Cloning of the rat insulin-like growth factor binding protein-1 gene and analysis of its 5' promoter region.

To understand specific mechanisms involved in the regulation of insulin-like growth factor binding protein-1 (IGFBP-1), an important modulator of IGF bioactivity, we cloned the rat IGFBP-1 gene and sequenced a 1.5 kb Sph1-Sph1 fragment containing 1110 bases upstream from the translation start site. Computer analysis reveals the presence of ATA, CACCC, and CCAAT elements, and putative homeodomain, AP-1, insulin and glucocorticoid response elements in the 5' promoter. Primer extension and ribonuclease protection studies reveal a single cap site in RNA from rat hepatoma cells and both control and diabetic rat liver.     

Adenoviral-mediated expression of human insulin-like growth factor-binding protein-3.

Insulin-like growth factors (IGFs) in the circulation are predominantly sequestered into ternary complexes comprising IGF, IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS). Besides its role in regulating IGF bioavailability in the circulation, IGFBP-3 has both IGF-dependent and IGF-independent actions on cell proliferation. As part of our studies into the structure-function relationships of the multifunctional IGFBP-3, we have evaluated the efficiency of an adenovirus-mediated expression system for rapid, medium-scale production of functional, glycosylated IGFBP-3. Replication-deficient adenovirus containing human IGFBP-3 cDNA was generated using standard techniques. Secreted, recombinant IGFBP-3 (IGFBP-3(Ad)) was purified from the culture medium of virus-infected cells by IGF-I affinity chromatography followed by reverse-phase HPLC. When analyzed by SDS-PAGE, IGFBP-3(Ad) was similar in size (43- to 45-kDa glycoform doublet) to IGFBP-3(Pl) derived from plasma. In addition, IGFBP-3(Ad) was detected by immunoblot using an antibody specific for human IGFBP-3 and by ligand blot using radiolabeled IGF-I. IGFBP-3(Ad) had similar affinities for IGF-I and ALS and an approximately 25% decreased affinity for IGF-II compared to IGFBP-3(Pl). IGFBP-3(Ad) showed no significant difference in its susceptibility to an IGFBP-3 protease present in medium conditioned by MCF-7 breast cancer cells compared to IGFBP-3(Pl), but appeared more resistant to the IGFBP-3 protease present in pregnancy serum. IGFBP-3(Ad) also exhibited increased binding to T47D cells which may be related to the glycosylation state of the protein.     

Isolation and molecular cloning of insulin-like growth factor-binding protein-6.

Insulin-like growth factors (IGFs) together with their binding proteins (BPs) are potential regulators of folliculogenesis in mammalian ovary. To identify the various species of IGFBPs present in the ovary, we have undertaken a comprehensive purification scheme using gel filtration, ligand-affinity chromatography, and several steps of reverse phase HPLC to isolate all of the BPs in pig ovarian follicular fluid. Our effort yielded five distinct IGFBPs, and upon analysis, they were found to correspond to the previously identified human and rat IGFBP-2, -3, -4, -5, and -6. IGFBP-1 was not found in the pig ovarian follicular fluid under our experimental procedure. Of the six known classes of IGFBPs, the complete primary structures of the first five have been determined, but not IGFBP-6. Using amino acid sequence information from a tryptic fragment of pig IGFBP-6 to prepare a probe, cDNA clones encoding rat and human IGFBP-6 have been isolated and characterized. The deduced amino acid sequence revealed that rat IGFBP-6 contains 201 amino acids with a calculated mol wt of 21,461, while the human homolog contains 216 amino acids with a calculated mol wt of 22,847. In addition, a distinctive feature of human and rat IGFBP-6 is that they lack, respectively, two and four of the 18 homologous cysteines that are present in all other five IGFBPs. The missing cysteines in IGFBP-6 resulted in the absence of the invariant Gly-Cys-Gly-Cys-Cys sequence in the amino-terminal region of the molecule. Human IGFBP-6 possesses a single Asn-linked glycosylation site near the carboxyl-terminal, whereas no potential Asn-linked glycosylation sites are present in the rat sequence. A single 1.3-kilobase IGFBP-6 mRNA was detected by Northern analysis in all rat tissues examined, including testis, intestine, adrenal, kidney, stomach, spleen, heart, lung, brain, and liver, indicating that this BP is a ubiquitous protein. The chromosome location of the IGFBP-6 gene in human has been determined using polymerase chain reaction on somatic cell hybrid DNAs of human and hamster, and the results showed that it is located on chromosome 12.     

Cloning and expression of the growth hormone-dependent insulin-like growth factor-binding protein

N-terminal as well as internal amino acid sequence data were obtained from the GH dependent, insulin-like growth factor (IGF) binding protein, BP-53, purified from human plasma. Based on these sequence data, full-length cDNA clones of BP-53 have been isolated, and the complete deduced sequence of BP-53 determined. This sequence contains a 27 amino acid putative signal sequence followed by a mature protein of 264 amino acids containing 18 cysteine residues clustered near the N- and C-terminus. The deduced protein sequence of BP-53 has 33% amino acid identity including conservation of all 18 cysteine residues with the recently cloned BP-28, a smaller human IGF-binding protein identified in amniotic fluid and also secreted by the cell line HEP G2. Expression of the cloned BP-53 cDNA in mammalian tissue culture cells results in secretion of the protein into the culture medium. This expressed protein is identical to plasma-derived BP-53 in its immunoreactivity, high affinity binding of IGF-I and IGF-II, and mobility on sodium dodecyl sulfate gel electrophoresis.     

Structure and localization of the human insulin-like growth factor-binding protein 2 gene.

Insulin-like growth factor binding proteins (IGFBPs) are extracellular proteins that specifically bind IGF and modulate their effects. The human IGFBP2 gene was studied and shown to be localized to chromosome 2 region q33-q34, by somatic cell hybrid analysis and in situ hybridization. Structural characterization of the gene showed that it consists of four exons with three introns of lengths 27.0, 1.0, and 1.9 kilobase-pairs. Comparison of the encoded protein sequence of each exon in IGFBP1, 2, and 3 reveals the highest amino acid identity, 28%, in exon 1, while the lowest was found in exon 2. However, pairwise sequence comparisons demonstrate 50% identity between the protein sequences encoded by exon 4 in IGFBP1 and 2, while their respective identities with IGFBP3 are only 25 and 30%.     

Pregnancy-associated plasma protein-A2 (PAPP-A2), a novel insulin-like growth factor-binding protein-5 proteinase

A novel metalloproteinase with similarity to pregnancy-associated plasma protein-A (PAPP-A), which we denoted PAPP-A2, has been identified. Through expression in mammalian cells we showed that recombinant PAPP-A2 polypeptide of 1558 residues resulted from processing of a 1791-residue prepro-protein. Unlike PAPP-A, PAPP-A2 migrated as a monomer (of 220 kDa) in non-reducing SDS-polyacrylamide gel electrophoresis. The prepro-parts of PAPP-A2 and PAPP-A are not homologous, but mature PAPP-A2 shares 45% of its residues with PAPP-A. Because PAPP-A specifically cleaves insulin-like growth factor-binding protein (IGFBP)-4, one of six known modulators of IGF-I and -II, we looked for a possible PAPP-A2 substrate among the members of this family. We showed that PAPP-A2 specifically cleaved IGFBP-5 at one site, between Ser-143 and Lys-144. In contrast to the cleavage of IGFBP-4 by PAPP-A that strictly requires the presence of IGF, the cleavage of IGFBP-5 by PAPP-A2 was IGF-independent. Recent data firmly establish PAPP-A and IGFBP-4 as an important functional pair in several systems. Because of its close relationship with PAPP-A, both structurally and functionally, PAPP-A2 is a likely candidate IGFBP-5 proteinase in many tissues and conditioned media where IGFBP-5 proteolysis has been reported.     

Proteomic identification of insulin-like growth factor-binding protein-6 induced by sublethal H2O2 stress from human diploid fibroblasts

Fibroblasts are the most ubiquitous cell types within our body. They produce various factors to maintain the texture and structure of a particular organ or tissue. To identify protein factors secreted by fibroblasts and alteration of these protein factors upon oxidative stress, HCA3 human skin diploid fibroblasts were exposed to a sublethal dose of H2O2, which induces a prematurely senescent phenotype. Conditioned media from prematurely senescent cells versus control cells were analyzed for proteins using an LC-MS/MS-based proteomic technique. Collagen alpha1(VI), collagen alpha2(I), fibronectin, lumican, and matrix metalloproteinase 2 were among the proteins consistently detected from control and H2O2-treated cells. Insulin-like growth factor-binding protein-6 (IGFBP-6) consistently showed up in the conditioned medium of H2O2-treated cells but not from untreated cells. Increased IGFBP-6 production due to H2O2 treatment was confirmed by RT-PCR and Western blot analyses. While H2O2 induced a dose-dependent elevation of IGFBP-6 mRNA, Western blot analyses detected elevated levels of IGFBP-6 protein in the conditioned medium of H2O2-treated cells. In comparison, fibronectin or matrix metalloproteinase 2 did not show changes at the mRNA level in cell lysates or at the protein level in the conditioned medium by H2O2 treatment. Using several types of toxins at sublethal doses, including cis-platin, hydroxyurea, colchicine, L-mimosine, rhodamine, dithiothreitol, or N-ethylmaleimide, we found that these agents induced increases of IGFBP-6 at mRNA and protein levels. An increased level of IGFBP-6 protein was detected in the plasma of aging mice and of young mice treated with doxorubicin. These data suggest that IGFBP-6 may serve as a sensitive biomarker of cell degeneration or injury in vitro and in vivo.