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.

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.

Multiple liver-enriched trans-acting factors interact with the glucocorticoid- (GRU) and cAMP-(CRU) responsive units within the h-IGFBP-1 promoter.

In response to hormonal control, serum concentrations of insulin-like growth factor-binding protein-1 (IGFBP-1) may vary as much as 10-fold, owing to strict control of its gene's expression in hepatocytes. IGFBP-1 gene transcription is increased by glucocorticoids and cAMP and inhibited by insulin. The effect of insulin is dominant since it suppresses constitutive and both glucocorticoid- and cAMP-stimulated transcription. Close examination of the human (h)IGFBP-1 promoter sequences showed that the glucocorticoid (GRE, nt -88 to -102) and cAMP (CRE, nt -259 to -264) response elements are 5'-flanked by an A/T-rich imperfect palindrome (nt -102 to -117 and -265 to -285, respectively). These A/T-rich motifs are putative cis-elements for liver-enriched trans-acting factors. Competition experiments in electrophoretic mobility shift assay were carried out using rat liver nuclear extracts and a set of synthetic oligonucleotides designed from hIGFBP-1 Glucorticoid and cAMP Response Units (GRU and CRU), the rat transthyretin HNF3 cis-element and the "D-site' of the mouse albumin promoter. The nucleotide motifs located between nt -108 and -121 of the GRU, interacted with the HNF3 family of trans-acting factors (alpha, beta, gamma), whereas those encompassing nt -81 to -104 bound DBP and/or nuclear proteins sharing similar sequence specificity (i.e. from the C/EBP family of bZIP proteins). We have also shown that the hIGFBP-1-GRE binds glucocorticoid receptor homodimers. In the case of the CRU, the cis-elements located between nt -249 and -285 bound DBP and/or nuclear proteins sharing similar sequence specificities. In addition, the nucleotide stretch lying between nt -256 and -275 was able to interact with the HNF3 family of trans-acting factors. Our results support the view that the dominant inhibitory effect of insulin over glucocorticoid- and cAMP- enhanced transcription may be mediated by different target sequences located 5'- of the GRE and CRE. In both cases, the mechanism would involve the interplay of common trans-acting factor(s), some of which are liver-enriched [HNF3, DBP or C/EBP related bZIP proteins] with their cognate target sequence.