Insulin-like Growth factor-I Protein Expression Research Articles

Deficiency of liver-derived insulin-like growth factor-I (IGF-I) does not interfere with the skin wound healing rate.

ObjectiveIGF-I is a growth factor, which is expressed in virtually all tissues. The circulating IGF-I is however derived mainly from the liver. IGF-I promotes wound healing and its levels are decreased in wounds with low regenerative potential such as diabetic wounds. However, the contribution of circulating IGF-I to wound healing is unknown.Here we investigated the role of systemic IGF-I on wound healing rate in mice with deficiency of liver-derived IGF-I (LI-IGF-I-/- mice) during normal (normoglycemic) and impaired wound healing (diabetes).MethodsLI-IGF-I-/- mice with complete inactivation of the IGF-I gene in the hepatocytes were generated using the Cre/loxP recombination system. This resulted in a 75% reduction of circulating IGF-I. Diabetes was induced with streptozocin in both LI-IGF-I-/- and control mice. Wounds were made on the dorsum of the mice, and the wound healing rate and histology were evaluated. Serum IGF-I and GH were measured by RIA and ELISA respectively. The expression of IGF-I, IGF-II and the IGF-I receptor in the skin were evaluated by qRT-PCR. The local IGF-I protein expression in different cell types of the wounds during wound healing process was analyzed using immunohistochemistry.ResultsThe wound healing rate was similar in LI-IGF-I-/- mice to that in controls. Diabetes significantly delayed the wound healing rate in both LI-IGF-I-/- and control mice. However, no significant difference was observed between diabetic animals with normal or reduced hepatic IGF-I production. The gene expression of IGF-I, IGF-II and IGF-I receptor in skin was not different between any group of animals tested. Local IGF-I levels in the wounds were similar between of LI-IGF-I-/- and WT mice although a transient reduction of IGF-I expression in leukocytes in the wounds of LI-IGF-I-/- was observed seven days post wounding.ConclusionDeficiency in the liver-derived IGF-I does not affect wound healing in mice, neither in normoglycemic conditions nor in diabetes.

Induction of insulin-like growth factor-I by interleukin-17F in bronchial epithelial cells

Increased expression of IL-17F has been noted in the airway of asthmatic patients, but its role in asthma has not been fully elucidated. Insulin-like growth FACTOR-I (IGF-I) is known to be involved in airway remodelling and inflammation, while its regulatory mechanisms remain to be defined. To further clarify the biological function of IL-17F, we investigated whether IL-17F is able to regulate the expression of IGF-I in bronchial epithelial cells. Bronchial epithelial cells were stimulated with IL-17F in the presence or absence of T-helper type 2 cytokines. Various kinase inhibitors were added to the culture to identify the key signalling events leading to the expression of IGF-I, in conjunction with the use of short interfering RNAs (siRNAs) targeting mitogen- and stress-activated protein kinase (MSK) 1, p90 ribosomal S6 kinase (p90RSK), and cyclic AMP response element-binding protein (CREB). IL-17F significantly induced IGF-I gene and protein expression, and co-stimulation with IL-4 and IL-13 augmented its production. MAP kinase kinase (MEK) inhibitors and the Raf1 kinase inhibitor significantly inhibited IGF-I production, and the combination of PD98059 and Raf1 kinase inhibitor showed further inhibition. Overexpression of Raf1 and Ras dominant-negative mutants inhibited its expression. MSK1 inhibitors significantly blocked IL17F-induced IGF-I expression. Moreover, transfection of the siRNAs targeting MSK1, p90RSK, and CREB blocked its expression. In bronchial epithelial cells, IL-17F is able to induce the expression of IGF-I via the Raf1-MEK1/2-ERK1/2-MSK1/p90RSK-CREB pathway in vitro.

Effect of circulating growth hormone on muscle IGF-I protein concentration in female mice with growth hormone receptor gene disruption

Growth hormone (GH) is a potent secretague for circulating insulin-like growth factor-I (IGF-I). The purpose of this study was to examine the effect of circulating GH on muscle IGF-I protein expression using GH transgenic animal models. Three different models were used: mice that overexpress bovine GH (bGH; n = 10), mice without a functional GH receptor (GHR-/-; n = 10), and wildtype mice ( n = 10). All mice were 16-week old females and each group differed in their basic phenotypic characteristics. Immediately after euthanization the triceps surae muscle group (soleus, plantaris, and gastrocnemius muscles) was removed. IGF-I was extracted from the muscle with an acid–ethanol solution (12.5% 2N hydrochloric acid and 87.5% ethanol, pH 1.5) followed by neutralization with Tris-base and subsequently quantified using a radioimmunoassay. Analysis revealed that bGH mice had significantly greater muscle IGF-I protein expression compared to GHR-/- and wildtype mice. No difference in IGF-I protein concentration was found between GHR-/- and wildtype animals. This study found that overexpression of GH leading to high circulating GH concentrations increase muscle IGF-I protein expression. However, the absence of a functional GHR did not affect muscle IGF-I protein expression compared to wildtype despite high circulating levels of GH and low circulating levels of IGF-I. In conclusion, it appears that at rest high circulating levels of GH augment muscle IGF-I protein expression only in the presence of an intact GHR but that the absence of a functional GH receptor does not affect basal levels of muscle IGF-I protein in female mice.

Adult Neurogenesis in the Hippocampus of Long-Lived Mice During Aging

Ames dwarf mice live considerably longer than normal animals, exhibit apparently normal cognitive functions, and maintain them into advanced age. Neurogenesis occurs throughout adult life span in the dentate gyrus of mammalian hippocampus and has been suggested to play an important role in cognitive function. We now report that the total number of bromodeoxyuridine (BrdU)-labeled cells in this brain region in aged Ames dwarf mice was not different from that in aged normal mice, whereas the fraction of newly generated neurons was significantly increased by monitoring BrdU labeling and cell marker expression. Evidence of activation of anti-apoptosis signal transduction cascade was also found in the hippocampus of aged dwarf mice. Together with previous findings, the results may suggest that an increase in hippocampal insulin-like growth factor-I protein expression and subsequent activation of antiapoptotic signaling might contribute to survival of newly born neurons and subsequently to the delay of cognitive loss during aging in these long-lived dwarf mice.

A novel selective progesterone receptor modulator asoprisnil (J867) down-regulates the expression of EGF, IGF-I, TGFβ3 and their receptors in cultured uterine leiomyoma cells

This study was conducted to evaluate the effects of a novel selective progesterone receptor modulator (SPRM) asoprisnil on the expression of growth factors and their receptors and on growth factor-induced proliferation of cultured uterine leiomyoma and matching myometrial cells. The expression of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) and transforming growth factor (TGFbeta3) was assessed by immunocytochemistry and semi-quantitative RT-PCR. The expression of phosphorylated EGF receptor (p-EGFR), IGF-I receptor alpha subunit (IGF-IRalpha) and phosphorylated TGFbeta receptor type II (p-TGFbeta RII) was assessed by Western blot analysis. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Treatment with 10(-7) M asoprisnil decreased EGF, IGF-I and TGFbeta3 mRNA and protein expression as well as p-EGFR, IGF-IRalpha and p-TGFbeta RII protein expression in leiomyoma cells cultured for 72 h. EGF (100 ng/ml), IGF-I (100 ng/ml) and TGFbeta3 (10 ng/ml) increased the number of viable leiomyoma cells cultured for 72 h, whereas the concomitant treatment with 10(-7) M asoprisnil antagonized the growth factor-induced increase in leiomyoma cell proliferation. In cultured myometrial cells, however, asoprisnil affected neither the growth factor and their receptor expression nor the cell proliferation. Asoprisnil inhibits the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured leiomyoma cells without affecting their expressions in myometrial cells.

Cellular and physiologic effects of exogenous liposomal IGF-1 cDNA gene transfer in acute wounds

The purpose of the present study was to examine the molecular interactions after liposomal cDNA gene transfer and whether the exogenous gene transfer is recognized by the cell and causes endogenous cellular and physiologic responses. Insulin-like growth factor-I (IGF-I) is known to cause, when administered as a protein, adverse side effects, due to lack of cellular responses. Therefore, we used IGF-I cDNA as a vector to study cellular and physiological effects after liposomal administration in an acute wound. Sprague-Dawley rats were inflicted an acute wound and divided into two groups to receive weekly subcutaneous injections of liposomes plus the Lac-Z gene (0.2 μg, vehicle), or liposomes plus the IGF-I cDNA (2.2 μg) and Lac Z gene (0.22 μg). Transfection was confirmed by histochemical assays for β-galactosidase. Planimetry, immunological assays, and histological and immunohistochemical techniques were used to determine molecular mechanisms after gene transfer, protein expression, and dermal and epidermal regeneration. IGF-I cDNA transfer increased IGF-I protein expression and caused concomitant cellular responses by increasing IGFBP-3 and decreasing IGFBP-1. IGF-I cDNA transfer increased IGF-I, FGF, KGF, and PDGF protein concentration, while it had no effect on TGF-β. IGF-I cDNA exerted promitogenic anti-apoptotic effects on basal keratinocytes, thus improving epidermal regeneration. IGF-I cDNA improved dermal regeneration by an increased collagen IV deposition. IGF-I cDNA increased VEGF concentrations and thus neovascularization. Exogenous administered IGF-I cDNA is recognized by the cell and leads to similar intracellular responses as the endogenous gene. Liposomal IGF-I gene transfer further leads to improved dermal and epidermal regeneration by interacting with other growth factors.

Bone elongation in rats with renal failure and mild or advanced secondary hyperparathyroidism

Impairment of growth in children with chronic renal failure may be due, in part to the insensitivity to the actions of growth hormone by insulin-like growth factor-I (IGF-I) because of accumulations of IGF binding proteins. There are a few studies describing the changes that occur in the growth plate in renal failure. None of these studies has simultaneously compared the modifications in the expression of selected markers of endochondral bone formation in renal failure with mild or advanced secondary hyperparathyroidism. Forty-six rats that underwent 5/6 nephrectomy (Nx) were fed either standard rodent diet (Nx-control) or high phosphorus diet to induce advanced secondary hyperparathyroidism (Nx-phosphorus) for 4 weeks. Sections of the tibia were obtained for growth plate histomorphometry, immunohistochemistry studies, and in situ hybridization experiments for selected markers of endochondral bone formation. Weight gain, gain in length, and tibial length were less in Nx animals. Serum parathyroid hormone (PTH) and phosphorus levels were higher and serum calcium levels were lower in the Nx-phosphorus group. The width of the growth plate was much shorter in the Nx-phosphorus group due to a decrease in both proliferative and hypertrophic zones. IGF-I protein and IGF binding protein-3 staining were diminished in both Nx groups without changes in the IGF-I receptor expression; the decline in IGF-I protein expression was much lower in the Nx-phosphorus group. PTH/PTH receptor protein (PTHrP) receptor mRNA transcripts decline and tartrate-resistant acid phosphastase (TRAP) staining increased only in the Nx-phosphorus group. The growth impairment in renal failure may be worsened by the severity of secondary hyperparathyroidism.

Progesterone down-regulates insulin-like growth factor-I expression in cultured human uterine leiomyoma cells.

Insulin-like growth factor-I (IGF-I) plays crucial roles in uterine leiomyoma cell growth through stimulating proliferation and inhibiting apoptosis. The present study was conducted to elucidate whether progesterone affects IGF-I and its receptor expression in cultured leiomyoma cells. Isolated leiomyoma cells were subcultured in Phenol Red-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 120 h and then stepped down to serum-free conditions for an additional 48 h in the presence or absence of 17beta-estradiol (E(2)) (10 ng/ml) or progesterone (100 ng/ml). IGF-I and its receptor mRNA and immunoreactive IGF-I in the cultured cells were assessed by quantitative RT-PCR with Southern blot analysis and by radioimmunoassay with Seppak C18 chromatography, respectively. The presence of estrogen receptor (ER) and progesterone receptor (PR) in cultured leiomyoma cells was immunocytochemically examined. Both treatment with progesterone alone and treatment with E(2) and progesterone combined significantly decreased IGF-I mRNA and protein expression in cultured leiomyoma cells compared with that in untreated cultures, but treatment with E(2) alone did not. IGF-I receptor mRNA expression in those cells was not affected by treatment with either E(2) or progesterone. Immunocytochemical analysis revealed that PR protein expression in cultured leiomyoma cells maintained in a serum-free condition for 48 h whereas ER protein expression in the cells remarkably decreased after 24 h culture under the serum-free condition. The present study provided evidence for the first time that progesterone down-regulates IGF-I mRNA and protein expression in cultured leiomyoma cells without affecting IGF-I receptor mRNA expression.

Insulin-like growth factor I (IGF-I) regulates endocrine activity of the embryonic testis in the mouse

Insulin-like growth factor I (IGF-I) is important for gonadal and reproductive functions in mammals, although the physiological role of this growth factor during gonadal development in rodents remains largely unknown. Here, we examined the steady-state levels of IGF-I mRNA by the reverse transcriptase polymerase chain reaction (RT-PCR). IGF-I protein expression was also detected by Western blot. The effect of IGF-I as promoter of 17α-hydroxylase/C17–20 lyase and 17β-hydroxysteroid dehydrogenase enzyme activity in vitro was evaluated by radioimmunoassay. Onset of IGF-I gene expression was on day E10 (urogenital ridge stage). IGF-I mRNA expression was markedly reduced on days E12 and E13 (testicular differentiation stage). IGF-I transcripts increased on day E14 and their transcription levels were maintained throughout the stages analyzed. Several IGF-I protein bands of 31–100 kDa were observed. Culture experiments demonstrated that 17α-hydroxyprogesterone and testosterone (T) secretion levels increased in the presence of IGF-I on days E11–E17. Additive effects of IGF-I plus (Bu) 2cAMP were also seen during testicular development. It is proposed that IGF-I regulates the expression of key steroidogenic enzymes important for endocrine activity of the testis during prenatal development leading to establishment of the male phenotype and fertility.

Effect of multiple gene transfers of insulinlike growth factor I complementary DNA gene constructs in rats after thermal injury.

Multiple subcutaneous injections of cholesterol-containing cationic liposomes encapsulating the complementary DNA (cDNA) gene for insulinlike growth factor I (IGF-I) increase the rate of transfected skin cells and result in increased IGF-I protein levels in the skin with subsequent improvement in wound healing when compared with a single injection. Laboratory. Twenty-four adult male Sprague-Dawley rats (350-375 g) received a full-thickness scald burn on 60% of their body surface. These rats were randomly divided to receive either 1 injection of liposomes containing 2.2 microg-cytomegalovirus-driven cDNA coding for IGF-I and 0.2 microg of the Lac Z gene cDNA construct, or 2 injections of liposomes containing 2.2 microg cytomegalovirus-driven cDNA coding for IGF-I and 0.2 microg of the Lac Z gene cDNA construct. Transfection rates and IGF-I protein levels in the skin and physiological responses to the IGF-I gene therapy, evaluated from changes in body weight, protein content in serum and liver, and the rate of burn wound healing. There was a significant decrease in transfection rate and IGF-I protein expression distal from the injection site in animals receiving 1 injection, as compared with a consistent increase in rats receiving multiple injections. Multiple injections improved the response to thermal trauma by increasing the extent of the healed burn wound 33 days after thermal injury (single injection, 31% +/- 1% vs multiple injections, 38% +/- 2%), total serum protein (single injection, 52 +/- 0.5 g/L vs multiple injections, 55 +/- 0.6 g/L), and total liver protein (single injection, 82.0 +/- 0.3 mg/mL vs multiple injections, 91.0 +/- 3.8 mg/mL), P<.05. Gene transfer rates can be increased by multiple injections of liposomes encapsulating IGF-I cDNA constructs. Increased transfer results in greater IGF-I protein skin concentrations, accelerated wound healing, and increased serum and liver protein concentrations. The clinical relevance of these findings is that liposomal gene constructs should be applied in well-defined distances to improve gene transfer in the skin, and thus clinical outcome after thermal injury.

Increased Expression of Insulin-like Growth Factor-I in Serum and Liver after Recombinant Human Growth Hormone Administration in Thermally Injured Rats

Background. Recombinant human growth hormone (rhGH) has been shown to modulate the hypermetabolic response and the hepatic acute-phase response after thermal injury. In vitro studies, however, demonstrated that rhGH activates insulin-like growth factor-I (IGF-I) gene transcription and production, suggesting that rhGH may exert some of its effects indirectly through IGF-I stimulation. The purpose of this study was to determine the effects of rhGH on serum and hepatic IGF-I in thermally injured rats.Methods. Sprague–Dawley rats (56 males) receiving a 60% TBSA third-degree scald burn were randomly divided to receive either rhGH (2.5 mg/kg/day im) or saline (control). Rats were sacrificed on postburn days 1, 2, 5, and 7 and serum IGF-I, hepatic IGF-I mRNA, and IGF-I protein concentration were measured. The physiologic response to changes in IGF-I levels was evaluated by measuring hepatocyte proliferation, total liver protein concentration, and muscle dry/wet weights.Results. Serum IGF-I was increased from postburn day 1 through day 7 in rats receiving rhGH compared to controls (P < 0.05). Hepatic IGF-I mRNA and IGF-I protein expression were increased from day 1 to 7 after burn in animals receiving rhGH when compared to controls (P < 0.05). Recombinant hGH increased hepatocyte proliferation at 5 days and total liver protein concentration at 5 and 7 days postburn compared to controls (P < 0.05). Muscle dry/wet weights increased in rats receiving rhGH at 7 days after burn compared to controls (P < 0.05).Summary. Liver and serum IGF-I levels decreased after a thermal injury. Recombinant hGH attenuated this decrease by stimulating hepatic IGF-I expression. Increases in IGF-I were associated with increases in hepatocyte proliferation and protein concentration in liver and muscle.Conclusion. We suggest that rhGH modulates the hypermetabolic response through IGF-I stimulation in the hepatic parenchyma.

Estrogen effects on insulin-like growth factor-I (IGF-I)-induced cell proliferation and IGF-I expression in native and allograft vessels.

Estrogen protects against cardiovascular disease in both patients and animal models and regulates insulin-like growth factor-I (IGF-I), an important cell-cycle progression factor. Smooth muscle cells and tissues were harvested from male recipient rabbits that 6 weeks earlier had received a cardiac allograft transplant consisting of a donor heart and ascending aorta. Segments of the ascending aorta from the native and allograft hearts from 9 placebo-treated and 8 estradiol-treated recipients were compared by using IGF-I-stimulated [3H]thymidine incorporation. The responses of the native vessel segments were similar (175.3+/-32% and 166.9+/-41%, respectively; P>.05) whether or not the recipients had been treated for 6 weeks with estradiol. In the grafts, however, estradiol markedly inhibited vascular cell thymidine incorporation (328.04+/-56% compared with 67.3+/-11%; P<.02). Smooth muscle cells were derived from the native aorta of the placebo-treated rabbits to study the effect of estradiol in vitro. IGF-I increased cell counts in a concentration-dependent manner. In serum-starved cells estradiol further decreased cell proliferation; this effect was blocked by the specific estrogen receptor antagonist ZK-119.010. Immunohistochemistry staining for IGF-I protein in the coronary arteries and ascending aorta of the cardiac allograft from the placebo-treated recipients revealed extensive IGF-I expression in the myointima. In contrast, IGF-I protein was not expressed in the coronary arteries and ascending aorta of the cardiac allograft from the estradiol-treated recipients. The IGF-I protein was extensively expressed only in the placebo-treated graft vessels. Myointimal thickening of the coronary arteries was significantly reduced by estradiol treatment (17.9+/-1.5% versus 44.3+/-3.7%; P<.02). In vivo estradiol treatment abolishes both IGF-I mitogenic effects and IGF-I protein expression in the vascular wall, which may be causally related to the inhibitory effect of estradiol on transplant arteriosclerosis.