Expression of insulin-like growth factor-1 and insulin-like growth factor-1 receptors in EL4 lymphoma cells overexpressing growth hormone

Almost all of the previous studies with growth hormone (GH) have been done with exogenously supplied GH and, therefore, involve actions of the hormone through its receptor. However, the actions of endogenous or lymphocyte GH are still unclear. In a previous study, we showed that overexpression of GH (GHo) in a lymphoid cell line resulted in protection of the cells to apoptosis mediated by nitric oxide (NO). In the present study, we show that the protection from apoptosis could be transferred to control cells with culture fluids obtained from GHo cells and blocked by antibodies to the insulin-like growth factor-1 (IGF-1) or antibodies to the IGF-1-receptor (IGF-1R). Northern and Western blot analysis detected significantly higher levels of IGF-1 in cells overexpressing GH. An increase in the expression of the IGF-1R in GHo cells was also detected by Western blot analysis, (125)I-IGF-1 binding and analysis of IGF-1R promoter luciferase constructs. Transfection of GHo cells with a dominant negative IGF-1R mutant construct blocked the generation of NO and activation of Akt seen in GHo cells compared to vector alone control EL4 cells. The results suggest that one of the consequences of the overexpression of GH, in cells lacking the GH receptor, is an increase in the expression of IGF-1 and the IGF-1R which mediate the protection of EL4 lymphoma cells from apoptosis.     

Replicated selection for insulin-like growth factor-1 and body weight in mice

Summary Five generations of divergent selection for plasma concentration of insulin-like growth factor-1 (IGF-1) and for 12-week body weight were carried out in mice, including randomly selected control lines for each trait. All lines were replicated once (12 lines in total). Each replicate line consisted of eight male and eight female parents per generation. Litter size was standardized to eight pups at birth. Mass selection was applied in the selected lines and within-family random selection in the control lines. Blood was taken from the orbital sinus of individual mice at 12 weeks of age for IGF-1 assay. Realized heritabilities were 0.10±0.01 for IGF-1 and 0.41 ± 0.02 for 12-week weight. The realized genetic correlation between IGF-1 and 12-week weight was 0.58 ± 0.01, with a phenotypic correlation of 0.38. Although the genetic correlation between IGF-1 and body weight in mice is moderately positive, 12-week weight responded 3.5 times as fast to weight selection as to selection for IGF-1.     

Responses to divergent selection for plasma concentrations of insulin-like growth factor-1 in mice

A divergent selection experiment with mice, using plasma concentrations of insulin-like growth factor-1 (IGF-1) at 42 days of age as the selection criterion, was undertaken for 7 generations. Lines were not replicated. To obtain sufficient plasma for the IGF-1 assay, blood from four individuals was volumetrically bulked to obtain a litter mean IGF-1 concentration. This necessitated the use of between family selection. Although inbreeding accumulated in a linear fashion in each of the high, control and low lines, the rates were different for each line (3.6, 1.6 and 5.3% per generation for the high, control and low lines, respectively). As a consequence, the effects of selection and inbreeding are confounded in this experiment. Divergence between the high and low lines in plasma concentrations of IGF-1 continued steadily until generation 5. In generations 6 and 7, there was a reduced degree of divergence and this contributed towards the low realized heritability value of 0.15 +/- 0.12. Six-week liveweight showed a steady positive correlated response to selection for or against plasma concentrations of IGF-1 until generation 4 (high-low difference = 1.7 g = 12%). In generation 5, a substantial drop in 6-week liveweight in the low line relative to both the high and control lines occurred (high-low difference, 3.9; g, 25%). This difference was maintained until generation 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

An experimental model of partial insulin-like growth factor-1 deficiency in mice

Insulin-like growth factor-1 (IGF-1) is responsible for many systemic growth hormone (GH) functions although it has an extensive number of inherent activities (anabolic, cytoprotective, and anti-inflammatory). The potential options for IGF-1 therapy arise as a promising strategy in a wide list of human diseases. However, deeper studies are needed from a suitable animal model. All human conditions of IGF-1 deficiency consist in partially decreased IGF-1 levels since total absence of this hormone is hardly compatible with life. The aim of this work was to confirm that heterozygous Igf-1 ^+/? mice (Hz) may be considered as an appropriate animal model to study conditions of IGF-1 deficiency, focusing on early ages. Heterozygous Igf-1 ^+/? mice were compared to homozygous Igf-1 ^+/+ by assessing gene expression by quantitative PCR, serum circulating levels by ELISA, and tissue staining. Compared to controls, Hz mice (25 days old) showed a partial but significant reduction of IGF-1 circulating levels, correlating with a reduced body weight and diminished serum IGFBP-3 levels. Hz mice presented a significant decrease of IGF-1 gene expression in related organs (liver, bone, testicles, and brain) while IGF-1 receptor showed a normal expression. However, gene expression of growth hormone receptor (GHR) was increased in the liver but reduced in the bone, testicles, and brain. In addition, a significant reduction of cortical bone thickness and histopathological alterations in the testicles were found in Hz mice when compared to controls. Finally, the lifelong evolution of IGF-1 serum levels showed significant differences throughout life until aging in mice. Results in this paper provide evidence for considering heterozygous mice as a suitable experimental model, from early stages, to get more insight into the mechanisms of the beneficial actions induced by IGF-1 replacement therapy.     

Identification of selective basophil chemoattractants in human nasal polyps as insulin-like growth factor-1 and insulin-like growth factor-2

In a search for novel leukocyte chemoattractants at sites of allergic inflammation, we found basophil-selective chemoattractant activity in extracts of human nasal polyps. The extracts were fractionated by reverse phase HPLC, and the resulting fractions were tested for leukocyte-stimulating activity using sensitive shape change assays. The basophil-selective activity detected was not depleted by a poxvirus CC-chemokine-binding protein affinity column. This activity was further purified by HPLC, and proteins in the bioactive fractions were analyzed by tandem electrospray mass spectrometry. Insulin-like growth factor-2 (IGF-2) was identified in these HPLC fractions, and the basophil-stimulating activity was inhibited by an anti-IGF-2-neutralizing Ab. Recombinant IGF-2 induced a substantial shape change response in basophils, but not eosinophils, neutrophils, or monocytes. IGF-2 stimulated chemokinesis of basophils, but not eosinophils or neutrophils, and synergized with eotaxin-1/CCL11 in basophil chemotaxis. IGF-2 also caused up-regulation of basophil CD11b expression and inhibited apoptosis, but did not stimulate degranulation or Ca(2+) flux. Recombinant IGF-1 exhibited similar basophil-selective effects as IGF-2, and both growth factors were detected in nasal polyp extracts by ELISA. This is the first demonstration of chemokinetic factors that increase the motility of basophils, but do not act on other granulocytes or monocytes. IGF-1 and IGF-2 could play a role in the selective recruitment of basophils in vivo.     

Variation in plasma concentration of insulin-like growth factor-1 and its covariation with liveweight in mice

Three experiments were undertaken to examine the degree and causes of variation in plasma concentrations of insulin-like growth factor-1 (IGF-1) in mice. The relationship between IGF-1 concentrations and liveweight was also examined. In all three experiments, a number of non-genetic factors were found to contribute significantly to the variation in IGF-1 concentrations, the most important of these being sex and litter size. In one experiment, where pups from 16 litters were cross-fostered to avoid the confounding of maternal and direct genetic effects, a heritability of 0.40 +/- 0.27 was estimated for plasma IGF-1 concentration at 35 days of age. To examine further the existence of genetic variation in plasma concentrations of IGF-1 and the genetic covariation between plasma IGF-1 levels and other body traits, a selection experiment with mice has been initiated. Moderate to strong phenotypic correlations between IGF-1 concentrations and weight at an early age have been found in all three experiments.     

Co-expression of insulin-like growth factor-1 and interleukin-4 in an in vitro inflammatory model

The ailment osteoarthritis (OA) has two aspects – inflammation and cartilage degradation – where combined transgene expression may offer an effective gene therapy. Our present study focuses on the co-expression of interleukin-4 (IL-4) and insulin-like-growth factor-1 (IGF-1), which specifically target inflammation and cartilage repair, respectively. In this study, we analyze the expression of IGF-1 and IL-4 from a single plasmid vector, where each gene is expressed through an independent promoter and enhancer sequence. Regenerative and anti-inflammatory effects of IGF-1 alone and of both IGF-1 and IL-4 were analyzed in an in vitro chondrocyte inflammatory model. Co-expression of both transgenes in primary chondrocytes was ascertained by immunoassays. Following stimulation with IL-1β and TNFα, pro-inflammatory mediators as well as IGF-binding proteins were down-regulated more effectively in the presence of both genes to levels comparable to the non-stimulated control. Further, cartilage regeneration proteins type II collagen and proteoglycans were up-regulated in stimulated cells transfected with IGF-1 alone and in combination with IL-4. The co-expression of IGF-1 and IL-4 shows that both transgenes complement each other by effectively triggering cartilage regeneration and reducing inflammation. Use of combinatorial transgene expression offers a promising avenue in the area of gene therapy in OA.     

Taurine fails to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced striatal dopamine depletion in mice

Taurine, a known antioxidant and neuroprotector has been investigated for its free radical scavenging action in vitro in isolated mitochondria, and tested whether it protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration in mice. Taurine (0.1-10 mM) did not affect 1-methyl-4-phenyl pyridinium-induced hydroxyl radical production in isolated mitochondria. Systemic administration of taurine (250 mg/kg, i.p.) caused a small, but significant loss of dopamine levels in the striatum of mice. Taurine failed to reverse MPTP-induced striatal dopamine depletion, but caused significant increase in dopamine turnover in these animals. In the light of the present study it may be suggested that consumption of taurine may neither help in scavenging of neurotoxic hydroxyl radicals in the brain mitochondria, nor would it help in blocking the process of neurodegeneration.     

Induction of autocrine epidermal growth factor receptor ligands in human keratinocytes by insulin/insulin-like growth factor-1

Autocrine activation of the epidermal growth factor (EGF) receptor on keratinocytes has been recognized as an important growth regulatory mechanism involved in epithelial homeostasis, and, possibly, hyperproliferative diseases. Insulin-like growth factor (IGF)-1 and insulin have been shown to be paracrine keratinocyte mitogens that bind to the type I IGF receptor which is expressed on actively proliferating keratinocytes in situ. In this report, we demonstrate that IGF-1/insulin induced production of keratinocyte-derived autocrine growth factors that bind to the EGF receptor. Increased steady-state mRNA levels for transforming growth factor alpha (TGF-α) and for amphiregulin (AR) were observed upon incubation of keratinocytes with mitogenic concentrations of IGF-1. IGF-1 also induced production and secretion of TGF-α and AR proteins as detected by immunoassays. An EGF receptor antagonistic monoclonal antibody abolished the mitogenic effect of IGF-1 on cultured keratinocytes. These results suggest that stimulation of keratinocyte growth by IGF-1 requires activation of an EGF receptor-mediated autocrine loop. © 1995 Wiley-Liss, Inc.     

Structural differences between insulin and somatomedin-C/insulin-like growth factor-1 receptors revealed by autoantibodies to the insulin receptor

Polyadenylated RNA prepared from first trimester human placenta was translated in a membrane-free cell-free system derived from wheat germ. Analysis of the [³⁵S]methionine-labeled products by SDS-polyacrylamide electrophoresis demonstrated two proteins with apparent M_rs of 14,500 and 16,000 that were specifically immunoprecipitated by antiserum to reduced and carboxylated bovine LH_α, and two different proteins with apparent M_rs of 18,500 and 21,000 that were specifically immunoprecipitated by antiserum to hCG_β. None of these products was sensitive to cleavage by endoglycosidase H, whereas the M_r 21,000 product precipitated by antisera to bovine LH_α and to hCG_α from translations supplemented by canine pancreatic microsomes was processed to a product with M_r 13,000 by endoglycosidase H. We suggest that the two forms of the α and β subunit precursors could arise from the translation of two distinct mRNAs encoding each subunit.     

Central depletion of dopamine in rats by 1-methyl-4-phenylpyridine

Effects of 1-methyl-4-phenylpyridine(MPP+), a putative neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP), on the contents of dopamine were examined in the various regions of the rat brain. Under anesthesia with pentobarbital sodium and flunitrazepam, MPP+ 150 micrograms/rat was intracerebroventricularly infused for 5 hours, at 30 micrograms/100 microliters/hr. Seven days later, the contents of dopamine, but not those of noradrenaline and activities of choline acetyl transferase in the brain were found to be significantly decreased, as compared to findings in the respective controls. The MPP+-induced depletion of dopamine was most evident in the striatum (38% of control). Contents of dopamine in the substantia nigra and ventral tegmental area were not significantly affected by MPP+. These results are interpreted to mean that intracerebroventricular continuous infusion of MPP+, in a relatively low concentration, induces a moderate but relatively specific disruption of central dopaminergic nerve terminals in rats, presumably by the selective accumulation of this neurotoxic agent into these nerve terminals.     

Modulation of chondrocyte migration and aggregation by insulin-like growth factor-1 in cultured cartilage

The effect of insulin-like growth factor-1 (IGF-1) on the behavior of rabbit chondrocytes in cultured collagen (CL) gels initially seeded with 2 × 10⁵ cells/ml was examined. On day 5, the frequency of migrating cells cultured in presence of 100 ng IGF-1/ml was 0.04, which was 54 % of the frequency in IGF-1-free culture. The presence of IGF-1 caused an increase in the frequency of dividing cells from 0.09 to 0.13. These results suggest that IGF-1 suppressed the migration of chondrocytes in the CL gels while stimulating cell division in the initial culture phase. The proteolytic migration of cells was thought to be suppressed by the down-regulation of membrane type 1 matrix metalloproteinase by IGF-1. This contributed to the formation of aggregates with spherical-shaped cells that produced collagen type II.     

Induction of hair growth by insulin-like growth factor-1 in 1,763 MHz radiofrequency-irradiated hair follicle cells

Radiofrequency (RF) radiation does not transfer high energy to break the covalent bonds of macromolecules, but these low energy stimuli might be sufficient to induce molecular responses in a specific manner. We monitored the effect of 1,763 MHz RF radiation on cultured human dermal papilla cells (hDPCs) by evaluating changes in the expression of cytokines related to hair growth. The expression of insulin-like growth factor-1 (IGF-1) mRNA in hDPCs was significantly induced upon RF radiation at the specific absorption rate of 10 W/kg, which resulted in increased expression of B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL-2) and cyclin D1 (CCND1) proteins and increased phosphorylation of MAPK1 protein. Exposure to 10 W/kg RF radiation 1 h per day for 7 days significantly enhanced hair shaft elongation in ex vivo hair organ cultures. In RF-exposed follicular matrix keratinocytes in the hair bulb, the expression of Ki-67 was increased, while the signal for terminal deoxynucleotidyl transferase dUTP nick end labeling was reduced. From these results, we suggest that 1,763 MHz RF exposure stimulates hair growth in vitro through the induction of IGF-1 in hDPCs.     

Human colon cancer stem cells are enriched by insulin-like growth factor-1 and are sensitive to figitumumab

Cancer stem cells (CSCs) are recognized as contributors to cancer progression and therapeutic resistance in liquid and solid malignancies. We analyzed a panel of human colon cancer cell lines for CSC populations by side population and aldehyde dehydrogenase activity. IGF-1 enriches these putative colon CSC populations in a β-catenin-dependent manner. Chemical inhibition of Akt depletes SP cells, and conversely, the overexpression of a constitutively active mutant version of Akt is sufficient to enrich CSC populations. CP-751,871, a fully human antibody with specificity to the IGF-1 receptor, is currently being tested in clinical trials for a variety of solid tumors. CP-751,871 reduces CSC populations in colon cancer cell lines in vitro and reduces tumor growth in vivo. We have identified a novel role for IGF-1 in the enrichment of chemoresistant CSC populations. Our results suggest that CP-751,871 has preferential activity against putative CSC populations and, therefore, may complement current standard chemotherapeutic regimens that target cycling cells.Key words: IGF-1, cancer stem cell, colon cancer, figitumumab     

Human colon cancer stem cells are enriched by insulin-like growth factor-1 and are sensitive to figitumumab

Cancer stem cells (CSCs) are recognized as contributors to cancer progression and therapeutic resistance in liquid and solid malignancies. We analyzed a panel of human colon cancer cell lines for CSC populations by side population and aldehyde dehydrogenase activity. IGF-1 enriches these putative colon CSC populations in a β-catenin-dependent manner. Chemical inhibition of Akt depletes SP cells, and conversely, the overexpression of a constitutively active mutant version of Akt is sufficient to enrich CSC populations. CP-751,871, a fully human antibody with specificity to the IGF-1 receptor, is currently being tested in clinical trials for a variety of solid tumors. CP-751,871 reduces CSC populations in colon cancer cell lines in vitro and reduces tumor growth in vivo. We have identified a novel role for IGF-1 in the enrichment of chemo-resistant CSC populations. Our results suggest that CP-751,871 has preferential activity against putative CSC populations and, therefore, may complement current standard chemotherapeutic regimens that target cycling cells.     

Regulation of insulin/insulin-like growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2

Insulin and insulin-like growth factor-1 (IGF-1) regulate metabolism and body growth through homologous receptor tyrosine kinases that phosphorylate the insulin receptor substrate (IRS) proteins. IRS-2 is an important IRS protein, as it mediates peripheral insulin action and beta-cell survival. In this study, we show that insulin, IGF-1, or osmotic stress promoted ubiquitin/proteasome-mediated degradation of IRS-2 in 3T3-L1 cells, Fao hepatoma, cells and mouse embryo fibroblasts; however, insulin/IGF-1 did not promote degradation of IRS-1 in 3T3-L1 preadipocytes or mouse embryo fibroblasts. MG132 or lactacystin, specific inhibitors of 26S proteasome, blocked insulin/IGF-1-induced degradation of IRS-2 and enhanced the detection of ubiquitinated IRS-2. Insulin/IGF1-induced ubiquitination and degradation of IRS-2 was blocked by inhibitors of phosphatidylinositol 3-kinase (wortmannin or LY294002) or mTOR (rapamycin). Chronic insulin or IGF-1 treatment of IRS-1-deficient mouse embryo fibroblasts inhibited IRS-2-mediated activation of Akt and ERK1/2, which was reversed by lactacystin pretreatment. By contrast, IRS-1 activation of Akt and ERK1/2 was not inhibited by chronic insulin/IGF-1 stimulation in IRS-2-deficient mouse embryo fibroblasts. Thus, we identified a novel negative feedback mechanism by which the ubiquitin/proteasome-mediated degradation of IRS-2 limits the magnitude and duration of the response to insulin or IGF-1.