Insulin-like Growth factor-I Messenger RNA Research Articles

Effects of Prednisolone on Serum and Tissue Fluid IGF-I Receptor Activation and Post-Receptor Signaling in Humans.

Short-term glucocorticoid exposure increases serum insulinlike growth factor I (IGF-I) concentrations but antagonizes IGF-I tissue signaling. The underlying mechanisms remain unknown. To identify at which levels glucocorticoid inhibits IGF-I signaling. Nineteen healthy males received prednisolone (37.5 mg/d) and placebo for 5 days in a randomized, double-blinded, placebo-controlled crossover study. Serum was collected on days 1, 3, and 5, and abdominal skin suction blister fluid (SBF; ~interstitial fluid) was taken on day 5 (n = 9) together with muscle biopsy specimens (n = 19). The ability of serum and SBF to activate the IGF-I receptor (IGF-IR) (bioactive IGF) and its downstream signaling proteins was assessed using IGF-IR-transfected cells. Prednisolone increased IGF-I concentrations and bioactive IGF in serum (P ≤ 0.001) but not in SBF, which, compared with serum, contained less bioactive IGF (~28%) after prednisolone (P < 0.05). This observation was unexplained by SBF concentrations of IGFs and IGF-binding proteins (IGFBPs) 1 to 4. However, following prednisolone treatment, SBF contained less IGFBP-4 fragments (P < 0.05) generated by pregnancy-associated plasma protein A (PAPP-A). Concomitantly, prednisolone increased SBF levels of stanniocalcin 2 (STC2) (P = 0.02) compared with serum. STC2 blocks PAPP-A from cleaving IGFBP-4. Finally, prednisolone suppressed post-IGF-IR signaling pathways at the level of insulin receptor substrate 1 (P < 0.05) but did not change skeletal muscle IGF-IR, IGF-I, or STC2 messenger RNA. Prednisolone increased IGF-I concentrations and IGF bioactivity in serum but not in tissue fluid. The latter may relate to a STC2-mediated inhibition of PAPP-A in tissue fluids. Furthermore, prednisolone induced post-IGF-IR resistance. Thus, glucocorticoid may exert distinct, compartment-specific effects on IGF action.

Development and Field Application of a Model Predicting Effects of Episodic Hypoxia on Short-Term Growth of Spot

AbstractIn North Carolina and elsewhere, there is concern that excessive nutrient loading and resulting hypoxic conditions in coastal ecosystems are adversely affecting the native fauna, but quantifying the effects on fish can be difficult. Hypoxia may reduce fish growth via direct exposure or indirectly (e.g., cost of low-oxygen avoidance, reduced food availability, and density-dependent effects in oxygenated refuges). Given the fine spatial and temporal scale of oxygen dynamics in estuarine habitats, evaluating the impacts of hypoxia on fish growth requires short-term growth indicators that integrate the effects of rapidly changing environmental conditions. To address this need, we experimentally determined the sensitivity and response time of a suite of bioindicators of recent growth (RNA:DNA ratio and RNA concentration in muscle tissue; insulin-like growth factor-I messenger RNA expression in the liver; hepatosomatic index; and Fulton’s condition factor K) to changes in the specific growth rate of juv...

Expression profile of IGF-I-calcineurin-NFATc3-dependent pathway genes in skeletal muscle during early development between duck breeds differing in growth rates.

The insulin-like growth factor I (IGF-I)-calcineurin (CaN)-NFATc signaling pathways have been implicated in the regulation of myocyte hypertrophy and fiber-type specificity. In the present study, the expression of the CnAα, NFATc3, and IGF-I genes was quantified by RT-PCR for the first time in the breast muscle (BM) and leg muscle (LM) on days 13, 17, 21, 25, and 27 of embryonic development, as well as at 7days posthatching (PH), in Gaoyou and Jinding ducks, which differ in their muscle growth rates. Consistent expression patterns of CnAα, NFATc3, and IGF-I were found in the same anatomical location at different development stages in both duck breeds, showing significant differences in an age-specific fashion. However, the three genes were differentially expressed in the two different anatomical locations (BM and LM). CnAα, NFATc3, and IGF-I messenger RNA (mRNA) could be detected as early as embryonic day 13 (ED13), and the highest level appeared at this stage in both BM and LM. Significant positive relationships were observed in the expression of the studied genes in the BM and LM of both duck breeds. Also, the expression of these three genes showed a positive relationship with the percentage of type IIb fibers and a negative relationship with the percentage of type I fibers and type IIa fibers. Our data indicate differential expression and coordinated developmental regulation of the selected genes involved in the IGF-I-calcineurin-NFATc3 pathway in duck skeletal muscle during embryonic and early PH growth and development; these data also indicate that this signaling pathway might play a role in the regulation of myofiber type transition.

Insulin‐like growth factor I messenger RNA and protein are expressed in the human lymph node and distinctly confined to subtypes of macrophages, antigen‐presenting cells, lymphocytes and endothelial cells

Insulin-like growth factor I (IGF-I) is a potent hormone that stimulates growth and differentiation and inhibits apoptosis in numerous tissues. Preliminary evidence suggests that IGF-I exerts differentiating, mitogenic and restoring activities in the immune system but the sites of synthesis of local IGF-I are unknown. Identification of these sites would allow the functional role of local IGF-I to be clarified. The presence of IGF-I in non-immune cells suggests that it acts as a trophic factor, while its occurrence in subtypes of lymphocytes or antigen-presenting cells indicates paracrine/autocrine direct regulatory involvement of IGF-I in the human immune response. The present study investigated the location of IGF-I messenger RNA and protein on archival human lymph node samples by in situ hybridization, immunohistochemistry and double immunofluorescence staining using an IGF-I probe and antisera specific for human IGF-I and CD3 (T lymphocytes), CD20 (B lymphocytes), CD68 (macrophages), CD21 (follicular dendritic cells), S100 (interdigitating dendritic cells) and podoplanin (fibroblastic reticular cells). Numerous cells within the B- and T-cell compartments expressed the IGF-I gene, and the majority of these cells were identified as macrophages. Solitary follicular dendritic cells exhibited IGF-I. A few T lymphocytes, and no B lymphocytes, contained IGF-I immunoreactive material. Furthermore, IGF-I immunoreactive cells outside the follicles that did not react with CD3, CD20, S100 or podoplanin markers were identified as high-endothelial venule (HEV) cells. From this we conclude that the main task of IGF-I in human non-tumoral lymph node may be autocrine and paracrine regulation of the differentiation, stimulation and survival of lymphocytes, antigen-presenting cells and macrophages and the differentiation and maintenance of HEV cells.

Stimulation of sensory neurons improves cognitive function by promoting the hippocampal production of insulin-like growth factor-I in mice

Calcitonin gene-related peptide (CGRP) increases the production of insulin-like growth factor-I (IGF-I) in the mouse brain. IGF-I exerts beneficial effects on the cognitive function by increasing synaptic transmission and by inducing angiogenesis and neurogenesis in the hippocampus. In the current study, we examined whether stimulation of sensory neurons by capsaicin improved the cognitive function by increasing the production of IGF-I in the hippocampus using wild-type (WT) and CGRP-knockout (CGRP-/-) mice. Significant increases of the hippocampal tissue levels of CGRP, IGF-I, and IGF-I messenger RNA (mRNA) were observed after capsaicin administration in WT mice (P < 0.01) but not in CGRP-/- mice. Increase in the expression of c-fos was also observed in the spinal dorsal horn, the parabrachial nuclei, and the hippocampus after capsaicin administration in WT mice but not in CGRP-/- mice. Significant enhancement of angiogenesis and neurogenesis was observed in the dentate gyrus of the hippocampus after capsaicin administration in WT mice (P < 0.01) but not in CGRP-/- mice. Although capsaicin administration improved spatial learning in WT mice, no such effect was observed in CGRP-/- mice. Capsaicin-induced improvement of the spatial learning was reversed by administration of an anti-IGF-I antibody and by that of a CGRP receptor antagonist CGRP (8-37) in WT mice. The administration of IGF-I improved the spatial learning in both WT and CGRP-/- mice. These observations strongly suggest that the stimulation of sensory neurons by capsaicin might increase IGF-I production via increasing the hippocampal tissue CGRP levels, and it may thereby promote angiogenesis and neurogenesis to produce improvement of the cognitive function in mice.

Effects of feeding l‐carnitine to gilts through day 70 of gestation on litter traits and the expression of insulin‐like growth factor system components and l‐carnitine concentration in foetal tissues

We investigated the influence of supplemental L-carnitine on foetal blood metabolites, litter characteristics, L-carnitine concentration in skeletal muscle and insulin-like growth factor (IGF) axis components in foetal hepatic and skeletal muscle tissues at day 40, 55 and 70 of gestating gilts. A total of 59 gilts (body weight = 137.7 kg) received a constant feed allowance of 1.75 kg/day and a top-dress containing either 0 or 50 ppm of L-carnitine starting on the first day of breeding through the allotted gestation length. Foetuses from the gilts fed diets with L-carnitine tended to be heavier (p = 0.06) and the circulating IGF-II tended to be lower (p = 0.09) at day 70, compared with the foetuses from the control gilts. Insulin-like growth factor-I messenger RNA (mRNA) was lower (p = 0.05) in hepatic tissue in the foetuses collected from gilts fed L-carnitine. Free and total carnitine concentration increased (p < 0.05) in the skeletal muscle from the foetuses collected from gilts fed supplemental L-carnitine. This study showed that L-carnitine had beneficial effects on the average foetal weight at day 70 of gestation, associated with changes in the foetal IGF system.

Suppressor of cytokine signaling-2: A growth hormone-inducible inhibitor of intestinal epithelial cell proliferation

Background & Aims: Growth hormone (GH) and insulin-like growth factor-I (IGF-I) increase intestinal growth. GH is thought to act indirectly via IGF-I. In several models, including rats given total parenteral nutrition (TPN), IGF-I more potently stimulates mucosal growth than GH, even when GH induces similar circulating IGF-I levels. These studies test the hypothesis that GH induces a suppressor of cytokine signaling (SOCS), which inhibits intestinal epithelial cell (IEC) proliferation. Methods: Rats on TPN received vehicle, GH, or IGF-I. Jejunal SOCS (SOCS-1, -2, -3, and cytokine-inducible SH2-domain-containing protein [CIS]) and IGF-I messenger RNA (mRNA) were quantified. Caco-2, IEC-6 cells, and SOCS-2 null and wild-type (WT) mice were used to examine the expression and functional role of SOCS-2. Results: As reported previously, IGF-I, but not GH, prevented mucosal atrophy during TPN, although GH elevated plasma IGF-I and increased body weight. GH, but not IGF-I, induced jejunal SOCS-2 mRNA. SOCS-2 mRNA levels in GH and IGF-I-treated rats inversely correlated with mucosal weight. SOCS-2 is expressed in Caco-2 cells, and elevated SOCS-2 expression in postconfluent cells is associated with reduced proliferative rates. SOCS-2 overexpression in Caco-2 cells inhibited cell proliferation and promoted differentiation. In IEC-6 cells, GH induced SOCS-2 and reduced basal or IGF-I-induced proliferation. GH also reduced proliferative activity in isolated crypts from WT but not SOCS-2 null mice, and SOCS-2 null crypts showed enhanced proliferative responses to GH and IGF-I. SOCS-2 null mice have increased intestinal weight and length. Conclusions: SOCS-2 is a GH-inducible, novel inhibitor of intestinal epithelial cell proliferation and intestinal growth.

Effects of flax supplementation and a combined trenbolone acetate and estradiol implant on circulating insulin-like growth factor-I and muscle insulin-like growth factor-I messenger RNA levels in beef cattle.

We evaluated effects of a 5% (dry matter basis) ground flaxseed supplement (flax) and a trenbolone acetate and estradiol-17beta implant, Revalor-S, on circulating IGF-I and muscle IGF-I messenger RNA (mRNA). Sixteen crossbred yearling steers (initial BW = 397 kg) were assigned randomly to one of four treatments: 1) flax/implant; 2) nonflax/implant; 3) flax/nonimplant; and 4) nonflax/nonimplant. Serum was harvested from blood collected on d 0 (before implant or flax addition), 14, and 28, and used in subsequent analyses of circulating IGF-I. Biopsy samples (0.5 g) were obtained from the longissimus muscle on d 0, 14, and 28. Total RNA was isolated from the muscle samples, and real-time quantitative-PCR was used to assess relative differences in IGF-I mRNA. Flax supplementation had no effect (P > 0.10) on circulating IGF-I concentrations. Following implantation, sera from implanted steers had 52 and 84% greater (P < 0.05) IGF-I concentrations than sera from nonimplanted steers on d 14 and 28, respectively. On d 28, local muscle IGF-I mRNA levels increased 2.4-fold (P < 0.01) in biopsy samples obtained from implanted compared with nonimplanted steers. Muscle biopsy samples from nonflax cattle had 4.4-fold higher (P < 0.01) levels of IGF-I mRNA than those from flax cattle on d 28. To determine whether a component of flax, alpha-linolenic acid (alphaLA), was directly responsible for IGF-I mRNA down-regulation, we incubated primary cultures of bovine satellite cells, from implanted and nonimplanted steers, in two concentrations of alphaLA (10 nM and 1 microM). An implant x dose interaction (P < 0.05) was observed for IGF-I mRNA concentrations in bovine satellite cells cultured for 72 h with alphaLA. Satellite cells from nonimplanted steers had similar (P > 0.10) IGF-I mRNA concentration regardless of the level of alphaLA exposure; however, satellite cells from implanted steers exposed to 10 nM and 1 microM alphaLA had 2.5- and 2.0-fold greater IGF-I mRNA levels, respectively, than cells from implanted steers that were not exposed to alphaLA (P < 0.05). Administration of a Revalor-S implant increased circulating IGF-I and local muscle IGF-I mRNA concentrations in finishing cattle. However, muscle IGF-I mRNA levels were decreased by flax supplementation. Muscle cell culture experiments suggested that alphaLA was not responsible for the IGF-I mRNA down-regulation.

Insulin-like growth factor I is a comitogen for hepatocyte growth factor in a rat model of hepatocellular carcinoma

Hepatocyte growth factor–scatter factor (HGF-SF) is a potent hepatic mitogen yet inhibits hepatocellular carcinoma (HCC) cell growth in vitro. Insulin-like growth factor I (IGF-I) is a pleiotropic growth factor shown to be important in cell growth and differentiation in other tumors. We hypothesized that IGF-I may play a role in regulating HGF-SF activity and HCC progression. Using an in vivo model of HCC, we showed elevated IGF-I messenger RNA (mRNA) expression in normal liver from tumor-burdened animals in the absence of changes in circulating IGF-I levels. Analysis of IGF-I receptor (IGF-IR) and HGF-SF (c-met) receptor expression showed significantly higher expression of both receptors in normal liver compared with an HCC specimen. Using cultured HCC cells from this model, we next showed that treatment with IGF-I led to significant increases in mitogen-activated protein kinase (MAPK) activity. Furthermore, we observed significant time-dependent increases in the expression of the c-fos and c-jun proto-oncogenes after addition of IGF-I (n = 5 per group, P < .05). Despite activation of a MAPK pathway and increased proto-oncogene expression, IGF-I failed to significantly affect cell mitogenesis. In contrast, HGF significantly inhibited cell mitogenesis in HCC lines (68.4% ± 9.4% vs. control, n = 4, P < .05). Pretreatment of HCC cells with IGF-I (60 minutes) led to significant HGF-SF stimulation of total cell mitogenesis dependent on both IGF-I and HGF-SF dose (194% ± 8% increase vs. control, n = 4, P < .05). In conclusion, tumor burden is important in altering intrahepatic growth factor synthesis. Signal cooperation between multiple cytokine pathways is an important factor in the progression of HCC. (H EPATOLOGY 2002;36:1089-1097.)

Cyclic Adenosine 3',5'-Monophosphate Inhibits Insulin-Like Growth Factor I Gene Expression in Rat Glioma Cell Lines: Evidence for Regulation of Transcription and Messenger Ribonucleic Acid Stability

cAMP inhibits growth and stimulates differentiation in glioma cells. We examined the effect of cAMP on insulin-like growth factor I (IGF-I) gene expression in the C6 cell line, a rat glioma cell line previously reported to grow in response to autocrine IGF-I. cAMP potently inhibited IGF-I messenger RNA (mRNA) and peptide secretion in C6 cells, associated with an attenuation of DNA synthesis. Exogenous IGF-I peptide at least partially prevented the inhibition of DNA synthesis, suggesting that the reduction in IGF-I biosynthesis may contribute to the inhibitory effect of cAMP on C6 cell growth. cAMP also inhibited IGF-I mRNA in rat RG2 glioma cells, but not in three other nonglioma tumor cell lines. The nuclear IGF-I pre-mRNA level and the half-life of mature IGF-I mRNA were both reduced by cAMP in C6 cells, suggesting effects on gene transcription and mRNA stability. However, cAMP had no effect on the activities of IGF-I exon 1 promoter-luciferase constructs. Protein synthesis inhibition partially reduced the inhibition of IGF-I mRNA by cAMP. Inhibition of cAMP-activated protein kinase A activity by H89 did not alter the inhibition of IGF-I gene expression in response to cAMP, suggesting that protein kinase A does not mediate the cAMP inhibitory effect on IGF-I gene expression.

Cyclic adenosine 3',5'-monophosphate inhibits insulin-like growth factor I gene expression in rat glioma cell lines: evidence for regulation of transcription and messenger ribonucleic acid stability.

cAMP inhibits growth and stimulates differentiation in glioma cells. We examined the effect of cAMP on insulin-like growth factor I (IGF-I) gene expression in the C6 cell line, a rat glioma cell line previously reported to grow in response to autocrine IGF-I. cAMP potently inhibited IGF-I messenger RNA (mRNA) and peptide secretion in C6 cells, associated with an attenuation of DNA synthesis. Exogenous IGF-I peptide at least partially prevented the inhibition of DNA synthesis, suggesting that the reduction in IGF-I biosynthesis may contribute to the inhibitory effect of cAMP on C6 cell growth. cAMP also inhibited IGF-I mRNA in rat RG2 glioma cells, but not in three other nonglioma tumor cell lines. The nuclear IGF-I pre-mRNA level and the half-life of mature IGF-I mRNA were both reduced by cAMP in C6 cells, suggesting effects on gene transcription and mRNA stability. However, cAMP had no effect on the activities of IGF-I exon 1 promoter-luciferase constructs. Protein synthesis inhibition partially reduced the inhibition of IGF-I mRNA by cAMP. Inhibition of cAMP-activated protein kinase A activity by H89 did not alter the inhibition of IGF-I gene expression in response to cAMP, suggesting that protein kinase A does not mediate the cAMP inhibitory effect on IGF-I gene expression.

The proliferative and antiapoptotic actions of growth hormone and insulin-like growth factor-1 are mediated through distinct signaling pathways in the Pro-B Ba/F3 cell line.

Biological actions of GH can be direct or mediated through insulin-like growth factor I (IGF-I). In the interleukin-3 (IL-3)-dependent Ba/F3 cell line, IGF-I induces cell cycle entry and proliferation. Ba/F3 cells expressing the rat GH receptor (Ba/F3 GHR cells) have been shown to escape from apoptosis and to proliferate under GH stimulation. Using the Ba/F3 GHR cell model, we sought to dissect the signals elicited specifically by IGF-I or GH. In contrast to IGF-I or IL-3, GH is able to maintain cell cycle entry of Ba/F3 GHR cells cultured for 7 days in the absence of serum. The presence of IGF-I messenger RNA was not detected by RT-PCR, and by RIA, IGF-I was not found in culture medium of Ba/F3 GHR cells, unstimulated or stimulated by GH. Moreover, the addition of an anti-IGF-I antibody that blocks IGF-I effects suggests that the actions of GH are not mediated by IGF-I, but appear to be direct. GH or IGF-I stimulation increased expression of cyclins A and D(1) with comparable kinetics, whereas expression of p21(waf1/cip1) seemed delayed in IGF-I-stimulated cells compared with that in GH-stimulated cells. Contrary to GH or IL-3, IGF-I did not induce nuclear factor-kappaB DNA-binding activity in Ba/F3 cells. Inhibition of nuclear factor-kappaB through expression of the mutant IkappaBalpha (A32/36) abrogated the GH-mediated survival signal, but did not result in alterations of the cell cycle in Ba/F3 GHR cells treated with IGF-I. Phosphatidylinositol 3-kinase was required for both survival and proliferative responses to IGF-I. Transfection of a dominant negative form of AKT (AH-AKT) resulted in suppression of IGF-I-mediated cell survival, but not of the antiapoptotic effect of GH in Ba/F3 GHR cells. Thus, GH and IGF-I are able to promote cell survival and proliferation through independent and different pathways in Ba/F3 cells.

Angiotensin II induces skeletal muscle wasting through enhanced protein degradation and down-regulates autocrine insulin-like growth factor I.

We previously showed that angiotensin II (ang II) infusion in the rat produces cachexia and decreases circulating insulin-like growth factor I (IGF-I). The weight loss derives from an anorexigenic response and a catabolic effect of ang II. In these experiments we assessed potential catabolic mechanisms and the involvement of the IGF-I system in these responses to ang II. Ang II infusion caused a significant decrease in body weight compared with that of pair-fed control rats. Kidney and left ventricular weights were significantly increased by ang II, whereas fat tissue was unchanged. Skeletal muscle mass was significantly decreased in the ang II-infused rats, and a reduction in lean muscle mass was a major reason for their overall loss of body weight. In skeletal muscles, ang II did not significantly decrease protein synthesis, but overall protein breakdown was accelerated; inhibiting lysosomal and calcium-activated proteases did not reduce the ang II-induced increase in muscle proteolysis. Circulating IGF-I levels were 33% lower in ang II rats vs. control rats, and this difference was reflected in lower IGF-I messenger RNA levels in the liver. Moreover, IGF-I, IGF-binding protein-3, and IGF-binding protein-5 messenger RNAs in the gastrocnemius were significantly reduced. To investigate whether the reduced circulating IGF-I accounts for the loss in muscle mass, we increased circulating IGF-I by coinfusing ang II and IGF-I, but this did not prevent muscle loss. Our data suggest that ang II causes a loss in skeletal muscle mass by enhancing protein degradation probably via its inhibitory effect on the autocrine IGF-I system.

Quantitative TaqMan PCR without a real-time thermal cycler: an assay for fish insulin-like growth factor I messenger RNA.

The reverse transcriptase-polymerase chain reaction, more commonly known as RT-PCR, has become a widely used tool in molecular biology and is now frequently used in monitoring gene expression levels. A number of variations in the RT-PCR technique now exist including TaqMan PCR (5' nuclease assay), which is a useful nonisotopic detection method for the quantification of PCR products. To monitor the formation of these fluorescent amplification products a "real-time" thermal cycler is normally required. In this study, repeated scanning of PCR products in a 96-well plate format showed that a conventional fluorescent plate reader can be used to generate similar results. To demonstrate the power of this approach, the nutritional regulation of insulin-like growth factor I (IGF-I) was investigated in a marine finfish, the snapper (Pagrus auratus). Hepatic IGF-I messenger RNA levels were shown to significantly decrease after 2 weeks of fasting and returned to fed control levels on refeeding. These results demonstrated that a real-time PCR machine was not required to generate this type of quantitative data and that this technology can be adapted for use in most molecular biology laboratories.

Double-Stranded Ribonucleic Acid Decreases C6 Rat Glioma Cell Numbers: Effects on Insulin-Like Growth Factor I Gene Expression and Action

Poly(IC), a synthetic double-stranded RNA copolymer of inosinic and cytidilic acids, decreases the growth of normal and tumorigenic cells. We tested the hypothesis that Poly(IC) decreases C6 glioma cell growth by disrupting an autocrine insulin-like growth factor I (IGF-I) growth loop. Addition of Poly(IC) decreased C6 cell number in confluent and sparse cultures in a dose-dependent manner. Addition of exogenous IGF-I partially compensated for the decrease in cell number caused by Poly(IC) in confluent and subconfluent cultures of C6 cells, suggesting that one mechanism of Poly(IC) action is through down-regulation of IGF-I gene expression and/or action. Treatment of confluent C6 cells with 10 and 200 μg/ml Poly(IC) for 24 h decreased IGF-I messenger RNA (mRNA) levels to 50% and 25% of the control value, respectively. Treatment of C6 cells with 200 μg/ml Poly(IC) for 24 h reduced IGF-I receptor mRNA levels to 50% of the control level. IGF-binding protein-1 (IGFBP-1), -2, and -6 mRNAs were not expressed in the C6 cells used in this study. Treatment of C6 cells with 200 μg/ml Poly(IC) for 24 h reduced IGFBP-4 mRNA and IGFBP-5 mRNA levels to 26% and 29% of the control level, respectively. There was no significant change in IGFBP-3, insulin receptor, or actin mRNA levels with Poly(IC) treatment. Treatment of confluent C6 cells with 200 μg/ml Poly(IC) for 24 h decreased levels of immunoreactive IGF-I in conditioned medium (CM) to 55% of the control value, decreased IGF-I receptor β-subunit levels to 28% of the control value, and decreased levels of IGFBP-3, IGFBP-4, and IGFBP-5 protein in CM to 45%, 50%, and 30% of the control values, respectively. There was no significant change in actin and tubulin protein levels with Poly(IC) treatment. These results suggest that IGF-I gene expression is down-regulated by Poly(IC) treatment and that IGF-I bioavailability and action in C6 cells are also altered due to decreases in IGF-I receptor and binding protein levels.

Double-stranded ribonucleic acid decreases C6 rat glioma cell numbers: effects on insulin-like growth factor I gene expression and action.

Poly(IC), a synthetic double-stranded RNA copolymer of inosinic and cytidilic acids, decreases the growth of normal and tumorigenic cells. We tested the hypothesis that Poly(IC) decreases C6 glioma cell growth by disrupting an autocrine insulin-like growth factor I (IGF-I) growth loop. Addition of Poly(IC) decreased C6 cell number in confluent and sparse cultures in a dose-dependent manner. Addition of exogenous IGF-I partially compensated for the decrease in cell number caused by Poly(IC) in confluent and subconfluent cultures of C6 cells, suggesting that one mechanism of Poly(IC) action is through down-regulation of IGF-I gene expression and/or action. Treatment of confluent C6 cells with 10 and 200 microg/ml Poly(IC) for 24 h decreased IGF-I messenger RNA (mRNA) levels to 50% and 25% of the control value, respectively. Treatment of C6 cells with 200 microg/ml Poly(IC) for 24 h reduced IGF-I receptor mRNA levels to 50% of the control level. IGF-binding protein-1 (IGFBP-1), -2, and -6 mRNAs were not expressed in the C6 cells used in this study. Treatment of C6 cells with 200 microg/ml Poly(IC) for 24 h reduced IGFBP-4 mRNA and IGFBP-5 mRNA levels to 26% and 29% of the control level, respectively. There was no significant change in IGFBP-3, insulin receptor, or actin mRNA levels with Poly(IC) treatment. Treatment of confluent C6 cells with 200 microg/ml Poly(IC) for 24 h decreased levels of immunoreactive IGF-I in conditioned medium (CM) to 55% of the control value, decreased IGF-I receptor beta-subunit levels to 28% of the control value, and decreased levels of IGFBP-3, IGFBP-4, and IGFBP-5 protein in CM to 45%, 50%, and 30% of the control values, respectively. There was no significant change in actin and tubulin protein levels with Poly(IC) treatment. These results suggest that IGF-I gene expression is down-regulated by Poly(IC) treatment and that IGF-I bioavailability and action in C6 cells are also altered due to decreases in IGF-I receptor and binding protein levels.

Activation of a uterine insulin-like growth factor I signaling pathway by clinical and environmental estrogens: requirement of estrogen receptor-alpha.

Recent data indicate that insulin-like growth factor I (IGF-I) may have a function in mediating the mitogenic effects of 17beta-estradiol (E2) in the uterus and in regulating the growth of uterine neoplasms. This study was designed to determine whether synthetic and plant-derived chemicals that interact with estrogen receptor-alpha (ERalpha) and elicit estrogenic responses also mimic E2 by activating the uterine IGF-I signaling pathway. Ovariectomized adult female mice were treated with both environmental and clinically relevant chemicals previously reported to display estrogenic and/or antiestrogenic properties, and their uteri were evaluated for an activated IGF-I signaling pathway. Diethylstilbestrol, 4-hydroxytamoxifen, the raloxifene analog LY353381, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), bisphenol A, and genistein were shown to mimic E2 in the uterus by increasing the level of IGF-I messenger RNA, inducing IGF-I receptor (IGF-IR) tyrosine phosphorylation, stimulating the formation of IGF-IR signaling complexes, and increasing both proliferating cell nuclear antigen expression and the number of mitotic cells in the epithelium. The dose of chemical necessary to activate IGF-I signaling varied, with the order of potency: E2 = diethylstilbestrol > LY353381 > 4-hydroxytamoxifen > genistein > HPTE > bisphenol A. Administration of the chemicals to ERalpha knockout mice did not activate IGF-IR, indicating that ERalpha is required for activation of uterine IGF-IR by these diverse chemicals. This study demonstrates that several chemicals shown previously to display estrogenic activities also mimic E2 by activating uterine IGF-I signaling.

Cell density influences insulin-like growth factor I gene expression in a cell type-specific manner.

The effect of cellular density on insulin-like growth factor I (IGF-I) gene expression was characterized in several tumor-derived cell lines. IGF-I messenger RNA (mRNA) transcripts increased more than 200-fold when C6 glioma cells grew to postconfluence. IGF-I receptor and beta-actin mRNAs were induced by 6- and 2-fold, respectively, as a function of confluence. IGF-I mRNA transcripts in GH3 and SK-N-MC cells increased about 4- to 5-fold in confluent cultures compared with sparse cultures. In OVCAR-3 cells, the IGF-I mRNA level remained constant as the cell density increased. Transient transfection experiments were performed with IGF-I exon 1 promoter/luciferase fusion constructs in C6 cells. The luciferase activity of a construct containing exon 1 sequence between +75 and +282 (the most 5' transcription initiation site was designated +1) was stimulated by 2.5-fold in dense cultures compared with that in sparse cultures of C6 cells. Luciferase activities of other constructs containing at least 282 bp of exon 1 sequence were also stimulated about 2- to 4-fold by cell density. However, 3' deletion to +192 led to loss of the cell density stimulatory effect. In contrast, luciferase activities of IGF-I promoter constructs were not altered by cell density in SK-N-MC cells. When the conditioned medium of low density C6 cultures was exchanged with that of high density cultures, the IGF-I mRNA level remained the same. In summary, cell density has a cell type- and gene type-specific effect on IGF-I gene expression. A cell density response element(s) may be located between +192 and +282 of the exon 1 promoter region in C6 cells.

Insulin-like growth factor–I in women with hydatidiform mole and in normal pregnancy

Objective: To compare molar and placental tissue insulin-like growth factor–I (IGF-I) levels and IGF-I messenger RNA (mRNA) expression and maternal serum IGF-I levels in molar and normal pregnancies of the same gestational length. Methods: Tissue and serum total IGF-I were measured by radioimmunoassay, and tissue IGF-I mRNA expression was measured by a solution hybridization technique in samples from seven patients with hydatidiform mole in gestational weeks 8 through 14 and from 12 women with normal placentas undergoing induced abortion during weeks 8 through 12. Results: Tissue and serum total IGF-I levels and tissue IGF-I mRNA expression were significantly lower in molar pregnancies than in pregnancies with normal placenta. Conclusion: The lower levels of both IGF-I mRNA and of the IGF-I protein itself in hydatidiform mole indicates that the regulation of expression of IGF-I is on the transcriptional level. This finding may reflect a decreased production of the placental growth hormone variant in molar tissue.

In vivo anabolic effects of parathyroid hormone (PTH) 28-48 and N-terminal fragments of PTH and PTH-related protein on neonatal mouse bones.

We developed a neonatal mouse model to investigate in vivo anabolic effects of intact PTH (1-84) and its two fragments PTH (1-34) and PTH (28-48) and of the N-terminal fragment of PTH-related peptide [PTHrP (1-34)]. Two-day-old mice were injected with low-dose (0.05 microg/g body weight) and high-dose (0.2 microg/g body weight) of each of these peptides daily for 6 or 16 consecutive days. Long bones (tibias and femurs) and mandibular condylar cartilages were harvested. Total DNA and protein were analyzed as parameters for anabolic effects. DNA was increased significantly in tibias only by low doses of PTH (1-84) and PTH (1-34), but by both doses of PTH (28-48). In the cartilages of the mandibular condyles, both doses of all three peptides increased DNA. Total protein was increased in the tibia by the low dose of the three peptides, whereas in the condylar cartilage high doses of PTH (1-34) and PTH (28-48) also caused a 2- to 4-fold increase. When the effects of PTH (1-34) and PTHrP (1-34) on the tibias were compared, it became apparent that PTH (1-34) was more effective than PTHrP (1-34) when injected in low doses, but the latter caused a severalfold increase in DNA and protein at both doses. The outstanding anabolic effect of PTH (28-48) was further investigated using [3H]thymidine autoradiography, analysis of insulin-like growth factor I (IGF-I) protein, and localization of IGF-I messenger RNA (mRNA) by in situ hybridization. PTH (28-48) increased by 3-fold the number of [3H]thymidine-labeled cells in the epiphyseal cartilage of tibias removed from 8-day-old injected mice, and in the proliferative zone of the epiphyseal growth plate of tibias removed from 18-day-old injected mice. Femurs from the latter showed a 20% increase in their IGF-I content. In parallel, only tibias from 18-day-old injected mice showed IGF-I mRNA localization in proliferating chondrocytes, whereas those from vehicle-injected control mice did not exhibit IGF-I mRNA. In summary, our study showed that the neonatal mouse is a sensitive model to examine anabolic effects of different PTH and PTHrP fragments. It also revealed that PTH (28-48) has strong anabolic effects on this model, and suggests that IGF-I might mediate the anabolic effects of PTH (28-48).