Src stimulates insulin-like growth factor I (IGF-I)-dependent cell proliferation by increasing IGF-I receptor number in human pancreatic carcinoma cells

We examined the potential function of Src in human pancreatic carcinoma. Overexpression of kinase-activated SrcY527F resulted in a significant increase of insulin-like growth factor I (IGF-I)-dependent cell proliferation in the cell line PANC-1. Western blotting and competition binding studies demonstrated 2.3 +/- 0.2-fold increase in IGF-I receptor expression and 2.8 +/- 0.4-fold increase in IGF-I-specific binding sites/cell. SrcY527F transfection alone did not change receptor affinity or basal receptor tyrosine phosphorylation, whereas IGF-I-stimulated receptor phosphorylation was increased by 2.1 +/- 0.5-fold. IGF-I mRNA expression and protein secretion did not change to exclude autocrine activation. We conclude that Src stimulates IGF-I-dependent proliferation of PANC-1 cells by increasing the number of IGF-I receptors/cell.     

Two different signal transduction pathways can be activated by transforming growth factor beta 1 in epithelial cells

Signal transduction initiated by transforming growth factor beta 1 (TGF beta 1) was studied in two sublines of the same colon carcinoma cell line, which respond in opposite ways to TGF beta 1, by proliferation or by growth inhibition. TGF beta 1 activates ras proteins within 5 min of addition when it acts to inhibit growth but not when it acts as a mitogen. In both cases TGF beta 1 also rapidly modulates the activities of three protein kinases, detected by their in gel kinase activity on the mitogen-activated protein kinase (MAP kinase) substrate, myelin basic protein (MBP). When TGF beta 1 acts as a mitogen for U9 cells, it increases the activity of MBP kinases of 57, 105, and 130 kDa within 10 min of the addition without detectably activating ras proteins. When TGF beta 1 inhibits the growth of HD3 cells, it activates ras proteins and the 57-kDa MBP kinase within 5 min but inhibits the activity of the 105- and 130-kDa MBP kinases. In HD3 cells ras activation occurred in two signal transduction pathways, one from TGF beta 1 leading to growth inhibition and one from epidermal growth factor (EGF) leading to proliferation. In addition to ras proteins, EGF activates a different set of MBP kinases in HD3 cells than does TGF beta 1, MBP kinases of 85, 57, and 44 kDa. The latter is likely to be the 44-kDa MAP kinase extracellular signal-regulated kinase (erk) 1, because EGF treatment of HD3 cells activates erk1 by increasing its phosphotyrosine level. Therefore, in two closely related epithelial cell lines TGF beta 1 activates two different signal transduction pathways, one ras-dependent and one ras-independent, and modulates the activities of a set of MBP kinases.     

Reconstitution of insulin signaling pathways in rat 3Y1 cells lacking insulin receptor and insulin receptor substrate-1. Evidence that activation of Akt is insufficient for insulin-stimulated glycogen synthesis or glucose uptake in rat 3Y1 cells

Rat 3Y1 cells have endogenous insulin-like growth factor-1 receptors and insulin receptor substrate (IRS)-2, but lack both insulin receptor (IR) and IRS-1. To investigate the role of IR and IRS-1 in effects of insulin, we transfected IR and IRS-1 expression plasmids into cells and reconstituted the insulin signaling pathways. 3Y1 cells stably expressing the c-myc epitope-tagged glucose transporter type 4 (3Y1-GLUT4myc) exhibit no effects of insulin, at physiological concentrations. The 3Y1-GLUT4myc-IR cells expressing GLUT4myc and IR responded to phosphatidylinositol 3,4, 5-trisphosphate (PI-3,4,5-P3) accumulation, Akt activation, the stimulation of DNA synthesis, and membrane ruffling but not to glycogen synthesis, glucose uptake, or GLUT4myc translocation. The further expression of IRS-1 in 3Y1-GLUT4myc-IR cells led to stimulation of glycogen synthesis but not to glucose uptake or GLUT4myc translocation in response to insulin, although NaF or phorbol 12-myristate 13-acetate did trigger GLUT4myc translocation in the cells. These results suggest that, in rat 3Y1 cells, (i) IRS-1 is essential for insulin-stimulated glycogen synthesis but not for DNA synthesis, PI-3,4,5-P3 accumulation, Akt phosphorylation, or membrane ruffling, and (ii) the accumulation of PI-3,4,5-P3 and activation of Akt are insufficient for glycogen synthesis, glucose uptake or for GLUT4 translocation.     

Identification of six novel autophosphorylation sites on fibroblast growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction

Fibroblast growth factor receptor (FGFR) activation leads to receptor autophosphorylation and increased tyrosine phosphorylation of several intra cellular proteins. We have previously shown that autophosphorylated tyrosine 766 in FGFR1 serves as a binding site for one of the SH2 domains of phospholipase Cy and couples FGFR1 to phosphatidylinositol hydrolysis in several cell types. In this report, we describe the identification of six additional autophosphorylation sites (Y-463, Y-583, Y-585, Y-653, Y-654 and Y-730) on FGFR1. We demonstrate that autophosphorylation on tyrosines 653 and 654 is important for activation of tyrosine kinase activity of FGFR1 and is therefore essential for FGFR1-mediated biological responses. In contrast, autophosphorylation of the remaining four tyrosines is dispensable for FGFR1-mediated mitogen-activated protein kinase activation and mitogenic signaling in L-6 cells as well as neuronal differentiation of PC12 cells. Interestingly, both the wild-type and a mutant FGFR1 (FGFR1-4F) are able to phosphorylate Shc and an unidentified Grb2-associated phosphoprotein of 90 kDa (pp90). Binding of the Grb2/Sos complex to phosphorylated Shc and pp90 may therefore be the key link between FGFR1 and the Ras signaling pathway, mito-genesis, and neuronal differentiation.     

Insulin-like growth factor I is an independent coregulatory modulator of natural killer (NK) cell activity

We aimed to investigate the natural killer (NK) cell activity in hGH-deficient adults and to analyze the effect of insulin-like growth factor (IGF)-I in vivo and in vitro on NK cell activity. NK cell activity was measured in a 4-h nonisotopic assay with europium-labeled and cryopreserved K-562 cells. NK-cell numbers were measured after incubation with murine monoclonal CD3 and CD16 antibodies by flow cytometry analysis. In a cross-sectional study, the basal and interferon-beta (IFN-beta) stimulated (1000 IU/ml) NK cell activity of 15 hGH-deficient patients and 15 age- and sex-matched controls was measured. The percentages and absolute numbers of CD3-/16+ NK-cells were not significantly different in the patient vs. control group. The basal and IFN-beta stimulated NK cell activity however was significantly decreased in the patient vs. control group at all effector/target (E/T) cell ratios from 12.5-100 (e.g. 17 +/- 3 vs. 28 +/- 3% lysis without IFN-beta, P < 0.05, and 42 +/- 4 vs. 57 +/- 4% lysis with IFN-beta, P < 0.05; both at E/T 50). IGF-I levels of patients and controls showed a significant positive correlation with NK cell activity (r = 0.37; P < 0.05). In an IGF-I in vitro study (IGF-I in vitro 250-1250 microg/L), the basal and IFN-beta stimulated NK cell activity of 13 hGH-deficient patients and of 18 normal subjects was significantly enhanced by IGF-I in vitro (e.g. GH-deficient patients: 9 +/- 2 vs. 10 +/- 2% lysis without IFN-beta, P < 0.05 and 25 +/- 4 vs. 30 +/- 4% lysis with IFN-beta, P < 0.005; and normal subjects: 15 +/- 3 vs. 23 +/- 3% lysis without IFN-beta, P < 0.001 and 35 +/- 4 vs. 44 +/- 5% lysis with IFN-beta, P < 0.001; both at IGF-I 500 microg/L). In summary, in our cross-sectional study, adult GH-deficient patients showed a significantly lower basal and IFN-beta stimulated NK cell activity than matched controls, despite equal NK cell numbers. IGF-I levels of patients and controls showed a weak positive correlation with NK cell activity. In an in vitro study, IGF-I significantly enhanced basal and IFN-beta stimulated NK cell activity of hGH-deficient patients and also of normal subjects. The decreased NK cell activity in GH-deficient patients may be caused at least in part by low serum IGF-I levels. IGF-I appears to be an independent coregulatory modulator of NK cell activity.     

Decrease in insulin and insulin-like growth factor I (IGF-I) binding to erythrocytes from patients with cystic fibrosis

Guidelines for a healthy diet often recommend limiting dietary sugars and fats. Some researchers have called these aims mutually incompatible, suggesting that fat and sugar intakes, when expressed as percent dietary energy, are inversely linked. Others have argued that sugar, more specifically sucrose, acts as a vehicle for dietary fat and serves to suppress the overall quality of the diet. This study examined the relationship between age, sucrose and fat intakes, body mass index (BMI), and measures of dietary diversity and variety in a community-based sample of 837 French adults. Consistent with other studies, high consumption of added sucrose (in g/day or g/1000 kcal per day) was associated with higher consumption of energy and fat and lower consumption of vegetables and fruit. However, eating patterns were strongly influenced by age. High-sucrose consumers were significantly younger and had lower BMI values than did low-sucrose consumers, who were both older and had higher BMIs. High-sucrose diets had minimal effect on the diet diversity score and were associated with more varied diets, as evidenced by a higher dietary variety score.     

Activation of OX40 signal transduction pathways leads to tumor necrosis factor receptor-associated factor (TRAF) 2- and TRAF5-mediated NF-kappaB activation

We investigated the intracellular signaling of OX40, a member of the tumor necrosis factor receptor family. Activation of NF-kappaB in OX40-transfected HSB-2 cells was detected by electrophoretic mobility shift assay within 30 min after the binding of the ligand gp34. In vitro binding experiments showed that tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2, TRAF3, and TRAF5 but not TRAF4 associated with glutathione S-transferase-OX40 fusion protein. The cotransfection experiments using human embryo kidney cell derived HEK 293T cells showed that TRAF2, TRAF3, and TRAF5 associated with OX40 in vivo. Studies with OX40 deletion mutants demonstrated that the cytoplasmic portion consisting of amino acid sequence 256-263 (GGSFRTPI) was required for the association with TRAFs and NF-kappaB activation. The introduction of the dominant negative mutants of TRAF2 and TRAF5 into HSB-2-OX40 cells suppressed NF-kappaB activation in a dose-dependent manner. In addition, the introduction of TRAF3 together with the dominant negative mutants of TRAF2 or TRAF5 further reduced NF-kappaB activation. These results indicate that the NF-kappaB activation resulting from OX40 stimulation is mediated by both TRAF2 and TRAF5, and is likely to be negatively modulated by TRAF3.     

Association and direct activation of signal transducer and activator of transcription1alpha by platelet-derived growth factor receptor

The binding parameters of [3H]nociceptin were examined in membrane preparations of rat heart and compared with those of [3H]dynorphin A-(1-13) ([3H]Dyn A-(1-13)). Scatchard analysis of [3H]nociceptin binding revealed the presence of two distinct sites: a high affinity (Kd: 583 nM) low capacity (Bmax: 132 pmol/mg protein) site and a low affinity (Kd: 10,316 nM) high capacity (1552 pmol/mg protein) site. Dyn A and related peptides were potent competitors of the binding to the high affinity site with the following rank order of potency: alpha-neo-endorphin > Dyn A-(2-13) = Dyn A-(3-13) > Dyn A-(5-13) > Dyn A-(1-13) > Dyn A > Dyn B > Dyn A-(6-10) > Dyn A-(1-8). Nociceptin was 6.7 times less potent than Dyn A with a Ki of 4.8 microM as compared with 0.72 microM for Dyn A. The order of potency of the various peptides in inhibiting [3H]nociceptin binding correlated well (r = 0.93) with their ability to complete with the binding of [3H]Dyn A-(1-13) (Dumont and Lemaire, 1993). In addition, the high affinity [3H]nociceptin and non-opioid [3H]Dyn A-(1-13) sites were both sensitive to NaCl (120 mM) and the phospholipase C (PLC) inhibitors, U-73122 and neomycin (100 microM). The binding activities were less affected by the weak PLC inhibitor, U-73343, and no effect was observed with the non-hydrolysable GTP analogs. Gpp(NH)p and GTP-gamma-S. Nociceptin (1-50 microM) was also shown to inhibit the uptake of [3H]noradrenaline ([3H]NA) by cardiac synaptosomal preparations. In spontaneously hypertensive rats (SHR), the potency of nociceptin in inhibiting [3H]NA uptake was increased by 1.6-fold as compared with Wistar Kyoto (WKY) control rats and such effect was accompanied by comparable increased levels of cardiac ORL1 mRNA and [3H]nociceptin high affinity sites. These changes correlated well with the previously observed increased levels of non-opioid cardiac [3H]Dyn A-(1-13) sites in SHR (1.3 times as compared with WKY) and increased potency of Dyn A-(1-13) in inhibiting [3H]NA uptake by cardiac synaptosomes in SHR (2.2-fold as compared with WKY) (Dumont and Lemaire, 1995). The results demonstrate that in rat heart the characteristics of the high affinity, low capacity [3H]nociceptin binding site are similar to those of the non-opioid Dyn binding site. The stimulation of this site by nociceptin, Dyn A or related peptides is more likely to produce a modulation of PLC activity and [3H]NA uptake and may participate to the pathophysiology of hypertension.     

Structural changes in insulin-like growth factor (IGF) I mutant proteins affecting binding kinetic rates to IGF binding protein 1 and IGF-I receptor

Ligand binding properties of five single amino acid substituted variants (V11A, D12A, Q15A, Q15E, and F16A) of human insulin-like growth factor I (IGF-I) were analyzed with respect to their binding affinities and binding kinetics to recombinant IGF binding protein 1 (IGFBP-1) and a soluble form of the IGF type I receptor (sIGF-I(R)), respectively. Side chains of the substituted residues are all predicted to be the most surface exposed in the alpha-helical portion of the B-region of the IGF-I molecule. The IGF-I variants were produced as fusion proteins to a IgG(Fc) binding protein domain, Z. Ligand binding kinetic rates were determined using BIAcore biosensor interaction analysis technology. All IGF-I variants showed altered binding affinities to both IGFBP- I and sIGF-I(R). Secondary structure content of the IGF-I variants was estimated using far-UV circular dichroism spectroscopy, followed by variable selection secondary structure calculations. The amount of calculated alpha-helicity is reduced for all the mutants, most predominantly for IGF-I(V11A) and IGF-I(F16A) proteins. Surprisingly, most of the effects of reduced binding affinities to both target proteins are attributed to lowered on-rates of binding, and these are correlated with the amount of alpha-helicity in each IGF-I variant. In addition, in some of the IGF-I variants, lowered off-rates of binding are observed. From the results, we propose that IGF-I is unusually sensitive to structural changes by surface amino acid substitutions in the B-region of the molecule. Therefore, biochemical or biological properties of amino acid substituted variants of IGF-I cannot be used in a straightforward way to dissect the direct involvement in binding of individual amino acid residues since structural changes may be involved.     

Stimulation of tumor growth by recombinant human insulin-like growth factor-I (IGF-I) is dependent on the dose and the level of IGF-I receptor expression

The insulin-like growth factors (IGF) I and II regulate metabolism, mitogenesis, differentiation, and apoptosis. The therapeutic uses of IGF-I have been discussed extensively; however, excessive activity of the IGF ligands and IGF-I receptor has been suggested as a factor in tumorigenesis. The inhibition of apoptosis by IGF-I is believed to be particularly important for the stimulation of tumor growth. This study examined whether systemic recombinant human IGF-I (rhIGF-I) therapy affects the growth of fibrosarcomas derived from fibroblasts expressing the IGF-I receptor at high or naturally occurring densities (1.9 x 10(5) compared with 1.6 x 10(4) IGF-I receptors/cell) in athymic nude mice. Treatment with 4 or 10 mg/kg rhIGF-I resulted in a marked reduction in the tumor latency and stimulated the growth of fibrosarcomas that overexpressed the IGF-I receptor. The latency and growth of fibrosarcomas expressing parental levels of the IGF-I receptor were not affected by rhIGF-I therapy. Analysis of mitosis by histone H3 mRNA in situ hybridization and of apoptosis by terminal deoxynucleotidyl transferase-mediated nick end labeling assay indicated that rhIGF-I-stimulated tumor growth was associated with a marked increase in mitogenesis; however, there was no evidence for any significant effect on apoptosis. These data imply that: (a) systemic rhIGF-I can stimulate the growth of tumors directly by stimulating mitosis; and (b) a reasonable level of IGF-I receptor expression is required for stimulation of tumor growth by systemic rhIGF-I.     

Synergistic activation of MAP kinase (ERK1/2) by erythropoietin and stem cell factor is essential for expanded erythropoiesis

Stem cell factor (SCF) and erythropoietin (EPO) work synergistically to support erythropoiesis, but the mechanism for this synergism is unknown. By using purified human erythroid colony-forming cells (ECFC), we have found that SCF and EPO synergistically activate MAP kinase (MAPK, ERK1/2), which correlates with the cell growth and thus may be responsible for the synergistic effects. Treatment of the cells with PD98059 and wortmannin, inhibitors of MEK and PI-3 kinase, respectively, inhibited the synergistic activation of MAPK and also the cell growth, further supporting this conclusion. Wortmannin only inhibits MAPK activation induced by EPO but not that by SCF, suggesting that SCF and EPO may activate MAPK through different pathways, which would facilitate synergy. Furthermore, EPO, but not SCF, led to activation of STAT5, whereas SCF and wortmannin had no effect on the EPO-induced STAT5 activation, suggesting that STAT5 is not involved in the synergistic action of SCF and EPO. Together, the data suggest that synergistic activation of MAPK by SCF and EPO is essential for expanded erythropoiesis.     

Recovery of growth hormone release from suppression by exogenous insulin-like growth factor I (IGF-I): evidence for a suppressive action of free rather than bound IGF-I

To determine the time course of recovery of GH release from insulin-like growth factor I (IGF-I) suppression, 11 healthy adults (18-29 yr) received, in randomized order, 4-h i.v. infusions of recombinant human IGF-I (rhIGF-I; 3 microg/kg-h) or saline (control) from 25.5-29.5 h of a 47.5-h fast. Serum GH was maximally suppressed within 2 h and remained suppressed for 2 h after the rhIGF-I infusion; during this 4-h period, GH concentrations were approximately 25% of control day levels [median (interquartile range), 1.2 (0.4-4.0) vs. 4.8 (2.8-7.9) microg/L; P < 0.05]. A rebound increase in GH concentrations occurred 5-7 h after the end of rhIGF-I infusion [7.6 (4.6 -11.7) vs. 4.3 (2.5-6.0) microg/L; P < 0.05]. Thereafter, serum GH concentrations were similar on both days. Total IGF-I concentrations peaked at the end of the rhIGF-I infusion (432 +/- 43 vs. 263 +/- 44 microg/L; P < 0.0001) and remained elevated 18 h after the rhIGF-I infusion (360 +/- 36 vs. 202 +/- 23 microg/L; P = 0.001). Free IGF-I concentrations were approximately 140% above control day values at the end of the infusion (2.1 +/- 0.4 vs. 0.88 +/- 0.3 microg/L; P = 0.001), but declined to baseline within 2 h after the infusion. The close temporal association between the resolution of GH suppression and the fall of free IGF-I concentrations, and the lack of any association with total IGF-I concentrations suggest that unbound (free), not protein-bound, IGF-I is the major IGF-I component responsible for this suppression. The rebound increase in GH concentrations after the end of rhIGF-I infusion is consistent with cessation of an inhibitory effect of free IGF-I on GH release.     

Insulin-like growth factor 1 (IGF-1) alters drug sensitivity of HBL100 human breast cancer cells by inhibition of apoptosis induced by diverse anticancer drugs

In this study, we tested the hypothesis that insulin-like growth factor-1 (IGF-1) modulates apoptosis in human breast cancer cells, HBL100, induced by diverse chemotherapeutic drugs. IGF-1 increased cell survival of HBL100 cells treated with 5-fluorouracil (antimetabolite), methotrexate (antimetabolite), tamoxifen (antiestrogen/antiproliferative), or camptothecin (topoisomerase 1 inhibitor) and after serum withdrawal. Elevated cell survival was not due to an increase in cell proliferation by IGF-1, but rather to an inhibition of apoptosis. Evidence for death by apoptosis was supported by cellular morphology and DNA fragmentation. There were no changes observed in Bcl-2 protein or bax mRNA levels. Extracellular matrix (ECM) is known to influence the apoptotic response of cells; therefore, the antiapoptotic effect of IGF-1 on breast cancer cells was examined using different ECMs: laminin, collagen IV, or Matrigel. IGF-1 protected cells from apoptosis induced by methotrexate on all ECMs tested, providing the first evidence that IGF-1 protects against apoptosis in three-dimensional culture systems. These data provide the rationale to search for drugs that lower serum IGF-1 in an effort to improve the efficacy of chemotherapeutic drugs for the treatment of breast cancer.     

Opposite regulation of tyrosine-phosphorylation of p130(Cas) by insulin and insulin-like growth factor I

To investigate the difference in signaling between insulin and insulin-like growth factor I (IGF-I), we studied the effects of these hormones on the phosphorylation state of Crk-associated substrate (Cas) in cells expressing human insulin receptor (HIRc). In the basal state, Cas was heavily tyrosine-phosphorylated, and insulin dephosphorylated Cas in a time- and dose-dependent manner. On the other hand, IGF-I phosphorylated rather than dephosphorylated Cas in HIRc cells. In HIRY/F2 cells expressing a mutant insulin receptor lacking a binding site of SHP-2, a protein-tyrosine phosphatase containing src homology 2 (SH2) regions, insulin accelerated phosphorylation of Cas, as did IGF-I. In HIRc cells expressing a mutant SHP-2 lacking a PTPase domain (DeltaPTP), which interfered with SHP-2 function, insulin failed to dephosphorylate Cas. In whole cell lysate obtained in the basal state, Cas bound to a glutathione-S transferase fusion protein containing SH2 domains of SHP-2 and dissociated from this GST protein in response to insulin. These results indicate that the opposite regulation of Cas phosphorylation by insulin and IGF-I may be mediated through different properties of their receptors, and that the interaction of the insulin receptor with SHP-2 may play an important role in determining the tyrosine-phosphorylation state of Cas.     

Growth inhibition by transforming growth factor beta (TGF-beta) type I is restored in TGF-beta-resistant hepatoma cells after expression of TGF-beta receptor type II cDNA

The growth of human hepatoma Hep 3B cells is potently inhibited by TGF-beta 1 (ID50 = 0.2 ng/ml, 8 pM). A mutant cell line was derived that was not inhibited in growth by TGF-beta 1 at 5 ng/ml (200 pM) and that lacked TGF-beta receptor type II (TGF-beta RII) gene. Transfection of the cloned cDNA for human TGF-beta RII to this mutant cell line restored receptor expression as well as the inhibition in growth by TGF-beta 1. In both wild-type and mutant cells stably transfected with TGF-beta RII cDNA, TGF-beta RII coimmunoprecipitated with TGF-beta receptor type I in the presence of ligand. These experiments provide direct evidence for the role of TGF-beta RII in the inhibitory effect of TGF-beta on growth and suggest that TGF-beta RII acts by means of a heteromeric surface complex with TGF-beta receptor type I.     

Antitumor effects elicited by antisense-mediated downregulation of the insulin-like growth factor I receptor (review)

The parasporal inclusion proteins of the type strain of Bacillus thuringiensis serovar higo (H44), that have moderate mosquitocidal activity, were characterized. The purified parasporal inclusions, spherical in shape, were examined for activity against the two mosquito species, Culex pipiens molestus and Anopheles stephensi and the moth-fly, Telmatoscopus albipunctatus. The LC50 values of the inclusion for the two mosquitoes were 3.41 and 0.15 microgram.ml-1, respectively. No mortality was shown for T. albipunctatus larvae by the inclusions at concentrations up to 1 mg ml-1. Solubilized parasporal inclusions exhibited no haemolytic activity against sheep erythrocytes. Parasporal inclusions consisted of eight proteins with molecular masses of 98, 91, 71, 63, 59, 50, 44 and 27 kDa. Of these, the 50 and 44 kDa proteins were the major components. Analysis with immunoblotting revealed that, among several inclusion proteins of B. thuringiensis serovar israelensis, only two proteins of 130 kDa and 110 kDa reacted weakly with antibodies against higo proteins. N-terminal amino acid sequences of the 98, 91, and 71 kDa proteins showed 85-100% identity to those of the two established Cry protein classes, Cry4A and Cry10A.