Biomedical subjects

D R Powell

Publications and source records attributed to D R Powell.

At least 19 recordsLinked to original sources

PubMed1992

Regulation of insulin-like growth factor-binding protein (IGFBP) expression by breast cancer cells: use of IGFBP-1 as an inhibitor of insulin-like growth factor action.

BACKGROUND: The insulin-like growth factors (IGFs) play an important role in normal growth and development. Evidence suggests they may also regulate the growth of several cancer cell types. This regulation is mediated by interactions between the receptors and ligands. There is now ample evidence to suggest that these interactions are also influenced by extracellular IGF-binding proteins (IGFBPs). Six different IGFBPs have been cloned. Some species may act to inhibit the mitogenic effects of the IGFs. Since breast cancer cells are responsive to the IGFs, it is possible that regulated expression of the IGFBPs affects tumor growth. Furthermore, inhibitory binding proteins could be used as neutralizers of IGF action. PURPOSE: We conducted this study to fully characterize the expression and hormonal regulation of IGF-binding protein expression in human MCF-7 breast cancer cells and to test the ability of purified IGFBP-1 to inhibit IGF-I action. METHODS: We used ribonuclease protection assays and Western ligand blotting to examine IGFBP expression in MCF-7 cells. The effect of IGF-I, IGFBP-1, and 17 beta-estradiol on serum-free cell growth was also studied. RESULTS: MCF-7 cells expressed IGFBP-2, IGFBP-4, and IGFBP-5 RNA and protein. These cells are dependent on estrogen for growth. In short-term culture, IGF-I can substitute for estrogen. Concomitant addition of IGF-I and estrogen enhanced stimulation above the level achieved by either factor alone. Estrogen also increased IGFBP production, making it unlikely that the IGFBPs induced by estrogen in MCF-7 cells could function as major inhibitors of IGF action. In contrast, exogenous addition of IGFBP-1 could block IGF-I-induced mitogenesis; this effect was reversible by excess IGF-I. CONCLUSIONS: The studies suggest that cancer cell growth may be regulated by endogenous IGFBP expression. Furthermore, the exogenous addition of the IGFBP-1 blocked IGF-I action and potentially could be used as a pharmacologic inhibitor of IGF action.

Blotting, Western↗

PubMed1992

Contiguous localization of the genes encoding human insulin-like growth factor binding proteins 1 (IGBP1) and 3 (IGBP3) on chromosome 7.

In extracellular fluids the insulin-like growth factors (IGFs) are bound to specific binding proteins (IGBPs). The genes for two members of this protein family have been mapped, the IGBP1 gene to human chromosomal region 7p14-p12 and the IGBP2 gene to region 2q33-q34. In this study, somatic cell hybrid analysis indicated that IGBP3 is also located on chromosome 7. Pulsed-field gel electrophoresis was used to demonstrate the close physical linkage between IGBP1 and IGBP3. Overlapping cosmid clones encompassing these genes were isolated, and restriction endonuclease mapping showed that the genes are arranged in a tail-to-tail fashion separated by 20 kb of DNA. Further characterization of the IGBP1 DNA sequence disclosed a duplication of the intron 3-exon 4 junction within the third intron. In addition, we report RFLPs for ApaLI and TaqI in the IGBP1 locus.

Amino Acid Sequence↗

PubMed1992

Effect of progestin, antiprogestin, and relaxin on the accumulation of prolactin and insulin-like growth factor-binding protein-1 messenger ribonucleic acid in human endometrial stromal cells.

Prolactin (PRL) and insulin-like growth factor-binding protein (IGFBP-1) are two major secretory proteins of human endometrial/decidual cells. We have characterized the mRNA of PRL and IGFBP-1 and studied the effect of progestin, medroxyprogesterone acetate (MPA), anti-progestin (RU486), and relaxin (RLX) on the levels of these two mRNA transcripts in a long-term culture of human endometrial stromal cells. Northern blot analysis showed that the size of PRL mRNA was 1.15 kb and that of IGFBP-1 mRNA, 1.6 kb. Primer extension of endometrial/decidual IGFBP-1 mRNA showed two transcription initiation sites identical to those found in HepG2 human hepatoma cell line. The levels of mRNA in control samples remained low, approximately 2 pg PRL and approximately 5 pg IGFBP-1/microgram RNA at various times of culture. When stromal cells were treated with MPA for 28 days, PRL mRNA gradually increased 100-fold whereas IGFBP-1 mRNA exponentially increased approximately 1000-fold compared to control values and leveled after 25 days in culture. The timing of maximal stimulation was shortened by withdrawing MPA or by replacing MPA with RU486. After removal of MPA, levels of both mRNAs increased and each peaked after approximately 10 days, with PRL showing a 2-fold and IGFBP-1 a 20-fold increase compared to cells treated with MPA continuously. Replacing MPA by RU486 caused a rapid increase of PRL mRNA (2-3-fold) in 2-3 days followed by a gradual reduction to less than 20% of peak levels over the next 3 days. IGFBP-1 mRNA levels increased 30- and 100-fold in 1-2 days followed by a reduction to less than 20% of peak levels over the next 24 h. The reduction of mRNA levels by RU486 was reversed when cells were rechallenged with MPA. Relaxin alone caused a transient stimulation of PRL and IGFBP-1 mRNA. Maximal stimulation occurred between 10 and 20 days of culture and was 100-fold for PRL and 1000-fold for IGFBP-1 relative to control values. Cells treated with MPA and RLX in sequence had higher mRNA levels than cells treated with MPA continuously or cells subjected to MPA withdrawal. Maximal mRNA levels reached 0.4 ng PRL and approximately 8 ng IGFBP-1/microgram total RNA, approximately 0.04% and 0.8% of cellular RNA. The mRNA levels under various hormonal manipulations were similar to the previously published synthesis and secretion patterns of PRL and IGFBP-1 proteins in this system.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1991

Insulin inhibits transcription of the human gene for insulin-like growth factor-binding protein-1.

Insulin rapidly lowers serum insulin-like growth factor-binding protein-1 (IGFBP-1) levels in vivo. In studies reported here, HEP G2 cells were used as a model system to investigate how insulin achieves this effect. When HEP G2 cells were incubated with 100 nM insulin for 6, 14, or 24 h, IGFBP-1 protein levels in conditioned medium fell to approximately 50% of control values. This apparently was due to a fall in the rate of IGFBP-1 protein synthesis, since HEP G2 cells incorporated 46% less [35S]methionine into IGFBP-1 during a 4-h incubation with 100 nM insulin. IGFBP-1 mRNA levels were similarly affected by 100 nM insulin, falling to 45% of control values after 2 h, and to 9% of control values after 4 h of incubation with this hormone. The fall in IGFBP-1 mRNA level is consistent with data from nuclear transcription assays. HEP G2 nuclei isolated from cells that were incubated with 100 nM insulin for 2 h synthesized only approximately 1/3 the number of IGFBP-1 transcripts as did control nuclei. Further evidence that insulin decreases IGFBP-1 gene transcription comes from transient transfections using chimeric IGFBP-1 promoter-chloramphenicol acetyltransferase reporter gene constructs. IGFBP-1 promoter activity fell to approximately 50% of control values when HEP G2 cells transfected with a construct containing the first 1205 base pairs of the IGFBP-1 promoter were incubated with 100 nM insulin for 6, 14, or 24 h. Insulin lowered both IGFBP-1 protein levels and promoter activity in a dose-dependent manner. A half-maximal effect was found at approximately 1 nM insulin and a maximal effect was found at approximately 10 nM insulin in each instance. Transfections with constructs containing smaller IGFBP-1 promoter fragments showed that the region spanning from 103 to 529 base pairs 5' to the IGFBP-1 mRNA cap site was necessary to demonstrate the inhibitory effect of insulin. These studies indicate that insulin lowers IGFBP-1 protein levels, at least in part, by rapidly decreasing the rate of IGFBP-1 gene transcription, and suggest that this insulin-mediated fall in transcription is conferred through a specific region of the IGFBP-1 promoter.

Carrier Proteins↗

PubMed1991

Identification of insulin-like growth factor binding proteins in breast cancer cells.

The insulin-like growth factors (IGFs) are potent mitogens for some breast cancer cell lines. Recent evidence suggests that IGF-induced mitogenesis may be influenced by specific IGF binding proteins (IGFBPs). In this study, breast cancer cell lines were examined for IGFBP protein and mRNA expression. Western ligand blot examination of conditioned media from breast cancer cell lines suggested that the IGFBP protein expression was heterogeneous. Although all breast cancer cell lines expressed a 24 kDa binding protein, MCF-7, an estrogen receptor positive (ER+) cell line, expressed a IGFBP compatible with reported sizes for IGFBP-2. Estrogen receptor negative (ER-) cells (MDA-MD-231, Hs578T) secreted IGFBPs consistent with sizes reported for IGFBP-1 and -3. Examination of mRNA expression supported these findings; IGFBP-2 was seen in all (4/4) ER+ cell lines while high levels of IGFBP-3 were found in ER- cell lines (3/5), although lower levels of IGFBP-3 mRNA could be found in some ER+ cell lines. In MCF-7 cells, steady state levels of IGFBP-3 mRNA were decreased by estradiol, while IGFBP-2 mRNA levels were slightly increased. These data suggest that IGFBP expression by breast cancer cells is heterogeneous, that the pattern of IGFBP expression is different between ER+ and ER- cell lines, and that in ER+ cells IGFBP mRNA may be regulated by estrogens. Thus, the IGFBPs may play an important role in mediating the mitogenic response of breast cancer cells to the IGFs.

Blotting, Northern↗

PubMed1991

Expression of the insulin like growth factor-binding protein 3 (IGFBP-3) gene is increased in human renal carcinomas.

After we had established that the IGFBP-3 gene is expressed in normal human kidney we examined renal adenocarcinoma tissue for alterations of the expression of this gene. For this purpose we prepared poly(A)+ RNA from normal kidney tissue and adjacent renal adenocarcinoma of 18 adult patients and compared the levels of IGFBP-3 mRNA by Northern analysis in both samples. The mean content by densitometry was markedly increased in the carcinoma tissues; in 17 of 18 patients the carcinoma contained significantly more IGFBP-3 mRNA than the normal kidney sample. The highest mRNA levels were found in patients with N2 and N3 lymph node extensions. Comparative Southern analysis of paired samples of four of these patients did not reveal amplification of the gene as the cause of these increased mRNA levels. In one patient, however, we identified a restriction fragment length polymorphism (RFLP) present in normal and malignant cellular DNA. This suggests a participation of the IGFBP-3 gene in the development of human renal cell cancer.

Aged↗

PubMed1991

Anthropometric characteristics as discriminators of body-building success.

A total of 36 non-elite male body builders were observed at the time of competition. Their mean physical characteristics (+/- S.D.) were: age, 24.6 +/- 4.8 years; height, 174.4 +/- 6.7 cm; bodyweight, 80.3 +/- 11.0 kg. Their body composition values were: percentage body fat, 9.3 +/- 1.6%; fat-free mass, 72.8 +/- 9.8 kg. The mean somatotype for all subjects was: endomorphy, 2.3 +/- 0.6; mesomorphy, 6.2 +/- 0.9; ectomorphy, 1.2 +/- 0.6. The body proportions (cm) included: biacromial diameter/bi-iliac diameter, 1.463 +/- 0.132; torso length/height, 0.468 +/- 0.018; chest circumference/abdominal circumference, 1.345 +/- 0.059. The body builders in the present study were younger, had lower bodyweights, lower fat-free mass, lower mesomorphy ratings, smaller circumferences, and smaller skeletal dimensions than elite body builders reported in the scientific literature. When the body builders in the present study were divided into successful and unsuccessful groups based on actual competition results, a multiple-discriminant analysis found that biacromial diameter/bi-iliac diameter, torso length/height, chest circumference/abdominal circumference, percentage body fat, height, and bodyweight accounted for 80.6% of the explained variance. These data indicate that the success of a body builder can be accounted for in large part by easily obtained physical variables.

Adult↗

PubMed1991

Biphasic contractile response of pulmonary artery to hypoxia.

Isolated perfused lungs exposed to low O2 exhibit a hypoxic pulmonary vasoconstriction response that is transient in nature. The purpose of this study was to determine whether the isolated pulmonary artery behaves similarly in response to hypoxia. Rat pulmonary arterial rings were placed in tissue baths (37 degrees C, air-5% CO2, pH = 7.4) and attached to force transducers. Maximum contractile responses (Po) to high K+ were elicited. After washout, arterial rings were submaximally contracted and made hypoxic (PO2 = 33.7 +/- 1.3, pH = 7.38 +/- 0.01). Aortic rings were used to obtain comparative data. The isolated pulmonary arterial hypoxic response was biphasic, displaying an initial rapid contraction of short duration (phase 1) then, before complete relaxation of this first response, a second slow but sustained contraction occurred (phase 2). Aortic rings did not exhibit a biphasic response, but showed only an initial short contraction followed by complete relaxation. The contractile response of the pulmonary artery was diminished when the endothelium was rendered nonfunctional. However, the phase 2 response was not endothelium dependent. Neither inhibitors of the lipoxygenase or cyclooxygenase pathways nor scavengers of extracellular reactive oxygen species had any effect on the biphasic hypoxic response. Pulmonary arterial hypoxic contractions were blunted when glucose was absent and appear to be dependent on glycolytic ATP. Results of this study show that hypoxia causes a biphasic contractile response of pulmonary arterial muscle and that two different mechanisms appear to be involved, since the transient phase 1 response is endothelium dependent, whereas the sustained contraction of phase 2 is endothelium independent.

Animals↗

PubMed1991

Regulation of insulin-like growth factor-binding protein messenger ribonucleic acid levels in sheep thyroid cells.

The insulin-like growth factors (IGFs) exist primarily bound to cell surface receptors or complexed to specific binding proteins (IGFBPs). The IGFBPs modulate the bioavailability of the IGFs and may enhance or inhibit IGF actions. Several distinct forms of IGFBPs have been described on the basis of size, immunological determinants, and distribution in biological fluids; the IGFBPs may differ as well in their biological function. Sheep thyroid cells produce IGFBPs under hormonal regulation. Cells grown in basal medium or with six-hormone (6H) medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) release nonglycosylated BPs that migrate at 24, 27, 29, and 32 kDa on Western ligand blot. Cells cultured with the thyroid mitogens epidermal growth factor and phorbol ester release additional glycosylated IGFBPs of 40-44 kDa. Immunoprecipitation experiments indicate that 29- and 32-kDa IGFBPs are antigenically related to IGFBP-2, and the 40- to 44-kDa proteins are related to IGFBP-3. Using specific cDNA probes IGFBP-1, -2, and -3, we examined the regulation of IGFBP mRNA levels in sheep thyroid cultures. The rat IGFBP-2 cDNA probe hybridized to an approximately 1.6-kilobase mRNA species in cells under all culture conditions. However, IGFBP-3 mRNA was detectable only in epidermal growth factor- or phorbol ester-treated cells and appeared within 4 h, preceding the release of IGFBP-3 protein into the medium. The 6H additives, which stimulate differentiated function in thyroid cells, inhibited the mRNA levels of both IGFBP-2 and IGFBP-3. IGFBP-1 mRNA was not detectable. The distinct regulation of these IGFBPs suggest that they may play different biological roles in modulating thyroid physiology.

Actins↗

PubMed1991

Insulin-like growth factor (IGF)-binding protein-3 blocks IGF-I-induced receptor down-regulation and cell desensitization in cultured bovine fibroblasts.

Insulin-like growth factor-I (IGF-I) initiates its diverse biological effects by binding to type I IGF receptors on cells. In addition, IGF-I associates with distinct proteins that can modulate its actions. One of these IGF-binding proteins, IGFBP-3, is the major circulating form in adults and is produced by many cells in culture. We investigated the effect of purified bovine IGFBP-3 on IGF-I binding and IGF-I stimulation of amino acid uptake and DNA synthesis in cultured bovine fibroblasts, a cell culture system highly suitable for these types of studies. Incubation of cells with IGF-I resulted in time- and dose-dependent decreases in [125I]IGF-I binding and IGF-I stimulated [3H]aminoisobutyric acid uptake and [3H]thymidine incorporation. Preincubation with 4 nM IGF-I resulted in a 50-60% decrease in IGF-I receptor binding, accompanied by marked decreases in IGF-I-stimulated [3H]aminoisobutyric acid uptake (50-60%) and [3H]thymidine incorporation (80-90%). Preincubation with the IGF-I analog [QAYL]IGF-I (4 nM) or with 100 nM insulin, growth factors that bind and activate type I IGF receptor signalling but have little or no affinity for IGFBP-3, had effects comparable to IGF-I, decreasing both IGF-I binding and action 50-95%. The addition of IGFBP-3 during the preincubation period with IGF-I blocked the decrease in receptor availability and prevented the cells from becoming desensitized. IGFBP-3 did not prevent the [QAYL]IGF-I- or insulin-induced receptor loss and cellular resistance to IGF-I. These data indicate that IGFBP-3 can prevent IGF-I-induced receptor down-regulation, a process that renders cells refractory to further stimulation by IGF-I. Thus, cell-derived IGFBP-3 may function in a buffering capacity to restrict IGF-I and target cell interaction, thereby modulating the biological response to changes in local IGF-I levels.

Aminoisobutyric Acids↗

PubMed1991

Insulin-like growth factors (IGFs) and IGF-binding proteins in the developing rhesus monkey.

Rhesus monkeys follow a developmental pattern of serum insulin-like growth factor-I (IGF-I) levels similar to that found in humans. In these monkeys, serum IGF-I levels peak during puberty (2.5-4.5 yr of age in males). We have examined the developmental pattern of IGF-binding protein-1 (IGFBP-1), -2, and -3 in serum by Western ligand blotting, the levels of IGFBP-3, IGF-I, and IGF-II in serum by RIA, and the IGFBP mRNA levels of IGFBP-1, -2, and -3 in the livers of rhesus monkeys from fetal life through adulthood by Northern analysis. The pattern of the serum levels of the IGFBPs reflected the liver mRNA levels of the IGFBPs. The IGFBP-1 and IGFBP-2 liver mRNA and serum levels were highest in the fetus and first year of life and were very low after 4 yr of age. Conversely, the IGFBP-3 liver mRNA and serum levels were relatively low early in life and peaked during puberty. The serum levels of IGF-I and IGF-II were strongly correlated with the level of IGFBP-3. We conclude that the developmental pattern of IGFBPs in the rhesus monkey is similar to that in the human, and that serum IGFBP levels are probably regulated by the rate of IGFBP mRNA synthesis.

Aging↗

PubMed1990

The promoter of the human gene for insulin-like growth factor binding protein-1. Basal promoter activity in HEP G2 cells depends upon liver factor B1.

Characterization of the human insulin-like growth factor binding protein-1 (IGFBP-1) promoter was initiated to facilitate study of developmental and hormonal factors regulating IGFBP-1 production. The region immediately 5' to the IGFBP-1 mRNA capsite is typical of a eukaryotic promoter, with a TATA sequence beginning 28 base pairs (bp) and a CCAAT promoter element beginning 72 bp upstream from this capsite. A 1.3-kilobase insert containing the IGFBP-1 capsite and 1205 bp of this putative IGFBP-1 promoter region directs expression of the reporter gene chloramphenicol acetyltransferase (CAT) in an orientation-specific manner in transfected HEP G2 cells, and the capsite identified for the CAT mRNA is identical to that identified for native IGFBP-1 mRNA. These observations suggest that the 1.3-kilobase insert contains the IGFBP-1 promoter. This promoter was further characterized by deletion analysis, site-directed mutagenesis, gel mobility shift assays, and DNaseI protection assays. These studies identify the CCAAT box region as the major cis element involved in basal IGFBP-1 promoter activity in HEP G2 cells, demonstrate that increased basal promoter activity is associated with the binding of at least one HEP G2 nuclear factor to the CCAAT box region, and indicate that the DNA binding factor(s) responsible for increased basal promoter activity is related to liver factor B1. These observations suggest that liver B1 is the major trans-acting factor stimulating basal IGFBP-1 promoter activity in HEP G2 cells.

Base Sequence↗

PubMed1990

Insulin-like growth factor binding protein-3. Organization of the human chromosomal gene and demonstration of promoter activity.

Insulin-like growth factor binding protein-3 (IGFBP-3) can modulate the mitogenic and metabolic effects of the insulin-like growth factors (IGFs). IGFBP-3 protein levels are developmentally regulated and influenced by a number of hormonal stimuli both in vitro and in vivo. As a first step toward understanding how hormonal and developmental factors regulate IGFBP-3 production, we are characterizing the human IGFBP-3 chromosomal gene and promoter. Southern analysis demonstrates a single copy of the IGFBP-3 gene in the human genome. This gene spans 8.9 kilobases; the protein-coding region is divided into four exons while a fifth exon contains the 3'-untranslated region. Primer extension studies locate the IGFBP-3 mRNA cap site 132 base pairs 5' to the ATG translation initiation codon. On the chromosomal gene, this cap site is located 30 base pairs 3' to the start of a TATA box and 97 base pairs 3' to a consensus GC upstream promoter element, an organization common to many eukaryotic promoters. When this potential IGFBP-3 promoter region is placed upstream to the chloramphenicol acetyltransferase reporter gene, it directs high-level production of chloramphenicol acetyltransferase in transfected COS-1 cells. These observations suggest an uncomplicated organization for the IGFBP-3 chromosomal gene and promoter in the human genome.

Amino Acid Sequence↗

PubMed1990

Structures of two isomeric hydroxamic acids: N-methyl-p-toluohydroxamic acid (MTH) and N-(4-methylphenyl)acetohydroxamic acid (MPA).

MTH, C9H11NO2, Mr = 165.19, monoclinic, P2(1)/n, a = 7.106 (2), b = 10.211 (3), c = 11.962 (2) A, beta = 97.99 (2) degrees, V = 859.5 A3, Z = 4, Dx = 1.276 Mg m-3, lambda(Mo K alpha) = 0.71069 A, mu = 0.054 mm-1, F(000) = 352, T = 138 K, final R = 0.042 for 1687 reflections with I greater than or equal to 2 sigma(I). MPA, C9H11NO2, Mr = 165.19, orthorhombic, Pbca, a = 9.300 (3), b = 9.463 (4), c = 19.340 (6) A, V = 1702.0 A3, Z = 8, Dx = 1.29 Mg m-3, lambda(Cu K alpha) = 1.5418 A, mu = 0.665 mm-1, F(000) = 704, T = 138 K, final R = 0.044 for 1362 reflections with I greater than or equal to 3 sigma(I). The hydroxamate group in each compound assumes the trans conformation as observed in other secondary hydroxamic acids. The interchange of the C and N substituents influences the planarity and dimensions (C = O, C-N bonds) of the hydroxamate moiety. In MTH the hydroxamate group shows significant deviation from planarity; the r.m.s. deviation of the four atoms, O = C-N-O(H), is 0.094 A, whereas in MPA it is only 0.004 A. This nonplanarity is due largely to out-of-plane bending at N, chi N = 23.9 (1) degree, as compared to chi N = 0.1 degree for MPA. The phenyl ring in MPA is nearly coplanar with the hydroxamate plane (dihedral angle of 10.3 degrees), but is significantly rotated from the hydroxamate plane in MTH (dihedral angle of 43.8 degrees). Electronic differences in the two molecules are considered.

Benzeneacetamides↗

PubMed1990

Insulin-like growth factor I expression by tumors of neuroectodermal origin with the t(11;22) chromosomal translocation. A potential autocrine growth factor.

Expression of insulin-like growth factor I (IGF-I) mRNA by some tumor cell lines of neuroectodermal origin has been described. To further explore the significance of IGF-I mRNA expression in these tumors, a more extensive analysis was performed. Most (9 of 10) neuroectodermal tumor cell lines with a t(11;22) translocation (primitive neuroectodermal tumor [PNET], Ewing's sarcoma, esthesioneuroblastoma) expressed IGF-I mRNA, whereas 0 of 15 cell lines without the translocation (PNET, neuroblastoma) expressed IGF-I. Furthermore, inasmuch as all neuroblastoma (12 of 12) cell lines examined expressed IGF-II RNA, the pattern of IGF expression could distinguish between these closely related tumors. CHP-100, a PNET cell line with the t(11;22) translocation, was shown to secrete both IGF-I protein and an IGF binding protein, IGFBP-2. This cell line also expressed the type I IGF receptor mRNA, and blockade of this receptor by a monoclonal antibody (alpha IR3) inhibited serum-free growth. These data demonstrate that IGF-I expression is a property of neuroectodermal tumors with a t(11;22) translocation and that interruption of an IGF-I autocrine loop inhibits the growth of these tumor cells.

Cell Division↗

PubMed1990

Structural and biological characterization of bovine insulin-like growth factor binding protein-3.

Insulin-like growth factor binding protein-3 (IGFBP-3) purified from bovine serum shares 19 of 25 amino-terminal amino acid residues with IGFBP-3 purified from human, rat, and porcine sources. A newly characterized bovine fibroblast model was used to investigate the biological effects of purified bovine IGFBP-3 (bIGFBP-3). Coincubation of insulin-like growth factor I (IGF-I) with increasing concentrations of bIGFBP-3 produced a dose-dependent inhibition of IGF-I-stimulated [3H]aminoisobutyric acid (AIB) uptake in cultured bovine fibroblasts. Inhibition was complete at equimolar concentrations of IGF-I and bIGFBP-3. Inhibition of IGF-I-stimulated [3H]AIB uptake paralleled the ability of bIGFBP-3 to prevent [125I]IGF-I cell surface binding. In contrast, preincubation with bIGFBP-3 resulted in a dose-dependent enhancement of IGF-I-stimulated [3H]AIB uptake; a 32-86% increase in IGF-I bioactivity was seen after a 24 h preexposure to 10 nM bIGFBP-3, and a 2- to 6-fold potentiation was seen after a 72 h preincubation. Preincubation with bIGFBP-3 increased both the sensitivity and maximal responsiveness of the cells to IGF-I. The potentiating effects of bIGFBP-3 were associated with increased [125I]IGF-I binding to cultured bovine fibroblasts. Affinity cross-linking experiments indicated that the increase in IGF-I binding was due to increased membrane-associated bIGFBP-3 rather than to a bIGFBP-3-induced increase in type I IGF receptors. bIGFBP-3 had no effect on insulin stimulation of [3H]AIB uptake under either experimental condition. These data suggest that soluble bIGFBP-3 inhibits IGF-I action by sequestering and preventing IGF-I receptor binding, whereas surface-associated bIGFBP-3 enhances the growth-promoting effects of IGF-I in bovine fibroblasts. We propose that IGFBP-3 serves a dual function in modulating IGF action in vivo.

Amino Acid Sequence↗

PubMed1990

The 25-kilodalton insulin-like growth factor (IGF)-binding protein inhibits both basal and IGF-I-mediated growth of chick embryo pelvic cartilage in vitro.

Insulin-like growth factor (IGF)-I stimulates the growth of many tissues, including growth plate cartilage. However, the role of IGF-binding proteins in the growth process is controversial. We purified a 25-kDa IGF-binding protein (BP-25) from amniotic fluid. We tested the effect of this BP-25 preparation on both basal and IGF-I-stimulated growth of chick embryo pelvic cartilages maintained in serum-free organ culture. Cartilage wet weight was 4.1 +/- 0.3 mg/cartilage initially; after 3 days, BP-25 inhibited both basal and IGF-I-stimulated growth. Control cartilages weighed 7.4 +/- 0.7 mg/cartilage, while those incubated with 100 nM BP-25 weighed 5.8 +/- 0.5 mg/cartilage (P less than 0.001 vs. control); BP-25 concentrations as low as 0.2 nM significantly inhibited basal cartilage growth. Cartilages incubated with 1.25 nM IGF-I weighed 10.4 +/- 0.8 mg/cartilage (P less than 0.001 vs. control), while those incubated with both 100 nM BP-25 and 1.25 nM IGF-I weighed 8.1 +/- 0.5 mg/cartilage (P less than 0.001 vs. cartilage incubated with IGF-I alone); BP-25 concentrations as low as 0.4 nM significantly inhibited IGF-I-stimulated cartilage growth. BP-25 also inhibited basal and IGF-I-stimulated increases in cartilage dry weight, [3H]thymidine incorporation into DNA, and 35SO4 incorporation into proteoglycan. A second BP-25 preparation, which in the presence of 1% platelet-poor plasma acts synergistically with IGF-I to stimulate DNA synthesis and cell replication of fibroblasts and smooth muscle cells in tissue culture, inhibited IGF-I-stimulated cartilage growth to the same degree as did our BP-25 preparation. In separate experiments, proteins present in serum-free medium conditioned for 3 days by chick cartilages were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and incubated with [125I]IGF-I. This medium was found to contain two IGF-binding proteins; one appeared to be the chick equivalent of BP-25, while the other had a molecular mass similar to that of a poorly characterized human 34-kDa IGF-binding protein. We conclude that purified BP-25 inhibits the growth of chick embryo pelvic cartilage in our serum-free organ culture system. Since conditioned medium from these cartilages contains both IGF-I-like peptides and IGF-binding proteins such as BP-25, we suggest that the IGF-binding proteins present may act to down-regulate the growth-promoting effects of the local IGF peptides.

Animals↗

PubMed1990

Characterization of insulin-like growth factor-binding proteins in human serum from patients with chronic renal failure.

Western ligand blotting of human serum reveals proteins of 41.5, 38, 33, 28, and 23 kD which will specifically bind [125I]insulin-like growth factor-I [( 125I]IGF-I). The relationship of these binding proteins (BPs) to the two serum BP-IGF complexes of 150 and 40 kD and their relationship to the two purified and well characterized IGF-BPs, BP-25 and BP-53, are not clear. The studies reported here used gel filtration, Western ligand blotting, deglycosylation, affinity cross-linking, and immunoprecipitation with antisera against BP-25 and BP-53 to explore the relationship among these IGF-BP forms. Serum from children with chronic renal failure was used for this study, since, by Western ligand blot, it was rich in all five IGF-BPs. The 28- and 33-kD BPs, which were hard to detect in normal serum, were elevated in chronic renal failure serum. Using this serum in each experiment, it was found that the large (150-kD) BP-IGF complex contained only the 41.5- and 38-kD BPs. Both of these BPs were precipitated by anti-BP-53 antiserum, but not by anti-BP-25 antisera, and both BPs were deglycosylated by endoglycosidase-F to a single 31-kD protein which retained the ability to bind IGF-I. This molecular mass of 31 kD is similar to the 28.7-kD mass for nonglycosylated BP-53 predicted by cDNA sequence analysis. In contrast, the 33-, 28-, and 23-kD BPs were only detected in the small (40-kD) BP-IGF complex. None of the three BPs was precipitated by anti-BP-53 antiserum, and none was digested by endoglycosidase-F. The 28-kD BP comigrated with pure BP-25 under all conditions and was precipitated by two independent anti-BP-25 antisera. The 33-kD BP was also precipitated by both anti-BP-25 antisera, while the 23-kD BP was not precipitated by any of the antisera tested. Thus, BP-53 consists of a core protein of 28.7 kD which is differentially glycosylated in serum to form two BPs of 41.5 and 38 kD; these BPs circulate exclusively as part of the large BP-IGF complex. BP-25 circulates in the small BP-IGF complex as a nonglycosylated protein with an apparent mol wt of 28 kD.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗