK activation (and c-Jun phosphorylation), which correlated with VSMC apoptosis. IGF-1 (13.3 nM), during apoptosis inhibition, transiently inhibited JNK activity at 1 h in a phosphatidylinositol 3-kinase (PI3-K)- and MEK-ERK-dependent manner, as wortmannin (100 nM) or PD98059 (30 M) partially attenuated the IGF-1 effect. PKC down-regulation had no effect on JNK inhibition by IGF-1. While IGF-1 alone produced a strong phosphorylation of Akt/PKB in hVSMCs up to 6 h, it was notably stronger and more sustained during ratmyc and hVSMCs apoptosis inhibition. Further, whereas transient expression of phosphorylated Akt protected VSMCs from apoptosis by nearly 50%, expression of dominant interfering alleles of Akt or PDK1 strongly inhibited IGF-1-mediated VSMC survival. These results demonstrate for the first time that transient inhibition of a pro-apoptotic stimulus in VSMCs may be sufficient to inhibit a programmed cell death and that sustained anti-apoptotic signals (Akt) elicited by IGF-1 are augmented during a death stimulus. Furthermore, PI3-K and ERK-MAPK pathways may cooperate to protect VSMCs from cell death.This work was supported by a grant from the Nebraska cancer and Smoking Related Disease Program, Department of Health, Nebraska, and National Institutes of Health Grants R01HL070885 (D.K.A.) and R01HL073349 (D.K.A.).     

IGF-1对细胞凋亡的抑制调控

胰岛素样生长因子-1（insulin—like growth factor,IGF—1）是胰岛素样生长因子家族中的一种,通过与IGF-1受体相结合产生生物学效应,是通过内分泌、自分泌和旁分泌的三种途径分泌的低分子多肽。近些年来,研究发现IGF-1不仅具有胰岛素类似的功能以及介导生长激素的作用,还是多种类型细胞凋亡的一个重要抑制因子。本文就IGF-1抑制细胞凋亡的信号转导途径和IGF-1对Bcl-2家族、caspases家族以及关键转录因子的调控机制作一综述。     

Transactivation of epidermal growth factor receptor by insulin-like growth factor 1 requires basal hydrogen peroxide

Insulin-like growth factor (IGF-1) plays an important role in prostate cancer development. Recent studies suggest that IGF-1 has mitogenic action through epidermal growth factor receptor (EGFR). However, the mechanism remains largely unknown. Here, we demonstrated in prostate cancer DU145 cells that IGF-1 induced EGFR transactivation, leading to ERK activation. Matrix metalloproteinase-mediated shedding of heparin-binding EGF is involved in this process. Antioxidants and catalase inhibited IGF-1-stimulated EGFR phosphorylation, indicating that H(2)O(2) is required for EGFR activation. However, exogenous H(2)O(2) did not activate EGFR or IGF-1R in DU145 cells. IGF-1 did not induced production of H(2)O(2) in DU145 cells. Our results suggest that transactivation of EGFR by IGF-1 requires basal intracellular H(2)O(2) in DU145 cells.     

Protein tyrosine phosphatase-1B and T-cell protein tyrosine phosphatase regulate IGF-2-induced MCF-7 cell migration

The protein tyrosine phosphatase-1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) have been implicated in down-regulation of tyrosine kinase receptors, conferring anti-oncogenic functions to these PTPases. However, recent work has shown that PTP1B is positively implicated in oncogenic properties of breast cancer cells by regulating the ERK pathway. Here, we studied the function of PTP1B and TC-PTP in IGF-2-induced growth, survival and migration of MCF-7 breast cancer cells. Using siRNA, we showed that reduction in the expression of these PTPases decreased cell growth and ERK phosphorylation. Reduction in the expression of these PTPases did not impair IGF-2 effects on cell survival to acute treatment with 4-OH Tamoxifen. In contrast, IGF-2-induced MCF-7 cell migration was markedly impaired by reduction of PTP1B or TC-PTP expression, independently of the ERK pathway. This novel finding reinforces the potential role of these PTPases as therapeutic targets for treatment of breast cancer.     

Evidence that insulin-like growth factor-1 requires protein kinase C-epsilon, PI3-kinase and mitogen-activated protein kinase pathways to protect human vascular smooth muscle cells from apoptosis

Insulin-like growth factor (IGF)-1 has been implicated in the development of occlusive vascular lesions. Although its role in vascular smooth muscle cell (VSMC) growth and migration are fairly well characterized, anti-apoptotic signals of IGF-1 in human VSMC remain largely unknown. In this study, we examined IGF-1 signals that protect human and rat VSMC from staurosporine (STAU)- and c-myc- induced apoptosis, respectively. Treatment with STAU resulted in apoptotic DNA fragmentation, phosphatidylserine externalization and cell shrinkage, but only occasional VSMC 'blebbing'. STAU-induced death and IGF-1-mediated survival were concentration dependent, while time-lapse video microscopy showed that IGF-1 inhibited c-myc-induced apoptosis by 90%. Pretreatment with mitogen-activated protein kinase/extracellular signal regulated kinase kinase (MEK) inhibitors UO126 and PD098059, or with the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, reversed IGF-1-mediated human VSMC survival by 25-27% and 66%, respectively. Translocation studies showed that IGF-1 activated protein kinase C (PKC)-epsilon, but not PKC-alpha or PKC-delta, even in the presence of STAU, while pharmacological PKC inhibition (Ro-318220 or Go6976) implicated PKC-zeta or a novel PKC isozyme in IGF-1-mediated survival. Transient expression of activated PKC-epsilon but not activated PKC-zeta decreased myc-induced apoptosis in rat VSMC. In human VSMC, antisense oligodeoxynucleotides to PKC-epsilon partially reversed IGF-1-induced survival. In addition, IGF-1 elicited a mild but sustained activation of extracellular signal regulated kinase (ERK)1/2 in human VSMC that was abolished after 1 h in the presence of STAU. PKC downregulation reversed both IGF-1- and PMA-induced ERK activity, but platelet-derived growth factor (PDGF)-induced activity was unchanged. These results indicate for the first time that IGF-1 can protect human VSMC via multiple signals, including PKC-epsilon, PI3-K and mitogen-activated protein kinase pathways.     

IGF-1 induces iNOS expression via the p38 MAPK signal pathway in the anti-apoptotic process in pulmonary artery smooth muscle cells during PAH

Abstract

Apoptosis and cell proliferation are two important cellular processes that determine the accumulation of pulmonary artery smooth muscle cells (PASMC) during pulmonary arterial hypertension (PAH). Insulin-like growth factor 1 (IGF-1) is an endocrine and autocrine/paracrine growth factor that circulates at high levels in the plasma and is expressed in most cell types. IGF-1 has major effects on development, cell growth and differentiation, also tissue repair. Inducible nitric oxide synthase (iNOS) has been shown to serve many vasoprotective roles in vascular smooth muscle cells (VSMCs) including inhibition of VSMC proliferation and migration and stimulation of endothelial cell growth. In this study, we investigated the involvement of iNOS in the process of IGF-1-induced inhibition of PASMC apoptosis. We also examined the role of p38 mitogen-activated protein kinase (MAPK) in the IGF-1-induced iNOS activation. Our results show that exogenous IGF-1 induced the up-regulation of iNOS in PASMC. Immunofluorescence of IGF-1 and iNOS showed a decreased immunostaining of both IGF-1 and iNOS in the cytoplasm and the perinucleus under serum deprivation condition. iNOS inhibition in PASMC in vitro markedly induced IGF-1-mediated anti-apoptosis as assessed by the cell viability measurement, Western blot, mitochondrial potential analysis and nuclear morphology determination. A p38 MAPK inhibitor blocked all the effects of IGF-1 on iNOS. Our findings suggest that IGF-1 inhibits cells apoptosis in PASMC by activating the p38 MAPK–iNOS transduction pathway. This mechanism may contribute to the accumulation of PASMC in early human PAH.     

The Interaction of Protein-tyrosine Phosphatase α (PTPα) and RACK1 Protein Enables Insulin-like Growth Factor 1 (IGF-1)-stimulated Abl-dependent and -independent Tyrosine Phosphorylation of PTPα

Protein tyrosine phosphatase α (PTPα) promotes integrin-stimulated cell migration in part through the role of Src-phosphorylated PTPα-Tyr(P)-789 in recruiting and localizing p130Cas to focal adhesions. The growth factor IGF-1 also stimulates PTPα-Tyr-789 phosphorylation to positively regulate cell movement. This is in contrast to integrin-induced PTPα phosphorylation, that induced by IGF-1 can occur in cells lacking Src family kinases (SFKs), indicating that an unknown kinase distinct from SFKs can target PTPα. We show that this IGF-1-stimulated tyrosine kinase is Abl. We found that PTPα binds to the scaffold protein RACK1 and that RACK1 coordinates the IGF-1 receptor, PTPα, and Abl in a complex to enable IGF-1-stimulated and Abl-dependent PTPα-Tyr-789 phosphorylation. In cells expressing SFKs, IGF-1-stimulated phosphorylation of PTPα is mediated by RACK1 but is Abl-independent. Furthermore, expressing the SFKs Src and Fyn in SFK-deficient cells switches IGF-1-induced PTPα phosphorylation to occur in an Abl-independent manner, suggesting that SFK activity dominantly regulates IGF-1/IGF-1 receptor signaling to PTPα. RACK1 is a molecular scaffold that integrates growth factor and integrin signaling, and our identification of PTPα as a RACK1 binding protein suggests that RACK1 may coordinate PTPα-Tyr-789 phosphorylation in these signaling networks to promote cell migration.     

Insulin-like growth factor-1 regulation of retinal progenitor cell proliferation and differentiation

Strategies to improve retinal progenitor cell (RPC) capacity to yield proliferative and multipotent pools of cells that can efficiently differentiate into retinal neurons, including photoreceptors, could be vital for cell therapy in retinal degenerative diseases. In this study, we found that insulin-like growth factor-1 (IGF-1) plays a role in the regulation of proliferation and differentiation of RPCs. Our results show that IGF-1 promotes RPC proliferation via IGF-1 receptors (IGF-1Rs), stimulating increased phosphorylation in the PI3K/Akt and MAPK/Erk pathways. An inhibitor experiment revealed that IGF-1-induced RPC proliferation was inhibited when the PI3K/Akt and MAPK/Erk pathways were blocked. Furthermore, under the condition of differentiation, IGF-1-pretreated RPCs prefer to differentiate into retinal neurons, including photoreceptors, in vitro, which is crucial for visual formation and visual restoration. These results demonstrate that IGF-1 accelerates the proliferation of RPCs and IGF-1 pretreated RPCs may have shown an increased potential for retinal neuron differentiation, providing a novel strategy for regulating the proliferation and differentiation of retinal progenitors in vitro and shedding light upon the application of RPCs in retinal cell therapy.     

Co-inhibition of epidermal growth factor receptor and type 1 insulin-like growth factor receptor synergistically sensitizes human malignant glioma cells to CD95L-induced apoptosis

Inhibition of epidermal growth factor receptor (EGFR) signaling sensitizes human malignant glioma cells to death ligand-induced apoptosis. However, tumor cells may compensate the loss of EGFR signaling by activation of the type 1 insulin-like growth factor receptor (IGF-1R). We here report that antagonism of the IGF-1R with the small-molecule inhibitor AG1024 in combination with inhibitors of the EGFR synergistically sensitizes human malignant glioma cells to CD95L-induced apoptosis. This cell death is p53-independent, but requires caspase 8 activity. The levels of the receptor, CD95, are not altered by the inhibitors alone or in combination. Analysis of the downstream signaling pathways reveals synergistic inhibition of ribosomal protein S6 phosphorylation by inhibitor co-treatment, suggesting an involvement of the mammalian target of rapamycin pathway. These findings suggest that adding inhibitors of IGF-1R may be a strategy to overcome escape from the anti-apoptotic effects of EGFR inhibition in malignant gliomas.     

GAIP-interacting protein,C-terminus is involved in the induction of zinc-finger protein 143 in response to insulin-like growth factor-1 in colon cancer cells

Previously, we reported that the expression of zinc-finger protein 143 (ZNF143) was induced by insulin-like growth factor-1 (IGF-1) via reactive oxygen species (ROS)- and phosphatidylinositide-3-kinase (PI3-kinase)-linked pathways in colon cancer cells. Here, we investigated whether GAIP-interacting protein, C-terminus (GIPC), a binding partner of IGF-1R, is involved in ZNF143 expression through IGF-1 and IGF-1R signaling in colon cancer cells. The knockdown of GIPC in colon cancer cells reduced ZNF143 expression in response to IGF-1. IGF-1 signaling through its receptor, leading to the phosphorylation and activation of the PI3-kinase-Akt pathway and mitogenactivated protein kinases (MAPKs) was unaffected by the knockdown of GIPC, indicating the independence of the GIPC-linked pathway from PI3-kinase- and MAPK-linked signaling in IGF-1-induced ZNF143 expression. In accordance with previous results in breast cancer cells (Choi et al., 2010), the knockdown of GIPC reduced ROS production in response to IGF-1 in colon cancer cells. Furthermore, the knockdown of GIPC reduced the expression of Rad51, which is regulated by ZNF143, in response to IGF-1 in colon cancer cells. Taken together, these data suggest that GIPC is involved in IGF-1 signaling leading to ZNF143 expression through the regulation of ROS production, which may play a role for colon cancer tumorigenesis.