Disrupted Iron Storage in Dental Fluorosis.

Enamel formation and quality are dependent on environmental conditions, including exposure to fluoride, which is a widespread natural element. Fluoride is routinely used to prevent caries. However, when absorbed in excess, fluoride may also lead to altered enamel structural properties associated with enamel gene expression modulations. As iron plays a determinant role in enamel quality, the aim of our study was to evaluate the iron metabolism in dental epithelial cells and forming enamel of mice exposed to fluoride, as well as its putative relation with enamel mechanical properties. Iron storage was investigated in dental epithelial cells with Perl's blue staining and secondary ion mass spectrometry imaging. Iron was mainly stored by maturation-stage ameloblasts involved in terminal enamel mineralization. Iron storage was drastically reduced by fluoride. Among the proteins involved in iron metabolism, ferritin heavy chain (Fth), in charge of iron storage, appeared as the preferential target of fluoride according to quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry analyses. Fluorotic enamel presented a decreased quantity of iron oxides attested by electron spin resonance technique, altered mechanical properties measured by nanoindentation, and ultrastructural defects analyzed by scanning electron microscopy and energy dispersive x-ray spectroscopy. The in vivo functional role of Fth was illustrated with Fth+/- mice, which incorporated less iron into their dental epithelium and exhibited poor enamel quality. These data demonstrate that exposure to excessive fluoride decreases ameloblast iron storage, which contributes to the defective structural and mechanical properties in rodent fluorotic enamel. They raise the question of fluoride's effects on iron storage in other cells and organs that may contribute to its effects on population health.

Dysregulation of energy homeostasis in mice overexpressing insulin-like growth factor-binding protein 6 in the brain.

AIMS/HYPOTHESIS: IGFs, IGF receptors and IGF binding proteins (IGFBPs) are widely expressed in the central nervous system. To investigate the physiological significance of IGFBP-6 in the brain we established two transgenic mouse lines overexpressing human (h)-IGFBP-6 under the control of glial fibrillary acidic protein promoter. Increasing evidence suggests that insulin/IGF signalling pathways could be implicated in the neuroendocrine regulation of energy homeostasis. We explored the impact of brain IGFBP-6 overexpression on the regulation of food intake and energy balance. METHODS: Transgenic mice were fed either a control diet or a high-fat diet for up to 3 months. Glucose and insulin tolerance tests were carried out before and after the diet period. Plasma parameters (insulin, leptin, glucose, NEFAs and triglycerides) were measured, and uncoupling protein 1 (UCP-1) expression was quantified in brown adipose tissue. Oxygen consumption was also measured in both groups. RESULTS: The transgenic mice fed a high-fat diet for 3 months developed obesity, showing increases in plasma leptin, glucose and insulin levels and mild insulin resistance. As compared with wild-type mice, no significant differences were found in the quantity of food intake. However, UCP-1 expression was down-regulated in the brown adipose tissue of the transgenic mice. CONCLUSIONS/INTERPRETATION: Our results show that brain IGFBP-6 has an impact on the regulation of energy homeostasis. These transgenic h-IGFBP-6 mice may be considered a new tool for studies of the involvement of the brain IGF system in metabolism control and obesity.

Insulin-like growth factor binding protein-6 inhibits neuroblastoma cell proliferation and tumour development.

In neuroblastoma cells, survival and proliferation are dependent upon the insulin-like growth factor (IGF) system. IGFs actively participate in cell growth, whereas IGFBP-6, is associated with the arrest of growth. With a view to blocking IGF-II action, we produced recombinant human IGFBP-6 capable of binding IGFs with affinities between 1.23 and 6.36 x 10(9) M(-1). Ex vivo mitogenic activities were tested on two human neuroblastoma cell lines, in which 100 ng/ml IGFBP-6 completely abolished the effects of both endogenous and exogenous IGF-II. In vivo, nude mice previously injected with neuroblastoma cells were submitted to either 15 daily injections of 4-20 microg IGFBP-6 or implantation of mini-pumps diffusing 20-100 microg IGFBP-6 over 2 weeks. The result was an average 18% reduction in the incidence and development of tumours. Delivery of the IGFBP-6 via mini-pumps also delayed tumour appearance by 6-15 days. Our results therefore show the involvement of IGFBP-6 in neuroblastoma cell growth, both ex vivo in terms of proliferation and in vivo in terms of tumour development.

The IGF system in neuroblastoma xenografts: focus on IGF-binding protein-6.

With a view to investigating the implication of IGF-binding protein-6 (IGFBP-6) in the growth of neuroblastomas, nude mice were injected with IGFBP-6-expressing or control IGR-N-91 human neuroblastoma cells and the resulting xenografts examined. Expression of IGFBP-3, IGFBP-4 and type 1 and type 2 IGF receptor messengers was similar in control tumours and equal-sized IGFBP-6-expressing tumours that had developed. IGF-II was more strongly expressed in control tumours, and IGFBP-6-expressing tumours contained less IGFBP-2 than controls. In both populations, there was a significant positive correlation between IGF-II and IGFBP-2 expression. In small IGFBP-6-expressing xenografts where tumour development had apparently been arrested, haematoxylin--eosin and TUNEL staining revealed numerous apoptotic cells. In situ hybridization indicated homogeneous distribution of the IGFBP-6 signal in test tumours. In cell culture, IGFBP-6-expressing cells expressed similar amounts of IGFBP-2, IGF-II and N-myc mRNAs as control cells; but media conditioned by IGFBP-6-expressing cells contained less intact IGFBP-2 protein, with no increase in its proteolytic fragment. In media treated with plasminogen, in which IGFBP-2 was proteolysed, IGFBP-6 was increased. With its especially strong affinity for IGF-II and its resistance to proteolysis, IGFBP-6 would act by sequestering IGF-II, hence inhibiting its mitogenic and anti-apoptotic effects. In excess, IGFBP-6 would displace IGF-II from IGFBP-2 whose potentiation of IGF-II action would cease and whose susceptibility to degradation would be increased. This study therefore shows that IGFBP-6 plays a role in neuroblastoma cell growth in vivo and in vitro and that stable overexpression of IGFBP-6 leads to alteration of the initial balance between the IGFBPs.

IGFBPs are involved in xenograft development in nude mice.

BACKGROUND: The insulin-like growth factors (IGFs) are involved in the growth and differentiation of neuroblastoma cells. In all biological fluids, they are non-covalently bound to high-affinity binding proteins (IGFBP-1 to -6) which modulate their bioavailability. We previously showed that IGFBP-6 expression is linked to the arrest of growth in neuroblastoma cells, whereas IGFBP-2 is associated with proliferation. PROCEDURE: To study the role of IGFBP-6 in cell growth, we stably IGR-N-91 neuroblastoma cells with a plasmid containing sequences coding for IGFBP-6 under the control of the cytomegalovirus (CMV) promoter. RESULTS: The incidence and size of tumors generated by injecting IGFBP-6-expressing cells into nude mice were reduced by factors of 2 and 5, respectively, as compared with those generated by injection by control cells. Northern blot analyses if xenografts revealed weaker expression of IGF-II, type 2 IGF receptor and IGFBP-2 mRNAs in IGFBP-6-expressing cthan in control xenografts. IGFBP-6 may therefore reduce the expression of IGF-II (which induces tumour development) at a transcriptional level. Conversely, containing IGFBP-2 cDNA under the control of CMV promoter grew three to four times as fast as normal control xenografts. Northern blot analyses revealed weaker expression of intact IGFBP-3 and IGFBP-1 in IGFBP-2-expressing than in control xenografts. CONCLUSIONS: IGFBP-1 and intact IGFBP-3 expression both enhance IGF bioavailability which promotes tumour growth. Although the mechanisms of action of IGFBP-2 and IGFBP-6 remain to be elucidated, an inverse relationship appears to exist between the two binding proteins, IGFBP-2 being involved in proliferation and IGFBP-6 in its arrest.

AUUUA sequences compromise human insulin-like growth factor binding protein-1 mRNA stability.

The instability of IGFBP-1 mRNA appears to play a role in regulating the expression of the IGFBP-1 gene, the 3' region of which contains five ATTTA sequences. We have studied the implication of these sequences for IGFBP-1 mRNA destabilization. Six plasmids were constructed, containing increasingly shorter lengths of IGFBP-1 cDNA, each with a successive ATTTA sequence deleted from the 3' end. These were stably transfected into two non-IGFBP-1-expressing (cervical carcinoma and neuroblastoma) cell lines. Kinetics studies following inhibition of transcription showed that (1) the half-life of the full-length messenger was 2.80 +/- 0.32 h; (2) deletion of each successive sequence (particularly the second and the fourth) yielded a transcript of increasing stability; and (3) the half-life of the AUUUA-free mRNA was 26.65 +/- 1.65 h. Although the primary source of IGFBP-1 is the liver, our results demonstrate that destabilization of its mRNA is not liver-specific. The ATTTA consensus sequences in the 3' untranslated region of the IGFBP-1 gene therefore provide a posttranscriptional regulation pathway that, combined with transcriptional regulation, may account for the variations in IGFBP-1 expression with developmental stage, nutritional status, and hormonal environment.

Multi-hormonal regulation of IGFBP-6 expression in human neuroblastoma cells.

Previous work has shown that neuroblastoma cells secrete IGFBP-2, -4 and -6 and that expression of these proteins is regulated by retinoic acid (at-RA) which promotes differentiation in these cells. Other agents also induce differentiation of neuroblastoma cells: these include the 9- cis and 13- cis isomers of at-RA, 1,25 dihydroxy- vitamin D3 (VD3), triidothyronine (T3) and 12-O-tetradecanoyl phorbol 13-acetate (TPA). Nine- cis and 13- cis isomers of at-RA increased IGFBP-6 expression, but decreased IGFBP-2 and IGFBP-4. VD3 stimulated IGFBP-6 and IGFBP-2 expression, whereas T3 inhibited IGFBP-6 expression without affecting IGFBP-2. TPA markedly enhanced expression of all three IGFBPs produced by SK-N-SH cells. Since IGFBP-6 secretion is associated with the arrest of proliferation in neuroblastoma cells and is regulated by the combined actions of differentiation factors, we subcloned the proximal promoter of human IGFBP-6 (nt -766/+1) into a pCAT expression vector so as to examine modulation of its transcriptional activity. VD3 and TPA were capable of stimulating promoter activity, T3 depressed it and at-RA and its 9- cis and 13- cis isomers had no effect. These results confirm the high sensitivity of IGFBP-6 expression to these differentiation agents, essentially at transcriptional level.

N-myc regulation of type I insulin-like growth factor receptor in a human neuroblastoma cell line.

Insulin-like growth factors I and II (IGF-I and IGF-II) stimulate proliferation and differentiation in many cell types, including cell lines derived from human neuroblastomas. Their effects are mediated via the IGF-I receptor (IGF-IR) that is essential for growth in these cells. Amplification of the N-myc oncogene is a marker for poor prognosis in neuroblastoma development, and it therefore seemed of interest to analyze the relationships that may exist between IGF-IR and N-myc. N-myc-deficient SK-N-SH neuroblastoma cells were used as an experimental model. After stable transfection with N-myc cDNA, Northern blotting revealed a marked increased in IGF-IR, IGF-II, IGF-binding protein (IGFBP)-2, and IGFBP-4 mRNA levels, whereas IGFBP-6 mRNA levels were clearly diminished. Western immunoblot analysis also demonstrated increased intact IGFBP-2 but decreased IGFBP-6 in the presence of N-myc oncogene. Parallel binding experiments using IGF-I missing the first 3 amino acids revealed a 47% increase in binding sites for IGF-I and an increase of at least 335% in DNA synthesis, as measured by labeled thymidine incorporation into DNA. s.c. injection of these cells into nude mice provoked xenograft development in 50-100% of cases (depending on the series of experiments). Control cells, in contrast, were not tumorigenic. In cells transfected with bp -420/+60 of the human IGF-IR promoter controlling expression of the luciferase reporter gene, promoter activity was stimulated by a factor of 3.8 +/- 0.6 (n = 6) in the presence of N-myc oncogene. This suggests transcriptional regulation of IGF-IR expression by N-myc. IGF-IR activity and N-myc amplification are two events that to date have been identified as independently instrumental in the etiology of human neuroblastoma. Our results provide the first evidence of a direct link between them and demonstrate the effects of the oncogene on components of the IGF system in neuroblastoma cell growth in vitro and in vivo.

Insulin-like growth factor (IGF) binding proteins modulate the glucocorticoid-dependent biological effects of IGF-II in cultured fetal rat hepatocytes.

The role of insulin-like growth factor binding proteins (IGFBPs) in regulation by IGF-II of glycogenesis and DNA synthesis was investigated in hepatocytes isolated from fetal rat livers at days 15 and 18 of gestation and grown in the presence or absence of cortisol. IGFBP-1 was clearly revealed by Western ligand blot and immunoblot analysis of IGFBPs secreted into conditioned media. Its production and cellular messenger RNA (mRNA) were positively regulated by cortisol and increased in older cells. In the absence of IGFBP (fresh medium), glycogenesis, and DNA synthesis were stimulated by IGF-II and insulin. In each case, cortisol enhanced this stimulation. In the presence of IGFBPs (cell-conditioned media), IGF-II stimulation of DNA synthesis and to a lesser extent glycogenesis was inhibited. The degree of inhibition was directly related to IGFBP-1 production. IGFBPs had no effect on stimulation of glycogenesis and DNA synthesis by des(1-6)IGF-II, a structural analog of IGF-II that does not bind to IGFBPs. Insulin, whose biological effects were not modified by conditioned media, inhibited IGFBP-1 production. Comparison of the dose dependence of the two bioactivities showed that DNA synthesis was more sensitive to IGF-II than glycogenesis. Our results suggest that in the case of DNA synthesis the effects of IGF-II are mediated via the IGF-I receptor and those of insulin via the insulin receptor, whereas in the case of glycogenesis both are mediated via the insulin receptor. In conclusion, IGF-II and insulin stimulation of glycogenesis and DNA synthesis in cultured fetal hepatocytes depends on the presence of glucocorticoid and the stage of development. IGF-II action is negatively regulated by IGFBP-1 whose synthesis increases in the presence of glucocorticoids.

Retinoic acid stimulates IGF binding protein (IGFBP)-6 and depresses IGFBP-2 and IGFBP-4 in SK-N-SH human neuroblastoma cells.

Insulin-like growth factors (IGF-I and IGF-II) stimulate proliferation and differentiation in many cell types. In biological fluids, they associate non-covalently with high-affinity binding proteins (IGFBPs) which control their bioavailability and modulate their action. We previously demonstrated that IGFBP-2, -4 and -6 are intimately involved in the growth of cells derived from human neuroblastomas. Here, we have investigated the effects of retinoic acid (RA), which induces differentiation in these cells, on the expression of IGFBPs secreted by SK-N-SH neuroblastoma cells. Analysis of transcriptional activity of the IGFBP-2, -4 and -6 genes in isolated nuclei (run-on experiments) showed that RA increased the transcriptional activity of the IGFBP-6 gene, reduced that of the IGFBP-4 gene and had no effect on that of the IGFBP-2 gene. Northern blot analysis following treatment with actinomycin D showed that RA increased the stability of IGFBP-6 mRNA by a factor of 2.6, decreased that of IGFBP-2 mRNA by a factor of 2.3 and failed to affect IGFBP-4 mRNA. Treatment of cells with cycloheximide indicated the involvement of labile proteins in the stabilization of these mRNAs the expression of which could be under the control of RA. The transcriptional and/or post-transcriptional mechanisms by which RA regulates each of the IGFBPs produced by SK-N-SH cells are therefore different. Such regulation may also reflect the state of differentiation of the neuroblastoma cells. With RA-induced differentiation, IGFBP-6 is strongly stimulated, whereas IGFBP-2 and IGFBP-4 are severely depressed, which would suggest that each IGFBP plays a specific role. Moreover, this regulation seems tissue-specific because it is different in other cell types.

IGFBP-2 expression in a human cell line is associated with increased IGFBP-3 proteolysis, decreased IGFBP-1 expression and increased tumorigenicity.

Insulin-like growth factors (IGF-I and -II) play an active role in cell proliferation. In biological fluids, they are non-covalently bound to high-affinity binding proteins (IGFBPs), at least 6 species of which have been identified to date, but with poorly defined functions. One of these IGFBPs, IGFBP-2, is secreted by most cell lines and appears to be involved in cell proliferation. A human epidermoid carcinoma cell line, KB 3.1, which produces IGFBP-1 and -3 and small amounts of IGFBP-4, but no IGFBP-2, was stably transfected with an expression vector comprising IGFBP-2 complementary DNA (cDNA), whose expression was placed under the control of the constitutive and ubiquitous cytomegalovirus promoter. After an s.c. injection of these IGFBP-2-expressing KB 3.1 cells into nude mice, tumours developed more quickly than in controls, they were 3 to 4 times larger and grew about 3 times as fast. Concomitant with IGFBP-2 expression in these tumours, were a decrease in IGFBP-1 expression and an increase in IGFBP-3 proteolysis, both of which increase the bioavailability of the IGF-II produced by the cells. The increased IGFBP-3 proteolysis most probably resulted from amplified expression of tissue-type plasminogen activator (t-PA) and depression of its inhibitor (PAI-I) observed in IGFBP-2-expressing xenografts. Our findings suggest that IGFBP-2 plays a role in this model of experimental tumorigenesis via a mechanism that remains unclear, but appears to involve increased protease activity and IGF-II bioavailability.

Expression of insulin-like growth factor-binding protein 6 complementary DNA alters neuroblastoma cell growth.

Insulin-like growth factors I and II (IGF-I and IGF-II) are actively involved in neuroblastoma cell growth. In all biological fluids, they are noncovalently bound to high-affinity binding proteins. At least six species of these IGF-binding proteins (IGFBPs) have been identified, but their precise roles remain unclear. One of them, IGFBP-6, is produced by neuroblastoma cells in culture under certain experimental conditions and seems to be associated with the arrest of cell growth. We stably transfected IGR-N-91 and SK-N-SH neuroblastoma cells with an expression vector comprising IGFBP-6 cDNA, whose expression was placed under the control of the constitutive and ubiquitous cytomegalovirus promoter. Analyses of the cell cycle (flux cytofluorometry), mitogenic activity (radiolabeled thymidine incorporation), and the number of viable cells (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test) showed that the mitogenic effects of serum, IGF-I, IGF-II, and des (1-3) IGF-I, a truncated IGF-I analogue with no affinity for IGFBP-6, were depressed in both transfected cell lines. With s.c. injection of transfected IGR-N-91 cells into nude mice, tumors developed in only 50-70% of cases, 1 or 2 weeks after those in controls, and were 60-90% smaller. Our findings show that IGFBP-6 influences neuroblastoma cell growth, both in vitro and in experimental xenograft development.

Liver-specific expression of human insulin-like growth factor binding protein-1 in transgenic mice: repercussions on reproduction, ante- and perinatal mortality and postnatal growth.

Study of the in vivo functions of the insulin-like growth factor binding proteins (IGFBPs) is complicated by their variety (six molecular species) and the differences in their expression related to tissue of origin and stage of development. To investigate the physiological role of IGFBP-1 in the bloodstream, we induced hepatic overexpression of IGFBP-1 in transgenic mice, placing human IGFBP-1 (hIGFBP-1) cDNA under the control of the alpha1-antitrypsin promoter so as to obtain liver-specific expression. Five transgenic founder mice were raised, only two of which (lines 124 and 149) produced transgenic offspring. Northern blotting revealed transgene expression exclusively in the liver during fetal life and unchanged through to adulthood, whereas expression of the endogenous gene was undetectable beyond 10-15 days postnatally. hIGFBP-1 was detected by western immunoblotting in the plasma of transgenic mice and IRMAs yielded mean concentrations of 2.41 +/- 0.33 ng/ml and 13.69 +/- 1.42 ng/ml in homozygous animals of lines 124 and 149, respectively. In the latter, IGFBP-1 levels were distinctly higher than in heterozygotes (2.99 +/- 0.39 ng/ml), P < 0.0001. These levels remained stable in each given animal and did not change with age. Plasma concentrations of IGF-I measured in line 149 exhibited the well-known profile of an increase from birth up to puberty. Values for heterozygotes were similar to those for wild-type mice, with adult levels (544 +/- 98 ng/ml) slightly below those of controls (630 +/- 56 ng/ml), P < 0.05. In homozygotes they were distinctly lower, with adult levels of 370 +/- 75 ng/ml, P = 0.001. In heterozygous and homozygous adults, there was a negative correlation between IGF-I and IGFBP-1 concentrations (r = 0.8, P < 0.0001), suggesting a link between transgene expression and IGF-I levels. Study of body weight gain in line 149 revealed growth retardation within the first weeks after birth, which was marked in homozygous males and females (P < 0.001) but also present in heterozygous males (P = 0.002), indicating some relationship with transgene expression. In addition, body weight in adult mice was negatively correlated to plasma concentrations of IGFBP-1 (r = 0.7, P < 0.0001). Reproductive function also appeared to be severely affected, especially in homozygous females: mating that failed to result in pregnancy in half of the homozygous females crossed with nontransgenic males, suggestive of impaired fertilization or implantation; interrupted or prolonged pregnancies with fetal and neonatal death. Litter size was reduced in transgenic females (by about half in homozygotes) and in nontransgenic females mated with homozygous males, resulting from pre- or neonatal mortality. Moreover, deaths occurred within the first 5 days of life, with an incidence of approximately 50% in the litters of homozygous females, 12-18% among heterozygotes mated with nontransgenic or heterozygous males, respectively, and 30% among those mated with homozygous males. These results, suggesting that fetal transgene expression largely accounted for ante- and perinatal mortality, were confirmed by the predominance of homozygotes among those that could be analyzed genetically. Similarly impaired reproductive function was seen in line 124, but to a lesser degree. Although the mechanisms responsible for these disorders remain to be determined, our results indicate that permanent and uncontrolled hepatic expression of IGFBP-1, even at low levels, affects fertility in females and both ante- and postnatal development.

Role of insulin-like growth factor binding protein-2 and its limited proteolysis in neuroblastoma cell proliferation: modulation by transforming growth factor-beta and retinoic acid.

Insulin-like growth factor (IGF) binding proteins (IGFBPs) modulate IGF action at cellular level through inhibition or, alternatively, potentiation, where their limited proteolysis is a contributory mechanism. Under basal conditions, neuroblastoma cells secrete IGFs (essentially IGF-II), IGFBPs (IGFBP-4 and predominantly IGFBP-2 that is partially proteolysed), and proteases, including tissue-type plasminogen (PLG) activator, whose activity is inhibited by PLG activator inhibitor-1. Neuroblastoma cells were used to investigate the influence of the plasmin system, transforming growth factor-beta retinoic acid on cell growth and the IGF system. In cells treated with 5 micrograms/ml PLG, proliferation was stimulated, an effect that was inhibited in the presence of either alpha IR-3 (which blocks the type 1 IGF receptor) or anti-IGF-II antibodies. There was a parallel increase in IGFBP-2 proteolysis, which resulted in a 5-fold loss of affinity for IGF-II. In the presence of 1 ng/ml transforming growth factor-beta, PLG-induced mitogenesis and IGFBP-2 proteolysis were reduced, and Northern blot analysis revealed increased PLG activator inhibitor-1 mRNA. Conversely, with 2 microM retinoic acid, the mitogenic effect of PLG, IGFBP-2 proteolysis, and tissue-type PLG activator mRNAs were increased. Therefore, IGF-II mediates autocrine proliferation in neuroblastoma cells under the control of IGFBPs secreted by the cells, its bioavailability being enhanced as a result of plasmin-induced IGFBP-2 proteolysis.

IGF-binding protein-6 is involved in growth inhibition in SH-SY5Y human neuroblastoma cells: its production is both IGF- and cell density-dependent.

The IGF system is involved in the growth and differentiation of neuroblastoma cells, but the precise roles played by the IGF-binding proteins (IGFBPs) remain unknown. We have examined the expression and functions of IGFBPs produced by the neuroblastoma cell line, SH-SY5Y, in the presence of: insulin, IGF-I, IGF-II, des(1-3)IGF-I (an IGF-I analogue with weak affinity for IGFBPs), acidic fibroblast growth factor, basic fibroblast growth factor, or nerve growth factor. Under basal conditions, SH-SY5Y cells in serum-free medium secreted IGF-II, and traces of IGF-I, IGFBP-2 and IGFBP-4. After 24 h of culture, comparative mitogenic potencies were: des(1-3)IGF-I > IGF-I > IGF-II > insulin. After 48 h, when IGFBP-2 and IGFBP-4 concentrations in the culture media had increased, des(1-3)IGF-I remained the most active, but the activity of insulin now equalled or exceeded that of IGF-I and IGF-II. This suggests a negative feedback mechanism involving partial sequestration of IGF-I and IGF-II by IGFBP-2 and IGFBP-4. At high cell density and with high concentrations of IGF-I, des(1-3)IGF-I (40 ng/ml) or IGF-II (80 ng/ml), the mitogenic activities of the IGFs diminished concomitantly with the appearance in the culture medium of an additional IGFBP identified as IGFBP-6, whose production depended on activation of the type 1 IGF receptor. These findings suggest that IGFBP-6 contributes as an autocrine inhibitor in the regulation of growth by the IGF system in these neuroblastoma cells.

Modulation by retinoic acid of insulin-like growth factor (IGF) and IGF binding protein expression in human SK-N-SH neuroblastoma cells.

Growth in neuroblastoma cells is regulated by insulin-like growth factors (IGFs) whose action is modulated by IGF binding proteins (IGFBPs). In this study, SK-N-SH neuroblastoma cells were shown to produce IGF-II, IGFBP-2, IGFBP-4 and small quantities of IGFBP-6. We have studied the effects of a natural morphogen, retinoic acid (RA), on growth and IGFBP expression in these cells. In all experiments, cells were cultured in serum-free medium and treated with 1 mumol/l RA for 12 h. Cell number increased by almost 50% during the first 24 h after the beginning of treatment. This stimulation was inhibited by 80% or more in the presence of the anti-type 1 IGF receptor antibody alpha-IR3 and anti-IGF-II antibody. The IGF-II concentrations in the culture media, measured after acidic gel filtration, increased about 1.5-fold and Northern blotting showed a concomitant increase in IGF-II mRNA levels. The mitogenic effect of RA therefore reflects its stimulation of IGF-II production. The availability of IGF-II to the cells may also be enhanced because of the proteolysis of IGFBP-2 to which it is bound. After this initial phase, proliferation ceased despite continued IGF-II production between 24 and 72 h. Both IGFBP-2 and IGFBP-4 production decreased, whereas that of IGFBP-6 increased. These changes appeared both in the protein quantities and in their mRNAs. Insulin-like growth factor binding protein 6 has a strong affinity for IGF-II, 5-10 times that of IGFBP-2 and at least 10 times that of the type I IGF receptor, and the arrested proliferation may result, at least in part, from sequestration by IGFBP-6 of the IGF-II secreted.

Transcriptional regulation of insulin-like growth factor binding protein-1 expression by insulin and cyclic AMP.

In biological fluids, insulin-like growth factor binding proteins (IGFBPs) interact with the IGFs and modulate their effects. In this study, changes in IGFBP-1 expression were investigated under the influence of insulin and cAMP which may regulate expression of the IGFBP-1 gene in vivo during the perinatal period. Western ligand blot analysis of IGFBPs secreted by HepG2 human hepatoma cells showed that 24 h treatment with forskolin increased IGFBP-1 secretion by approximately 100%, whereas similar treatment with insulin resulted in a 50% reduction. After 24 h, the amounts of IGFBP-1 mRNA (measured by Northern blotting) were increased 2.5 times by forskolin and decreased by 65% by insulin. Transient transfection experiments showed that forskolin enhanced IGFBP-1 promoter activity by 70%, suggesting that stimulation of IGFBP-1 gene expression by cAMP is transcriptional, via a protein recognizing the cAMP responsive element (CRE) consensus sequence (nt -268 to -248). In contrast, modulation of gene expression by insulin is more complex, probably involving several levels of regulation. Complementary experiments (site-directed mutagenesis and/or use of a heterologous promoter) will be needed to confirm the functionality of the proteins interacting with the IRE (nt -285 and -276) and the CRE (between nt -268 and -248) described.

Interactions between liver nuclear proteins and the human insulin-like growth factor binding protein 1 promoter in the course of development.

Insulin-like growth factor binding proteins (IGFBPs) modulate the bioavailability of the IGFs. Among the six IGFBPs known to date, IGFBP-1 is the most tissue-specific, its expression being limited to the liver and the endometrium. In the liver, IGFBP-1 gene expression is maximal during the perinatal period, with its peak corresponding to a transient rise in gene transcription activity. In this study, interactions between rat liver nuclear proteins and the human IGFBP-1 promoter have been analysed in the course of development, using in vitro DNase I protection and mobility shift assays. Only the interactions between DNA and proteins localized between nt -305 and -268 varied through the period studied (16 days in utero to 70 days postnatally). Three proteins, named Pa, PC1 and PC2, interacted with sequences between nt -295 and -285, nt -305 and -295 and nt -285 and -268, respectively. There was a marked perinatal increase in phenotype expression of Pa, which was parallel to that in IGFBP-1 gene transcription activity. In addition, DNA-Pa interactions and DNA-PC2 interactions were mutually exclusive. These results suggest that the interaction of Pa with its target sequence(s) prevent PC2 binding and thereby contribute towards increased IGFBP-1 gene transcription.

Interplay of the liver-enriched trans-acting factors, DBP and HNF1, in the transactivation of human IGFBP-1 promoter.

In the liver, expression of insulin-like growth factor binding protein-1 (IGFBP-1) is regulated essentially at the transcriptional level, at least in part by HNF1. In this study, the functional role of DBP and C/EBP (which have several potential binding sites on the IGFBP-1 proximal promoter) have been investigated. Transient co-transfection of the reporter plasmid, pBP-1341 and eukaryotic expression vectors which code for DBP and C/EBP in human cell lines Hep3B, HepG2 and C33 showed that IGFBP-1 promoter activity was unchanged by C/EBP, but increased between 2 and 7 times by DBP (depending on the cell line). In addition, DBP and HNF1 were capable of functional co-operation in activating the IGFBP-1 promoter. Our results support the notion of DBP being involved in limited tissue specificity of IGFBP-1 expression.

Expression of insulin-like growth factor binding protein-1 and -2 genes through the perinatal period in the rat.

Insulin-like growth factor binding proteins (IGFBPs) are essential mediators of the bioavailability and biological effects of the IGFs. Liver expression of the rat (r) IGFBP-1 and rIGFBP-2 genes has been characterized between day 16 in utero (16 diu) and 16 days postnatally (+16 dpn). Run-on experiments showed transcriptional activity of the rIGFBP-1 and rIGFBP-2 genes at birth (B) to be 25 and 5 times that at 16 diu, respectively. After B, transcriptional activity of the rIGFBP-1 gene remained high (140% B at +6 dpn), but that of the rIGFBP-2 gene dropped to 70% B by +6 dpn. Northern blot analysis done simultaneously showed rIGFBP-1 messenger RNA (mRNA) levels to increase approximately 50-fold between 16 diu and B, whereas rIGFBP-2 mRNA increased only 5- to 10-fold. rIGFBP-1 mRNA levels decreased after birth, reaching about 20% B by +6 dpn; rIGFBP-2 mRNA, however, remained stable (about 80% B) at least up to +6 dpn. Parallel Western ligand blot and immunoblot analyses of serum rIGFBPs revealed rIGFBP-1 and rIGFBP-2 concentrations to be increased 3- and 2-fold, respectively between 20 diu and B. Maximal expression of rIGFBP-1 was at +1 dpn (220% B), and of rIGFBP-2, at B. Both rIGFBPs then decreased, reaching about 5% B at adulthood. All these data indicate that increased transcriptional activity of the rIGFBP-1 and rIGFBP-2 genes at birth would determine the increased synthesis in the liver and circulating levels of these proteins. In addition, it would seem that post-transcriptional events (reduced half-life of the rIGFBP-1 messenger after birth, translation efficiency of the rIGFBP-2 messenger) modulate transcriptional regulation.