ABSTRACT
Insulin-like growth factor binding protein-2 (IGFBP-2) as one of the most important IGFBPs has never been assessed in the intracellular compartment in vivo. Since there is evidence for novel intracellular functions of distinct IGFBPs, we investigated the presence of IGFBP-2 inside the cell. In peri/nuclear fractions of various tissues isolated from IGFBP-2 transgenic and non-transgenic mice we were able to show the presence of intact IGFBP-2. In addition, we demonstrate the presence of a highly conserved carboxyl-terminal IGFBP-2 fragment in the peri/nuclear fraction by using different peptide-induced antibodies. In pancreatic sections, confocal microscopy revealed the presence of IGFBP-2 on the nuclear surface but not within the nucleus. Our findings suggest novel functions of intact IGFBP-2 and IGFBP-2 fragments within the cell.
Subject(s)
Cell Nucleus/metabolism , Insulin-Like Growth Factor Binding Protein 2/chemistry , Amino Acid Sequence , Animals , Blotting, Western , Centrifugation, Density Gradient , Immunoprecipitation , Insulin-Like Growth Factor Binding Protein 2/metabolism , Ligands , Mice , Mice, Transgenic , Microscopy, Confocal , Microscopy, Fluorescence , Molecular Sequence Data , Peptides/chemistry , Propidium/pharmacology , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Tissue DistributionABSTRACT
BACKGROUND: We investigated the effects of androgens, estradiol (E2) and insulin-like growth factor (IGF)-I on IGF-II, insulin-like growth factor binding protein (IGFBP)-2, -3 and -5 and mRNA in genital fibroblasts (GF) from patients with complete androgen insensitivity (CAIS) and normally virilized males (C). METHODS: Proteins were measured by specific RIA and Western ligand blot, and specific mRNA levels by RT-PCR normalized by GAPDH levels. RESULTS: Secretion of IGF-II was lowered in CAIS (p<0.001) GF and by testosterone + IGF-I in C GF. Secretion of IGFBP-2 was higher (p<0.001) in CAIS GF and IGFBP-2 mRNA levels were increased by E2 in C GF (p<0.05). E2 stimulated IGFBP-2, -3 and -5 expression in CAIS GF. CAIS GF also secreted more IGFBP-3 (p<0.001) and accumulated 3-5 times more IGFBP-5 mRNA than C GF (p<0.001). CONCLUSION: In contrast to C GF, the availability of IGF-II in CAIS GF is apparently decreased by two facts: by the decreased expression and by increased expression of IGFBP-2, -3 and -5. Furthermore, E2 and IGF-I modulate the expression of IGF-II and IGFBP in GF. This may play a role in the failure to develop male external genitals in CAIS patients.
Subject(s)
Androgen-Insensitivity Syndrome/genetics , Fibroblasts/metabolism , Genitalia, Male , Insulin-Like Growth Factor Binding Proteins/metabolism , Skin/metabolism , Blotting, Western , Humans , Insulin-Like Growth Factor Binding Protein 2/metabolism , Insulin-Like Growth Factor Binding Protein 3/metabolism , Insulin-Like Growth Factor Binding Protein 5/metabolism , Insulin-Like Growth Factor I/metabolism , Insulin-Like Growth Factor II/metabolism , Male , RNA, Messenger/metabolism , Radioimmunoassay , Receptors, Androgen/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Using insulin-like growth factor-binding protein-2 (IGFBP-2) transgenic mice (D mice) as a model of elevated IGFBP-2 expression, which is often found in unphysiological conditions, we found association of IGFBP-2 to purified plasma membranes of many organs. To determine whether the RGD (Arg-Gly-Asp) motif of IGFBP-2 mediates cell surface binding in vivo, we mutated the RGD motif of IGFBP-2 into an RGE (Arg-Gly-Glu) sequence and produced transgenic mice (E mice) which express elevated amounts of mutated IGFBP-2. Our data demonstrate that in vivo IGFBP-2 cell surface association is not dependent on the RGD motif and that mutation of this sequence does not alter growth inhibitory effects of IGFBP-2.
Subject(s)
Body Weight/physiology , Insulin-Like Growth Factor Binding Protein 2/metabolism , Membrane Proteins/metabolism , Oligopeptides/metabolism , Amino Acid Motifs/genetics , Amino Acid Motifs/physiology , Animals , Body Weight/genetics , Cell Membrane/metabolism , Insulin-Like Growth Factor Binding Protein 2/blood , Insulin-Like Growth Factor Binding Protein 2/genetics , Insulin-Like Growth Factor I/analysis , Insulin-Like Growth Factor I/metabolism , Insulin-Like Growth Factor II/analysis , Insulin-Like Growth Factor II/metabolism , Membrane Proteins/blood , Membrane Proteins/genetics , Mice , Mice, Transgenic/growth & development , Mice, Transgenic/physiology , Oligopeptides/genetics , Organ Size/genetics , Organ Size/physiology , Point MutationABSTRACT
The action of androgen by way of the AR is required for the development of male gonads and external genitalia. The interplay between androgens and the somatotropic axis, in particular the IGFs in sexual development, is currently under thorough investigation. The IGF system is thought to mediate the androgen action in androgen-responsive cells. To investigate the interaction of androgens with the IGF system, we compared the expression of IGFs and IGF-binding proteins in cultured genital skin fibroblasts from nine patients with the syndrome of complete androgen insensitivity with that in genital skin fibroblasts from 10 normally virilized males. Mutations in the AR gene and/or abnormalities of the AR protein in the immunoblot were detected in all complete androgen insensitivity genital skin fibroblast strains. They caused a complete failure of DHT binding. RIA and RT-PCR demonstrated that the genital skin fibroblast strains expressed IGF-II, IGF-binding protein-2, and IGF-binding protein-3, but no IGF-I. Most strikingly, complete androgen insensitivity genital skin fibroblast strains produced significantly lower IGF-II (P < 0.001; 42.2 +/- 9.7 vs. 106.9 +/- 11.8 ng/mg protein) and IGF-II mRNA (P < 0.01, by RT-PCR) than control genital skin fibroblast strains. The production of IGF-binding protein-2 was also decreased (P < 0.03) in complete androgen insensitivity genital skin fibroblasts, whereas that of IGF-binding protein-3 did not differ. Furthermore, high levels of IGF-binding protein-5 mRNA were detected in all genital skin fibroblast strains, whereby the 28-kDa band in the ligand blot, probably representing IGF-binding protein-5, was more abundant in complete androgen insensitivity genital skin fibroblasts. Exposure of the genital skin fibroblasts to T (5 x 10(-8) M) had only weak effects on the expression of IGFs and IGF-binding proteins. In conclusion, although the mechanism underlying these differences requires further study, it is conceivable that in addition to the endocrine actions of IGF-I, IGF-II and IGF-binding protein-2, as local growth factors, are involved in the mediation of androgen action and growth of genital tissues.
Subject(s)
Androgen-Insensitivity Syndrome/metabolism , Genitalia, Male/metabolism , Insulin-Like Growth Factor Binding Protein 2/biosynthesis , Insulin-Like Growth Factor II/biosynthesis , Cells, Cultured , Fibroblasts/metabolism , Humans , Insulin-Like Growth Factor Binding Protein 2/genetics , Insulin-Like Growth Factor II/genetics , Male , RNA, Messenger/analysis , Receptors, Androgen/chemistryABSTRACT
Many cancers produce high amounts of the insulin-like growth factor binding protein (IGFBP)-2, which can influence the tumorigenicity and growth of tumor cells. In order to study the possible cause of elevated expression of IGFBP-2 in tumors, we investigated the transcriptional regulation by IGF of a 633-bp fragment of the human IGFBP-2 promoter in a transiently transfected choriocarcinoma (JAR) and a leukemic T-cell line (Molt-4) that express IGFBP-2 highly, and in a leukemic B-cell line (Raji) that expresses little IGFBP-2. Strong basal promoter activity, i.e. luciferase activity was measurable in all of the tumor cell lines. The introduction of equal amounts of normal IGF-I and IGF-II stimulated the transcription of IGFBP-2 only slightly. Synthetic IGF analogues with increased biological activity, however, caused a specific 2.0-3.3-fo1d transactivation of the promoter, as well as a 25% increase in IGFBP-2 mRNA. Synchronously, IGF analogues caused a decrease in the level of IGFBP-3 mRNA of about 45%, while the production of IGFBP-2 as measured by RIA increased in relation to IGFBP-3 by up to 15 times. Blocking with the IGF antagonist JB1 revealed partial involvement of the IGF-I receptor in the regulation of IGFBP-2 expression by locally produced IGF. We conclude, that the reduced ability of IGF analogues to form complexes with locally produced IGFBP may account for their increased biological activity in the stimulation of expression of IGFBP-2 and of cell growth. Since increased biological activity had also been demonstrated for natural pro-IGF forms often produced by tumors, pro-IGFs may be involved in the mechanism leading to elevated IGFBP-2 expression of tumors in vivo.
Subject(s)
Insulin-Like Growth Factor Binding Protein 2/genetics , Tumor Cells, Cultured/metabolism , Humans , Insulin-Like Growth Factor Binding Protein 2/metabolism , Insulin-Like Growth Factor Binding Protein 3/genetics , Insulin-Like Growth Factor Binding Protein 3/metabolism , Promoter Regions, Genetic/drug effects , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Somatomedins/pharmacology , Transcriptional Activation , TransfectionABSTRACT
Human central nervous system tumors and glioma cell lines highly express the insulin-like growth factor-binding protein (IGFBP)-2. As IGFBP-2 can affect tumor growth, we studied the relationship between IGFBP-2 expression and the malignancy of brain tumors in vivo. To do so, we investigated by immunohistochemistry the accumulation of IGFBP-1, -2, and -3 in 50 human gliomas classified by the WHO Malignancy Scale. Double labeling using anti-CD68 (monocytes/macrophages), antiglial fibrillary acidic protein, and anti-CD3 (T cells) antibodies was performed to further characterize the IGFBP-1, -2, and -3(+) cells. The expression of IGFBP messenger RNAs (mRNAs) was tested by RT-PCR in tumor samples from nine gliomas of different grades and in eight cell lines representing the cellular composition of human glioma. As controls, the accumulation of IGFBP-2 was investigated in normal brain and in the rat C6 glioblastoma model. IGFBP-1 and -3 accumulated in endothelial and macrophage/microglial cells. IGFBP-2(+) macrophage/microglial and glioma cells clustered in the immediate vicinity of focal necrosis of the human gliomas as well as of the rat C6 glioblastoma. The labeling score of IGFBP-1 accumulation in endothelial cells correlated negatively (P: = 0.0229), and that of IGFBP-2 accumulation in glioma cells correlated positively (P: < 0.0006) with the tumor grade of the gliomas. In addition, RT-PCR analysis confirmed mRNA expression of IGFBP-1, -2, and -3 by the gliomas and glial cells. Small amounts of IGFBP-1 and -3 mRNA, but high amounts of IGFBP-2 mRNA, were detectable in macrophage-like and glioma cell lines. The results suggest cell type-specific accumulation of IGFBP-1, -2, and -3 in human glial tumors of the brain. The increase in IGFBP-2 expression with this malignancy suggests a role of IGFBP-2 in the biology of human gliomas.
