Rat Insulin-like Growth Factor Binding Research Articles

Interstitial tissue-specific gene expression in mouse testis by intra-tunica albuguineal injection of recombinant baculovirus

The purpose of this study is to establish a gene delivery system for interstitial tissue-specific protein expression in mice testes using modified recombinant baculovirus. Green fluorescent protein (GFP)-expressing recombinant baculovirus (GFP-baculovirus), in which the insect cell-specific polyhedron promoter was replaced by the cytomegalovirus (CMV)-IE promoter, was used to transfect testicular cells in vitro, and for intra-tunica albuguineal injection of the interstitial tissue of the testis. GFP expression was monitored in frozen testes sections by fluorescence microscopy. Expression of GFP in testicular tissues was also assessed by reverse transcription polymerase chain reaction (RT-PCR), and protein expression was assessed by Western blot. Testicular cells in vitro were infected efficiently by modified recombinant GFP-baculovirus. Intra-tunica albuguineal injection of GFP-baculovirus into the mouse testis resulted in a high level of GFP expression in the interstitial tissues. RT-PCR analysis clearly showed GFP gene expression in the testis, particularly interstitial tissues. Intra-tunica albuguineal injection of a modified baculovirus that encoded recombinant rat insulin-like growth factor binding protein (IGFBP)-5 resulted in an increase in IGFBP-5 in testis and semen. In conclusion, we have developed an efficient delivery system for gene expression in vivo in testicular cells, particularly cells of the interstitial tissue using intra-tunica albuguineal injection of a modified recombinant baculovirus. This method will be particularly relevant for application that requires gene delivery and protein expression in the testicular cells of the outer seminiferous tubule of the testis.

The in Vivo Phosphorylation and Glycosylation of Human Insulin-like Growth Factor-binding Protein-5

Mass spectrometry is often used to determine post-translational modifications by analysis of tryptic digests of proteins. Here we demonstrate that the analysis of tryptic peptides together with analysis of the full-length protein provided optimal characterization of insulin-like growth factor-binding protein-5 (IGFBP-5) phosphorylation and glycosylation. IGFBP-5 binds insulin-like growth factors with high affinity and has important roles in cell survival, differentiation, and apoptosis. Until now, the primary structure of IGFBP-5 has been incompletely defined. We analyzed human IGFBP-5 from T47D cells by mass spectrometry to determine all of the in vivo post-translational modifications. In full-length IGFBP-5, 31% of the protein was unmodified, 37% was monophosphorylated, and 4% was diphosphorylated with no other modification. The remaining 27% was glycosylated, more than half of which was also monophosphorylated. The major phosphorylation site was Ser(96) in the central domain, and a minor phosphorylation site was Ser(248) near the C terminus. Neither site was phosphorylated in vitro by casein kinase 2, ruling it out as the in vivo kinase. An in vivo phosphorylation site was also found in IGFBP-2 at an analogous position, Ser(106). IGFBP-5 was heterogeneously O-glycosylated mainly by sialylated core 1 type glycans. The most abundant structure contained N-acetylhexosamine, hexose, and two N-acetylneuraminic acid carbohydrates. A small amount of sialylated core 2 type glycan was also present. Phosphorylation and O-glycosylation both affected IGFBP-5 binding to heparin but not insulin-like growth factor binding or ternary complex formation with the acid-labile subunit. The results reveal the first description of the in vivo phosphorylation of IGFBP-5 and its glycan composition.

Identification of N-linked oligosaccharides of rat insulin-like growth factor binding protein-4.

Insulin-like growth factor binding protein-4 (IGFBP-4) is, like the other five IGFBPs, a critical regulator of the activity of insulin-like growth factor (IGF)-I and IGF-II. Whereas IGFBP-1 and IGFBP-2 are not glycosylated, IGFBP-3 and IGFBP-4 are N-glycosylated and IGFBP-5 and IGFBP-6 are O-glycosylated. In this study we identified the glycosylation of IGFBP-4 using a nanoflow LC/MS/MS techniques. Although N-linked oligosaccharides are structurally diverse, their variants are well reported in the literature. Based on the molecular weight of the possible oligosaccharide moieties, we identified five different glycosylation isoforms of the protein. Identified glycans were biantennary and differ in the number of sialic acid terminal residues and/or core modification with fucose.

A new radioimmunoassay to measure rat insulin-like growth factor binding protein-4 (IGFBP-4) in serum, wound fluid and conditioned media.

Insulin-like growth factor binding protein-4 (IGFBP-4) is, like the other five IGFBPs, a critical regulator of the activity of insulin-like growth factor-I (IGF-I) and IGF-II. Because of the possible role of IGFBP-4 in multiple systems including reproduction, pregnancy, bone formation and wound healing, we developed a radioimmunoassay (RIA) to measure the concentration of IGFBP-4 in the serum, extracellular fluid and conditioned media of cells of rodents so that these animal models could be better exploited. Rat IGFBP-4 was expressed in Escherichia coli and purified to homogeneity. The recombinant material was used to raise a rabbit polyclonal antibody against rat IGFBP-4 and for iodination and standards. The RIA developed was sensitive to less than 1 ng/mL of rat IGFBP-4 and IGF-I did not interfere. There was no cross-reactivity with other rat IGFBPs on immuno-Western analysis of serum and wound fluid. IGFBP-4 from mouse serum did cross-react in our assay; however, serum from horse, pig or human showed no cross-reaction. Human IGFBP-1 and IGFBP-3 showed a very weak cross-reactivity. Serum IGFBP-4 levels showed no gender difference but did reveal a significant 66% increase in older rats. During the course of wound healing, which is IGF-I dependent, IGFBP-4 showed no changes. In conclusion an RIA for rat IGFBP-4 has been developed that specifically measures the concentration of IGFBP-4 in the sera, extracellular fluid and conditioned media of rodents. With this assay, the role of IGFBP-4 and IGFs in growth, development and the function of multiple systems can be further investigated using rodent models.

Glucose regulated production of human insulin in rat hepatocytes.

Insulin gene therapy requires that insulin secretion be coupled to metabolic requirements. To this end, we have developed an insulin transgene whose transcription is stimulated by glucose and inhibited by insulin. Glucose- and insulin-sensitive promoters were constructed by inserting glucose-responsive elements (GlREs) from the rat L-pyruvate kinase (L-PK) gene into the insulin-sensitive, liver-specific, rat insulin-like growth factor binding protein-1 (IGFBP-1) promoter. Glucose (5 to 25 mM) stimulated, and insulin (10-10 to 10-7 M) inhibited, luciferase expression driven by these promoters in primary cultured rat hepatocytes. The capacity of transfected hepatocytes to secrete mature, biologically active insulin was demonstrated using a human proinsulin cDNA (2xfur), modified to allow protein processing by endogenous endopeptidase activity. Medium conditioned by insulin-producing hepatocytes contained greater than 300 microU/ml immunoreactive insulin, while denaturing SDS-PAGE of an anti-insulin immunoprecipitate revealed bands with the mobilities of insulin A, and B chains. Biological activity of hepatocyte-produced insulin was demonstrated in a transfection assay, in which medium conditioned by insulin-producing hepatocytes exerted an effect similar to 10-7 M insulin. We then combined the glucose- and insulin-sensitive promoter with the modified human proinsulin cDNA to create a metabolically sensitive insulin transgene ((GlRE)3BP-1 2xfur). In both H4IIE hepatoma cells stably transfected with this construct, and normal rat hepatocytes (GlRE)3BP-1 2xfur-mediated insulin secretion increased in response to stimulation by glucose. Moreover, a capacity to decrease insulin production in response to diminishing glucose exposure was also demonstrated. We conclude that the transcriptional regulation of insulin production using these glucose- and insulin-sensitive constructs meets the requirements for application in a rodent model of insulin gene therapy. Gene Therapy (2000) 7, 205-214.

Expression of rat insulin-like growth factor binding protein-6 in the brain, spinal cord, and sensory ganglia

Insulin-like growth factors (IGFs) are important trophic factors during development as well as in the adult or damaged nervous system. Their trophic actions are modulated by interactions with six distinct IGF binding proteins. The mRNA expression profiles of binding proteins 2, 4 and 5 in the normal developing and adult CNS are well characterized and are shown to have distinctive, non-overlapping distributions. The IGF binding protein-6 (BP6) is also expressed in the CNS, however, details regarding its mRNA expression distribution in the developing and adult nervous system is limited. BP6 has the unique property of preferentially binding the IGF-II ligand. Coupled with the fact that this ligand is the most abundantly expressed IGF in the adult CNS, this suggests that the IGF-II/BP6 complex has a unique role in modulating IGF-II function in the adult brain. In this report the anatomical distribution of BP6 messenger RNA in the developing and adult rat nervous system is presented. In the embryonic animal the CNS expression is tightly restricted to trigeminal ganglia and, relative to the rest of the embryo, this structure has the highest expression. The expression in the forebrain and cerebellum does not occur until after postnatal day 21 and then is primarily associated with GABAergic interneurons. The highest levels of expression in the adult animal are in the hindbrain, spinal cord, cranial ganglia, and dorsal root ganglia. These nuclei in the hindbrain and periphery that express BP6 are all associated with the coordination of sensorimotor function in the cerebellum, which indicates an important role for the BP6/IGF-II complex in the function and maintenance of these systems.

Amino acids within the extracellular matrix (ECM) binding region (201-218) of rat insulin-like growth factor binding protein (IGFBP)-5 are important determinants in binding IGF-I.

The highly conserved N-and C-terminal domains of IGFBPs are believed to participate in IGF binding, but only recently have some of the critical residues in the IGFBP sequence involved in ligand binding been identified. Here we describe two highly conserved amino acids in the C-terminal domain of rat IGFBP-5 that are involved in binding IGF-I. Site-directed mutagenesis was used to produce two mutants, G203K and Q209A, of rIGFBP-5. Relative to wild-type rIGFBP-5, an 8-fold reduction in affinity for human IGF-I was found for recombinant G203K protein in both IGF-I ligand blots and solution phase ligand binding assays, and a 7-and 6-fold reduction for Q209A respectively. This shows that Gly203 and Gln209 in IGFBP-5 are important determinants in binding IGF-I, and due to their complete conservation in all IGFBP sequences, we suggest that they are likely to be involved in binding IGF-I in all six binding proteins. In addition, these two non-basic residues lie within the ECM binding region (201-218) of IGFBP-5, demonstrating that the C-terminus contains partially overlapping IGF-I and ECM binding sites. We therefore propose that heparin binding to basic amino acids in IGFBP-5 between 201-218 may physically occlude subsequent interaction between IGF-I and Gly203/Gln209, and that this may explain previous work of others showing reduced affinity of ECM bound IGFBP-5 for IGF-I.

Identification and characterization of the putative retinoblastoma control element of the rat insulin-like growth factor binding protein-2 gene

The authors previously identified a silencer of the rat IGFBP-2 gene. Sequence examination of the silencer has revealed that it contains the target sequence for the pRb (retinoblastoma) tumour suppressor gene, referred to as the retinoblastoma control element (RCE) which is frequently found in the regulatory element of cellular oncogenes and growth factors. The presence of RCE suggests that the IGFBP-2 gene may be regulated by the pRb tumour suppressor gene. An in vitro gel retardation assay has shown that the putative RCEs from the IGFBP-2 gene are complexed with multiple nuclear factors from the rat liver BRL-3A cells. These DNA–protein complexes were not detected with the nuclear extracts from the cells that were growth arrested at the G1/S border of the cell cycle by high cell density. Using specific antibodies, Sp1 was shown to be one of the components for the multiple DNA–protein complex while pRb does not appear to be directly involved in the formation of the complex.

Cell-density (cycle) Dependent Silencer of the Rat Insulin-like Growth Factor Binding Protein-2 (IGFBP-2) Gene

An element which has a negative effect on transcription has been identified in the 5′-flanking region of the rat insulin-like growth factor binding protein-2 (IGFBP-2) gene. This element was confirmed to be a silencer by truncation or by linking it to a heterologous promoter. The silencer activity disappeared in the growth-arrested BRL-3A cells at the G1/S border by high cell density where the IGFBP-2 production is highly elevated. This observation may represent a novel mechanism through which gene expression is controlled by modulation of a silencer. Taken together, these results suggest a regulatory link between cell growth and IGFBP-2 expression regulated by its silencer.

Phosphorylation of Rat Insulin-like Growth Factor Binding Protein-1 Does Not Affect its Biological Properties

Insulin-like growth factors (IGFs) I and II stimulate growth and expression of specific genes through binding to cell membrane receptors. IGF binding proteins also bind IGF-I with higher affinity than the receptor. They are found in the circulation and tissues and can modulate IGF actions. Human IGFBP-1 is phosphorylated on serine residues, which increases its affinity for IGF-I. An acidic, presumably phosphorylated, form of human IGFBP-1 inhibits IGF-I-stimulated DNA synthesis in cultured cells, while a less acidic, unphosphorylated form potentiates this function. Phosphorylation of human IGFBP-3, however, does not affect its affinity for IGF-I. Previously we found that multiple forms of rat IGFBP-1 are obtained by anion-exchange chromatography, raising the possibility that it also is phosphorylated, which led us to examine its properties. Phosphopeptide analysis of32P-labeled, immunoprecipitated rat IGFBP-1 synthesized by H-4-II-EC3 rat hepatoma cells indicated that it is phosphorylated on two sites that were deduced to be ser107 and ser132 in the central nonconserved domain. Dephosphorylation of purified phosphorylated rat IGFBP-1 did not affect its affinity for IGF-I or its specific binding activity, and the dephosphorylated form inhibited DNA synthesis in 3T3 cells. Incubation of cells labeled with radioactive proline in the presence of monensin and brefeldin A, which inhibit secretion at different sites, led to intracellular accumulation of the least phosphorylated form of rat IGFBP-1, but prevented further phosphorylation. The results suggested that phosphorylation occurs at two sites in cells, thecis-Golgi and thetrans-Golgi network. In summary, these studies have shown that rat IGFBP-1 is phosphorylated on two sites by reactions that occur in different secretory organelles and that similar to human IGFBP-3, but unlike human IGFBP-1, phosphorylation does not affect its affinity for IGF-I.

Competitive binding assay for determination of rat insulin-like growth factor binding protein-3.

We describe a novel competitive assay for rat insulin-like growth factor (IGF)-binding protein-3 (rIGFBP-3) based on the ability of IGFBP-3 to form a ternary complex with the acid labile subunit (ALS) in the presence of IGF. Human (h)ALS was bound to test tubes pre-coated with anti-human ALS antibody. The assay depends on competition between a covalent complex of 125I-hIGF-I and hIGFBP-3, added as tracer, and hIGFBP-3 or rIGFBP-3 in standards and test samples, for binding to the immobilized hALS. Purified natural hIGFBP-3 served as standard. Human IGFBP-3 and rIGFBP-3 were able to compete for tracer binding in the presence, but not in the absence, of IGF-I. Before assay, rat serum samples were acidified to denature endogenous ALS. Standards ranged from 0.10 (lower detection limit) to 20 ng/tube. Rat serum, semipurified rIGFBP-3, human serum and purified hIGFBP-3 diluted in parallel. The level of rIGFBP-3 was 1.63+/-0.28 mg/l (mean+/-SEM) in young rats and increased to 3.41+/-0.26 mg/l (p < 0.05) in old rats (n = 5-6). Fasting for 3 days reduced rIGFBP-3 from 2.41+/-0.27 to 1.33+/-0.14 mg/l (p < 0.05). Levels of rIGFBP-3 were reduced in hypophysectomized (0.16+/-0.04 mg/l; p < 0.05) and diabetic rats (1.04+/-0.30 mg/l; p < 0.05), and normal in insulin-treated diabetic rats (2.49+/-0.04 mg/l; ns), when compared to controls (2.79+/-0.22 mg/l). Changes in levels of IGFBP-3 parallelled those of immunoreactive rALS. We conclude that this assay provides a novel method of quantitating functional IGFBP-3 in rat serum.

Hepatocyte nuclear factor 1 and the glucocorticoid receptor synergistically activate transcription of the rat insulin-like growth factor binding protein-1 gene.

The insulin-like growth factor (IGF) binding proteins (IGFBPs) are a family of proteins that bind IGF-I and IGF-II and modulate their biological activities. IGFBP-1 is distinctive among the IGFBPs in its rapid regulation in response to metabolic and hormonal changes. The synthetic glucocorticoid, dexamethasone, increases IGFBP-1 mRNA abundance and gene transcription in rat liver and in H4-II-E rat hepatoma cells. A glucocorticoid response element (GRE) located at nucleotide (nt) -91/-77 is required for dexamethasone to stimulate rat IGFBP-1 promoter activity in transient transfection assays in H4-II-E cells. In addition to the GRE, three accessory regulatory sites [a putative hepatocyte nuclear factor-1 (HNF-1) site (nt -62/-50), an insulin-response element (nt -108/-99), and an upstream site (nt -252/-236)] are involved in dexamethasone stimulation under some, but not all, circumstances. The present study begins to address the mechanism by which transcription factors bound to the putative HNF-1 site act synergistically with the glucocorticoid receptor (GR) bound to the GRE. In gel shift assays, HNF-1alpha and HNF-1beta in H4-II-E extracts bind to the palindromic HNF-1 site. Both half-sites are required. Overexpression of HNF-1beta enhances dexamethasone-stimulated promoter activity. Both the HNF-1 site and the GRE must be intact for stimulation to occur. By contrast, overexpression of HNF-1alpha does not enhance dexamethasone-stimulated promoter activity, although, as also observed with overexpression of HNF-1beta, it inhibits basal promoter activity. Thus, the synergistic effects of HNF-1beta and the GR on dexamethasone-stimulated promoter activity require that they are bound to the HNF-1 site and the GRE, respectively, and may involve protein-protein interactions between the transcription factors, or between them and the basal transcription machinery or a steroid receptor coactivator. Synergy between the ubiquitously expressed GR and HNF-1, which is developmentally regulated and expressed in a limited number of tissues, provides a possible mechanism for tissue- and development-specific regulation of glucocorticoid action.

Interleukin-15 Stimulates C2 Skeletal Myoblast Differentiation

Interleukin-15 (IL-15) is a cytokine which is highly expressed in skeletal muscle, and which stimulates muscle protein accretion in cultured skeletal muscle fibers. Using parental C2 skeletal myoblasts, no significant effects of IL-15 on skeletal muscle differentiation were observed. To test the hypothesis that IL-15 may stimulate skeletal muscle differentiation if the strong differentiation-inducing effects of autocrine insulin-like growth factor (IGF) production were inhibited, a C2 myoblast subline (C2-pBP4) was stably transfected with an expression vector for rat IGF binding protein-4 (IGFBP-4). Differentiation responses to autocrine and exogenous IGFs in C2-BP4 myoblasts were reduced 3- to 4-fold in C2-BP4 cultures compared to C2-pLXSN cultures, a subline transfected with a control plasmid. Addition of IL-15 to C2-pBP4 myoblasts doubled the number of differentiated muscle cells which arose. These findings indicate that IL-15 can stimulate myogenic differentiation in conditions in which the strongly differentiative effects of the IGFs are inhibited. The differentiative activity of IL-15 may be of physiological significance in conditions in which IGF concentrations are low or in which the IGFs are sequestered by binding proteins.

Dexamethasone stimulation of rat insulin-like growth factor binding protein-1 promoter activity involves multiple cis-elements.

Insulin-like growth factor binding protein-1 (IGFBP-1) modulates the mitogenic actions of IGF-I and IGF-II. Dexamethasone increases IGFBP-1 mRNA abundance and gene transcription in rat liver and in H4-II-E rat hepatoma cells. A glucocorticoid response element (GRE) located at nucleotide (nt) -91/-77 is required for dexamethasone to stimulate rat IGFBP-1 promoter activity in transient transfection assays in H4-II-E cells. Mutagenesis of nt -108/-99 (the M4 region of the insulin response element), however, decreased dexamethasone-stimulated promoter activity despite the presence of an intact GRE, suggesting that regulatory sites in addition to the GRE were required for optimal dexamethasone stimulation. To identify these sites, we introduced 5'-deletion and substitution mutations into rat IGFBP-1 promoter fragments coupled to a luciferase reporter gene and transfected these constructs into H4-II-E cells. Three sites are required for optimal basal promoter activity: a site (nt -62/-50) that binds the liver-enriched transcription factor, hepatocyte nuclear factor-1 (HNF-1), the M4 site, and a putative binding site for transcription factor AP-2 (nt -293/-286). The HNF-1 and M4 sites and an upstream site (nt -252/-236) are also involved in dexamethasone stimulation under some, but not all, circumstances. Mutation of either the HNF-1 site or the M4 site decreased dexamethasone stimulation by more than 80% in constructs whose 5'-end was at nt -92, -135, or -235 but not if the 5' -end was at nt -278 or -327. These results suggest that the nt -278/-236 region can compensate for the loss of the HNF-1 site or the M4 site but that the HNF-1 and M4 sites do not compensate for each other in constructs whose 5'-end was at nt -135 or -235, which lack the nt -278/-236 region. The site within the nt -278/-236 region was localized to nt -252/-236 by deoxyribonuclease I protection and transfection assays. Thus, several cis-elements in the rat IGFBP-1 promoter cooperate, in varying combinations, with the low-affinity GRE to allow optimal dexamethasone-stimulated promoter activity.

Dexamethasone stimulation of rat insulin-like growth factor binding protein-1 promoter activity involves multiple cis-elements

Dexamethasone stimulation of rat insulin-like growth factor binding protein-1 promoter activity involves multiple cis-elements

Impaired estrogen action in the uterus of insulin-like growth factor binding protein-1 transgenic mice.

Insulin-like growth factor-I (IGF-I) has been implicated as autocrine/paracrine mediator of estrogen action in the rodent uterus. Here, we examined the effects of 17-beta estradiol (E2), epidermal growth factor (EGF), and IGF-I on DNA synthesis in the uterus of ovariectomized (ovex) transgenic mice (Tg), which overexpress rat insulin-like growth factor binding protein-1 (IGFBP-1). Litter size was significantly reduced in Tg mice compared with wild-type mice. Immunohistochemical studies localized the expression of the transgene to the luminal and glandular epithelium. In addition, rat IGFBP-1 immunoreactivity was present in luminal secretions. E2-induced uterine DNA synthesis as measured by methyl-3H thymidine incorporation, was significantly reduced in ovex Tg mice; 4.77 +/- 0.59 and 4.97 +/- 0.53 for Tg strains 57C and 277A, respectively, compared with 8.65 +/- 0.73 fmol/microgram of DNA for Wt mice. Similarly, uterine weight after three daily injections of E2 was reduced in Tg mice compared with Wt mice; 2.85 +/- 0.39 vs. 4.23 +/- 0.26 mg/g BW, P < 0.01. Semiquantitative RT-PCR assays were used to demonstrate changes in uterine IGF-I messenger RNA (mRNA) and EGF mRNA abundance after administration of E2. An approximately 3-fold increase in IGF-I mRNA abundance was seen 6 h after E2 in both Tg and Wt mice. Over the same time course, little change was seen in EGF mRNA levels, which were similar in Tg and Wt mice. After 3 days of E2 treatment, an increase in EGF mRNA was apparent in Wt mice but not in Tg mice. The uterine DNA response to both IGF-I and EGF was significantly attenuated in Tg mice compared with Wt mice. The data reported here together with previous reports of E2 regulation of IGF-I and IGFBP-1 expression in uterus support the hypothesis that the IGF-I is a mediator of estrogen action in the uterus. In addition attenuation of the EGF response in the uterine tissue of Tg mice suggests that this response is also mediated, in part, by IGF-I.

Impaired estrogen action in the uterus of insulin-like growth factor binding protein-1 transgenic mice

Insulin-like growth factor-I (IGF-I) has been implicated as autocrine/paracrine mediator of estrogen action in the rodent uterus. Here, we examined the effects of 17-beta estradiol (E2), epidermal growth factor (EGF), and IGF-I on DNA synthesis in the uterus of ovariectomized (ovex) transgenic mice (Tg), which overexpress rat insulin-like growth factor binding protein-1 (IGFBP-1). Litter size was significantly reduced in Tg mice compared with wild-type mice. Immunohistochemical studies localized the expression of the transgene to the luminal and glandular epithelium. In addition, rat IGFBP-1 immunoreactivity was present in luminal secretions. E2-induced uterine DNA synthesis as measured by methyl-3H thymidine incorporation, was significantly reduced in ovex Tg mice; 4.77 +/- 0.59 and 4.97 +/- 0.53 for Tg strains 57C and 277A, respectively, compared with 8.65 +/- 0.73 fmol/microgram of DNA for Wt mice. Similarly, uterine weight after three daily injections of E2 was reduced in Tg mice compared with Wt mice; 2.85 +/- 0.39 vs. 4.23 +/- 0.26 mg/g BW, P < 0.01. Semiquantitative RT-PCR assays were used to demonstrate changes in uterine IGF-I messenger RNA (mRNA) and EGF mRNA abundance after administration of E2. An approximately 3-fold increase in IGF-I mRNA abundance was seen 6 h after E2 in both Tg and Wt mice. Over the same time course, little change was seen in EGF mRNA levels, which were similar in Tg and Wt mice. After 3 days of E2 treatment, an increase in EGF mRNA was apparent in Wt mice but not in Tg mice. The uterine DNA response to both IGF-I and EGF was significantly attenuated in Tg mice compared with Wt mice. The data reported here together with previous reports of E2 regulation of IGF-I and IGFBP-1 expression in uterus support the hypothesis that the IGF-I is a mediator of estrogen action in the uterus. In addition attenuation of the EGF response in the uterine tissue of Tg mice suggests that this response is also mediated, in part, by IGF-I.

Growth retardation and hyperglycemia in insulin-like growth factor binding protein-1 transgenic mice.

Transgenic mice that constitutively overexpressed rat insulin-like growth factor binding protein-1 (IGFBP-1) were generated to determine the effects of overexpression of IGFBP-1 on growth and development. In offspring of three of the founders that showed high levels of expression, birth weight was significantly reduced to approximately 83-92% of the weight of their nontransgenic littermates. The transgenic mice gained less weight and were approximately 3.5-8 g lighter than nontransgenic littermates at 40 days of age. The difference in body weight between transgenic and wild-type mice was most apparent around the time of weaning when transgenic mice showed a more marked growth deceleration than wild-type mice. No significant catch-up growth was apparent over the first 3 months of life. In addition, offspring of all three high-expressing founders demonstrated fasting hyperglycemia. The transgene was highly expressed in the brain, uterus, lung, kidney, and heart, but little expression was detected in the liver. The weight of the brain relative to body weight was significantly reduced in transgenic mice compared with wild-type mice, whereas the relative weight of most other organs was similar to wild-type mice. These data demonstrate that IGFBP-1 may function to inhibit IGF action in vivo and that this inhibition selectively impairs development of organs such as the brain.

Growth retardation and hyperglycemia in insulin-like growth factor binding protein-1 transgenic mice

Transgenic mice that constitutively overexpressed rat insulin-like growth factor binding protein-1 (IGFBP-1) were generated to determine the effects of overexpression of IGFBP-1 on growth and development. In offspring of three of the founders that showed high levels of expression, birth weight was significantly reduced to approximately 83-92% of the weight of their nontransgenic littermates. The transgenic mice gained less weight and were approximately 3.5-8 g lighter than nontransgenic littermates at 40 days of age. The difference in body weight between transgenic and wild-type mice was most apparent around the time of weaning when transgenic mice showed a more marked growth deceleration than wild-type mice. No significant catch-up growth was apparent over the first 3 months of life. In addition, offspring of all three high-expressing founders demonstrated fasting hyperglycemia. The transgene was highly expressed in the brain, uterus, lung, kidney, and heart, but little expression was detected in the liver. The weight of the brain relative to body weight was significantly reduced in transgenic mice compared with wild-type mice, whereas the relative weight of most other organs was similar to wild-type mice. These data demonstrate that IGFBP-1 may function to inhibit IGF action in vivo and that this inhibition selectively impairs development of organs such as the brain.

Dexamethasone stimulation of rat insulin-like growth factor binding protein-1 (IGFBP-1) promoter activity involves the interaction of multiple transcription factors

Using an improved procedure for transient transfection of H4-II-E rat hepatoma cells, we characterized the cis elements in the proximal promoter of the rat insulin-like growth factor binding protein-1 (rat IGFBP-1) gene that are required for basal (unstimulated) and dexamethasone-stimulated promoter activity. Three sites are required for optimal basal promoter activity: an AP-2 site (nt −286 to −293), the M4 region of the insulin response element (nt −108 to −99), and a hepatocyte nuclear factor-1 (HNF-1) site (nt −62 to −50). In addition to the glucocorticoid response element (nt −91 to −77), participation of two of three accessory sites is required for optimal stimulation by dexamethasone: the M4 and HNF-1 sites, and a third site located between nt −252 and −236. Further study will focus on how the interactions of tissue-specific and hormonally-responsive transcription factors are integrated.