Unbalancing p53/Mdm2/IGF-1R axis by Mdm2 activation restrains the IGF-1-dependent invasive phenotype of skin melanoma.

Melanoma tumors usually retain wild-type p53; however, its tumor-suppressor activity is functionally disabled, most commonly through an inactivating interaction with mouse double-minute 2 homolog (Mdm2), indicating p53 release from this complex as a potential therapeutic approach. P53 and the tumor-promoter insulin-like growth factor type 1 receptor (IGF-1R) compete as substrates for the E3 ubiquitin ligase Mdm2, making their relative abundance intricately linked. Hence we investigated the effects of pharmacological Mdm2 release from the Mdm2/p53 complex on the expression and function of the IGF-1R. Nutlin-3 treatment increased IGF-1R/Mdm2 association with enhanced IGF-1R ubiquitination and a dual functional outcome: receptor downregulation and selective downstream signaling activation confined to the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. This Nutlin-3 functional selectivity translated into IGF-1-mediated bioactivities with biphasic effects on the proliferative and metastatic phenotype: an early increase and late decrease in the number of proliferative and migratory cells, while the invasiveness was completely inhibited following Nutlin-3 treatment through an impaired IGF-1-mediated matrix metalloproteinases type 2 activation mechanism. Taken together, these experiments reveal the biased agonistic properties of Nutlin-3 for the mitogen-activated protein kinase pathway, mediated by Mdm2 through IGF-1R ubiquitination and provide fundamental insights into destabilizing p53/Mdm2/IGF-1R circuitry that could be developed for therapeutic gain.

Chapter Seven - When Phosphorylation Encounters Ubiquitination: A Balanced Perspective on IGF-1R Signaling.

Cell-surface receptors govern the critical information passage from outside to inside the cell and hence control important cellular decisions such as survival, growth, and differentiation. These receptors, structurally grouped into different families, utilize common intracellular signaling-proteins and pathways, yet promote divergent biological consequences. In rapid processing of extracellular signals to biological outcomes, posttranslational modifications offer a repertoire of protein processing options. Protein ubiquitination was originally identified as a signal for protein degradation through the proteasome system. It is now becoming increasingly recognized that both ubiquitin and ubiquitin-like proteins, all evolved from a common ubiquitin structural superfold, are used extensively by the cell and encompass signal tags for many different cellular fates. In this chapter we examine the current understanding of the ubiquitin regulation surrounding the insulin-like growth factor and insulin signaling systems, major members of the larger family of receptor tyrosine kinases (RTKs) and key regulators of fundamental physiological and pathological states.

Dietary protein deprivation decreases the serine phosphorylation of insulin receptor substrate-1 in rat skeletal muscle.

Evidence has shown that protein malnutrition tends to increase peripheral insulin sensitivity, but the molecular mechanism underlying this increase is not yet clear. Here we show that, in rat muscle, the state of insulin receptor (IR) substrate-1 (IRS-1), a pivotal component of the signaling pathway of the IR, changes drastically according to protein supply. After rats were fed a protein-free diet (PF) or a 12% casein diet for 1 week, their IR and IRS-1 states were analyzed by immunoblotting using various antibodies. PF slightly increased the amount of IR without affecting the state of IR tyrosine phosphorylation. In contrast, PF decreased the amount of IRS-1 and markedly increased phosphorylation of IRS-1 tyrosine residues after insulin injection. Moreover, IRS-1 in PF rats exhibited faster mobility in SDS-PAGE as well as far less phosphorylation of Ser612 and Ser307, indicating hypophosphorylation on its serine residues. Results of additional experiments using energy-restricted (pair-fed) rats and streptozotocin-induced diabetic rats suggest that dietary protein deficiency by itself alters serine phosphorylation of IRS-1, while the up-regulation of tyrosine phosphorylation requires other factors, such as a reduction in basal plasma insulin. The serine dephosphorylation followed by up-regulation of insulin-dependent IRS-1 tyrosine phosphorylation in skeletal muscle of PF rats in vivo is similar to a phenomenon observed in cultured cells under restriction of amino acids in the medium. With these findings, it could be inferred that the reduction of serine phosphorylation contributes to the sensitization of IRS-1 to IR tyrosine kinase under protein malnutrition.

Long-term hormonal regulation of the cAMP-specific phosphodiesterases in cultured FRTL-5 thyroid cells.

Thyrotropin (TSH) and pharmacological agents that elevate intracellular cAMP concentrations potentiate the mitogenic response of FRTL-5 thyroid cells to insulin-like growth factor-I (IGF-I). This study was undertaken to determine the role of cAMP phosphodiesterases (PDEs) in this TSH-dependent regulation. Incubation of FRTL-5 cells with TSH, forskolin, or dibutyryl cAMP gradually induced the PDE activity, and treatment for 24 h produced a marked increase in type 4 high affinity cAMP PDEs. Under basal conditions, transcripts corresponding to PDE4A, PDE4B, PDE4C, and PDE4D were present. Stimulation for 24 h by TSH, forskolin or dibutyryl cAMP induced an increase in mRNA levels of PDE4B, PDE4D, and PDE4C. To understand the role of this cAMP-dependent PDE regulation in the potentiation of the mitogenic response to IGF-I, thymidine incorporation into DNA in response to IGF-I and TSH was measured in the absence or presence of PDE inhibitors. Exposure of the cells to 3-isobutyl-1-methylxanthine (IBMX) or RO 20-1724 had opposing effects on thymidine incorporation into DNA, depending on the stimulus applied. When IGF-I was used alone, both IBMX and RO 20-1724 potentiated IGF-I-stimulated thymidine incorporation. However, when IGF-I and TSH at high concentrations were used in combination, these PDE inhibitors blocked thymidine incorporation into DNA. In addition, these inhibitors depressed the synergistic increase in cyclin D1 and cyclin D- or cyclin E-associated cyclin-dependent kinase (CDK) activity that is induced by TSH and IGF-I. Increased CDK activities have been shown to play a crucial role in progression through the G(1)/S phase of the cell cycle. These data demonstrate that TSH produces marked changes in the cAMP degradative pathway of FRTL-5 cells by regulating the expression of cAMP PDEs. The regulation of the intracellular cAMP levels by this mechanism may contribute to the TSH- and IGF-I-dependent control of the entry into the S phase of the cell cycle through changes in the cyclin/CDK system in FRTL-5 cells.

Dietary restriction of single essential amino acids reduces plasma insulin-like growth factor-I (IGF-I) but does not affect plasma IGF-binding protein-1 in rats.

The effects of dietary restriction of a single essential amino acid (EAA) on insulin-like growth factor-I (IGF-I) and IGF-binding protein (IGFBP)-1 were investigated in rats. Rats were fed experimental diets containing amino acid (AA) mixtures in which the concentrations of all EAA were at levels recommended by the National Research Council (control), in which a single EAA was restricted to 20% of that of the control diets (Leu(-), Lys(-), Met(-) or Thr(-)), or in which the diet was devoid of amino acids (AA(-)). To eliminate the effect of differences in energy intake, rats were fed the mean amount of food as consumed by the AA(-) group on the previous day. Growth was significantly retarded in rats fed diets restricted in just one EAA compared with that of rats fed the control diet, and further growth retardation was observed in rats fed the AA(-) diet. On the other hand, the plasma IGF-I concentrations in the groups with a single EAA restriction or in the AA(-) group were 66% (P: < 0. 05) and 50% (P: < 0.05) of that of the control group, respectively. The effect of any single EAA restriction was not significantly different from that of total AA deprivation. The plasma IGFBP-1 concentration in the control group did not differ from that of rats fed diets with the single EAA restrictions except for methionine restriction, but it was approximately 6-fold greater in the AA(-) group. Differences in plasma IGFBP-1 concentration under these conditions could be explained by differences in hepatic IGFBP-1 mRNA contents. Based on these results, we conclude that restriction of single EAA does not affect IGFBP-1 synthesis in vivo, although the deprivation of a single EAA has been reported to increase IGFBP-1 production in hepatocyte cultures. Our results also indicated that a single EAA restriction decreased IGF-I production but did not affect IGFBP-1 production. The present study suggests that not only plasma IGF-I, but also IGFBP-1, affects the magnitude of growth retardation in vivo.

Tyrosine kinase and phosphatidylinositol 3-kinase activation are required for cyclic adenosine 3',5'-monophosphate-dependent potentiation of deoxyribonucleic acid synthesis induced by insulin-like growth factor-I in FRTL-5 cells.

In previous studies, we showed that pretreatment of rat FRTL-5 thyroid cells with TSH, or other agents that increased intracellular cAMP, markedly potentiated DNA synthesis in response to insulin-like growth factor-I (IGF-I). In addition, we found that TSH pretreatment caused an increase in tyrosine phosphorylation of intracellular proteins including an unidentified 125-kDa protein that was well correlated with the TSH-potentiating effect on DNA synthesis induced by IGF-I. These results suggested that cAMP amplified IGF-I-dependent signals for cell growth through changes of cAMP-dependent tyrosine phosphorylation. The present studies were undertaken to determine how tyrosine kinase activation followed by an increase in tyrosine phosphorylation is required for cAMP-dependent potentiation of DNA synthesis induced by IGF-I in this cell line. First of all, we measured tyrosine kinase or protein-tyrosine phosphatase activities in the cell lysates by the in vitro assay. Chronic treatment with TSH or (Bu)2-cAMP stimulated tyrosine kinase activity in the particulate fraction and protein-tyrosine phosphatase activity in the soluble fraction, suggesting that tyrosine kinase plays more important roles for a cAMP-dependent increase in tyrosine phosphorylation of intracellular proteins. The increased tyrosine kinase activity was sensitive to genistein, a potent tyrosine kinase inhibitor. Genistein abolished both the cAMP-dependent increase in tyrosine phosphorylation of the 125-kDa protein and the enhanced DNA synthesis induced by IGF-I in a similar concentration-dependent manner. The only tyrosine-phosphorylated protein associated with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase in response to cAMP was 125 kDa. In addition, we found that PI 3-kinase activity bound to p85 subunit significantly increased after (Bu)2cAMP treatment. These results suggested that cAMP stimulates PI 3-kinase through tyrosine phosphorylation of the 125-kDa protein. We then measured DNA synthesis in cells pretreated for 24 h with TSH or (Bu)2cAMP in the absence or presence of LY294002, a PI 3-kinase inhibitor, followed by treatment with IGF-I for 24 h. Presence of LY294002 during TSH or (Bu)2cAMP pretreatment completely abolished cAMP-dependent potentiation of DNA synthesis induced by IGF-I. These results suggest that in FRTL-5 cells cAMP activates genistein-sensitive tyrosine kinases that in turn activate PI 3-kinase activity. These mechanisms appear to be necessary for cAMP-dependent potentiation of the DNA synthesis induced by IGF-I.

Amino acid regulation of gene transcription of rat insulin-like growth factor-binding protein-1.

To investigate the molecular mechanisms of increased transcription of the insulin-like growth factor-binding protein-1 (IGFBP-1) gene in dietary protein-deprived animals, the cis-acting sequence that is involved in this regulation was analyzed. We first showed that IGFBP-1 gene transcription was up-regulated by amino acid deprivation in cultured liver cell lines: H4IIE and HuH-7. Since HuH-7 cells showed a greater increase in IGFBP-1 mRNA in response to amino acid deprivation, this cell line was used in further experiments. Using a promoter function assay, we found that up-regulation of promoter activity responding to amino acid deprivation was abolished by deleting the region between -112 and -81 bp from the cap site from the gene construct. This cis-acting region includes the insulin-responsive element (IRE) and glucocorticoid responsive element (GRE) of IGFBP-1. In summary, the present observation suggests that the 32-bp (-112 to -81) in the IGFBP-1 gene 5' promoter region is involved in the induction of the IGFBP-1 gene in response to amino acid deprivation.

Short term feedback regulation of cAMP in FRTL-5 thyroid cells. Role of PDE4D3 phosphodiesterase activation.

Together with a transient accumulation of intracellular cAMP, thyrotropin (TSH) stimulation of the FRTL-5 thyroid cell induces phosphorylation and activation of a cAMP-specific phosphodiesterase (PDE4D3). Here we have investigated the impact of PDE4D3 activation on hormone responsiveness. Stimulation of FRTL-5 cells with TSH caused an increase in PDE activity within 3 min, with a maximal stimulation reached after 5 min. Preincubation with the protein kinase A (PKA) inhibitor H89 or (R(p))-cAMPS, but not with the inactive isomer H85, blocked this activation. Preincubation with PKA inhibitors also blocked the shift in mobility of the PDE4D3 protein. Under these conditions, H89, but not H85, potentiated the cAMP accumulation induced by TSH. Incubation of FRTL-5 cells with the PKA activator 8-(4-chlorophenylthio)adenosine-cAMP caused an increase in PDE activity and a decrease in the endogenous cAMP, confirming the presence of a PKA-PDE feedback loop. MA-10 Leydig tumor cells stably transfected with either a wild type PDE4D3 or a PDE4D3 with mutations in the PKA phosphorylation sites showed an increase in PDE activity when compared with control cells. Human choriogonadotropin or Bt(2)cAMP treatment induced a stimulation of PDE activity in cells transfected with wild type PDE4D3, whereas the activation was absent in mutant- and control-transfected cells. The increase in cAMP accumulation elicited by human choriogonadotropin was reduced in cells transfected with the wild type PDE4D3, but not in cells transfected with the mutant PDE. Rolipram, a specific inhibitor of PDE4, restored the cAMP accumulation in the PDE4D3-transfected cells. These data provide evidence that a rapid activation of PDE4D3 is one of the mechanisms determining the intensity of the cAMP signal.

Insulin-like growth factor-I potentiates protein synthesis induced by thyrotropin in FRTL-5 cells: comparison of induction of protein synthesis and DNA synthesis.

In FRTL-5 cells, we and others have shown that TSH and insulin-like growth factor-I (IGF-I) stimulate DNA synthesis synergistically. The present study was undertaken to determine whether interaction between TSH and IGF-I also affects protein synthesis in this cell line, and if so by what mechanism. Quiescent cells were treated with TSH and/or IGF-I and [3H]valine incorporation into the acid-insoluble fraction was measured as a parameter of protein synthesis. Similar to their effects on cell proliferation, TSH or IGF-I alone induced protein synthesis only slightly, but treatment with a combination of TSH and IGF-I (or insulin with about a 100-fold higher concentration than IGF-I) greatly increased protein synthesis. The presence of IGF-I potentiated a TSH-concentration-dependent increase in protein synthesis and in DNA synthesis. In addition, we observed this potentiation when the cells were treated with other cAMP-generating agents and cAMP analogues instead of TSH. We have shown that priming with TSH potentiated DNA synthesis induced by IGF-I, whereas pretreatment with IGF-I enhanced protein synthesis induced by TSH. This observation suggested that protein synthesis and DNA synthesis were potentiated through different mechanisms. From an analysis of cAMP production, it appears that the potentiation of protein synthesis may be explained by an IGF-I-dependent increase in cAMP production induced by TSH at least in part. On the other hand, IGF-I and TSH stimulated (alpha-aminoisobutyric acid (AIB) uptake synergistically, but RNA synthesis induced by IGF-I was depressed by TSH. From these results, we concluded that in FRTL-5 cells, IGF-I potentiated protein synthesis induced by TSH by means of complex mechanisms and the interaction between IGF-I and cAMP-dependent pathways may also have a physiological meaning in regulating protein anabolism.

Influence of fabrication errors on wölter mirror imaging performance.

The resolution of the Wölter mirror, which is utilized as an objective in soft-x-ray microscopes, is limited by fabrication errors. We studied the relation between fabrication errors and imaging performance of the Wölter mirror to determine how this performance could be improved. Figure errors, which are characterized by low spatial frequency, were analyzed by ray tracing, and surface roughness, characterized by high spatial frequency, was analyzed by modified ray tracing. Modified ray tracing was based on ray tracing but took scattering into account. The results of these analyses were compared with experimental data. As a result, we obtained a simple and practical fabricating tolerance criterion that may be employed to obtain higher Wölter mirror resolution. Additionally, we discuss problems in current Wölter mirror fabrication techniques and the changes that might be made in both the design and the fabrication process to improve imaging performance.

Interaction between cAMP-dependent and insulin-dependent signal pathways in tyrosine phosphorylation in primary cultures of rat hepatocytes.

The present studies were undertaken to determine whether the interaction between cAMP-dependent and insulin-dependent pathways in primary cultures of rat hepatocytes affects biological functions and tyrosine phosphorylation. Quiescent hepatocytes were pretreated with dibutyryl cAMP or cAMP-generating agents such as glucagon, and then treated or not with insulin. Preincubation for 6 h with dibutyryl cAMP or glucagon enhanced the effect of insulin on DNA synthesis, but not the effect of insulin on amino acid transport or glycogen and protein synthesis. Tyrosine phosphorylation of intracellular proteins was determined by immunoblot analysis using an anti-phosphotyrosine antibody. Maximum tyrosine phosphorylation of a 195 kDa protein, which may be a substrate of insulin receptor kinase, of 175-180 kDa proteins, including insulin receptor substrate (IRS)-1, and of 90-95 kDa proteins, including the insulin receptor beta-subunit, was reached within 30 s of incubation with insulin. Pretreatment for about 3 h with dibutyryl cAMP or cAMP-generating agents clearly increased insulin-dependent tyrosine phosphorylation of the 195 kDa protein, but not IRS-1, IRS-2 or the insulin receptor beta-subunit. Because dibutyryl cAMP and cAMP-generating agents did not increase insulin receptor number or its kinase activity, the effect of cAMP on this potentiation of tyrosine phosphorylation is assumed to be exerted at a step distal to insulin receptor kinase activation. The potentiation by cAMP pretreatment of insulin-stimulated tyrosine phosphorylation may in part be secondary to inhibition of phosphotyrosine phosphatase activity, because cAMP pretreatment blunted the effect of Na3VO4 on the net tyrosine phosphorylation of the 195 kDa protein as compared with cells pretreated with no additive. In summary, the interactions between cAMP-dependent and insulin-dependent pathways that lead to augmentation of DNA synthesis appear to parallel the changes in tyrosine phosphorylation. Further studies will be required to determine whether there is a causal relationship between these phenomena.

Effect of protein restriction on the messenger RNA contents of bone-matrix proteins, insulin-like growth factors and insulin-like growth factor binding proteins in femur of ovariectomized rats.

It has been reported that loss of ovarian oestrogen after menopause or by ovariectomy causes osteoporosis. In order to elucidate the effect of dietary protein restriction on bone metabolism after ovariectomy, we fed ovariectomized young female rats on a casein-based diet (50 g/kg diet (protein restriction) or 200 g/kg diet (control)) for 3 weeks and measured mRNA contents of bone-matrix proteins such as osteocalcin, osteopontin and alpha 1 type I collagen, insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) in femur. Ovariectomy decreased the weight of fat-free dry bone and increased urinary excretion of pyridinium cross-links significantly, although dietary protein restriction did not affect them. Neither ovariectomy nor protein restriction affected the content of mRNA of osteopontin and osteocalcin; however, ovariectomy increased and protein restriction extensively decreased the alpha 1 type I collagen mRNA content in bone tissues. Ovariectomy increased IGF-I mRNA only in the rats fed on the control diet. Conversely, protein restriction increased and ovariectomy decreased the IGF-II mRNA content in femur. Furthermore, the contents of IGFBP-2, IGFBP-4 and IGFBP-5 mRNA increased, but the content of IGFBP-3 mRNA decreased in femur of the rats fed on the protein-restricted diet. In particular, ovariectomy decreased the IGFBP-2 mRNA content in the protein-restricted rats and the IGFBP-6 mRNA content in the rats fed on the control diet. These results clearly show that the mRNA for some of the proteins which have been shown to be involved in bone formation are regulated by both quantity of dietary proteins and ovarian hormones.

Familial dysautonomia (Riley-Day syndrome).

Familial dysautonomia, also known as Riley-Day syndrome, is a disorder of autonomic nervous system with an autosomal recessive mode of inheritance. Reduction and/or loss of unmyelinated and small myelinated fibers is found, as reduction of dopamine beta-hydroxylase in blood. The diagnosis is based on clinical features: diminished lacrimation, insensitivity to pain, poor temperature control, abolished deep tendon reflexes, postural hypotension, vomiting attacks, poor motor coordination, and mental retardation. The treatment is symptomatic and many children die during the first years of life, usually as a result of repeated aspiration pneumonia. We report the case of a 1 year-old child with familial dysautonomia.

Creation of an autocrine model of insulin-like growth factor-I action in transfected FRTL-5 cells.

Although there is much evidence that insulin-like growth factor-I (IGF-I) is delivered to its target tissues via the circulation from distal sites of synthesis, many other observations suggest that it is synthesized in or near its target tissues and acts by autocrine and/or paracrine modalities. Studies of the mechanisms of such local actions, however, have been problematic, because in vivo studies of a single tissue are technically difficult and confounded by many variables, whereas in vitro studies of autocrine/paracrine actions have been limited by low levels of IGF-I expression and/or lack of dramatic or clearly defined responses to IGF-I. We, therefore, set about to create IGF-I expression in FRTL-5 cells, a diploid nontransformed line of rat thyroid follicular cells that have been extensively studied as a model of TSH action. The modest increase in thymidine incorporation stimulated by TSH in wild type FRTL-5 cells is markedly increased in the presence of exogenous IGF-I. By transfecting these cells with a chimeric IGF-IA gene, driven either by the mouse metallothionein-1 or IGF-II 5' genomic regulatory regions, we were able to generate stable cell lines that synthesize and secrete mature IGF-I. This was demonstrated by RIA, by Northern analysis, and by polyacrylamide gel electrophoresis characterization of the radiolabeled intracellular and extracellular products that reacted with an IGF-I antibody. The mitogenic responses to TSH in IGF-I-expressing transfected FRTL-5 cells were indistinguishable from those stimulated by TSH and IGF-I in wild type or control-transfected cells (FRTL-5 cells stably transfected with a similar transgene that does not encode IGF-I). Basal DNA synthesis was higher and the peak of thymidine incorporation was earlier in IGF-I-expressing transfected FRTL-5 cells than in wild type or control cells (18-24 h vs. 30-36 h). The concentrations of TSH that maximally stimulate the incorporation of thymidine were not altered by IGF-I expression, and transfected cells did not appear to be transformed, as judged by their inability to form colonies in soft agar. TSH-stimulated DNA synthesis was blocked in IGF-I-expressing FRTL-5 cell by a monoclonal antibody to IGF (Sm 1.2). Thus, secretion of IGF-I appears to be required for the autocrine effects observed. These IGF-I-expressing FRTL-5 cell lines provide a model in vitro system to study the intracellular processing of IGF-I and the mechanisms by which IGF-I acts in an autocrine manner.

Microinjection of endogenous and exogenous proteins into primary cultures of rat hepatocytes and the degradation of the injected proteins.

1. A method to microinject proteins into cells through packaging proteins to erythrocyte ghosts (erythrocyte-mediated microinjection) was modified partially in order to apply the method to primary cultures of rat hepatocytes. 2. Degradation of the microinjected proteins was examined employing the improved method. The mean half-life of the injected endogenous liver protein was 20 hr. The data suggested that the injected proteins are degraded through both lysosomal and non-lysosomal proteolytic pathways probably depending on their structure. 3. The present method to microinject exogenous proteins into primary cultures of rat hepatocytes can be employed usefully for the investigations of protein metabolism in liver.