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The mouse IGF-II receptor (Igf2r) and its antisense transcript Air are reciprocally imprinted in most normal tissues. Several mechanisms have been hypothesized to explain Igf2r-Air imprinting, including Igf2r silencing by Air, and transcriptional repression of Igf2r-Air by two differentially methylated regions (DMR1 and DMR2). We employed Mus musculus x Mus spretus interspecific mice and chromatin immunoprecipitation (ChIP) to investigate allele-specific histone modifications in the two DMRs. We show that, in both DMRs, the active alleles of both Igf2r, and Air are associated with acetylated histones (H3, and H4), acetyl lysine 9 of histone H3 (H3 K9-Ac), and methyl lysine 4 of histone H3 (H3 K4-Me). The silenced alleles are associated with methylated DNA, deacetylated H3 K9, and unmethylated H3 K4. Allele-specific histone modifications are present in the DMR2 that is established in the gametes and represents the DNA gametic-imprint of the Igf2r. In the DMR2 from liver, kidney, and central nervous system tissues, H3 K9 methylation is associated exclusively with the silenced allele, and H3 S10 phosphorylation with the active alleles. Treatment of fibroblast cells with 5-aza-deoxycytidine and/or Trichostatin A led to partial reactivation of the silenced allele, which correlates with biallelic histone acetylation. In central nervous system, despite the presence of imprinted Air transcripts, biallelic expression of Igf2r occurs. The tissue-specific relaxation of Igf2r imprinting correlates with biallelic histone acetylation, and biallelic H3 K4 methylation in the promoter region of Igf2r (DMR1). We propose a model of the histone code for Igf2r, and Air imprinting that defines histone modifications specific for the putative gametic imprint DMR2, and explains the tissue-specific imprinting of Igf2r in the mouse and the absence of IGF2R imprinting in human.

The adjacent IGF2 and H19 genes are imprinted in most normal mouse and human tissues, but imprinting is often lost in tumors. Mouse models suggest that parental-allele specific CCCTC-binding factor (CTCF) binding at the IGF2/H19 imprinting control region (ICR) regulates the expression of these two genes. Using chromatin immunoprecipitation and PCR, we show that in several normal and neoplastic human tissues, CTCF consistently binds unmethylated ICR elements, but CTCF binding does not result in predictable gene expression. In the fetal brain, CTCF binding is monoallelic and specific for the unmethylated ICR, yet IGF2/H19 expression is biallelic. In osteosarcoma tumors, aberrant methylation of the IGF2/H19 ICR results in equally aberrant CTCF binding, yet expression of these genes does not correlate with CTCF binding. This is the first description of chromatin immunoprecipitation for CTCF binding at the human IGF2/H19 ICR, and the results demonstrate that CTCF binding at the IGF2/H19 ICR is insufficient to regulate the expression of IGF2/H19 in many human tissues.

The mouse insulin-like growth factor II receptor (Igf2r) gene encodes two reciprocally imprinted RNA transcripts: paternally imprinted Igf2r sense and maternally imprinted Igf2r antisense. Although DNA methylation has been implicated in the initiation and maintenance of genomic imprinting, acetylation of core histones has recently been appreciated as another important factor that regulates gene expression. To determine whether histone acetylation participates in the regulation of Igf2r imprinting, we examined the relative abundance of acetylated histones in interspecific mice (M. spretus × C57BL/6). Oligonucleosomes derived from liver were immunoprecipitated with acetyl-histone antiserum and were analyzed for the allelic distribution of DNA from the region of the sense and antisense Igf2r promoters. In nucleosomes associated with the Igf2r sense promoter, histone acetylation was demonstrated on the maternal allele, which is transcriptionally active. There was much less histone acetylation on the suppresse...

IGF binding protein (IGFBP)-2 is one of the most significant genes in the signature of major aggressive cancers. Previously, we have shown that IGFBP-2 enhances proliferation and invasion of neuroblastoma cells, suggesting that IGFBP-2 activates a protumorigenic gene expression program in these cells. Gene expression profiling in human neuroblastoma SK-N-SHEP (SHEP)-BP-2 cells indicated that IGFBP-2 overexpression activated a gene expression program consistent with enhancement of tumorigenesis. Regulation was significant for genes involved in proliferation/survival, migration/adhesion, and angiogenesis, including the up-regulation of vascular endothelial growth factor (VEGF) mRNA (>2-fold). Specific transcriptional activation of the VEGF gene by IGFBP-2 overexpression was demonstrated via cotransfection of a VEGF promoter Luciferase construct in SHEP-BP-2. Cotransfection of VEGF promoter Luciferase construct with IGFBP-2 protein in wild-type SHEP cells indicated that transactivation of VEGF promoter only occurs in the presence of intracellular IGFBP-2. Cell fractionation and immunofluorescence in SHEP-BP-2 cells demonstrated nuclear localization of IGFBP-2. These findings suggest that transcriptional activation of VEGF promoter is likely to be mediated by nuclear IGFBP-2. The levels of secreted VEGF (up to 400 pg/106 cells) suggested that VEGF might elicit angiogenic activity. Hence, SHEP-BP-2 cells and control clones cultured in collagen sponge were xenografted onto chick embryo chorioallantoic membrane. Neomicrovascularization was observed by 72 h, solely in the SHEP-BP-2 cell xenografts. In conclusion, our data indicate that IGFBP-2 is an activator of aggressive behavior in cancer cells, involving nuclear entry and activation of a protumorigenic gene expression program, including transcriptional regulation of the VEGF gene and consequent proangiogenic activity of NB cell xenografts in vivo.

Normal somatic growth requires that both the thyroid hormone axis and GH axis be intact. Thyroid hormone stimulates GH secretion, and many thyroid hormone actions on the insulin-like growth factor (IGF) system can be explained by this mechanism. We have previously described distinct changes in IGF binding protein (IGFBP) expression in experimental hypothyroidism in the rat; these changes could be completely corrected by thyroid hormone replacement. To see if the effects of thyroid hormone on IGFBP expression are, in fact, indirect GH effects, we rendered both newborn and adult rats hypothyroid with methimazole treatment, and investigated whether we could correct the resulting IGF and IGFBP changes with GH replacement. The prolonged high expression of serum IGFBP-2 and liver IGFBP-2 messenger RNA (mRNA) during the perinatal period in hypothyroid rat pups could not be normalized by GH therapy, although serum IGF-I values (reduced to 54% of control levels in the hypothyroid animals) were brought up to control level. In adult hypothyroid rats, serum IGF-I concentrations (51% of control levels), were increased up to 79% of control levels, but not totally corrected, by GH therapy. Reduced IGFBP-3 expression (80% of control serum and 50% of control liver mRNA levels) in adult hypothyroid animals was normalized by GH, but there was no correction of the reduced IGFBP-4 serum levels (50% of control levels). Hepatic mRNA levels for the type 1 and 2 IGF receptors were not altered by hypothyroidism, or by thyroid or GH replacement. Somatic growth in hypothyroid pups and adults was only partially corrected by GH therapy. We conclude that GH treatment of hypothyroid animals normalized serum IGF-I levels in the hypothyroid rat pup, but did not correct their prolonged IGFBP-2 expression. In the mature animal, serum IGF-I levels were partially corrected and IGFBP-3 levels were normalized by GH, but no change could be induced in the reduced serum IGFBP-4 levels. All the above changes were normalized by thyroid hormone replacement. Thus, the effects of thyroid hormone on serum IGF levels and IGFBP-3 expression seem to be mediated indirectly via GH. The effects on IGFBP-2 ontogeny, and IGFBP-4 expression in the mature animal, however, are either direct thyroid hormone effects, or mediated by some other route, independent of GH, IGFs, or IGF receptors.

Rat pituitary GH3 tumor cells express GH and insulin-like growth factor-I (IGF-I) mRNAs and possess specific IGF-I and IGF-II receptors which mediate the inhibitory effect of IGF on GH secretion. T3 increases the rate of cell replication and GH gene transcription, and causes an increase in IGF-I binding to cell membranes. Since the IGFs circulate in association with specific binding proteins (IGFBPs) that appear to modulate the access of IGFs to their receptors, we have investigated the effect of T3 treatment on the expression of IGFBPs in GH3 cells. Cells were grown in serum-free defined medium, and IGFBP secretion was determined by Western ligand blotting of conditioned medium after the addition of T3 and/or various peptides for 48-72 h. The conditioned medium of GH3 cells revealed a complex of bands migrating at 40-45 Mr, a pattern typical of rat (r) IGFBP-3. T3 treatment resulted in an increase in rIGFBP-3. IGF-I, IGF-II, and insulin did not alter rIGFBP-3 levels. After concentrating (10-fold) conditioned medium samples, two additional bands at 24K and 28K mol wt were also seen. These bands corresponded in size to rIGFBP-4 (24K) and its glycosylated form (28K). The mRNAs for both rIGFBP-3 and rIGFBP-4 were present in GH3 cells; T3 treatment increased steady state levels of rIGFBP-3 mRNA, but did not affect BP-4 mRNA levels. To learn whether the increased expression of IGFBPs could be responsible for the increased IGF-I binding seen after T3 treatment, [125I]IGF-I was cross-linked to GH3 membranes, and the proteins were separated on a 5-15% gradient sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. T3 treatment induced a large increase in the intensity of bands migrating at 135K and 280K, representing the alpha-subunit of the IGF-I receptor and an incompletely reduced alpha-alpha-dimer, respectively. No membrane-associated IGFBPs were detected. In conclusion, GH3 cells express two IGFBPs, rIGFBP-3 and rIGFBP-4, which are differentially regulated by T3. The increased binding of IGF-I to GH3 cell membranes after T3 treatment indicates that thyroid hormone induces an up-regulation of IGF-I receptors and that the increased IGF-I binding to GH3 membranes is not due to increased expression of membrane-associated IGFBPs.

The isolation and hormonal regulation of two low molecular weight insulin-like growth factor binding proteins (IGFBPs) present in the conditioned medium (CM) of the rat neuroblastoma cell line B104 cells has been performed. IGFBPs were purified by ZnSO4 precipitation, insulin-like growth factor-I 1IGF-I) affinity chromatography, and reverse phase HPLC. Final isolation and N-terminal analysis was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotting to polyvinylidene difluoride membranes, and sequencing of the bound proteins. Two IGFBPs, with apparent Mr of 28K and 24K were coisolated and sequenced. Both proteins had identical N-terminal sequences and appear to be two forms of IGFBP-4. Treatment of the IGFBPs with endoglycosidase-F caused a shift in the apparent Mr of the 28K IGFBP to 24K. However, there was no change in the apparent Mr of the 24K IGFBP. The data from this study suggest that the IGFBP-4 exists as both a glycosylated and nonglycosylated protein. Treatment of B104 cells with IGF-I increased the expression of both the 24K and 28K IGFBPs and also resulted in the appearance of IGFBP-3 and an unknown IGFBP at 29K. When added to subconfluent cells, IGF-I was also mitogenic in B104 cells. Similar to IGF-I, IGF-II treatment increased cell number and resulted in the appearance of IGFBP-3 and the 29K IGFBP. However, IGF-II treatment resulted in a significant decrease (approximately 50%) in the 24K IGFBP and also decreased the 28K IGFBP. This decrease in the expression of the 24K and 28K IGFBPs was dose-dependent and was blocked by addition of IGF-I to the cells. When an IGF-II receptor antibody was added to the cells it mimicked the effects of IGF-II on B104 cells, suggesting that the inhibitory effects of IGF-II are mediated through the type II IGF receptor. Although both IGF-I and IGF-II affected the amount of the 24K IGFBP in the CM, neither peptide affected the expression of the messenger RNA for the 24K IGFBP. In conclusion, we have isolated two IGFBPs from the CM of B104 cells. Both the 24K and 28K IGFBPs appear to be isoforms of the same protein, and sequence data suggest these proteins are two forms of IGFBP-4. IGF-I increases the expression of both of these IGFBPs, whereas IGF-II decreases their expression.(ABSTRACT TRUNCATED AT 400 WORDS)

Specific receptors for insulin and the somatomedin peptides insulin-like growth factors I and II (IGF-I and IGF-II) have been characterized on three separate cloned strains of rat pituitary tumor cells (GH3, GH1, and GC). Binding of 125I-labeled peptides was time, temperature, and pH dependent for all three cell lines. Specific binding of [125I]insulin, which was extremely low in normal rat adenohypophyseal cells, was demonstrable for all three lines, with the Kd for the high affinity receptor ranging from 10(-10) to 4 X 10(-10) M/liter. A specific high affinity IGF-I receptor was also identified, with a Kd of approximately 10(-9) M/liter. IGF-II and insulin were, respectively, 10% and 1% as potent as IGF-I in competing for this receptor. When [125I]insulin and [125I]IGF-I were cross-linked to GH3 cells with disuccinimidyl suberate, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both receptors were found to have an apparent mol wt greater than 300,000 in the unreduced state, with subunits of apparent mol wt 125,000 after reduction. A third discrete receptor, which bound [125I]IGF-II, was also identified on all three cell lines. IGF-I was only 10% as potent as IGF-II at displacing [125I]IGF-II, and insulin was virtually unreactive. When [125I]IGF-II was cross-linked to GH3 cells and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two receptors were identified. One had an apparent mol wt of 205,000 unreduced and 250,000 upon reduction, and presumably represents the type II receptor. Additionally, a band was observed at an apparent mol wt greater than 300,000 unreduced and 125,000 upon reduction, probably representing IGF-II binding to the IGF-I or insulin receptor. The presence of specific high affinity receptors for insulin, IGF-I, and IGF-II in these transformed cell lines is consistent with previous observations in normal rat and human pituitary cells and suggests a role for these peptides in the modulation of pituitary function.

Catecholestrogens are biologically active metabolites of estrogen which are synthesized by estrogen-2-hydroxylase in the liver, brain, and other organs. Although catecholestrogens are inactivated by enzymatic O-methylation, demethylation of the 2-methoxyestrogens to reform catecholestrogens also occurs. A sensitive and specific radioenzymatic assay for rat liver 2-methoxyestrogen demethylase has been developed which makes it possible to characterize its substrate requirements and to study the influence of various hormones on catecholestrogen formation. 2-Methoxyestrogen demethylase activity is localized in rat liver microsomes. The apparent Km for 2-methoxyestrone (2MeOE1) is 12 muM and that for 2-methoxyestradiol (2MeOE2) is 3 umM. The two most prevalent 2-methoxyestrogens, 2MeOE1 and 2 MeOE2, appear to be demethylated by different enzymes. The enzymes have absolute requirements for NADPH, an their activities are inhibited by CO and SKF-525A, indicating that they are cytochrome P450 dependent. 2MeOE2 demethylation but not 2MeOE1 demethylation exhibits substrate inhibition. 2MeOE1 demethylase activity in the female rat liver is only one third that in the male rat liver, but sexual dimorphism was not found in 2MeOE2 demethylation. Thyroidectomy and estradiol treatment of the male rat resulted in diminished 2MeOE1, but not 2MeOE2, demethylation.

Hypothalamic-pituitary-end-organ axes are frequently controlled by long loop negative feedback homeostatic mechanisms. Insulin-like growth factor I (IGF-I), IGF-II, and insulin receptors have recently been described in normal and neoplastic rat and acromegalic human pituitary cells, a finding which suggests the possibility that somatomedins might exert feedback at the level of the anterior pituitary. To study the kinetics of this feedback response, we used perifused dispersed rat anterior pituitary cells to learn if somatomedins or insulin could inhibit GH-releasing hormone (GHRH)-stimulated GH secretion. Cells were exposed to hourly boluses of 1 nM GHRH with or without varying doses of IGF or insulin. IGF-I inhibited GHRH-elicited GH release with an IC50 of 6.5 nM; maximal inhibition (approximately 67%) was achieved with 10 nM IGF-I. IGF-II was a less potent hormone, with 10 nM inhibiting about 30% of GHRH-stimulated GH release. Slight inhibition of stimulated GH release (less than 15%) was seen when cells were treated with insulin, but only when doses of insulin of 10 nM or more were used. In conclusion, nanomolar concentrations of IGF-I and IGF-II inhibited GHRH-elicited GH release from perifused rat pituitary cells in a dose-dependent manner; and insulin was not an effective inhibitor of stimulated GH release at physiological peptide concentrations. In conjunction with our previous findings that the concentrations of IGF-I and IGF-II receptors greatly exceed that of insulin receptors on normal rat pituitary cells, we hypothesize that the GH-inhibiting action of high dose insulin is mediated through an IGF receptor.

Preincubation of rat pituitary cells in primary culture with rat GH-releasing factor (rGRF) resulted in substantial desensitization to subsequent GRF stimulation. rGRF-directed GH release and intracellular cAMP accumulation decreased in the desensitized cells. Whereas prior treatment of rat pituitary cells caused partial depletion of intracellular GH levels, diminished cellular reserves could not entirely account for the decreased GH release. Cells that had been preexposed to 10 nM rGRF for 4 h demonstrated at 30-50% depletion of intracellular GH; subsequent stimulation of those cells with 10 nM rGRF elicited GH release which was only 5% of that seen in cells that were not desensitized [control, 112 +/- 3.2 ng/well (+/- SEM); GRF-stimulated, 435 +/- 32 ng/well; GRF-pretreated, control, 63 +/- 3 ng/well, GRF-pretreated, GRF-stimulated, 73 +/- 3.4 ng/well]. Despite the marked depletion of cellular GH stores and the greatly diminished rGRF-stimulated GH release in cells that had been preexposed to rGRF, both forskolin and (Bu)2cAMP were able to induce a 2-fold stimulation of GH release. Incubation of the rGRF-pretreated cells with fresh medium which lacked rGRF resulted in gradual recovery of the ability of rGRF to stimulate GH release without complete reconstitution of the intracellular GH stores. These results indicate that exposure of rat pituitary cells to rGRF results in 1) partial depletion of intracellular GH stores; 2) a diminished ability of a subsequent rGRF challenge to elicit GH secretion and intracellular cAMP accumulation, and 3) a sustained ability of forskolin and (Bu)2cAMP to stimulate GH release, indicating that rGRF desensitization occurs in vitro.

The ability of ovine corticotropin-releasing factor (CRF) to stimulate both ACTH release and intracellular cAMP accumulation is rapidly and reversibly desensitized when rat anterior pituitary cells are cultured in the absence of added glucocorticoids. Since this desensitization has not been readily apparent in vivo, where initial CRF exposure results in high levels of ambient glucocorticoids, we examined the effect of glucocorticoids on the desensitization process in vitro. Rat anterior pituitary cells were cultured for 3-5 days in the absence of added steroid hormones. Dexamethasone was then added to some culture wells 24 h before the desensitization experiment began. Desensitization of CRF was achieved by preincubating the cells for 4 h with varying concentrations (10(-11)-10(-7) M) of ovine CRF, washing the cells with medium alone, and then reexposing the cells to CRF. In the absence of glucocorticoid, the ED50 for CRF desensitization (the preincubation dose causing 50% desensitization of subsequent ACTH release) was 3 X 10(-10) M, but cells that had been preexposed to dexamethasone desensitized less readily. With concentrations of dexamethasone of 10(-8) M or greater, no desensitization occurred. When cells were incubated in the absence of added glucocorticoids, CRF-stimulated intracellular cAMP accumulation was diminished by prior exposure to CRF. No decrease in intracellular cAMP accumulation was seen in those cells that had been preincubated with dexamethasone, however. Similar changes in CRF desensitization of ACTH release were observed when cells were incubated with corticosterone, but not with 10(-8) M testosterone, progesterone, aldosterone, or estradiol. These data demonstrate that glucocorticoids profoundly alter the development of CRF desensitization in vitro and suggest that high ambient glucocorticoid concentrations prevent the development of substantial CRF desensitization in vivo.

Primary cultures of rat anterior pituitary cells were assessed for the presence of specific receptors for insulin and for the somatomedin peptides, insulin-like growth factors I and II (IGF-I and IGF-II). Specific binding per 100,000 pituitary cells averaged 9.45 +/- 1.69% (mean +/- SD) for [125I]IGF-II, 0.83 +/- 0.06% for [125I]IGF-I, and only 0.11% for [125I]insulin, IGF-II was twice as potent as IGF-I in displacing [125I]IGF-II, while insulin was totally nonreactive, IGF-I was 5-fold more potent than IGF-II at displacing [125I]IGF-I and 1000-fold more potent than insulin. Scatchard analysis of [125I]IGF-II binding revealed a curvilinear plot, which could be resolved into a high affinity receptor with a Ka of 7.0 X 10(8) M-1 and 120,000 receptor sites/cell, and a low affinity receptor with a Ka of 1.1 X 10(8) M-1 and 720,000 receptor sites/cell. The existence of abundant high affinity somatomedin receptors (especially for IGF-II) on rat anterior pituitary cells is consistent with a potential role for these peptides in the regulation of GH secretion.