Biomedical subjects

R G Rosenfeld

Publications and source records attributed to R G Rosenfeld.

At least 19 recordsLinked to original sources

PubMed1992

Preferential binding of insulin-like growth factor-II (IGF-II) to a putative alpha 2 beta 2 IGF-II receptor type in C2 myoblasts.

We have studied insulin-like-growth-factor (IGF) binding in two subclones of the C2 myogenic cell line. In the permissive parental subclone, myoblasts differentiate spontaneously into myotubes in medium supplemented with fetal calf serum. Unlike permissive myoblasts, inducible myoblasts require high concentrations of insulin (1.6 microM) or lower concentrations of IGF-I (25 nM) to differentiate, and expression of MyoD1 is not constitutive. IGF receptors were studied in microsomal membranes of proliferating and quiescent myoblasts and myotubes. IGF-II binding was also studied in inducible myoblasts transfected with the MyoD1 cDNA (clone EP5). Both inducible and permissive cells exhibited a single class of binding sites with similar affinity for IGF-I (Kd 0.8-1.2 nM). Affinity cross-linking of [125I]IGF-I to microsomal membranes, under reducing conditions, revealed a binding moiety with an apparent molecular mass of 130 kDa in permissive cells and 140 kDa in inducible cells, which corresponded to the alpha subunit of the IGF-I receptor. In permissive quiescent myoblasts, linear Scatchard plots suggested that [125I]IGF-II bound to a single class of binding sites (Kd 0.6 nM) compatible with binding to the IGF-II/M6P receptor. This was confirmed by affinity cross-linking experiments showing a labeled complex with an apparent molecular mass of 260 kDa and 220 kDa when studied under reducing and non-reducing conditions, respectively. In contrast, competitive inhibition of [125I]IGF-II binding to inducible quiescent myoblasts generated curvilinear Scatchard plots which could be resolved into two single classes of binding sites. One of them corresponded to the IGF-II/M6P receptor (Kd 0.2 nM) as evidenced by cross-linking experiments. The second was the binding site of highest affinity (Kd 0.04 nM) which was less inhibited by IGF-I than by IGF-II and was not inhibited by insulin. It migrated in SDS/PAGE at a position equivalent a molecular mass of 140 kDa, under reducing conditions, and at approximately 300 kDa, under non-reducing conditions. The labeling of this atypical binding moiety was not inhibited by anti(IGF-II/M6P-receptor) immunoglobulin. It was also observed in permissive and inducible myoblasts at proliferating stage. It was absent for permissive quiescent myoblasts and from permissive and inducible myotubes. Forced expression of MyoD1 in inducible cells (EP5 cells) dramatically reduced [125I]IGF-II binding to this atypical receptor. It emerges from these experiments that C2 cells express a putative alpha 2 beta 2 IGF-II receptor structurally related to the insulin/IGF-I receptor family. It is present in myoblasts but not in myotubes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

The insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptor mediates IGF-II-induced motility in human rhabdomyosarcoma cells.

Insulin-like growth factor-II (IGF-II) is an autocrine growth and motility factor for human rhabdomyosarcoma. It interacts with three different receptors: the IGF-I, the IGF-II, and the insulin receptor. A specific function of the IGF-II receptor in mediating IGF-II responses has not been defined. In this report we investigate the mechanism of IGF-II-mediated motility in rhabdomyosarcoma cells. We demonstrate that IGF-II and [Leu27]IGF-II, an analog selective for the IGF-II receptor, stimulate motility at concentrations in which they interact only with their own receptor. An antibody that blocks the IGF-I receptor does not inhibit either peptide activity, while an antibody specific for the IGF-II receptor suppresses the IGF-II-induced motility. This antibody does not interfere with rhabdomyosarcoma cell proliferation. We conclude that in rhabdomyosarcoma cells IGF-II stimulates two different responses mediated by distinct receptors: 1) a mitogenic response through the type I receptor and 2) a motility response through the type II receptor.

Affinity Labels↗

PubMed1992

Mutation creating a new splice site in the growth hormone receptor genes of 37 Ecuadorean patients with Laron syndrome.

Laron syndrome is an autosomal recessive condition characterized by resistance to growth hormone. We sought to determine the molecular basis of this condition in an Ecuadorean population with a high incidence of affected individuals. Growth hormone receptor gene sequences from an obligate heterozygote were amplified by the polymerase chain reaction and screened for mutations using denaturing gradient gel electrophoresis. Only exon 6 revealed homo- and heteroduplexes on denaturing gradient gels. Sequencing revealed a substitution of guanine for adenine in the third position of codon 180 that did not change the amino acid encoded. Sequencing of the exon 6-exon 7 splice junction from RNA-polymerase chain reaction amplified cellular RNA of an affected individual revealed that the substitution activates a 5' splice site 24 nucleotides upstream from the normal exon 6-intron 6 boundary. Splicing in two probands' lymphoblasts occurred virtually exclusively at the abnormal 5' splice site created by the codon 180 substitution. Exon 6 sequences from 38 patients and 47 relatives were amplified and analyzed by sequencing or dot-blot hybridization with allele-specific oligonucleotides. The substitution was detected in 74 of 76 Laron syndrome patients' GH-receptor alleles. All 26 parents and 12 of 21 unaffected siblings were heterozygous for this mutation. It was absent in 61 unrelated unaffected control individuals. We conclude that the codon 180 nucleotide substitution probably causes Laron syndrome as translation of the observed, abnormally spliced growth hormone receptor transcript would lead to the synthesis of a receptor protein with an 8 amino acid deletion from the extracellular domain.

Alternative Splicing↗

PubMed1992

Six-year results of a randomized, prospective trial of human growth hormone and oxandrolone in Turner syndrome.

Seventy girls with Turner syndrome, verified by karyotype, were randomly assigned to observation or treatment with human growth hormone (hGH), oxandrolone, or a combination of hGH plus oxandrolone for a period of 12 to 24 months, to assess the effect of treatment on growth velocity and adult height. Subsequently, all subjects received either hGH alone or hGH plus oxandrolone. Data are presented for 62 subjects treated for a period of 3 to 6 years. When compared with the anticipated growth rate in untreated patients, the growth rate after treatment with hGH, both alone and in combination with oxandrolone, showed a sustained increase for at least 6 years. Treatment is continuing in over half of the subjects; at present, 14 (82%) of 17 girls receiving hGH alone and 41 (91%) of 45 girls receiving combination therapy exceeded their expected adult heights. Thirty girls have completed treatment; mean height for these 30 patients is 151.9 cm, compared with their mean original projected adult height of 143.8 cm. We conclude that therapy with hGH, alone and in combination with oxandrolone, can result in a sustained increase in growth rate and a significant increase in adult height for most prepubertal girls with Turner syndrome.

Age Determination by Skeleton↗

PubMed1992

Cardiovascular evaluation in Turner syndrome: utility of MR imaging.

Forty patients with karyotypically proven Turner syndrome were prospectively studied using magnetic resonance imaging (MRI) and echocardiography in order to determine the frequency of cardiovascular anomalies and to assess the utility of both imaging modalities as methods for cardiovascular evaluation in Turner syndrome. Cardiovascular anomalies were found in 45% of patients. A high absolute prevalence of bicuspid aortic valve (17.5%) and aortic coarctation (12.5%) were observed relative to comparable series. Of clinically significant abnormalities, three of five aortic coarctations and four of five ascending aortic dilatations were solely MRI detected and not evident at echocardiographic examination. MRI is thus seen as a valuable adjunct to echocardiography in the cardiovascular evaluation of Turner syndrome patients. The usefulness of MRI primarily relates to its ability to provide excellent visualisation of the entire thoracic aorta where a large proportion of clinically significant anomalies occur in Turner syndrome.

Adolescent↗

PubMed1992

Platelet-derived growth factor isoforms decrease insulin-like growth factor I gene expression in rat vascular smooth muscle cells and selectively stimulate the biosynthesis of insulin-like growth factor binding protein 4.

Platelet-derived growth factor (PDGF) is believed to be a critical mediator of vascular smooth muscle cell (SMC) proliferation. Because insulin-like growth factor (IGF) I (IGF-I) functions as a progression factor for the mitogenic effects of PDGF, we hypothesized that IGF-I gene expression and the production of IGF binding proteins (IGFBPs) by cultured rat aortic SMCs might be regulated by one or more of the three isoforms of PDGF: PDGF-AA, -BB, and -AB. IGF-I gene expression was highly dependent on cell density: IGF-I mRNA transcripts decreased markedly as a function of cell confluence. IGF-I mRNA content was inhibited to a similar degree by PDGF-AA, -BB, and -AB through a mechanism requiring protein synthesis. The inhibition was readily apparent at 4 hours, reaching approximately 25% of control levels after 24 hours. Radioimmunoassayable IGF-I was only barely detectable in SMC-conditioned serum-free medium and not significantly modulated by PDGF. Western ligand blot revealed that vascular SMCs release 30-kd and 24-kd IGFBP into serum-free conditioned medium. PDGF isoforms did not significantly alter release of the 30-kd IGFBP but evoked a fivefold to sixfold increase in the 24-kd IGFBP. The 24-kd IGFBP was found to comigrate with IGFBP-4, a recently identified binding protein that inhibits IGF action. The 30-kd protein was not merely a glycosylated form of IGFBP-4, because it was not sensitive to N-glycanase digestion. PDGF-AA, -BB, and -AB markedly induced expression of IGFBP-4 mRNA in a time- and concentration-dependent fashion. Vascular SMCs also express IGFBP-2 mRNA, but its abundance was not induced by PDGF. In conclusion, PDGF evokes a complex pattern of regulation of genes in the IGF/IGFBP system. By inhibiting IGF-I production and specifically inducing biosynthesis of the inhibitory binding protein IGFBP-4, PDGF may set in motion mechanisms to limit the final magnitude of the mitogenic response.

Animals↗

PubMed1992

Decreased serum insulin-like growth factor-I associated with growth failure in newborn lambs with experimental cyanotic heart disease.

To determine whether chronic hypoxemia results in alterations in endocrine function that may contribute to growth failure, we measured growth hormone (GH), somatomedins (insulin-like growth factors I and II, IGF-I and IGF-2), hepatic growth hormone receptors, and circulating IGF-binding proteins IGFBP-3 and IGFBP-2 in 12 newborn lambs with surgically created pulmonic stenosis and atrial septal defect, and in 10 controls. During chronic hypoxemia (oxygen saturation of 60-74% for 2 wk), weight gain was 60% of control (hypoxemic, 135 +/- 20 vs. control, 216 +/- 26 g/d, P less than 0.02). IGF-I was decreased by 43% (hypoxemic 253.6 +/- 29.3 SE vs. control 448.0 +/- 75.5 ng/ml, P = 0.01), whereas GH was unchanged (19.9 +/- 5.1 vs. 11.9 +/- 3.0 ng/ml, NS). The increase in IGF-1 was associated with a decrease in IGFBP-3 (hypoxemic, 5.09 +/- 1.25 vs. control, 11.2 +/- 1.08 arbitrary absorbency units per mm (Au.mm), P less than 0.01), and increase in IGFBP-2 (0.47 +/- 0.03 vs. 0.19 +/- 0.13 Au.mm, P less than 0.05), but no significant downregulation of hepatic GH receptors (hypoxemic, 106.1 +/- 20.1 vs. control, 147.3 +/- 25.9 fmol/mg, NS). Thus, chronic hypoxemia in the newborn is associated with a decrease in IGF-I and IGFBP-3 in the face of normal GH. This suggests peripheral GH unresponsiveness, similar to protein-calorie malnutrition or GH receptor deficiency dwarfism, but mediated at a level distal to the hepatic GH receptor.

Animals↗

PubMed1992

Granulosa cell-derived insulin-like growth factor (IGF) binding proteins are inhibitory to IGF-I hormonal action. Evidence derived from the use of a truncated IGF-I analogue.

An increasing body of information now suggests that insulin-like growth factor (IGF) binding proteins (BPs) may serve as antigonadotropins at the level of the ovary. It is the objective of the present communication to evaluate the functional role of endogenous (granulosa cell-derived) IGFBPs by exploiting the unique properties of des(1-3)IGF-I, a naturally occurring IGF-I analogue characterized as a weak ligand of IGFBPs but not of type I IGF receptors. Given IGFBP-replete circumstances, des(1-3)IGF-I proved more potent (10-fold) than its intact counterpart in promoting the follicle stimulating hormone (FSH)-stimulated accumulation of progesterone by cultured rat granulosa cells. In contrast, des(1-3)IGF-I proved virtually equipotent to the unmodified principle under IGFBP-deplete circumstances. Taken together, these findings are in keeping with the notion and that the apparently enhanced potency of des(1-3)IGF-I (under IGFBP-replete conditions) is due to its diminished affinity for endogenously generated IGFBPs and that rat granulosa cell-derived IGFBPs are inhibitory to IGF (and thus inevitably to gonadotropin) hormonal action. Accordingly, the reported ability of gonadotropins to attenuate IGFBP release by granulosa cells may be designed to enhance the bioavailability of endogenously generated IGFs in the best interest of ovarian steroidogenesis.

Animals↗

PubMed1992

Effects of continuous infusion of insulin-like growth factor I and II, alone and in combination with thyroxine or growth hormone, on the neonatal hypophysectomized rat.

In this study, we examined the effects of systemically administered insulin-like growth factor (IGF)-I and -II on growth of the hypophysectomized (Hx) neonatal rat. Neonatal Wistar rats were Hx or sham Hx on postnatal day (PND) 6 and implanted sc with Alzet pumps on PND 10. Recombinant human IGF-I or -II were infused between PND 10 and 18 at an average dose of 1.9 micrograms/g body weight (BW) per day. In addition, some groups received daily sc injections of recombinant human GH or thyroxine (T4) at 2.5 micrograms and 25 ng/g BW per day, respectively. Pups were sacrificed on PND 18 and serum IGF levels determined. Despite restoration of serum IGF-I levels to sham control values in the Hx pups infused with IGF-I, no significant increase in BW occurred, although some increase in individual organ growth was observed (spleen, kidney, lung). Similarly, administration of IGF-II proved ineffective as a growth promoter in the neonatal Hx rat. In contrast, GH alone stimulated BW gain (P less than 0.001). T4 proved most potent in increasing skeletal growth (50% increase over Hx controls, P less than 0.001), without increasing serum IGF-I or -II levels. IGF-I and GH were equally effective in promoting a small yet statistically significant (17% over Hx controls, P less than 0.05) increase in skeletal growth. A synergistic effect on BW was observed with combined administration of T4 plus IGF-I to the Hx pups (P less than 0.05). The effects of hormonal therapy on serum IGF binding proteins (IGFBPs) was assessed by Western ligand blots. Administration of IGF-I, but not GH, resulted in increased levels of IGFBP-3, the predominant IGFBP of the adult rat. We conclude that systemically administered IGFs in doses that result in normalization of serum levels are ineffective promoters of somatic growth in neonatal rats. While normalization of serum IGF-I levels does result in modest skeletal growth, selective organ growth and increased serum IGFBP-3, growth stimulation does not equal that seen with GH (body weight) or thyroid hormone (skeletal growth). Differences in IGFBP profiles fail to account for the increased potency of GH as a promoter of BW gain. Thus, our data do not support a major endocrine role for IGF-I or -II in neonatal growth, but are consistent with an autocrine/paracrine action of IGF in the mediation of neonatal mammalian growth.

Animals↗

PubMed1992

Insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP-4) concentration and stimulate IGFBP-3 independently of IGF receptors in human fibroblasts and epidermal cells.

Recent studies have provided a consensus that insulin-like growth factor-I (IGF-I) stimulates IGF-binding protein-3 (IGFBP-3) in vivo and in vitro. While it also appears well established that IGFBP-1 is inversely related to insulin concentrations, evidence regarding regulation of other IGFBP is inconclusive. Using immunoprecipitation and Western ligand blot, we have characterized the IGFBPs released into conditioned medium (CM) by cells from the adult human fibroblast cell line N3652 and the human epidermal squamous cell carcinoma line SCL-1. N3652 cells expressed IGFBP-3, IGFBP-2, a 24-kilodalton (kDa) IGFBP presumed to be IGFBP-4, and IGFBPs at 30 and 28 kDa. SCL-1 expressed IGFBP-3 and a putative IGFBP-4, with intermediate bands at 34 and 30 kDa. As determined by ligand blot of CM from confluent cells 72 h after the addition of peptides to serum-free medium, IGF-I and IGF-II potently stimulated IGFBP-3 in both cell lines, but otherwise IGFBP regulation in the two cells diverged. In N3652 cells, IGFBP-3 concentrations in CM increased to 700% and 800% of basal levels in the presence of IGF-I and IGF-II (at 100 ng/ml; n = 5 experiments), respectively. IGFBP-3 was not affected by insulin up to 10 micrograms/ml. In contrast, IGFBP-4 levels were diminished 54% and 73% by 100 ng/ml IGF-I and IGF-II, respectively, with no response to insulin. In SCL-1 cells, IGF-I and IGF-II were virtually identical in stimulating a mean 200% increase in IGFBP-3 (n = 5 experiments). Insulin was less potent, but caused a significant stimulation of IGFBP-3 levels. IGF-I, IGF-II, and insulin all stimulated an approximately 50% increase in IGFBP-4 concentrations. To test the hypothesis that IGF-induced alterations in IGFBP-3 and IGFBP-4 concentrations were regulated via the type 1 IGF receptor, we attempted to block IGFBP changes with type 1 IGF receptor antibody alpha IR-3 and to induce IGFBP changes with an IGF-II analog, [Leu27]IGF-II, with little affinity for the type 1 receptor. alpha IR-3 failed to block either the IGF-induced rise in IGFBP-3 in each cell line or the decline in IGFBP-4 in N3652 CM. [Leu27]IGF-II was as potent as IGF-II or IGF-I in inducing changes in IGFBP-3 and IGFBP-4 concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Carcinoma, Squamous Cell↗

PubMed1992

Regulation of insulin-like growth factor-binding protein expression by thyroid hormone in rat GH3 pituitary tumor cells.

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.

Animals↗

PubMed1992

Sensitive colorimetric bioassays for insulin-like growth factor (IGF) stimulation of cell proliferation and glucose consumption: use in studies of IGF analogs.

We have established two in vitro bioassay systems for quantification of insulin-like growth factor (IGF) bioactivity. The first assay was used to quantitate mitogenic activity and the second was used to quantitate metabolic activity. Both assays use BALB/c 3T3 fibroblasts grown under serum-free conditions; detection of bioactivity in assays was performed colorimetrically and did not require the use of radioisotopes. The mitogenic bioassay, which requires 48 h for detection, quantitates changes in cell number and provides an index for determining the mitogenic activity of growth factors. Changes in cell number were measured by the enzymatic reduction of exogenously added MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide] to MTT-formazan by mitochondrial enzymes, which was directly correlated to cell number. The metabolic bioassay, which requires 22 h for detection, measures glucose consumption by detecting changes from the initial glucose concentration of conditioned medium after addition of various growth factors. When appropriate standards were established for these bioassays, they demonstrated a high level of reproducibility (coefficients of variation were 0.085-0.096 for the mitogenic bioassay and 0.120-0.191 for the metabolic bioassay). Both assays can be performed in 96-well microtiter plates, without the use of radioisotopes, or the limitations of conventional glucose, amino acid, or thymidine incorporation studies. In initial experiments for assay specificity, epidermal growth factor had no measurable effect in either assay. However, IGF-I, IGF-II, and insulin demonstrated effects on both metabolism and mitogenesis. In the case of the mitogenic bioassay, the maximum mitogenic activation by these growth factors was approximately 180% of control, and these factors demonstrated parallel sigmoidal dose-response curves, ranging from 0.02-2 ng/ml for IGF-I and from 2-200 ng/ml for both IGF-II and insulin. In the metabolic bioassay, in contrast to the mitogenic bioassay, insulin showed a dose-response curve whose shape was different from those of IGF-I and IGF-II. IGF-I and IGF-II stimulated glucose consumption in dose-dependent ranges of 0.02-3 ng/ml and 0.4-40 ng/ml, respectively. However, the dose-response effect of insulin was wider, ranging from 0.1-2000 ng/ml. When these assays were used to measure the bioactivity of IGF analogs, a des(1-3)-IGF-I, which has decreased affinity for IGF binding proteins, demonstrated activity equivalent to IGF-I, a [Leu27]IGF-II, which has markedly diminished affinity for the type 1 IGF receptor, exhibited approximately 0.07% of the potency of IGF-I and 1% of the potency of IGF-II.

3T3 Cells↗

PubMed1992

Rat ovarian insulin-like growth factor binding protein-3: a growth hormone-dependent theca-interstitial cell-derived antigonadotropin.

To further the identification and characterization of insulin-like growth factor binding proteins at the level of the immature rat ovary, we have set out to study the ovarian expression, cellular localization, and hormonal regulation of the insulin-like growth factor binding protein (IGFBP)-3. To this end, use was made of a solution hybridization/RNAse protection assay wherein ovarian total RNA from immature (21-23 days old) female rats was hybridized with a 343 bases-long [32P]-labeled rat IGFBP-3 riboprobe. As in liver, a single protected fragment (315 bases-long) corresponding to IGFBP-3 transcripts was identified in whole ovarian material. Cellular localization studies revealed the IGFBP-3 gene to be exclusively expressed in the theca-interstitial rather than the granulosa cell compartment. To confirm presence and cellular distribution of the IGFBP-3 protein, media conditioned by cultured granulosa cells, theca-interstitial cells, and whole ovarian dispersates were subjected to Western Ligand Blotting. Importantly, media conditioned by cultured theca-interstitial (but not granulosa) cells displayed an IGFBP the size of rat IGFBP-3 (46kDa) as determined by comigration with a rat serum standard. A similarly-sized band was apparent in media conditioned by cultured whole ovarian dispersates reflecting in all likelihood the contribution of the theca-interstitial cell component. Significantly, deglycosylation of media conditioned by cultured theca-interstitial cells revealed the glycosylated nature of the 46kDa IGFBP species as judged by the apparent reduction in its molecular size to 35kDa. Similar alterations were noted in corresponding rat serum samples. Hypophysectomy of immature rats resulted in a modest but statistically insignificant decrease in the relative (densitometrically-quantified) abundance of ovarian IGFBP-3 transcripts, an effect further augmented by the systemic provision of either FSH or diethylstilbestrol (DES). In contrast, systemic treatment of hypophysectomized rats with GH produced a marked (3.2-fold) increase (P less than 0.05) in the steady state levels of ovarian (as well as hepatic) IGFBP-3 transcripts. However, the concurrent provision of either FSH or DES resulted in substantial (P less than 0.05) attenuation (78 and 57% inhibition, respectively) of the upregulatory GH effect. These findings document the highly compartmentalized expression of the IGFBP-3 gene at the level of the immature rat ovary, implicate the theca-interstitial cell as the sole source of its generation, reveal its pituitary dependence, and disclose its diametrically-opposed (indeed antagonistic) regulation by FSH (or estrogens) and GH.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Not all insulin-like growth factor-binding proteins (IGFBPs) are detectable by western ligand blotting: case studies of PC12 pheochromocytoma and rat anterior pituitary IGFBPs and proteolyzed IGFBP-3.

We studied the limitations of the Western ligand blot (WLB) for detecting insulin-like growth factor-binding proteins (IGFBPs). PC12 rat pheochromocytoma cells and rat anterior pituitary cells (AP) secrete IGFBPs that cannot be detected by WLB. We used affinity labeling, WLB, dot blotting, competitive binding, ion exchange chromatography, and deglycosylation to characterize these IGFBPs. These IGFBPs were compared with pregnancy protease-derived IGFBP-3 fragments that also bind insulin-like growth factors (IGFs), but are not detectable by WLB. We showed that PC12 IGFBP is cationic, not glycosylated, with 25,500 mol wt reduced (18,500 unreduced), with high affinity for IGF-II and low affinity for IGF-I. It cannot be detected by WLB and is not a proteolytic derivative of other IGFBPs or IGF-II receptors. Its binding activity is not destroyed by sodium dodecyl sulfate (SDS) and heating. It binds to nitrocellulose and IGF-II after dot blotting, but not to IGF-II during WLB. AP also secrete an IGFBP(s) that was not detectable by WLB. AP IGFBPs, unlike those of PC12, have a higher mol wt, and at least one component is glycosylated. The failure of WLB to detect these proteins remains unexplained. Pregnancy protease-derived IGFBP-3 fragments also bind IGFs and are not detectable by WLB. However, they do electrotransfer to nitrocellulose. The failure of WLB to detect these fragments is probably due to proteolysis rendering the binding site susceptible to irreversible denaturation (under conditions of WLB) during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that WLB, while valuable, may have significant limitations in specific cases. Other techniques must complement WLB for detection of IGFBPs in conditioned media and other biological specimens.

Affinity Labels↗

PubMed1992

Expression of a lactogen-dependent insulin-like growth factor-binding protein in cultured mouse mammary epithelial cells.

The ability of normal mouse mammary epithelial cells (MECs) to express insulin-like growth factor-binding proteins (IGFBPs) was examined. MECs were isolated from day 11 pregnant mice and cultured on floating collagen gels in serum-free basal medium. After 24 h, the medium was replaced with fresh medium with/or without mouse PRL (mPRL), mouse placental lactogen-I (mPL-I), mPL-II, mouse GH (mGH), IGF-I, and IGF-II, either alone or in combinations. The MECs were cultured for an additional 5 days before collection of conditioned medium (CM). The relative amount of IGFBPs present in the CM was determined by Western ligand blotting, and alpha-lactalbumin content was determined with a specific RIA. The CM of the MECs contained two IGFBPs, with approximate mol wt of 29K and 40-45K. The 40-45K IGFBP appears to be the mouse equivalent of IGFBP-3, but the identity of the 29K IGFBP is not presently known. The 29K IGFBP was not N-glycosylated and did not cross-react with antiserum to rodent IGFBP-2 or human IGFBP-1. Basal IGFBP expression was very low, but the addition of mPL-I, or mPL-II stimulated a marked increase in the amount of 29K IGFBP that was released into the CM and a lesser increase in the release of IGFBP-3. This increase in the release of 29K IGFBP was dose dependent, with increases found at concentrations as low as 1 ng/ml lactogen. mGH also stimulated the release of 29K IGFBP, but was less potent than any of the three lactogens. Treatment of MECs with either IGF-I or IGF-II increased the amount of both the 29K IGFBP and IGFBP-3 in the CM, with relative potencies similar to those of the lactogenic hormones. However, when either IGF-I or IGF-II was added together with one of the lactogenic hormones, the release of 29K IGFBP was increased in an additive manner. While the IGFs acted additively with the lactogenic hormones on the expression of 29K IGFBP, they did not stimulate alpha-lactalbumin production by the MECs or act to enhance the effects of the lactogenic hormones in stimulating alpha-lactalbumin production. This study demonstrates that IGFBPs are expressed in normal mouse MECs, and the release of these IGFBPs into the CM is hormonally regulated by both lactogenic hormones and IGFs.

Animals↗

PubMed1992

Insulin-like growth factor binding protein expression in the hypothyroid rat is age dependent.

Thyroid hormone is essential for normal growth and development. For certain T4 effects, there is a critical period during ontogeny when normal T4 levels are required, and thyroid replacement after that period cannot correct the changes in hypothyroid animals. We have previously described a prolonged high expression of serum insulin-like growth factor binding protein (IGFBP)-2 during the perinatal period in congenitally hypothyroid rats. To see if this effect was confined only to a certain period during rat ontogeny, we made rats hypothyroid with methimazole treatment either prenatally, or at different postnatal ages from 1 to 14 days of life, and at adult age. Serum IGF-I levels were reduced by approximately 30% in all the 18-day-old hypothyroid animals, and did not correlate with the duration of the hypothyroid state. Serum IGF-I levels in the adult animals were 50% of control levels. At the age of 18 days, control animals had only very low levels of IGFBP-2 demonstrable by western ligand blotting, whereas the congenitally hypothyroid animals had elevated levels. Pups placed on methimazole treatment since the first day of life showed higher IGFBP-2 levels at the age of 18 days, although the change was not as prominent as in the congenitally hypothyroid animals (200% vs. 500% of control levels, respectively). Binding protein changes were approximately 2-fold at the mRNA level. Rats started on methimazole after the first 5 days of life showed normal low levels of IGFBP-2 at the age of 18 days. Abnormal IGBFP-2 expression in congenitally or neonatally hypothyroid animals could be corrected by thyroid hormone replacement, if started during the first week of the life, but not later. In adult hypothyroid animals, there was no induction of IGFBP-2 expression, but the levels of IGFBP-3 and -4 were decreased to 80% and to 30% of control levels, respectively. IGFBP-3 messenger RNA (mRNA) levels were decreased to 50% of control levels but IGFBP-4 mRNA levels were paradoxically increased in the hypothyroid animals. All these changes could be corrected by T4 replacement. In conclusion, there exists a critical period during the perinatal development of the rat, when thyroid hormone is essential for a subsequent normal IGFBP-2 ontogenic pattern. Adult animals show a completely different IGFBP response to hypothyroidism, with a decrease of IGFBP-3 and -4 levels. Thus, the effects of thyroid hormone on IGF-IGFBP axis regulation depend on the developmental stage of the animal.

Aging↗

PubMed1992

Measurement and characterization of insulin-like growth factor binding protein-3 in human biological fluids: discrepancies between radioimmunoassay and ligand blotting.

The inability to detect insulin-like growth factor binding protein-3 (IGFBP-3) in some circumstances by Western ligand blot analysis has emphasized the need to characterize IGFBPs by both ligand binding and immunological techniques. In this study, we have: 1) characterized and quantified IGFBP-3 in nonpregnancy, pregnancy, and fetal cord serum, follicular, peritoneal, and amniotic fluid, seminal plasma, cerebrospinal fluid (CSF), and urine; 2) established a new IGFBP-3 RIA that detects both intact and fragments of IGFBP-3; 3) identified both intact and fragments of IGFBP-3 by Western immunoblot techniques; and 4) addressed the discordance between Western ligand blot analysis and RIA by assessing fluids for IGFBP proteolytic activity. All fluids examined, except pregnancy serum, CSF, and amniotic fluid, displayed a 44-34-kilodalton (kDa) IGFBP-3 doublet by Western ligand blot analysis. Western immunoblot analysis using specific IGFBP-3 antiserum showed a 44-34-kDa IGFBP-3 doublet and a 28-kDa fragment in nonpregnancy serum, fetal cord serum, follicular fluid, and peritoneal fluid. The immunoreactive 42-38-kDa doublet was faint in urine and seminal plasma. IGFBPs in CSF did not cross-react with IGFBP-3 antiserum. Pregnancy serum and amniotic fluid contained only the 28-kDa fragment when compared against equal volumes of nonpregnancy serum. With the development of an IGFBP-3 RIA, IGFBP-3 could be accurately measured; urine, CSF, and seminal plasma contained the lowest levels of IGFBP-3 at 27 +/- 3 ng/ml (mean +/- SEM), 110 +/- 26 ng/ml, and 209 +/- 56 ng/ml, respectively. In increasing concentration: fetal cord serum contained 753 +/- 101 ng/ml; peritoneal fluid, 1124 +/- 130 ng/ml; follicular fluid, 2356 +/- 211 ng/ml; nonpregnancy serum, 3556 +/- 508 ng/ml; pregnancy serum, 3718 +/- 842 ng/ml; and amniotic fluid, 5150 +/- 688 ng/ml. The measurable concentrations of IGFBP-3 in CSF and the high concentrations measured in pregnancy serum and amniotic fluid conflicted with Western blot analysis. Thus, fluids were assessed for IGFBP proteolytic activity by incubation with a source of IGFBP-3, either nonpregnancy serum or purified IGFBP-3. All fluids displayed some proteolytic activity with either assay. Fluids with little protease activity (nonpregnancy serum, follicular fluid, and urine) showed a close relationship between immunoassayable IGFBP-3 by RIA and IGFBP-3 band intensity by Western ligand blot. Fluids with high proteolytic activity (pregnancy serum, CSF, seminal plasma, peritoneal fluid, and amniotic fluid) gave discrepant IGFBP-3 values between RIA and Western ligand blot.(ABSTRACT TRUNCATED AT 400 WORDS)

Amniotic Fluid↗

PubMed1992

Non-receptor mediated, post-transcriptional regulation of insulin-like growth factor binding protein (IGFBP)-3 in Hs578T human breast cancer cells.

Hs578T human breast cancer cells secrete insulin-like growth factor binding protein 3 (IGFBP-3) as the major BP species. In addition, cell surface-associated IGFBP-3 is demonstrable by the use of cell monolayer affinity cross-linking or immunoperoxidase staining of the cell surface with a specific polyclonal anti-human IGFBP-3 antibody (alpha IGFBP-3 gamma 1). In this study, we have demonstrated that regulation of Hs578T IGFBP-3 by IGF peptides is specific, non-receptor mediated, and post-translational by showing: 1) dose-dependent increase of IGFBP-3 in conditioned media (CM) following addition of IGF-I and -II (maximum 13 fold increase at 100 ng/ml), but not by insulin up to 1 mg/ml; 2) no change in CM IGFBP-3 level by [Gln3,Ala4,Tyr15,Leu16] IGF-I, which has decreased affinity for IGFBPs; 3) no change in IGFBP-3 mRNA following addition of IGFs; 4) release of cell surface-associated IGFBP-3 into CM by the addition of IGFs, but not by [Gln3,Ala4,Tyr15,Leu16]IGF-I. These studies demonstrate that IGF peptides regulate CM concentrations of IGFBP-3 through non-receptor mediated dissociation of cell surface-associated IGFBP-3.

Blotting, Northern↗