Vincenzo C. Russo

Co-ordinated and cellular specific induction of the components of the IGF/IGFBP axis in the rat brain following hypoxic-ischemic injury

Insulin-like growth factor 1 (IGF-1) is induced after hypoxic-ischemic (HI) brain injury, and therapeutic studies suggest that IGF-1 may restrict delayed neuronal and glial cell loss. We have used a well-characterised rat model of HI injury to extend our understanding of the modes of action of the IGF system after injury. The induction of the IGF system by injury was examined by in situ hybridization, immunohistochemistry, Northern blot analysis, RNase protection assay and reverse transcriptase-polymerase chain reaction (RT-PCR). IGF-1 accumulated in blood vessels of the damaged hemisphere within 5 h after a severe injury. By 3 days, IGF-1 mRNA was expressed by reactive microglia in regions of delayed neuronal death, and immunoreactive IGF-1 was associated with these microglia and reactive astrocytes juxtaposed to surviving neurones surrounding the infarct. Total IGF-1 receptor mRNA was unchanged by the injury. IGFBP-2 mRNA was strongly induced in reactive astrocytes throughout the injured hemisphere, and IGFBP-3 and IGFBP-5 mRNA were moderately induced in reactive microglia and neurones of the injured hippocampus, respectively. IGFBP-6 mRNA was induced in the damaged hemisphere by 3 days and increased protein was seen on the choroid plexus, ependyma and reactive glia. In contrast, insulin II was not induced. These results indicate cell type-specific expression for IGF-1, IGFBP-2,3,5 and 6 after injury. Our findings suggest that the IGF-1 produced by microglia after injury is transferred to perineuronal reactive astrocytes expressing IGFBP-2. Thus, modulation of IGF-1 action by IGFBP-2 might represent a key mechanism that restricts neuronal cell loss following HI brain injury.

Endogenous IGF-1 regulates the neuronal differentiation of adult stem cells.

Stem cells from the adult forebrain of mice were stimulated to form clones in vitro using fibroblast growth factor‐2 (FGF‐2). At concentrations above 10 ng/ml of FGF‐2, very few clones gave rise to neurons; however, if FGF‐2 was removed after 5 days, 20–30% of clones subsequently gave rise to neurons. The number of neuron‐containing clones and the number of neurons per clone was significantly enhanced, if insulin‐like growth factor (IGF)‐1 or heparin were added subsequent to FGF‐2 removal. The spontaneous production of neurons after FGF‐2 removal was shown to be due to endogenous IGF‐1, since antibodies to IGF‐1 and an IGF‐1 binding protein totally inhibited neuronal production. Similarly, these reagents also abrogated the neuron‐promoting effects of heparin. Thus, it appears that endogenous IGF‐1 may be a major regulator of stem cell differentiation into neurons. Furthermore, it was found that high levels of IGF‐1 or insulin promoted the maturation and affected the neurotransmitter phenotype of the neurons generated. J. Neurosci. Res. 59:332–341, 2000

Neuronal protection from glucose deprivation via modulation of glucose transport and inhibition of apoptosis: a role for the insulin-like growth factor system

Glucose is the brains major energy source; therefore, loss of neuronal cells is a potential consequence of hypoglycaemia. Since apoptosis is a major mechanism of neuronal loss following a range of insults, we explored potent anti-apoptotic systems (IGF-I and bcl-2) as means of enhancing neuronal survival in the face of glucose deprivation. Human neuroblastoma cells (SH-SY5Y, SHEP and SHEP-bcl-2) were exposed to low glucose as a model of glucopenia-induced neuronal damage. Administration of IGF-I and/or over-expression of the survival gene bcl-2 were exploited to attempt to limit neuronal loss. Neuronal survival mechanisms and interactions between these systems were investigated. Low glucose (0.25-2.5 mM) adversely affected cell growth and survival; however, IGF-I ameliorated these outcomes. Over-expression of bcl-2 blunted low glucose-induced apoptosis and up-regulated IGF-I receptor, with the effect of IGF-I addition being negligible on apoptosis, while significantly enhancing mitochondrial activity. In SH-SY5Y cells, IGF-I significantly changed >two-fold mRNA levels of the apoptosis-related genes gadd45, fas, iNOS, NFkB, TRAIL, without further affecting bcl-2 expression. In low glucose, IGF-I acutely enhanced glucose transport and translocation of GLUT1 protein to the cell membrane. GLUT1 mRNA expression was up-regulated by both IGF-I and bcl-2. The potent anti-apoptotic systems IGF-I and bcl-2 are both thus able to enhance cell survival in a glucose-deprived human neuronal model. Although we clearly show evidence of positive cross-talk via bcl-2 modulation of IGF-I receptor, IGF-I also has enhancing effects on mitochondrial function outside the bcl-2 pathway. The common effect of both systems on enhancement of GLUT-1 expression suggests that this is a key mechanism for enhanced survival. These studies also point to the potential use of IGF-I therapy in prevention or amelioration of hypoglycaemic brain injury.

The role of the insulin-like growth factor system in the developing brain.

Insulin-like growth factors (IGFs) play a central role in brain growth and development, with IGF-1, its receptors and binding proteins (IGFBPs) being highly expressed, particularly in the olfactory bulb (OB), representing a local paracrine system. A potential role of IGFBPs in transporting and targeting IGFs to their receptors is supported by the finding that abundant IGFBP-2 is associated with cell surface proteoglycans. An OB organ culture system expressing IGF-1 and IGFBPs represents an in vitro injury model. Although IGF-1 is potently antiapoptotic for neurons and oligodendrocytes, marked gliosis and IGFBP-3 expression are seen, similar to in vivo injury responses, where intraventricular injection of IGF-1 postinjury enhances neuronal survival by blocking apoptosis. In contrast, des (1–3) IGF-1, which has low affinity for IGFBPs, is ineffective, supporting a transport or receptor targeting role for local IGFBPs. We conclude that the IGF system plays a crucial cell survival and growth role in the developing and injured brain and that IGFs have significant therapeutic potential.

IGFBP-2 Enhances VEGF Gene Promoter Activity and Consequent Promotion of Angiogenesis by Neuroblastoma Cells

IGF binding protein (IGFBP)-2 is one of the most significant genes in the signature of major aggressive cancers. Previously, we have shown that IGFBP-2 enhances proliferation and invasion of neuroblastoma cells, suggesting that IGFBP-2 activates a protumorigenic gene expression program in these cells. Gene expression profiling in human neuroblastoma SK-N-SHEP (SHEP)-BP-2 cells indicated that IGFBP-2 overexpression activated a gene expression program consistent with enhancement of tumorigenesis. Regulation was significant for genes involved in proliferation/survival, migration/adhesion, and angiogenesis, including the up-regulation of vascular endothelial growth factor (VEGF) mRNA (>2-fold). Specific transcriptional activation of the VEGF gene by IGFBP-2 overexpression was demonstrated via cotransfection of a VEGF promoter Luciferase construct in SHEP-BP-2. Cotransfection of VEGF promoter Luciferase construct with IGFBP-2 protein in wild-type SHEP cells indicated that transactivation of VEGF promoter only occurs in the presence of intracellular IGFBP-2. Cell fractionation and immunofluorescence in SHEP-BP-2 cells demonstrated nuclear localization of IGFBP-2. These findings suggest that transcriptional activation of VEGF promoter is likely to be mediated by nuclear IGFBP-2. The levels of secreted VEGF (up to 400 pg/10(6) cells) suggested that VEGF might elicit angiogenic activity. Hence, SHEP-BP-2 cells and control clones cultured in collagen sponge were xenografted onto chick embryo chorioallantoic membrane. Neomicrovascularization was observed by 72 h, solely in the SHEP-BP-2 cell xenografts. In conclusion, our data indicate that IGFBP-2 is an activator of aggressive behavior in cancer cells, involving nuclear entry and activation of a protumorigenic gene expression program, including transcriptional regulation of the VEGF gene and consequent proangiogenic activity of NB cell xenografts in vivo.

Interactions between Bcl-2 and the IGF system control apoptosis in the developing mouse brain

The IGF system and the pro-survival Bcl-2 proteins protect cells from apoptosis and play a key role in brain development. In order to examine a possible relationship between these two potent anti-apoptotic systems, we utilised two transgenic mice models overexpressing either Bcl-2 or IGF-I proteins in olfactory bulb (OB) or cerebellar neurons, respectively. We have demonstrated that while the organization of the defined layers of the OB from the bcl-2 transgenic and wildtype mice cultured in serum free medium (SF) was similarly poor, the mitral cell layer from the transgenic mice was expanded and their neurons were well preserved. Addition of IGF-I improved the definition of the layers normally present within the OB, in both wildtype and bcl-2 transgenic mice, and restored wildtype mitral cell layer structure and neuronal survival similar to that in bcl-2 mice, whose mitral cell survival was not further enhanced by IGF-I. Immunoreactivity for IGF-I and IGFBP-2 was markedly increased in these Bcl-2-expressing mitral cells compared to wildtype mice. In newborn IGF-I transgenic mice, cerebellar Purkinje cells overexpressing IGF-I showed markedly increased immunoreactivity for Bcl-2 and IGFBP-2. These studies indicate that in the developing brain IGF-I modulates expression of its major binding protein IGFBP-2, as well as the Bcl-2 protein. In addition apoptosis caused by culturing OBs in SF medium, is inhibited by expression of Bcl-2 in the mitral neurons and is associated with enhanced expression of the IGF system, including IGF-I and IGFBP-2. The later may thus play a role in IGF targeting. This complex interaction between the two potent anti-apoptotic systems is likely to provide a robust system of cell protection during brain development and repair.

Expression of insulin-like growth factor binding protein-3 (IGFBP-3) in human keratinocytes is regulated by EGF and TGF?1

Insulin‐like growth factor‐I (IGF‐I) is essential for normal epidermal homeostasis; however, the role of IGF binding proteins (IGFBPs), regulators of IGF action, remains unclear. Here we examine the regulation of human keratinocyte‐produced IGFBPs by epidermal growth factor (EGF), transforming growth factor beta 1 (TGFβ1), and IGF‐I, growth factors known to be active in skin. In the absence of added growth factors, IGFBP‐3 was the major binding protein secreted into the medium by primary keratinocytes. Addition of EGF or TGFβ1 to keratinocyte cultures resulted in a significant decrease in IGFBP‐3 abundance in conditioned medium when compared with control, untreated cells. Specifically, EGF (50 ng/ml) and TGFβ1 (50 ng/ml) reduced IGFBP‐3 abundance to 15 ± 6% and 22 ± 9%, respectively. Using Northern blot analysis, we found EGF and TGFβ1 (50 ng/ml) to reduce IGFBP‐3 mRNA levels in keratinocytes to 51 ± 12% and 50 ± 38%, respectively, when compared with control, untreated cells. Treatment with IGF‐I or its analogue des(1‐3)IGF‐I did not lead to any consistent change in IGFBP‐3 abundance. However, both IGF‐I and des(1‐3)IGF‐I at 100 ng/ml led to a modest increase in IGFBP‐3 mRNA levels in keratinocytes, suggesting posttranscriptional regulation of IGFBP‐3 abundance. We propose that local modulation of IGFBP‐3 abundance may represent another level of regulation of growth factor action in the epidermis, where EGF and TGFβ1 and possibly other local growth factors specifically regulate the availability of IGF‐I to its keratinocyte receptors. J. Cell. Physiol. 179:201–207, 1999.

Leptin Enhances Insulin Sensitivity by Direct and Sympathetic Nervous System Regulation of Muscle IGFBP-2 Expression: Evidence From Nonrodent Models

Leptin is produced from white adipose tissue and acts primarily to regulate energy balance. Obesity is associated with leptin resistance and increased circulating levels of leptin. Leptin has recently been shown to influence levels of IGF binding protein-2 (IGFBP-2), a protein that is reduced in obesity and type 2 diabetes. Overexpression of IGFBP-2 protects against obesity and type 2 diabetes. As such, IGFBP-2 signaling may represent a novel pathway by which leptin regulates insulin sensitivity. We sought to investigate how leptin regulates skeletal muscle IGFBP-2 levels and to assess the impact of this on insulin signaling and glucose uptake. In vitro experiments were undertaken in cultured human skeletal myotubes, whereas in vivo experiments assessed the effect of intracerebroventricular leptin on peripheral skeletal muscle IGFBP-2 expression and insulin sensitivity in sheep. Leptin directly increased IGFBP-2 mRNA and protein in human skeletal muscle through both signal transducer and activator of transcription-3 and phosphatidylinositol 3-kinase signaling, in parallel with enhanced insulin signaling. Silencing IGFBP-2 lowered leptin- and insulin-stimulated protein kinase B phosphorylation and glucose uptake. In in vivo experiments, intracerebroventricular leptin significantly increased hind-limb skeletal muscle IGFBP-2, an effect completely blocked by concurrent peripheral infusion of a β-adrenergic blocking agent. Sheep receiving central leptin showed improvements in glucose tolerance and circulating insulin levels after an iv glucose load. In summary, leptin regulates skeletal muscle IGFBP-2 by both direct peripheral and central (via the sympathetic nervous system) mechanisms, and these likely impact on peripheral insulin sensitivity and glucose metabolism.

IGFBP-2 nuclear translocation is mediated by a functional NLS sequence and is essential for its pro-tumorigenic actions in cancer cells

IGFBP-2 is highly expressed in both the serum and tumor tissues of most cancers, and is considered one of the most significant genes in the signature of major cancers. IGFBP-2 mainly modulates IGF actions in the pericellular space; however, there is considerable evidence to suggest that IGFBP-2 may also act independently of the IGFs. These IGF-independent actions of IGFBP-2 are exerted either via interactions at the cell surface or intracellularly, via interaction with cytoplasmic or nuclear-binding partners. The precise mechanism underlying the intracellular/intranuclear localization of IGFBP-2 remains unclear. In this study, we investigated IGFBP-2 nuclear localization in several common cancer cells with the aim of dissecting the mechanism of its nuclear trafficking. IGFBP-2 is detected in the nuclei of common cancer cells, including breast, prostate and several neuroblastoma cell lines, using cell fractionation and confocal microscopy. Via nuclear import assays, we show that nuclear entry of IGFBP-2 is mediated by the classical nuclear import mechanisms, primarily through importin-α, as demonstrated by the use of blocking, competition and co-immunoprecipitation assays. Bioinformatics analysis of the IGFBP-2 protein sequence with PSORT II identified a classical nuclear localization signal (cNLS) sequence at 179PKKLRPP185, within the IGFBP-2 linker domain, mutagenesis of which abolishes IGFBP-2 nuclear import. Accordingly, the NLSmutIGFBP-2 fails to activate the VEGF promoter, which would otherwise occur in the presence of wild-type IGFBP-2. As a consequence, no activation of angiogenic processes were observed in NLSmutIGFBP-2 expressing SHEP cells when implanted onto our in vivo quail chorio-allantoic membrane model. Taken together, these data show for the first time that IGFBP-2 possesses a functional NLS sequence and that IGFBP-2 actively translocates into the nucleus by a classical nuclear import mechanism, involving formation of IGFBP-2 complexes with importin-α. Nuclear IGFBP-2 is required for the activation of VEGF expression and consequent angiogenesis.

Activation of a prometastatic gene expression program in hypoxic neuroblastoma cells

The hypoxia inducible factor-1alpha (HIF1alpha) is a key regulator of oxygen homeostasis, modulating cell survival, and growth in cells exposed to hypoxia. In this study, neuroblastoma (NB) cells SH-SY5Y and SK-N-MC were employed to determine the mechanisms regulating adaptation to hypoxia. NB cells were cultured in a serum-free medium in the presence or absence of CoCl(2) (100 muM, hypoxia mimic) for up to 48 h. SH-SY5Y and SK-N-MC cell numbers were not affected by CoCl(2) treatment, while mitochondrial activity was reduced by approximately 50% in SH-SY5Y cells and by approximately 70% in SK-N-MC cells. Intracellular accumulation of HIF1alpha protein was detected as early as 30 min of post-hypoxia, followed by the increase of mRNA for vascular endothelial growth factor (VEGF) and nuclear accumulation of the ID1-2 transcription factors by 4 h. In hypoxic SH-SY5Y NB cells, real-time PCR analysis showed that the genes involved in maintenance of cell-cell and cell-matrix interactions (i.e. adenomatosis polyposis coli, E-cadherin, catenin, EphB2, fibronectin-1, HTATIP2, tissue inhibitor of metalloprotease-4) were down-regulated by up to 90%, while genes involved in enhancement of metastatic behavior (integrin a7b1, hepatocyte growth factor receptor, transforming growth factor-beta1, VEGF, kisspeptin, interleukin-1beta) were dramatically up-regulated above 200%. These changes were all consistent with the induction of epithelial-mesenchymal transition. We have thus demonstrated that NB cell adaptation to hypoxia, in addition to the modulation of HIF1alpha and VEGF expression and nuclear translocation of ID1 and ID2 transcription factors, involve in the activation of a gene expression program consistent with the pro-metastatic events. These processes are probably responsible for the NB cell transition from an adherent phenotype to a highly migratory, invasive and aggressive NB cell type.