Insulin-like Growth factor-I mRNA Abundance Research Articles

Glucocorticoid receptor-mediated insulin-like growth factor-I transcriptional regulation in BeWo trophoblast cells before and after syncytialisation

Prenatal exposure to excessive glucocorticoids (GCs) leads to intrauterine growth retardation and fetal programming of adult health and disease through deregulation of placental functions. Placental secretion of insulin-like growth factor-I (IGF-I) plays a critical role in the regulation of placental development and function. However, it remains elusive whether GCs affect placental functions through glucocorticoid receptor (GR)-mediated transcriptional regulation of IGF-I gene. In this study, human placental choriocarcinoma (BeWo) cells before and after syncytialization were used as cytotrophoblast and syncytiotrophoblast models, respectively, to explore the effects of dexamethasone (Dex) on transcriptional regulation of IGF-I gene at both stages. Dex significantly inhibited (P<0.05) cell proliferation in cytotrophoblasts and down-regulated amino acid transporter SLC7A5 in syncytiotrophoblasts. Concurrently, the abundance of IGF-I mRNA and its transcript variants, together with IGF-I level in culture media, were significantly reduced, in association with significantly enhanced (P<0.05) GR phosphorylation. GR antagonist RU486 was able to abolish all these effects. Two glucocorticoid response elements (GREs) were predicted in the promoter regions of IGF-I gene. GR binding to GRE1 was significantly enriched (P<0.05) in both cytotrophoblasts and syncytiotrophoblasts, but that to GRE2 was significantly diminished (P<0.05) in cytotrophoblasts but not in syncytiotrophoblasts, in response to Dex treatment. IGF-I supplementation completely rescued Dex-induced cell cycle arrest but not SLC7A5 down-regulation, indicating different regulatory mechanisms. Taken together, our results suggest that GR-mediated transcriptional regulation of IGF-I is involved in Dex-induced inhibition of placental cell proliferation and function.

Developmental changes of &amp;lt;i&amp;gt;GHR&amp;lt;/i&amp;gt; and &amp;lt;i&amp;gt;IGF-I&amp;lt;/i&amp;gt; mRNA expression in lamb rumen

Abstract. Lambs from birth (0-day-old) to 56-day-old were selected in present study to investigate developmental changes of growth hormone receptor (GHR) and insulin-like growth factor I (IGF-I) mRNA expression in their rumen tissue. Forty-five lambs (5 lambs per group) were slaughtered at 0, 7, 14, 21, 28, 35, 42, 49, 56 days of age respectively for sampling the tissue of the rumen dorsal sac. The abundance of GHR and IGF-I mRNA were detected through real-time quantitative PCR method. The results indicated that the expression levels of GHR and IGF-I mRNA had similar change tendency in rumen tissue that the GHR and IGF-I mRNA abundance decreased with age from birth to 56-day-old. There was significant positive correlation between the two gene mRNA expression levels. The results suggested that GHR and IGF-I gene expression levels had the specific developmental pattern in rumen tissue.

Genetic merit for fertility traits in Holstein cows: III. Hepatic expression of somatotropic axis genes during pregnancy and lactation

The objective of this study was to characterize the circulating concentrations of insulin-like growth factor-I (IGF-I) and the hepatic expression of key genes regulating the somatotropic axis in cows divergent in genetic merit for fertility traits but with similar genetic merit for milk production traits. A total of 11 cows with good genetic merit for fertility (Fert+) and 12 cows with poor genetic merit for fertility (Fert−) underwent liver biopsy by percutaneous punch technique on d 20 (±6.7 d) prepartum and on d 2 (±1.5 d), d 58 (±3.7 d), d 145 (±13 d), and d 245 (±17.1 d) postpartum. Total RNA was isolated and the mRNA expression of growth hormone receptor (GHR 1A and GHRtot), IGF-I, janus tyrosine kinase 2 (JAK2), signal transducer and activator of transcription 5B (STAT5B), suppressor of cytokine signaling 3 (SOCS-3), acid-labile subunit (ALS), and IGF-binding proteins (IGFBP1 to IGFBP6) were measured by real-time quantitative PCR. During lactation, the circulating concentrations of IGF-I were 34% greater in Fert+ cows. The Fert+ cows had increased mean expression of IGF-I mRNA during the study; however, the difference in IGF-I mRNA abundance between Fert+ and Fert− cows was most pronounced at d 145 and 245. The expression of IGFBP3 and ALS transcript was similar in Fert+ and Fert− cows for the duration of the study. The Fert− cows, however, had greater expression of IGFBP2, IGFBP4, IGFBP5, and IGFBP6. Genotype had no effect on mRNA abundance of GHR 1A, STAT5B, JAK2, or SOCS-3. Genetic merit for fertility traits affects hepatic expression of key genes of the somatotropic axis regulating the synthesis, bioavailability, and stability of circulating IGF-I.

Effects of Dietary Lysine on Growth Performance, Serum Concentrations of Insulin-Like Growth Factor-I (IGF-I) and IGF-I mRNA Expression in Growing Rabbits

The effects of dietary lysine on production performance, serum concentrations of metabolites, growth hormone (GH), insulin-like growth factor-I (IGF-I) and IGF-I mRNA expression in growing rabbits were examined. One hundred weaned New Zealand rabbits were allocated to individual cages and randomly offered a diet containing 5.5 (L1), 6.5 (L2), 7.5 (L3), 8.5 (L4), or 9.5 g (L5) lysine per kg diet. The results showed that the average daily gain (ADG) of the rabbits from L3, L4 or L5 was higher than those from L1 or L2 ( P < 0.05). The feed gain ratio (F/G) in the rabbits from L4 or L5 was lower than those from L1 or L2 ( P < 0.05). Dietary lysine did not affect serum concentrations of total protein (TP), glucose, insulin (INS), and growth hormone (GH) ( P > 0.05). The quadratic effects of lysine on the serum urea nitrogen (SUN) concentration was detected ( P = 0.035). Serum IGF-I concentrations had a trend to increase quadratically with the increasing dietary lysine ( P = 0.07). A significant correlation was found between serum IGF-I concentrations ( x, ng mL −1) and ADG ( y, g kg −1): y = −0.017 x 2 + 1.984 x + 20.87 ( R 2 = 0.8982, P = 0.003). The relative abundance of hepatic and muscular IGF-I mRNA tended to increase with increasing dietary lysine levels ( P = 0.053 and 0.082, respectively). Providing the diets mainly consisted of corn, wheat bran and peanut vine, the most appropriate dietary lysine level for growing meat rabbits from weaning to 70 d old was found to be 8.5 g kg −1, and IGF-I may be an important factor controlling growth of weaned rabbits.

Increased degradation of insulin-like growth factor-I in serum from feed-deprived steers

Severe feed restriction decreases serum insulin-like growth factor I (IGF-I) concentration in animals, and this decrease is thought to be due to reduced IGF-I production in the liver. The objective of this study was to determine whether feed deprivation also increases degradation of serum IGF-I and serum levels of IGF binding protein 3 (IGFBP-3) and acid-labile subunit (ALS), which inhibit IGF-I degradation and increase IGF-I retention in the blood by forming a ternary complex with IGF-I, in cattle. Five steers had free access to pasture, and another five were deprived of feed for 60 h. Serum concentration of IGF-I and liver abundance of IGF-I mRNA at the end of the 60-h period were 50% and 80% lower, respectively, in feed-deprived steers than in fed steers. Less 125I-labeled IGF-I remained intact after a 45-h incubation in sera of feed-deprived steers than in sera of fed steers, suggesting that serum IGF-I is more quickly degraded in feed-deprived animals. Serum levels of IGFBP-3 and ALS were decreased by 40% and 30%, respectively, in feed-deprived steers compared with fed steers. These decreases were associated with more than 50% reductions in IGFBP-3 and ALS mRNA in the liver, the major source of serum IGFBP-3 and ALS. Taken together, these results suggest that feed deprivation reduces serum concentration of IGF-I in cattle not only by decreasing IGF-I gene expression in the liver, but also by increasing IGF-I degradation and reducing IGF-I retention in the blood through decreasing IGFBP-3 and ALS production in the liver.

Acute growth hormone administration induces antidiuretic and antinatriuretic effects and increases phosphorylation of NKCC2

Growth hormone (GH) has antidiuretic and antinatriuretic effects in rats and humans, but the molecular mechanisms responsible for these effects are unknown. The aim of this study was to investigate the mechanisms behind the acute renal effects of GH in rats. Female rats received rat (r)GH (2.8 mg/kg sc) or saline and were placed in metabolic cages for 5 h. Urinary excretion of electrolytes and urinary volume were reduced after rGH injection, while urine osmolality was increased. Creatinine and lithium clearance remained unchanged, suggesting that rGH increases reabsorption in segments distal to the proximal tubule. Total plasma insulin-like growth factor I (IGF-I) levels did not change, while cortical IGF-I mRNA abundance was increased. The relative abundance of total and Ser(256)-phosphorylated aquaporin 2 was found to be unchanged by immunoblotting, whereas a significant increase of Thr(96) and Thr(101)-phosphorylated NKCC2 (renal Na(+), K(+), 2Cl(-) cotransporter) was found in the inner stripe of outer medulla thick ascending limbs (mTAL). Additionally, an increased NKCC2 expression was observed in the cortical region. Immunohistochemistry confirmed these findings. The density of NKCC2 molecules in the apical membrane of mTAL cells appeared to be unchanged after rGH injection evaluated by immunoelectron microscopy. Basolateral addition of rGH or IGF-I to microperfused rat mTAL segments did not change transepithelial voltage. In conclusion, GH appears to exert its acute antinatriuretic and antidiuretic effects through indirect activation of NKCC2 in the mTAL.

Temperature affects insulin-like growth factor I and growth of juvenile southern flounder, Paralichthys lethostigma

Temperature profoundly influences growth of heterothermic vertebrates. However, few studies have investigated the effects of temperature on growth and insulin-like growth factor I (IGF-I) in fishes. The aim of this study was to examine effects of temperature on growth and establish whether IGF-I may mediate growth at different temperatures in southern flounder, Paralichthys lethostigma. In two experiments, juvenile flounder were reared at 23 and 28 degrees C and growth was monitored for either 117 or 197 days. Growth was similar across treatments in both experiments until fish reached approximately 100 mm total length. Body size then diverged with fish at 23 degrees C ultimately growing 65-83% larger than those at 28 degrees C. Muscle IGF-I mRNA, plasma IGF-I, and hepatosomatic index (HSI) were significantly higher in flounder at 23 degrees C, whereas hepatic IGF-I mRNA abundance did not differ with treatment. Muscle IGF-I mRNA was correlated with HSI, while plasma IGF-I was correlated with body size, hepatic IGF-I mRNA, and HSI. These results demonstrate a strong effect of temperature on flounder growth and show that temperature-induced variation in growth is associated with differences in systemic IGF-I and local (i.e., muscle) IGF-I mRNA levels. The results also support the use of plasma IGF-I and HSI as indicators of flounder growth status.

Genetic Increase in Serum Insulin-Like Growth Factor-I (IGF-I) in C3H/HeJ Compared with C57BL/6J Mice Is Associated with Increased Transcription from the IGF-I Exon 2 Promoter

C3H/HeJ (C3H) mice exhibit 30-40% higher serum IGF-I than do C57BL/6J (B6) mice, in association with increased bone mineral density and strength. These differences are inherited and thus provide a model for determining molecular mechanisms for genetic variation of serum IGF-I and downstream actions. We now report that increased serum IGF-I in C3H mice is associated with increased transcription from the minor exon 2 promoter in liver from female and male mice. The increase in hepatic IGF-I gene expression caused by increased abundance of IGF-I mRNA transcribed from the exon 2 promoter can quantitatively account for the increased serum IGF-I in C3H mice. Also, levels of both Ea and Eb IGF-I mRNAs are increased in livers of male C3H mice. Fasting lowered serum IGF-I and liver IGF-I mRNA levels in female mice of both strains. However, serum IGF-I and liver IGF-I mRNA levels remained higher in fasted C3H mice compared with fasted B6 mice. Levels of IGF-I transcripts initiated from exon 2 are also significantly increased in skeletal muscle, fat, ovaries, and kidneys of C3H mice. IGF binding protein (IGFBP)-5 mRNA levels are significantly higher in muscle and fat of C3H mice than in B6 mice. Levels of exon 1-containing transcripts are increased in whole femurs of male and female C3H mice. We conclude that increased transcription of the IGF-I gene occurs in a promoter- and tissue-specific manner in C3H mice. The increased IGF binding protein-5 mRNA levels in fat and muscle suggest that IGF-I signaling is increased in these tissues in C3H mice.

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.

Effect of dietary supplementation of chitosan and galacto-mannan-oligosaccharide on serum parameters and the insulin-like growth factor-I mRNA expression in early-weaned piglets

The study was to determine effects of dietary supplementation of chitosan (COS) and galacto-mannan-oligosaccharids (GMOS) on some serum biochemical indices, serum growth hormone (GH) and insulin-like growth factor-I (IGF-I) levels, and hepatic and long gissimus muscle IGF-I mRNA expression in early-weaned piglets. Twenty six Duroc × Landrace × Yorkshire piglets at the age of 15 days were used. The piglets had access to creep feed during the suckling. Six piglets were sacrificed for sampling at the beginning of the study. The other 20 piglets were individually housed in metabolic cages and randomly allotted to four corn and soybean meal-based diets including the control group, the antibiotic group with 110 mg lincomycin/kg diet, the COS group containing 0.025% COS, and the GMOS group with 0.20% GMOS, respectively, in a 2-week feeding experiment. Blood urea nitrogen (BUN) level was reduced whereas serum total protein concentration was increased ( P < 0.05) in responses to the COS and GMOS supplementation. Dietary supplementation of COS and GMOS also increased ( P < 0.05) the serum GH and IGF-I levels along with enhanced hepatic and the muscle IGF-I mRNA abundance. Dietary supplementation of oligosaccharides such as COS and GMOS may improve growth and feed conversion efficiency by increasing plasma GH and IGF-I levels, in the early-weaned piglets.

Differential Regulation of Porcine Hepatic IGF-I mRNA Expression and Plasma IGF-I Concentration by a Low Lysine Diet

The influence of dietary lysine on hepatic insulin-like growth factor-I (IGF-I) gene expression and plasma IGF-I level was investigated. Two male 6-wk-old pigs from each of six litters were used. Each littermate was assigned to one of two diets, control or low lysine (LL), that were isoenergetic and similar in protein content and provided 14.3 MJ digestible energy/kg for both diets, 185 g protein/kg for the control diet and 180 g protein/kg for the LL diet. The control diet contained all essential amino acids in the recommended amounts, including 11.5 g lysine/kg. The LL diet was similar but contained only 7 g lysine/kg. Pigs were pair-fed these diets for 3 wk. Growth rates and feed efficiencies of pigs fed the LL diet were significantly lower than those of pigs fed the control diet (P < 0.01). Plasma IGF-I levels in pigs fed the LL diet were 52% lower than in those fed the control diet (P < 0.01), and the LL group also had lower plasma IGF-binding protein-3 (IGFBP3) levels (P < 0.05). Despite the strikingly lower plasma IGF-I in pigs fed the LL diet, hepatic IGF-I mRNA abundance did not differ between the two treatment groups. We conclude that the reduction in plasma IGF-I caused by reduced dietary lysine may have been due in part to suppression of post-transcriptional events in IGF-I expression. The lower plasma IGFBP3 in pigs fed the LL diet suggests that increased clearance rates of circulating IGF-I may have been involved in this response.

Chronic salinity adaptation modulates hepatic heat shock protein and insulin-like growth factor I expression in black sea bream.

Black sea bream ( Mylio macrocephalus) hepatic heat shock proteins hsp90, hsp70, and hsp60 were found to be thermally and reversibly inducible as they were elevated 2.0, 3.2, and 2.1 fold, respectively, on acute heat shock and returned to pre-heat-shock levels after a 40-hour recovery period. To establish whether salinity plays a role in regulating heat shock protein (hsp) and insulin-like growth factor-I (IGF-I) expression in a euryhaline marine fish, we adapted groups of juvenile black sea bream to salinities of 50 ppt (hypersaline), 33 ppt (seawater), 12 ppt (isoosmotic), and 6 ppt (hypoosmotic) for 8 months. The lowest levels of hsps were found in fish reared in an isoosmotic salinity and the highest in those adapted to hypersaline and hypoosmotic salinities. Hepatic beta-actin messenger RNA abundance remained unchanged in all groups during salinity adaptation, whereas IGF-I mRNA abundance was highest in isoosmotic adapted black sea bream. This study is the first report of an effect of salinity ranging from hypersaline to hypoosmotic on the expression of different hsp forms and IGF-I in fish, and the possible relationship between environmental salinity, hepatic IGF-I expression, and hsp regulation is discussed.

Nutritional regulation of visceral markers in rat liver and cultured hepatoma cells.

Protein malnutrition in humans and other animals is consistently associated with a decreased concentration of circulating serum albumin, transthyretin (TTR), and insulin-like growth factor-I (IGF-I). The molecular mechanisms for regulation of the three polypeptides by dietary protein remain to be completely elucidated. The abundance of albumin, TTR and IGF-I mRNA is decreased in liver of juvenile rats consuming insufficient amounts of protein. Moreover, protein restriction specifically decreases the abundance of albumin and TTR nuclear transcripts, indicating that the reduction in mRNA levels for these two genes is caused at least partly by a decrease in gene transcription. Expression of several other genes transcribed at a high level in the liver is also decreased under conditions of dietary protein restriction, suggesting that the level/functional activity of liver-enriched transcription factor(s) might be decreased under these conditions. Limitation of cultured hepatoma cells for a single amino acid also selectively decreases the mRNA levels of several genes with liver-enriched expression, including albumin and TTR. The decrease in albumin mRNA is caused partly by decreased albumin gene transcription and partly by destabilization of albumin mRNA.

Insulin-like growth factor-I gene expression patterns during spontaneous repair of acute articular cartilage injury

This study evaluated the constitutive insulin-like growth factor-I (IGF-I) gene expression pattern in spontaneously healing cartilage defects over the course of 16 weeks, and correlated the tissue morphology and matrix gene expression with IGF-I mRNA levels. Full-thickness 15 mm cartilage defects were debrided in the femoral trochlea of both femoropatellar joints of 8 horses and the healing defects examined 2, 4, 8, or 16 weeks after surgery. Samples were harvested for histologic assessment of tissue healing using H&E staining, toluidine blue histochemical reaction for proteoglycan deposition, and in situ hybridization and immunohistochemistry procedures to demonstrate collagen type II mRNA and protein expression. Total RNA was isolated for Northern analysis to measure cartilage matrix molecule expression, and for semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) to determine IGF-I gene expression patterns in healing cartilage defects. Full-thickness cartilage defects in horses were slow to heal compared to smaller lesions in similar locations in other animals. However, a progressive decline in tissue cellularity and vascularity, and increased tissue organization were observed on H&E stained specimens over the 16-week experiment. Evidence of early chondrogenic repair was detected through collagen type II in situ hybridization and immunohistochemistry. However, levels of collagen type II and aggrecan mRNA in lesions were not abundant on Northern analysis indicating incomplete chondrogenesis. IGF-I message expression followed a cyclic pattern with low levels at 2 weeks, followed by an increase at 4 and 8 weeks, and a subsequent decline at 16 weeks. There was no direct correlation between the stage of healing and cartilage matrix message expression, and the abundance of IGF-I mRNA in the healing lesions. In conclusion, this study demonstrated that the spontaneous healing of articular defects was accompanied by a temporal fluctuation in IGF-I gene expression which was discoordinate to the steady rise in expression of cartilage matrix molecules such as procollagen type II.

Control of ovine hepatic growth hormone receptor and insulin-like growth factor I by thyroid hormones in utero.

By use of RNase protection assays, hepatic growth hormone receptor (GHR) and insulin-like growth factor I (IGF-I) mRNA abundances were measured in sheep fetuses after experimental manipulation of fetal plasma thyroid hormone concentrations by fetal thyroidectomy (TX) and exogenous infusion of triiodothyronine (T(3)) and cortisol. TX abolished the normal prepartum rise in hepatic GHR abundance but had little effect on hepatic GHR gene expression at 127-130 days (term 145 +/- 2 days). By contrast, it upregulated basal IGF-I expression in immature fetal liver by increasing both Class 1 and Class 2 transcript abundance but had no further effects on IGF-I gene mRNA levels at 142-145 days. Raising plasma T(3) to prepartum values by exogenous infusion of either T(3) or cortisol into immature intact fetuses prematurely raised hepatic GHR and IGF-I mRNA abundances to values similar to those seen in intact fetuses at 142-145 days. In TX fetuses, cortisol infusion increased hepatic GHR mRNA but not total IGF-I mRNA abundance at 127-130 days. These findings show that thyroid hormones have an important role in the regulation of hepatic GHR and IGF-I gene expression in fetal sheep during late gestation and suggest that T(3) mediates the maturational effects of cortisol on the hepatic somatotropic axis close to term.

Effects of dietary protein and tumor necrosis factor on components of the insulin-like growth factor-I pathway in the colon and small intestine in protein-depleted rats

Intestinal cell growth is markedly affected by nutrient intake and the presence of cytokines. Since insulin-like growth factor-I (IGF-I) is an important hormone regulator of intestinal proliferation, this study examined the effects of dietary protein content and tumor necrosis factor (TNF) on mRNA levels of IGF-I, IGF-I receptor, IGF binding protein-3 (IGFBP-3), and IGFBP-4 and on the histology of the colon, jejunum, and ileum in protein-malnourished rats. After 2 weeks of feeding a 2% casein diet, rats continued on the 2% casein diet or were refed with a 20% casein diet and received daily intraperitoneal injections of either TNF (50 μg/kg) or saline for 4 days. The abundance of mRNA in the intestine was determined by RNA dot-blot analysis, and morphology measurements were performed by light microscopy. Simultaneous refeeding with the 20% casein diet and administration of TNF led to a modest increase in IGF-I and IGFBP-4 mRNA abundance in the colon. However, in the jejunum and ileum, refeeding had no effect but TNF caused a decrease in IGF-I and IGFBP-3 mRNA levels in malnourished rats. Refeeding with the 20% casein diet resulted in relatively modest histologic changes, which were greater in the colon versus the small intestine. The decreased magnitude of histologic changes in the order of the colon, ileum, and jejunum may reflect a response to a gradient of amino acid availability from intraluminal nutrients. These data demonstrate that TNF has distinct effects on colon and small intestine mRNA, but these mild changes had only a slight impact in the colon and did not translate into identifiable histologic changes in the small intestine. Combined protein restrictio and TNF administration had only a modest effect on intestinal mRNA levels and mucosal histology.

CD9 is involved in extravillous trophoblast invasion

Prenatal exposure to excessive glucocorticoids (GCs) leads to intrauterine growth retardation and fetal programming of adult health and disease through deregulation of placental functions. Placental secretion of insulin-like growth factor-I (IGF-I) plays a critical role in the regulation of placental development and function. However, it remains elusive whether GCs affect placental functions through glucocorticoid receptor (GR)-mediated transcriptional regulation of IGF-I gene. In this study, human placental choriocarcinoma (BeWo) cells before and after syncytialization were used as cytotrophoblast and syncytiotrophoblast models, respectively, to explore the effects of dexamethasone (Dex) on transcriptional regulation of IGF-I gene at both stages. Dex significantly inhibited (P < 0.05) cell proliferation in cytotrophoblasts and down-regulated amino acid transporter SLC7A5 in syncytiotrophoblasts. Concurrently, the abundance of IGF-I mRNA and its transcript variants, together with IGF-I level in culture media, were significantly reduced, in association with significantly enhanced (P < 0.05) GR phosphorylation. GR antagonist RU486 was able to abolish all these effects. Two glucocorticoid response elements (GREs) were predicted in the promoter regions of IGF-I gene. GR binding to GRE1 was significantly enriched (P < 0.05) in both cytotrophoblasts and syncytiotrophoblasts, but that to GRE2 was significantly diminished (P < 0.05) in cytotrophoblasts but not in syncytiotrophoblasts, in response to Dex treatment. IGF-I supplementation completely rescued Dex-induced cell cycle arrest but not SLC7A5 down-regulation, indicating different regulatory mechanisms. Taken together, our results suggest that GR-mediated transcriptional regulation of IGF-I is involved in Dex-induced inhibition of placental cell proliferation and function.

Prostaglandin A2 Specifically Represses Insulin-Like Growth Factor-I Gene Expression in C6 Rat Glioma Cells

The cyclopentenone PGs (PGA and PGJ series) inhibit tumor cell proliferation in vitro and tumorigenesis in vivo via mechanisms that are at present poorly understood. The C6 rat glioma cell line synthesizes and secretes insulin-like growth factor-I (IGF-I), which is believed to act as an autocrine factor for these cells. PGA2 inhibits the proliferation of the C6 cells and causes an increase in the fraction of cells in the G1 phase of the cell cycle. The inhibition of cell proliferation by PGA2 is accompanied by a decrease in the abundance of IGF-I messenger RNA (mRNA). This regulation of IGF-I gene expression is specific, as the abundance of hypoxanthine-guanine phosphoribosyl transferase (HPRT) and ubiquitin mRNA is not significantly affected by PGA2. The repression of IGF-I gene expression is observed at PGA2 concentrations as low as 10 μm and is evident within 4 h after treatment of the C6 cells with PGA2. In addition to specifically regulating the expression of the IGF-I gene, PGA2 also decreases the abundance of cyclin D1 mRNA and increases the abundance of Waf1 mRNA. The inhibition of cell proliferation by PGA2 is partially reversed by coaddition of IGF-I, indicating partial dominance of IGF-I action over PGA2 action. To investigate the molecular basis for the regulation of IGF-I gene expression by PGA2, we developed a sensitive RT-PCR assay for IGF-I nuclear transcripts. A similar assay was developed for quantifying HPRT transcripts, which were used as a control. Treatment of the C6 cells with 20 μm PGA2 resulted in approximately a 6-fold decrease in IGF-I mRNA and IGF-I nuclear transcripts. In contrast, HPRT mRNA and nuclear transcript levels were not significantly affected by PGA2. These results indicate that the decrease in IGF-I mRNA abundance that occurs in response to PGA2 is caused largely by a decrease in IGF-I nuclear transcript levels. To identify the cis-acting element that mediates the effect of PGA2 on IGF-I transcription, C6 cells were transiently transfected with IGF-I/luciferase expression constructs in which luciferase transcription is driven by IGF-I P1 promoter fragments extending from −1711 to +328 or from −1114 to +328 relative to the beginning of exon 1. Treatment of cells with PGA2 in these transient transfection assays did not decrease luciferase activity. These results suggest that the cis-acting regulatory element required for the response to PGA2 is located outside the −1711 to +328 promoter interval.

Prostaglandin A2 specifically represses insulin-like growth factor-I gene expression in C6 rat glioma cells.

The cyclopentenone PGs (PGA and PGJ series) inhibit tumor cell proliferation in vitro and tumorigenesis in vivo via mechanisms that are at present poorly understood. The C6 rat glioma cell line synthesizes and secretes insulin-like growth factor-I (IGF-I), which is believed to act as an autocrine factor for these cells. PGA2 inhibits the proliferation of the C6 cells and causes an increase in the fraction of cells in the G1 phase of the cell cycle. The inhibition of cell proliferation by PGA2 is accompanied by a decrease in the abundance of IGF-I messenger RNA (mRNA). This regulation of IGF-I gene expression is specific, as the abundance of hypoxanthine-guanine phosphoribosyl transferase (HPRT) and ubiquitin mRNA is not significantly affected by PGA2. The repression of IGF-I gene expression is observed at PGA2 concentrations as low as 10 microM and is evident within 4 h after treatment of the C6 cells with PGA2. In addition to specifically regulating the expression of the IGF-I gene, PGA2 also decreases the abundance of cyclin D1 mRNA and increases the abundance of Waf1 mRNA. The inhibition of cell proliferation by PGA2 is partially reversed by coaddition of IGF-I, indicating partial dominance of IGF-I action over PGA2 action. To investigate the molecular basis for the regulation of IGF-I gene expression by PGA2, we developed a sensitive RT-PCR assay for IGF-I nuclear transcripts. A similar assay was developed for quantifying HPRT transcripts, which were used as a control. Treatment of the C6 cells with 20 microM PGA2 resulted in approximately a 6-fold decrease in IGF-I mRNA and IGF-I nuclear transcripts. In contrast, HPRT mRNA and nuclear transcript levels were not significantly affected by PGA2. These results indicate that the decrease in IGF-I mRNA abundance that occurs in response to PGA2 is caused largely by a decrease in IGF-I nuclear transcript levels. To identify the cis-acting element that mediates the effect of PGA2 on IGF-I transcription, C6 cells were transiently transfected with IGF-I/luciferase expression constructs in which luciferase transcription is driven by IGF-I P1 promoter fragments extending from -1711 to -328 or from -1114 to +328 relative to the beginning of exon 1. Treatment of cells with PGA2 in these transient transfection assays did not decrease luciferase activity. These results suggest that the cis-acting regulatory element required for the response to PGA2 is located outside the -1711 to +328 promoter interval.

Decreased hepatic insulin-like growth factor (IGF)-I and increased IGF binding protein-1 and -2 gene expression in experimental uremia.

The imbalance between normal insulin-like growth factor-I (IGF-I) and markedly increased IGF binding protein (IGFBP) plasma levels plays a pathogenic role for growth retardation and catabolism in children with chronic renal failure. To investigate the mechanism of these alterations, experiments were performed in an experimental model of uremia in rats (5/6 nephrectomy) and in pair-fed and ad libitum-fed sham-operated controls Using a specific solution hybridization/RNase protection assay, we observed a marked reduction of hepatic IGF-I messenger RNA (mRNA) abundance at steady state in uremic animals (37 +/- 5% of control) compared both with pair-fed (65 +/- 10%) and ad libitum-fed controls (100 +/- 11%) (P < 0.001). Reduced IGF-I gene expression was clearly organ-specific; it was most pronounced in liver (significant vs., pair-fed controls) and lung and muscle tissue (significant vs., ad libitum-fed controls); no change was observed in kidney and heart tissue. To determine a potential mechanism of reduced hepatic IGF-I gene expression in uremia, the hepatic GH receptor gene expression in the same experimental animals was analyzed by specific solution hybridization/RNase protection assay. Uremic animals had a 20-30% reduction of hepatic GH receptor mRNA abundance compared with controls. Hepatic GHBP expression in uremia was decreased in parallel. Despite the reduction of hepatic IGF-I mRNA abundance, plasma IGF-I levels in uremia were not different from ad libitum-fed controls. This discrepancy is explained by an increased concentration of IGFBPs in uremic plasma. By RIA, plasma IGFBP-1 levels in uremia were increased 4-fold; by Western immunoblot, plasma IGFBP-2 levels were increased 7-fold and plasma IGFBP-4 levels were increased 2-fold compared with both control groups. Intact IGFBP-3 (M(r), approximately 48 kDa) and low molecular IGFBP-3 fragments were not significantly different among the three groups. By Northern blot analysis, hepatic IGFBP-1 mRNA levels in uremia were 2-fold higher than in controls. IGFBP-2 mRNA abundance in liver tissue was increased 4-fold, whereas in kidney there was a significant reduction of IGFBP-2 mRNA (30% of control). IGFBP-4 mRNA was increased by 50% in kidney but not in liver. Plasma insulin and corticosterone levels were not different among the groups. Our study shows that hepatic IGF-I gene expression was specifically reduced in uremia, partially as the consequence of a reduced hepatic GH receptor gene expression. One of the mechanisms contributing to increased IGFBP levels in uremia is increased hepatic gene expression of IGFBP-1 and IGFBP-2. The imbalance between reduced hepatic IGF-I production and increased hepatic IGFBP-1 and 2 production is likely to play a pathogenic role for catabolism and growth failure in CRF.