Rat Insulin-like Growth factor-I Research Articles

Rejuvenating Effect of Long-Term Insulin-Like Growth Factor-I Gene Therapy in the Hypothalamus of Aged Rats with Dopaminergic Dysfunction.

The aging female rat constitutes an interesting model of spontaneous and progressive age-related dopaminergic dysfunction as it allows assessing new therapeutic strategies for Parkinson's disease. Insulin-like growth factor I (IGF-I) is emerging as a powerful neuroprotective molecule, strongly induced in the central nervous system after different insults. We constructed a helper-dependent recombinant adenoviral vector (HDRAd-IGFI) harboring the gene for rat IGF-I. This was used to implement long-term IGF-I gene therapy in the hypothalamus of aged female rats, which display hypothalamic dopaminergic (DA) dysfunction and, as a consequence, chronic hyperprolactinemia. Rejuvenating long-term IGF-I gene therapy was implemented in young (3 months) and aged (24 months) female rats, which received a single intrahypothalamic injection of 4 × 109 viral particles of either HD-RAd-IGFI or HD-RAd-DsRed (control vector) and were sacrificed 119 days postinjection. In the young animals, neither vector modified serum prolactin (PRL) levels, but in the RAd-IGFI-injected aged rats a nearly full reversion of their hyperprolactinemic status was recorded. Morphometric analysis revealed a significant increase in the total number of tyrosine hydroxylase (TH)-positive cells in the hypothalamus of experimental compared with control aged animals (5874 ± 486 and 3390 ± 498, respectively). Our results indicate that IGF-I gene therapy in aged female rats is highly effective in rejuvenating the hypothalamic DA neuron groups.

Insulin-like growth factor-I gene therapy increases hippocampal neurogenesis, astrocyte branching and improves spatial memory in female aging rats.

In rats, learning and memory performance decline during aging, which makes this rodent species a suitable model to evaluate therapeutic strategies of potential value for correcting age-related cognitive deficits. Some of these strategies involve neurotrophic factors like insulin-like growth factor-I (IGF-I), a powerful neuroprotective molecule in the brain. Here, we implemented 18-day long intracerebroventricular (ICV) IGF-I gene therapy in 28months old Sprague-Dawley female rats, and assessed spatial memory performance in the Barnes maze. We also studied hippocampal morphology using an unbiased stereological approach. Adenovectors expressing the gene for rat IGF-I or the reporter DsRed were used. Cerebrospinal fluid (CSF) samples were taken and IGF-I levels determined by radioimmunoassay. At the end of the study, IGF-I levels in the CSF were significantly higher in the experimental group than in the DsRed controls. After treatment, the IGF-I group showed a significant improvement in spatial memory accuracy as compared with DsRed counterparts. In the dentate gyrus (DG) of the hippocampus, the IGF-I group showed a higher number of immature neurons than the DsRed controls. The treatment increased hippocampal astrocyte branching and reduced their number in the hippocampal stratum radiatum. We conclude that the ependymal route is an effective approach to increase CSF levels of IGF-I and that this strategy improves the accuracy of spatial memory in aging rats. The favorable effect of the treatment on DG neurogenesis and astrocyte branching in the stratum radiatum may contribute to improving memory performance in aging rats.

Restorative effect of intracerebroventricular insulin-like growth factor-I gene therapy on motor performance in aging rats

Insulin-like growth factor-I (IGF-I) is a powerful neuroprotective molecule in the brain and spinal cord. We have previously shown that intracerebroventricular (i.c.v.) IGF-I gene therapy is an effective strategy to increase IGF-I levels in the cerebrospinal fluid (CSF). Since aging in rats is associated with severe motor function deterioration, we implemented i.c.v. IGF-I gene therapy in very old rats (30–31 months) and assessed the beneficial impact on motor performance. We used recombinant adenovectors (RAds) expressing either green fluorescent protein (GFP) or rat IGF-I. Injection in the lateral or fourth ventricle led to high transgene expression in the ependymal cell layer in the brain and cervical spinal cord. RAd-IGF-I-injected rats but not RAd-GFP-injected controls, showed significantly increased levels of CSF IGF-I. Motor tests showed the expected age-related decline in aged rats. Seventeen-day IGF-I gene therapy induced a significant improvement in motor performance in the aged but not in the young animals. These results show that IGF-I is an effective restorative molecule in the aging brain and spinal cord. The data also reveal that the ependymal route constitutes a promising approach for implementing protective IGF-I gene therapy in the aging CNS.

Dispersed Chromosomal Stat5b-binding Elements Mediate Growth Hormone-activated Insulin-like Growth Factor-I Gene Transcription

The growth hormone (GH)-insulin-like growth factor-I (IGF-I) axis regulates somatic growth during childhood and orchestrates tissue repair throughout the life span. Recently described inactivating mutations in Stat5b in humans with impaired growth have focused attention on this transcription factor as a key agent linking GH-stimulated signals to IGF-I gene expression, and several putative Stat5b sites have been identified in the IGF-I gene. Here, we define and characterize potential GH- and Stat5b-activated chromosomal enhancers that can regulate IGF-I gene transcription. Of 89 recognizable Stat5 sequences in 200 kb centering on the rat IGF-I gene, 22 resided within conserved regions and/or were identical among different species. Only 15 of these sites, organized into 7 distinct domains, were found to bind Stat5b by quantitative chromatin immunoprecipitation assays in liver chromatin of rats, but only after acute GH treatment. These sites could bind Stat5b in vitro, and individual domains could mediate GH- and Stat5b-stimulated IGF-I promoter activity in cultured cells. Further analyses revealed that four Stat5b domains possessed chromatin signatures of enhancers, including binding of co-activators p300 and Med1, and RNA polymerase II. These modifications preceded GH-stimulated recruitment of Stat5b, as did lysine 4 monomethylation of histone H3, which was enriched in 6/7 Stat5b-binding elements. In contrast, histone acetylation was induced by GH but was limited to Stat5b binding domains found within the IGF-I transcription unit. We conclude that GH stimulates recruitment of Stat5b to multiple dispersed regions within the igf1 locus, including several with properties consistent with long range transcriptional enhancers that collectively regulate GH-activated IGF-I gene transcription.

Insulin-like growth factor-I gene therapy reverses morphologic changes and reduces hyperprolactinemia in experimental rat prolactinomas.

BackgroundThe implementation of gene therapy for the treatment of pituitary tumors emerges as a promising complement to surgery and may have distinct advantages over radiotherapy for this type of tumors. Up to now, suicide gene therapy has been the main experimental approach explored to treat experimental pituitary tumors. In the present study we assessed the effectiveness of insulin-like growth factor I (IGF-I) gene therapy for the treatment of estrogen-induced prolactinomas in rats.ResultsFemale Sprague Dawley rats were subcutaneously implanted with silastic capsules filled with 17-β estradiol (E2) in order to induce pituitary prolactinomas. Blood samples were taken at regular intervals in order to measure serum prolactin (PRL). As expected, serum PRL increased progressively and 23 days after implanting the E2 capsules (Experimental day 0), circulating PRL had undergone a 3–4 fold increase. On Experimental day 0 part of the E2-implanted animals received a bilateral intrapituitary injection of either an adenoviral vector expressing the gene for rat IGF-I (RAd-IGFI), or a vector (RAd-GFP) expressing the gene for green fluorescent protein (GFP). Seven days post vector injection all animals were sacrificed and their pituitaries morphometrically analyzed to evaluate changes in the lactotroph population. RAd-IGFI but not RAd-GFP, induced a significant fall in serum PRL. Furthermore, RAd-IGFI but not RAd-GFP significantly reversed the increase in lactotroph size (CS) and volume density (VD) induced by E2 treatment.ConclusionWe conclude that IGF-I gene therapy constitutes a potentially useful intervention for the treatment of prolactinomas and that bioactive peptide gene delivery may open novel therapeutic avenues for the treatment of pituitary tumors.

Restorative effect of insulin-like growth factor-I gene therapy in the hypothalamus of senile rats with dopaminergic dysfunction

Insulin-like growth factor-I (IGF-I) is emerging as a powerful neuroprotective molecule that is strongly induced in the central nervous system after different insults. We constructed a recombinant adenoviral vector (RAd-IGFI) harboring the gene for rat IGF-I and used it to implement IGF-I gene therapy in the hypothalamus of senile female rats, which display hypothalamic dopaminergic (DA) neurodegeneration and as a consequence, chronic hyperprolactinemia. Restorative IGF-I gene therapy was implemented in young (5 months) and senile (28 months) female rats, which received a single intrahypothalamic injection of 3 x 10(9) plaque-forming units of RAd-betagal (a control adenoviral vector expressing beta-galactosidase) or RAd-IGFI and were killed 17 days post-injection. In the young animals, neither vector modified serum prolactin levels, but in the RAd-IGFI-injected senile rats a nearly full reversion of their hyperprolactinemic status was recorded. Morphometric analysis revealed a significant increase in the total number of tyrosine hydroxylase-positive cells in the hypothalamus of experimental as compared with control senile animals (5874+/-486 and 3390+/-498, respectively). Our results indicate that IGF-I gene therapy in senile female rats is highly effective for restoring their hypothalamic DA dysfunction and thus reversing their chronic hyperprolactinemia.

Insulin-like Growth Factor-1 Gene Transfer Augments Muscle IGF Protein Content Despite Cast Immobilization

Insulin-like growth factor I (IGF-I) is a potent myogenic factor that has been shown to play a critical role in muscle regeneration and muscle hypertrophy. Viral-mediated gene transfer of IGF-I appears to facilitate muscle hypertrophy and improve muscle function, yet it is unclear whether the benefits of gene transfer are maintained during cast immobilization or with subsequent restoration of muscle function following cast immobilization. PURPOSE: To investigate the potential of virus-mediated gene transfer of IGF-1 to augment IGF-1 protein levels in skeletal muscle under normal loading/activity conditions and whether elevations are maintained during and after cast immobilization with subsequent reloading. METHODS: The anterior compartments of the left hindlimb of 43 young adult C57BL6 mice were injected with a recombinant adeno-associated virus vector for IGF-I (rAAV-1). The construct consisted of the entire rat IGF-I cDNA which encodes for IGF-I, a Myosin Light Chain (MLC) 1/3 promoter and enhancer, and SV40 polyadenlyation sequence. At 20 weeks of age, 35 of these mice were immobilized in a bilateral cast for 2 weeks with ∼60° of dorsiflexion to maximize anterior compartment muscle atrophy. After cast immobilization, the tibialis anterior muscle was removed from both legs at 0 days and 1,3,5, and 10 weeks of free cage reambulation. Eight mice remained uncasted for comparison. RT-PCr was performed to quantify IGF-1 mRNA levels. IGF-1 protein levels were quantified using a mouse IGF-1 immunoassay. RESULTS: IGF-1 mRNA levels were consistently elevated in injected limbs. In addition, there was a 27 fold increase in tibialis anterior IGF-1 protein content of the injected limb in uncasted mice (85.2+32.9ng/g injected; 3.2+0.48ng/g uninjected) (p<0.05). Significant differences in absolute values of IGF-1 protein content from injected to uninjected limbs persisted with cast immobilization and free cage reambulation (range: 14–19 fold differences) (p<0.05). When comparing the relative magnitude of side-to-side differences in IGF protein content across all timepoints, there was a significantly greater difference in IGF-1 protein content before cast immobilization (p<0.05). CONCLUSION: Muscle specific adeno-associated virus overexpression of IGF-I not only increased IGF-1 mRNA levels, but also resulted in markedly higher IGF-1 protein levels that remained elevated despite cast immobilization. Our findings suggest that gene transfer of IGF-1 may be able to guard skeletal muscle from the deleterious impact of immobilization/disuse and enhance the subsequent restoration of muscle function.

Insulin-like Growth Factor-1 Gene Transfer Augments Muscle Protein Synthesis Rate And Size In Adult Mice

Insulin-like growth factor I (IGF-I) is a potent myogenic factor that has been shown to play a critical role in muscle regeneration and muscle hypertrophy. Viral-mediated gene transfer of IGF-I upregulates muscle size and increases muscle strength. It is not clear whether IGF-I-induced muscle hypertrophy is mediated by an increase in protein synthesis and/or a decrease in proteolysis rates in existing myofibers and satellite cells. PURPOSE The objective of this study was to investigate the effect of IGF-I overexpression on in vivo muscle protein synthesis rate. METHODS The left hindlimb of young adult C57BL6 mice was injected with a recombinant adeno-associated virus vector for IGF-I (rAAV-1). The construct consisted of the entire rat IGF-I cDNA which encodes for IGF-I, a Myosin Light Chain (MLC) 1/3 promoter and enhancer, and SV40 polyadenlyation sequence. Six mice (age=3 weeks) were injected with 80ul of 10% glycerol/PBS containing approximately 1010 rAAV particles into the interstitial space of the anterior compartment of one hindlimb, targeting the extensor digitorum longus (EDL). Three months after transfection, in vivo mixed muscle protein synthesis rates were measured using the incorporation of intravenously administered L- [13C]-phe into EDL muscle proteins. [13C]-phe abundance in the structural proteins was quantitated using gas chromatography-combustion-isotope ratio mass spectrometry. Paired samples t-tests were used for comparisons between transfected and contralateral hindlimbs. RESULTS rAAV-IGF1 transfection resulted in a significant increase (mean ± SD) (12.1 ± 8.5%) in muscle wet weight and cross-sectional area (9.9 ± 9.1%)(p<0.05). In vivo mixed muscle protein synthesis rate was 26.9 ± 15.6% higher in the rAAV-IGF1 injected limbs compared to the contralateral control limb (p<0.05). Average EDL muscle protein synthesis rate was 10.5 ± 2.8%/day in the rAAV-IGF1 injected limb and 8.2 ± 1.9%/day in the contralateral limb. CONCLUSIONS These results demonstrate that muscle specific adeno-associated virus overexpression of IGF-I increased mixed muscle protein synthesis rate and this contributed to muscle hypertrophy. Further research will determine EDL contractile properties, and the components of the IGF-I signaling pathway that are activated in satellite cells and mature myofibers. Supported by R01-HD042955, NCRR-000954.

Identification of a Distal STAT5-binding DNA Region That May Mediate Growth Hormone Regulation of Insulin-like Growth Factor-I Gene Expression

Growth hormone (GH) regulates insulin-like growth factor-I (IGF-I) gene expression through signal transducer and activator of transcription 5b (STAT5b) and STAT5a. The objective of this study was to identify the cis-regulatory DNA region involved in this process. By cotransfection analyses of shotgun DNA fragments of a bacterial artificial chromosome sequence containing the entire human IGF-I gene and a large 5'-flanking region, a approximately 700-bp DNA region approximately 75 kb 5' to the IGF-I gene was found to have the ability to enhance gene expression from both heterologous and homologous promoters in the presence of constitutively active STAT5a or STAT5b. This 700-bp DNA region contains two closely located consensus STAT5-binding sites, and its sequence appears to be evolutionarily conserved. Electrophoretic mobility shift assays verified the ability of the two putative STAT5-binding sites to bind to STAT5a and STAT5b. Cotransfection analyses confirmed that both STAT5-binding sites were necessary for the 700-bp DNA region to mediate STAT5a or STAT5b activation of gene transcription. Chromatin immunoprecipitation assays demonstrated that the chromosomal region containing these two STAT5-binding sites was bound by constitutively active STAT5b protein in HepG2 cells and that the binding was accompanied by increased expression of IGF-I mRNA. In reconstituted GH-responsive cells, this 700-bp DNA region was able to mediate GH-induced STAT5a or STAT5b activation of gene expression. These results together suggest that this STAT5-binding site-containing distal 5'-flanking region of IGF-I gene may be an enhancer mediating GH-induced STAT5 activation of IGF-I gene transcription.

Insulin-like Growth Factor-I Stimulates Acromegaly-like Specific Mandibular Enlargement in Rats

To help us investigate the time course of mandibular enlargement in acromegaly or acrogiantism to determine the most suitable period for occlusal treatment in this disease, our aim was to develop a rat model of acromegaly (acrogiantism). In this study, prominent mandibular enlargement was induced by continuous subcutaneous infusion of human recombinant insulin-like growth factor-I (IGF-I) (640 microg/day) in 10-week-old male rats for 4 weeks (n = 6); the control sham-operated group was injected with saline alone (n = 6). Circulating human IGF-I was clearly detectable in the IGF-I group during the four-week administration period, while endogenous rat IGF-I levels decreased. Total IGF-I (human + rat) increased significantly during administration, returning to control levels afterwards. The length of every bone examined (mandible, maxilla, and femur) showed a significant increase compared to control rats, especially the mandible. Although the mandible did not continue to grow after discontinuation of IGF-I administration, it did not return to control size, unlike the maxilla and femur, and disharmonious jaw size (between maxilla and mandible) persisted even after circulating IGF-I levels normalized. These findings in our rat model suggest that mandibular occlusal treatment should only be considered for acromegalic (acrogiantic) patients after serum IGF-I levels have normalized and bone growth has ceased.

Effects of oxidative stress, insulin and IGF-I on p70S6 kinase in rat thecal-interstitial cells

Appropriate regulation of ovarian thecal-interstitial proliferation and steroidogenesis is essential for normal ovarian function. Previously, we have shown that insulin, insulin-like growth factor-I (IGF-I) and moderate oxidative stress, all stimulate proliferation and steroidogenesis of theca-interstitial cells. We have also observed that the effects of the combination of these agents are not additive indicating a possibility of a common saturable step(s) of their signal transduction pathways. Recently, we have shown that oxidative stress and insulin/IGF-I stimulate the MAPK pathway. In contrast, insulin and IGF-I, but not oxidative stress, stimulate the PI3K/Akt pathway. In the present study we proposed that both oxidative stress and insulin/IGF-I signaling pathways converge at the level of p70S6 kinase (p70S6K). In vitro evaluation of human endometrial cell proliferation. Purified rat theca-interstitial cells were cultured in chemically defined media without (control) or with IGF-I (10 nM), insulin (100 nM), hypoxanthine + xanthine oxidase (X/XO; 1 mM/30 μU/mL), hydrogen peroxide (100 nM), wortmannin (100 nM) and/or PD98059 (10 μM). The above concentrations of X/XO and hydrogen peroxide have been shown previously to induce moderate oxidative stress and to promote theca-interstitial proliferation and steroidogenesis. Phosphorylation of p70S6 kinase was determined by western blot of p70S6K[pT/pS421/424]. Quantitative data was analyzed by ANOVA followed by post-hoc pairwise comparisons. In rat theca-interstitial cells, insulin and IGF-I stimulated p70S6K phosphorylation at tyrosine/serine 421/424 with a maximum effect after 10 minutes of exposure. Insulin and IGF-I increased phosphorylation by 63±13% (mean±SEM; p<0.001) and 51±13% (p<0.001), respectively, above control levels. Moderate oxidative stress induced by X/XO and hydrogen peroxide stimulated p70S6K phosphorylation by 60±25% (p<0.05) and 29±8% (p<0.01), respectively. Wortmannin, an inhibitor of PI3K, decreased insulin-induced p70S6K phosphorylation by 53±2%. Similarly, PD98059, a selective inhibitor of MAPK, decreased insulin-induced p70S6K phosphorylation by 44±3%. The present findings indicate that in thecal-interstitial cells, both PI3K and MAPK pathways activate p70S6K. A convergence of actions of insulin/IGF-I and oxidative stress at the level of p70S6K may explain the comparable effects of the above agents on proliferation and testosterone production. We propose that excessive activation of this kinase by oxidative stress and hyperinsulinemia may promote hyperplasia of ovarian mesenchyme in conditions such as polycystic ovary syndrome (PCOS).

Mechanisms of Growth Hormone (GH) Action: IDENTIFICATION OF CONSERVED Stat5 BINDING SITES THAT MEDIATE GH-INDUCED INSULIN-LIKE GROWTH FACTOR-I GENE ACTIVATION

Many of the actions of growth hormone (GH) on somatic growth and tissue maintenance are mediated by insulin-like growth factor-I (IGF-I), a secreted protein whose gene expression is rapidly and potently induced by GH by unknown mechanisms. Recent studies implicating Stat5a and Stat5b in the growth response to GH in mice and observations linking Stat5b to control of IGF-I gene transcription in rats have prompted the current investigations into the molecular determinants of a putative regulatory network extending from GH through Stat5b to IGF-I. Here we characterize as critical components of this hormone-activated transcriptional pathway two adjacent Stat5 binding sites in the second intron of the rat IGF-I gene located within a conserved region previously found to undergo acute and reversible changes in chromatin structure after in vivo GH treatment. As assessed by chromatin immunoprecipitation assays, GH rapidly induced binding of Stat5 to this DNA segment in the liver of GH-deficient rats, just prior to the onset of transcription from both major and minor IGF-I gene promoters. Biochemical reconstitution experiments showed that the two intronic Stat5 DNA elements were able to bind Stat5b in vitro after GH treatment could transmit GH- and Stat5b-dependent transcriptional responsiveness to the major IGF-I promoter and to a minimal neutral gene promoter and were required for full stimulation of reporter gene activity by GH. Taken together, these results identify an intronic enhancer as a key mediator of GH-induced IGF-I gene transcription working through Stat5b and provide new insight into the molecular architecture of this transcriptional pathway.

A new animal model for evaluation of long-term growth rate over one month by rhGH/PLGA microcapsule formulations.

A new animal model to evaluate the long-term growth rate produced by a sustained-release formulation of recombinant human growth hormone (rhGH) over one month was developed and the usefulness of our microcapsule formulations was demonstrated in this model. Long-term pharmacological effects by subcutaneous injection of microcapsules for sustained release of rhGH were evaluated in hypophysectomized (Hpx) rats treated with immunosuppressive agent along with hormone supplement. Copoly(DL-lactic/glycolic)acid (PLGA) microcapsules for sustained release of rhGH, a two-week sustained-release formulation (rhGH-SR-2W) and a one-month sustained-release formulation (rhGH-SR-1M), were prepared by a solid-in-oil-in-water emulsion solvent evaporation technique. Body-weight gain, body-length gain and serum levels of rat insulin-like growth factor-I (rIGF-I) induced by subcutaneous injection of rhGH-SR were compared with those by daily injections of rhGH solution in Hpx rats for 35 days. Serum IGF-I levels in Hpx rats after the injection of rhGH-SR2W microcapsules were higher than those after daily injections of rhGH solution. Body-length gain, a new parameter, after single injection of rhGH-SR-1M microcapsules demonstrated the higher growth rate than that after daily injections of rhGH solution for 35 days. Thus, single injection of rhGH-SR microcapsules demonstrated long-term pharmacological effects greater than those by daily injections of rhGH solution in a newly developed model, immunosuppressed Hpx rats.

IGF-I Transcript Levels in Whole-Liver Tissue, in Freshly Isolated Hepatocytes, and in Cultured Hepatocytes from Lean and Obese Zucker Rats

Background: The mechanisms underlying the maintenance of normal to high rates of linear growth and plasma insulin-like growth factor I (IGF-I) levels in spite of a low growth hormone secretion in obese children remain unknown. Among the animal models of early-onset obesity, obese Zucker (fa/fa) rats (which are homozygous for an inactivating missense mutation in the leptin receptor) are particularly appropriate, because their linear growth shows this growth hormone independence. Methods: To study the regulation of IGF-I synthesis in this model, we have established primary cultures of hepatocytes derived from 12-week-old Zucker male obese and lean rats. The rat IGF-I gene contains six exons, and alternative splicing generates different mRNAs, one of which (called IGF-1B) has been shown to be decreased by fasting. We report steady state mRNA levels for IGF-I (all transcripts) and for IGF-IB in hepatocytes after 3 days in culture, in freshly isolated hepatocytes, and in whole-liver tissue. RT-PCRs using primers specific for IGF-I or IGF-IB were performed with two different internal competitors for quantification. Results: In primary cultures of hepatocytes, the IGF-IB mRNA was increased by >50-fold (p = 0.01) in cells derived from obese animals as compared with cells from lean animals. However, these transcript levels were not significantly different when measured in freshly isolated hepatocytes or in whole-liver tissue. Conclusions: Increased IGF-IB transcription could be an intrinsic characteristic of cultured hepatocytes harbouring leptin receptors that bear the fa mutation. However, the modulation of this characteristic by cell-cell interactions and by in vivo hormone and metabolic status remains to be studied.

Stimulatory effect of zinc on insulin-like growth factor-I and transforming growth factor-beta1 production with bone growth of newborn rats.

The effect of zinc, an essential trace element, on insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 production was investigated to determine the role of this metal in bone growth of newborn rats. Femoral-diaphyseal and metaphyseal tissues were obtained between 1 and 28 days after birth of newborn rats, and cultured for 24 h in a serum-free Dulbecco's modified Eagle's medium containing either vehicle or zinc sulfate (10(-6) - 10(-4) M). Protein concentration in the medium was significantly increased by culture with bone tissues of newborn rats with increasing age (14 and 21 days). Medium IGF-I and TGF-beta1 concentration was gradually reduced with increasing age after birth. The presence of zinc (10(-5) and 10(-4) M) caused a significant increase in protein, IGF-I, and TGF-beta1 concentrations in the medium cultured with the diaphyseal or metaphyseal tissues obtained at 7 and 14 days after birth. The expression of IGF-I and TGF-beta1 mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) analysis in the diaphyseal and metaphyseal tissues cultured for 24 h using rat IGF-I or TGF-beta1-specific primers. These expressions were significantly raised in the presence of zinc (10(-4) M) in culture medium. The present study demonstrates that zinc has a stimulatory effect on IGF-I and TGF-beta1 production in the femoral tissues with bone growth of newborn rats.

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.

Developmental regulation of insulin-like growth factor-I and growth hormone receptor gene expression

During development, the insulin-like growth factor I (IGF-I) gene is expressed in a tissue specific manner; however, the molecular mechanisms governing its developmental regulation remain poorly defined. To examine the hypothesis that expression of the growth hormone (GH) receptor accounts, in part, for the tissue specific expression of the IGF-I gene during development, the developmental regulation of IGF-I and GH receptor gene expression in rat tissues was examined. The level of IGF-I and GH receptor mRNA was quantified in RNA prepared from rats between day 17 of gestation (E17) and 17 months of age (17M) using an RNase protection assay. Developmental regulation of IGF-I gene expression was tissue specific with four different patterns of expression seen. In liver, IGF-I mRNA levels increased markedly between E17 and postnatal day 45 (P45) and declined thereafter. In contrast, in brain, skeletal muscle and testis, IGF-I mRNA levels decreased between P5 and 4M but were relatively unchanged thereafter. In heart and kidney, a small increase in IGF-I mRNA levels was observed between the early postnatal period and 4 months, whereas in lung, minimal changes were observed during development. The changes in GH receptor mRNA levels were, in general, coordinate with the changes in IGF-I mRNA levels, except in skeletal muscle. Interestingly, quantification of GH receptor levels by Western blot analysis in skeletal muscle demonstrated changes coordinate with IGF-I mRNA levels. The levels of the proteins which mediate GH receptor signaling (STAT1, -3, and -5, and JAK2) were quantified by Western blot analysis. These proteins also are expressed in a tissue specific manner during development. In some cases, the pattern of expression was coordinate with IGF-I gene expression, whereas in others it was discordant. To further define molecular mechanisms for the developmental regulation of IGF-I gene expression, protein binding to IGFI-FP1, a protein binding site that is in the major promoter of the rat IGF-I gene and is important for basal promoter activity in vitro, was examined. Gel shift analyses using a 34-base pair oligonucleotide that contained IGFI-FP1 did not demonstrate changes in protein binding that paralleled those in IGF-I gene expression, suggesting that protein binding to IGFI-FP1 does not contribute to the developmental regulation of IGF-I gene expression, at least in brain and liver. In summary, the present studies demonstrate coordinate expression of the IGF-I gene and GH receptor during development and suggest that GH receptor expression contributes to the tissue specific expression of the IGF-I gene during development.

Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I.

The molecular mechanisms underlying myogenic induction by insulin-like growth factor I (IGF-I) are distinct from its proliferative effects on myoblasts. To determine the postmitotic role of IGF-I on muscle cell differentiation, we derived L6E9 muscle cell lines carrying a stably transfected rat IGF-I gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC-IGF-I exclusively in differentiated L6E9 myotubes, which express the embryonic form of myosin heavy chain (MyHC) and no endogenous IGF-I, resulted in pronounced myotube hypertrophy, accompanied by activation of the neonatal MyHC isoform. The hypertrophic myotubes dramatically increased expression of myogenin, muscle creatine kinase, beta-enolase, and IGF binding protein 5 and activated the myocyte enhancer factor 2C gene which is normally silent in this cell line. MLC-IGF-I induction in differentiated L6E9 cells also increased the expression of a transiently transfected LacZ reporter driven by the myogenin promoter, demonstrating activation of the differentiation program at the transcriptional level. Nuclear reorganization, accumulation of skeletal actin protein, and an increased expression of beta1D integrin were also observed. Inhibition of the phosphatidyl inositol (PI) 3-kinase intermediate in IGF-I-mediated signal transduction confirmed that the PI 3-kinase pathway is required only at early stages for IGF-I-mediated hypertrophy and neonatal MyHC induction in these cells. Expression of IGF-I in postmitotic muscle may therefore play an important role in the maturation of the myogenic program.

CCAAT/Enhancer-binding Protein δ Is a Critical Regulator of Insulin-like Growth Factor-I Gene Transcription in Osteoblasts

Insulin-like growth factor-I (IGF-I) plays a major role in promoting skeletal growth by stimulating bone cell replication and differentiation. Prostaglandin E2 and other agents that induce cAMP production enhance IGF-I gene transcription in cultured rat osteoblasts through a DNA element termed HS3D, located in the proximal part of the major rat IGF-I promoter. We previously determined that CCAAT/enhancer-binding protein delta (C/EBPdelta) is the key cAMP-stimulated regulator of IGF-I transcription in these cells and showed that it transactivates the rat IGF-I promoter through the HS3D site. We now have defined the physical-chemical properties and functional consequences of the interactions between C/EBPdelta and HS3D. C/EBPdelta, expressed in COS-7 cells or purified as a recombinant protein from Escherichia coli, bound to HS3D with an affinity at least equivalent to that of the albumin D-site, a known high affinity C/EBP binding sequence, and both DNA elements competed equally for C/EBPdelta. C/EBPdelta bound to HS3D as a dimer, with protein-DNA contact points located on guanine residues on both DNA strands within and just adjacent to the core C/EBP half-site, GCAAT, as determined by methylation interference footprinting. C/EBPdelta also formed protein-protein dimers in the absence of interactions with its DNA binding site, as indicated by results of glutaraldehyde cross-linking studies. As established by competition gel-mobility shift experiments, the conserved HS3D sequence from rat, human, and chicken also bound C/EBPdelta with similar affinity. We also found that prostaglandin E2-induced expression of reporter genes containing human IGF-I promoter 1 or four tandem copies of the human HS3D element fused to a minimal promoter and show that these effects were enhanced by a co-transfected C/EBPdelta expression plasmid. Taken together, our results provide evidence that C/EBPdelta is a critical activator of IGF-I gene transcription in osteoblasts and potentially in other cell types and species.

Identification of stretch-responsive genes in pulmonary artery smooth muscle cells by a two arbitrary primer-based mRNA differential display approach.

Physical forces induce profound changes in cell phenotype, shape and behavior. These changes can occur in vascular structures as a result of pressure overload and their effects can be seen in atherosclerotic vessels in which smooth muscle cells have undergone hyperplastic and hypertrophic changes. At the molecular level, mechanical stimuli are converted into chemical ones and lead to modulation of gene expression and/or the activation of a new repertoire of genes whose encoded proteins help the cells to adapt to their microenvironment. In this study, we have used a two primer-based mRNA differential display technique to identify candidate mechano-responsive genes in pulmonary artery smooth muscle cells. As compared to the original method described by Liang and Pardee, this technique uses two arbitrary primers instead of an anchored oligo(dt) plus an arbitrary primer in the polymerase chain reaction. The chief advantages of these modifications are an increase in the efficiency of the amplification and in the identification of differentially expressed clones. Using this approach, we compared the pattern of expressed genes in cells cultured under static conditions with those in cells that were mechanically stretched (1 Hz) for 24 h in a well-defined in vitro mechanical system. Three candidate genes that showed reproducible differences were chosen for further characterization and cloning. One clone was under expressed in stretched cells and had a DNA sequence with 90% homology to the human fibronectin gene. Two other clones were highly expressed in stretched cells and had a 92% and a 83% sequence homology with human platelet-activating factor (PAF) receptor and rat insulin-like growth factor-I (IGF-I) genes respectively. Northern blot analysis confirmed low levels of fibronectin mRNA transcripts in stretched cells. In contrast, accumulation of PAF receptor mRNA occurred 30 min after mechanical stretch was initiated whereas IGF-I mRNA levels peaked at 8 h. Both mRNA levels were sustained for up to 24 h of mechanical stretching. These results demonstrate the usefulness of the two primer-based mRNA differential display that enabled us to identify and characterize alterations at the level of gene expression among matrix proteins, G-protein coupled receptors and growth factors, each of whose response to mechanical strain is different. A more complete understanding of these responses will provide further insight into the pathologic processes associated with hypertension and atherosclerosis.