Insulin-like Growth factor-I Pathways Research Articles

Carthamus tinctorius L. extract activates insulin-like growth factor-I receptor signaling to inhibit FAS-death receptor pathway and suppress lipopolysaccharides-induced H9c2 cardiomyoblast cell apoptosis.

Carthamus tinctorius L. (Compositae) is used in Chinese medicine to treat heart disease and inflammation. In our previous study, we found that C. tinctorius L. inhibited lipopolysaccharides (LPS)-induced tumor necrosis factor-alpha (TNF-α) activation, JNK expression, and apoptosis in H9c2 cardiomyoblast cells. The present study was performed to investigate the protective effect of C. tinctorius extract (CTF) on LPS-challenged H9c2 myocardioblast cell and to explore the possible underlying mechanism. Cell viability assay showed that LPS treatment decreased the cell viability of H9c2 cell, whereas CTF treatment reversed LPS cytotoxicity in a dose-dependent manner, especially in the LPS + CTF 25 (μg/mL) group. LPS treatment-induced apoptosis was determined by transferase-mediated dUTP nick end labeling assay, and by Western blot. LPS-induced apoptotic bodies were decreased following CTF treatment. Expression of TNF-α, FAS-L, FAS, FADD, caspase-8, BID, and t-BID was significantly increased in LPS-treated H9c2 cells. In contrast, it was significantly suppressed by the administration of CTF extract. In addition, CTF treatment activates antiapoptotic proteins, Bcl-2 and p-Bad, and downregulates Bax, cytochrome-c, caspase-9, caspase-3, and apoptosis-inducing factor expression. Furthermore, CTF exerted cytoprotective effects by activating insulin-like growth factor-I (IGF-I) signaling pathway leading to downregulation of the apoptotic proteins involved in FAS death receptor pathway. In addition, AG1024 and IGF-I receptor (IGF-IR) inhibitor and siRNA silencing reverses the effect of CTF implying that CTF functions through the IGF-IR pathway to inhibit LPS-induced H9c2 apoptosis. These results suggest that treatment with CTF extract prevented the LPS-induced apoptotic response through IGF-I pathway.

Expression of Insulin-like Growth Factor Binding Protein-3 and Regulation of the Insulin-like Growth Factor-I Axis in Pig Testis

The stem cell niche is a complex unit comprising key components, such as the extracellular matrix and various paracrine factors, which regulate the differentiation of adult stem cells. In our previous study, we established pig spermatogonial stem cells (pSSCs) in culture and identified the expression of insulin-like growth factor binding protein-3 (IGFBP-3) in pSSCs. The present study investigated not only the expression of IGFBP-3, but also its possible role in pSSCs. In this study, IGFBP-3-expressing cells responded positively to protein gene product 9.5 (PGP9.5), which is a marker for pig spermatogonia. IGFBP-3 expression was significantly increased in 60-dayold pig testes. Additionally, the expression levels of insulinlike growth factor I (IGF-I) and its receptor (IGF-IR) were observed in pSSCs and pig Sertoli cells (pSCs). Furthermore, IGF-I treatment enhanced the proliferation of pSCs and pSSCs when they were co-cultured. Blocking the IGF-I pathway using a specific IGF-IR inhibitor dramatically reduced the proliferation of pSCs. In addition, when heparan sulfate was used to sequester IGFBP-3 from IGF-I binding, a significant increase in the proliferation of pSCs was observed. Exogenous IGF-I treatment also increased the expression level of IGFBP-3 in cultured pSSCs. Furthermore, pSSCs grew well in IGF-I-treated pSC conditioned media. In summary, IGF-I and IGF-IR signaling are important for the proliferation of pSCs, and the germ cell-derived IGFBP-3 had an inhibitory effect on the mitotic activity of IGF-I in pSCs.

Research Article Genotypes within the prolactin and growth hormone insulin-like growth factor-I pathways associated with milk production in heat stressed Holstein cattle: Genotypes and milk yield in heat stressed Holstein cows

The association of single nucleotide polymorphisms (SNPs) within genes of the prolactin (PRL), growth hormone (GH) and insulin-like growth factor-I (IGF1) pathways with 305-day milk yield (MY305) were evaluated in heat-stressed Holstein cows. Ambient temperature and relative humidity were used to calculate a temperature-humidity index (THI), which revealed that heat stress conditions existed in the Yaqui Valley, Sonora, Mexico from May through October of 2011 and 2012. Cows (n = 573) were genotyped for 179 tag SNPs within 43 candidate genes in the PRL and GH/IGF1 pathways. Seven SNPs within 7 genes (AVPR1A, Furin, IGFBP5, IGFBP6, PMCH, PRLR and STAT5B) were found to be associated with MY305 (P<0.05); therefore, their effects were used to estimate a molecular breeding value (MBV). The correlation between the MBV and MY305 was 0.21 (P<0.001) and the adjusted coefficient of determination (R2) was 4%, whereas correlation between MBV and the estimated breeding value for MY305 was 0.25 (P<0.001) with a R2 of 6%. Heritability estimate for MY305 was 0.41 ± 0.12. The MBV estimated was positive, but weakly associated with MY305. The small amount of additive and phenotypic variation explained by the MBV was most likely due to the few number of SNPs and the complexity of the trait, particularly under extreme weather conditions. In conclusion, SNPs within the PRL and GH/IGF1 pathways were associated with MY305; however, the MBV estimated with these SNPs was not yet suitable to use in genetic selection procedures due to the small amount of variation explained for MY305.

Insulin-like growth factor-I inhibition with pasireotide decreases cell proliferation and increases apoptosis in pre-malignant lesions of the breast: a phase 1 proof of principle trial

IntroductionEstrogen inhibition is effective in preventing breast cancer in only up to 50% of women with precancerous lesions and many experience side effects that are poorly tolerated. As insulin-like growth factor I (IGF-I) underlies both estrogen and progesterone actions and has other direct effects on mammary development and carcinogenesis, we hypothesized that IGF-I inhibition might provide a novel approach for breast cancer chemoprevention.MethodsIn total, 13 women with core breast biopsies diagnostic of atypical hyperplasia (AH) were treated for 10 days with pasireotide, a somatostatin analog which uniquely inhibits IGF-I action in the mammary gland. They then had excision biopsies. 12 patients also had proliferative lesions and one a ductal carcinoma in situ (DCIS). Primary outcomes were changes in cell proliferation and apoptosis after treatment. Expression of estrogen receptor (ER), progesterone receptor (PR), and phosphorylated Insulin-like growth factor I receptor (IGF-1R), protein kinase B (AKT) and extracellular signal-regulated kinases 1/2 (ERK1/2) were also assessed. Core and excision biopsies from 14 untreated patients served as non-blinded controls. Hyperglycemia and other side effects were carefully monitored.ResultsPasireotide decreased proliferation and increased apoptosis in all AH (from 3.6 ± 2.6% to 1.3 ± 1.2% and from 0.3 ± 0.2% to 1.5 ± 1.6%, respectively) and proliferative lesions (from 3.8 ± 2.5% to 1.8 ± 1.8% and from 0.3 ± 0.2% to 1.3 ± 0.6%, respectively). The DCIS responded similarly. ER and PR were not affected by pasireotide, while IGF-1R, ERK1/2 and AKT phosphorylation decreased significantly. In contrast, tissue from untreated controls showed no change in cell proliferation or phosphorylation of IGF-1R, AKT or ERK 1/2. Mild to moderate hyperglycemia associated with reduced insulin levels was found. Glucose fell into the normal range after discontinuing treatment. Pasireotide was well tolerated and did not cause symptoms of estrogen deprivation.ConclusionsIGF-I inhibition by pasireotide, acting through the IGF-1R, was associated with decreased proliferation and increased apoptosis in pre-malignant breast lesions and one DCIS. Assuming hyperglycemia can be controlled, these data suggest that inhibiting the IGF-I pathway may prove an effective alternative for breast cancer chemoprevention.Trial registrationNCT01372644 Trial date: July 1, 2007.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0463-1) contains supplementary material, which is available to authorized users.

Cytohesins/ARNO: The Function in Colorectal Cancer Cells

Epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) are critical regulators of cell differentiation, survival, proliferation, and migration in cancers. This study found that ARNO (cytohesin-2), an activator of the EGF and IGF-I pathways, was more highly expressed in colorectal cancer tissue than in benign adjacent colorectal tissue. When ARNO-siRNA or the chemical inhibitor SecinH3 blocked ARNO, the downstream of the EGF and IGF-I pathways decreased in colorectal cell lines HT29 and HCT116. This blocking also weakened cell proliferation, invasion, and migration in vitro. Furthermore, EGF receptor (EGFR)-dependent colorectal tumor xenografts in nude mouse exerted anti-proliferative and growth suppression effects by injecting secineH3. These data suggested that inhibiting cytohesins or ARNO as cytoplasmic activators of EGFR and IGF-I in colorectal cancer resulted in anti-proliferation, reduced invasion, decreased migration, and suppressed growth in vivo and in vitro. Therefore, cytohesins or ARNO may be a potential therapy target for some colorectal cancer.

P2-03-03: An Insulin-Like Growth Factor I (IGF-I)-Induced Gene, Solute Carrier Family 7 Member 11 (SLC7A11)/xCT, Mediates IGF-I-Induced Biological Behaviors in Breast Cancer Cells.

Abstract Our laboratory studied the gene expression profiles of a series of T47D variant cell lines with differential insulin receptor substrate (IRS) adaptor protein expression to develop predictive IGF-I pathway biomarkers. We identified an IGF-I-induced gene, SLC7A11 (or xCT), which is specifically regulated through IRS-1. xCT encodes the cystine/glutamate transporter subunit of the heterodimeric amino acid transport system xc- which is a major plasma membrane transporter for the cellular uptake of cystine in exchange for intracellular glutamate. xCT is involved in the regulations of proliferation, metastasis, and drug resistance in various cancers. However, to date, the linkage between xCT and the IGF-I signaling pathway has not been described. To study the role for xCT in mediating IGF-I-induced biology in breast cancer cell lines, we examined xCT mRNA expression upon IGF-I stimulation in two breast cancer cell lines; the MCF-7 (IRS-1 activated) and MDA-MB-231 (IRS-2 activated) cells. Significant increased xCT expression was observed only in MCF-7 cells after IGF-I treatment. Immunoblots showed that xCT protein expression was elevated after IGF-I treatment and induced glutamate/cystine exchange in MCF-7 cells. shRNA was used to downregulate xCT in MCF-7 and MDA-MB-231 cells. In MCF-7, IGF-I-stimulated cell monolayer growth was suppressed by xCT shRNA or by the xCT inhibitor sulfasalazine (SASP). In MDA-MB-231 cells, xCT downregulation did not affect IGF-mediated Boyden chamber migration. Thus, IGF-I induction of cellular xCT levels is associated with cell growth in the IRS-1 activated MCF-7 cells, while MDA-MB-231 cells were not affected by downregulation of this gene. Therefore, our data imply that xCT may mediate IGF-I induced biological functions in breast cancer cell lines through an IRS-1 dependent pathway. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-03-03.

Aromatase inhibitor-induced loss of grip strength is body mass index dependent: hypothesis-generating findings for its pathogenesis

BackgroundOur preliminary results showed that tenosynovial changes and decrease in grip strength are associated with the aromatase inhibitor-induced musculoskeletal syndrome (AIMSS). Here, we report the final results and assess the relationship between grip strength and body mass index (BMI). Patients and methodsWe conducted a prospective study including postmenopausal early breast cancer patients receiving either an aromatase inhibitor (AI) or tamoxifen. Primary end point was change from baseline in tenosynovial abnormalities. Secondary end points were changes from baseline in morning stiffness, intra-articular fluid and grip strength and its association with BMI. ResultsAfter 6 months of therapy, 74% [95% confidence interval (CI) 51% to 89%] of AI-treated patients had worsened tenosynovial abnormalities, 56% (95% CI 34% to 75%) had increased intra-articular fluid, and 22% (95% CI 9% to 45%) had increased morning stiffness. Grip strength decreased 8% for the left hand (95% CI 2% to 21%) and 11% for the right (95% CI 4% to 17%). Regression analysis suggested that grip strength decreased more for subjects with high or with low BMI. ConclusionsAIMSS is characterized by tenosynovial changes, intra-articular fluid and morning stiffness. We hypothesize that the quadratic association between BMI and loss of grip strength reflects AI-induced changes on the endocrine control of the growth hormone insulin-like growth factor-I pathway.

Comprehensive analysis of common genetic variation in 61 genes related to steroid hormone and insulin-like growth factor-I metabolism and breast cancer risk in the NCI breast and prostate cancer cohort consortium†

There is extensive evidence that increases in blood and tissue concentrations of steroid hormones and of insulin-like growth factor I (IGF-I) are associated with breast cancer risk. However, studies of common variation in genes involved in steroid hormone and IGF-I metabolism have yet to provide convincing evidence that such variants predict breast cancer risk. The Breast and Prostate Cancer Cohort Consortium (BPC3) is a collaboration of large US and European cohorts. We genotyped 1416 tagging single nucleotide polymorphisms (SNPs) in 37 steroid hormone metabolism genes and 24 IGF-I pathway genes in 6292 cases of breast cancer and 8135 controls, mostly Caucasian, postmenopausal women from the BPC3. We also imputed 3921 additional SNPs in the regions of interest. None of the SNPs tested was significantly associated with breast cancer risk, after correction for multiple comparisons. The results remained null when cases and controls were stratified by age at diagnosis/recruitment, advanced or nonadvanced disease, body mass index, with or without in situ cases; or restricted to Caucasians. Among 770 estrogen receptor-negative cases, an SNP located 3' of growth hormone receptor (GHR) was marginally associated with increased risk after correction for multiple testing (P(trend) = 1.5 × 10(-4)). We found no significant overall associations between breast cancer and common germline variation in 61 genes involved in steroid hormone and IGF-I metabolism in this large, comprehensive study. Although previous studies have shown that variations in these genes can influence endogenous hormone levels, the magnitude of the effect of single SNPs does not appear to be sufficient to alter breast cancer risk.

Pancreatic cancer cells activate CCL5 expression in mesenchymal stromal cells through the insulin-like growth factor-I pathway

Mesenchymal stromal cells (MSCs) have a critical role in cancer progression and metastasis. Despite extensive studies of the physiological responses in cancer cells, the molecular mechanisms regulating gene expression in MSCs by cancer cells remain undefined. Here we demonstrate that CC chemokine ligand 5 (CCL5) expression was increased in MSCs co-cultured with pancreatic cancer cells (PCCs), and this activation was dependent on extracellular insulin-like growth factor (IGF-I). Moreover, CCL5 induction in MSCs was required for the activation of IGF-I pathway in PCCs. These results reveal a link between the IGF-I pathway in PCCs and CCL5 pathway in MSCs through the interaction of those cells.

Insulin-like Growth Factor-I Receptor Blockade Improves Outcome in Mouse Model of Lung Injury

The insulin-like growth factor-I (IGF-I) pathway is an important determinant of survival and proliferation in many cells. However, little is known about the role of the IGF-I pathway in lung injury. We previously showed elevated levels of IGF-I in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome. Furthermore, immunodepletion of IGF from acute respiratory distress syndrome bronchoalveolar lavage increased fibroblast apoptosis. We examined the effect of blockade of type 1 IGF tyrosine kinase receptor (IGF-IR) in a murine model of bleomycin-induced lung injury and fibrosis. Mice were treated with a monoclonal antibody against the IGF-I receptor (A12) or vehicle after intratracheal bleomycin instillation. Mice treated with A12 antibody had significantly improved survival after bleomycin injury compared with control mice. Both groups of mice had a similar degree of fibrosis on days 7 and 14, but by Day 28 the A12-treated group had significantly less fibrosis. Delayed treatment with A12 also resulted in decreased fibrosis. A12-treated mice had significantly decreased apoptotic cells on Day 28 compared with control mice. We confirmed that A12 treatment induced mouse lung fibroblast apoptosis in vitro. In addition, IGF-I increased lung fibroblast migration. The primary pathway activated by IGF-I in lung fibroblasts was the insulin receptor substrate-2/phosphatidylinositol 3-kinase/Akt axis. IGF-I regulated survival and migration of fibrogenic cells in the lung. Blockade of the IGF pathway increased fibroblast apoptosis and subsequent resolution of pulmonary fibrosis. Thus, IGF-IR may be a potential target for treatment of lung injury and fibrosis.

Developmental reprogramming of IGF signaling and susceptibility to endometrial hyperplasia in the rat

In rodents, a brief neonatal exposure of the developing reproductive tract to the xenoestrogen, diethylstilbestrol (DES) reprograms developing tissues to increase susceptibility to tumorigenesis in adult animals, including uterine adenocarcinoma. Progression from a normal endometrium to carcinoma occurs via the intermediate stage of endometrial hyperplasia. We previously reported that endometrial hyperplasia in postmenopausal women is linked to abnormal insulin-like growth factor-I (IGF-I) signaling. To identify early events involved in the development of hyperplasia in the endometrium, we examined expression and activation of IGF-I pathway components in endometrium of rats exposed to DES. By 5 months of age, 36/60 (60%) of rats exposed to DES on days 3–5 after birth developed endometrial hyperplasia compared to 0% of vehicle-treated controls. Consistent with activation of a mitogenic signaling pathway, Ki67-positive cells increased in DES-exposed endometrium despite compromised ovarian function and hypoestrogenic milieu characteristic of DES-exposed animals. The endometrium of DES-exposed rats overexpressed IGF-II and insulin receptor substrate-1 (IRS-1) and exhibited elevated Akt expression and activation (as judged by phosphorylation) and mTOR signaling (phosphorylation of S6) compared to vehicle-treated endometrium. In contrast to vehicle-treated endometrium, in which negative feedback to IRS-1 was observed (phosphorylation of S636/639), negative feedback to IRS-1 was absent in DES-exposed endometrium. These data support a central role for IGF-I signaling in the development of both human and rodent endometrial hyperplasia. Furthermore, both global activation of IGF-IR signaling and abrogation of negative feedback to IRS-1 appear to be reprogrammed by DES in endometrial hyperplasia, implicating for the first time loss of negative feedback to IRS-1 in development of a preneoplastic lesion.

ANABOLIC AGENTS FOR IMPROVING MUSCLE REGENERATION AND FUNCTION AFTER INJURY

1. In the present review, we describe how muscles can be injured by external factors, internal factors or during the performance of some actions during sports. In addition, we describe the injury to a muscle that occurs when its blood supply is interrupted, an occurrence common in clinical settings. An overview of muscle regeneration is presented, as well as a discussion of some of the potential complications that can compromise successful muscle repair and lead to impaired function and permanent disability. 2. Improving muscle regeneration is important for hastening muscle repair and restoring muscle function and the present review describes ways in which this can be achieved. We describe recent advances in tissue engineering that offer considerable promise for treating muscle damage, but highlight the fact that these techniques require rigorous evaluation before they can become mainstream clinical treatments. 3. Growth-promoting agents are purported to increase the size of existing and newly regenerating muscle fibres and, therefore, could be used to improve muscle function if administered at appropriate times during the repair process. The present review provides an update on the efficacy of some growth-promoting agents, including anabolic steroids, insulin-like growth factor-I (IGF-I) and beta(2)-adrenoceptor agonists, to improve muscle function after injury. Although these approaches have clinical merit, a better understanding of the androgenic, IGF-I and b-adrenoceptor signalling pathways in skeletal muscle is important if we are to devise safe and effective therapies to enhance muscle regeneration and function after injury.

Temporal analysis of E2 transcriptional induction of PTP and MKP and downregulation of IGF-I pathway key components in the mouse uterus

17beta-Estradiol (E2) is well known to be associated with uterine cancer, endometriosis, and leiomyomas. Although insulin-like growth factor I (IGF-I) has been identified as a mediator of the uterotrophic effect of E2 in several studies, this mechanism is still not well understood. In the present study, identification of the genes modulated by a physiological dose of E2, in the uterus, has been done in ovariectomized mice using Affymetrix microarrays. The E2-induced genomic profile shows that multiple genes belonging to the IGF-I pathway are affected after exposure to E2. Two phases of regulation could be identified. First, from 0 to 6 h, the expression of genes involved in the cell cycle, growth factors, protein tyrosine phosphatases, and MAPK phosphatases is quickly upregulated by E2, while IGF-I receptor and several genes of the MAPK and phosphatidylinositol 3-kinase pathways are downregulated. Later, i.e., from 6 to 24 h, transporters and peptidases/proteases are stimulated, whereas defense-related genes are differentially regulated by E2. Finally, cytoarchitectural genes are modulated later. The present data show that a physiological dose of E2 induces, within 24 h, a series of transcriptional events that promote the uterotrophic effect. Among these, the E2-mediated activation of the IGF-I pathway seems to play a pivotal role in the uterotrophic effect. Furthermore, the protein tyrosine phosphatases and MAPK phosphatases are likely to modulate the estrogenic uterotrophic action by targeting, at different steps, the IGF-I pathway.

Disturbed Cross Talk between Insulin-Like Growth Factor I and AMP-Activated Protein Kinase as a Possible Cause of Vascular Dysfunction in the Amyloid Precursor Protein/Presenilin 2 Mouse Model of Alzheimer's Disease

Cerebrovascular dysfunction appears to be involved in Alzheimer's disease (AD). In double mutant amyloid precursor protein/presenilin 2 (APP/PS2) mice, a transgenic model of AD, vessel homeostasis is disturbed. These mice have elevated levels of vascular endothelial growth factor (VEGF) and increased brain endothelial cell division but abnormally low brain vessel density. Examination of the potential involvement of insulin-like growth factor I (IGF-I) in these alterations revealed that treatment with IGF-I, a potent vessel growth promoter in the brain that ameliorates cognitive dysfunction in APP/PS2 mice, counteracted vascular dysfunction as follows: VEGF levels and endothelial cell proliferation were reduced, whereas vascular density was normalized. Notably, abnormally elevated brain IGF-I receptor levels in APP/PS2 mice were also normalized by IGF-I treatment. Analysis of possible processes involved in these alterations indicated that AMP-activated protein kinase (AMPK), a cell energy sensor that intervenes in angiogenic signaling and interacts with IGF-I, was also abnormally activated in APP/PS2 brains. Examination of the consequences of AMPK activation on cultured brain endothelial cells revealed increased VEGF levels together with enhanced endothelial cell proliferation and metabolism. Although these effects were also independently elicited by IGF-I, when both IGF-I and AMPK pathways were simultaneously activated on brain endothelial cells, VEGF production and endothelial cell proliferation ceased while cells remained metabolically activated (glucose use, peroxide production, and mitochondrial activity were elevated) and became more resistant to oxidative stress. Therefore, high IGF-I receptor and phosphoAMPK levels in APP/PS2 brains may reflect imbalanced IGF-I and AMPK angiogenic cross talk that could underlie vascular dysfunction in this model of AD.

Overexpression of the Insulin-Like Growth Factor I Receptor and Activation of the AKT Pathway in Hyperplastic Endometrium

Although there is considerable information on the molecular aberrations associated with endometrial cancer, very little is known of the changes in gene expression associated with endometrial hyperplasia. To address this, we have compared the level of expression of estrogen-regulated genes and components of the insulin-like growth factor I (IGF-I) signaling pathway in endometrial biopsies from subjects with normal endometrium, complex atypical endometrial hyperplasia, and endometrial adenocarcinoma (type I). There was a significant increase in the expression of the IGF-I receptor (IGF-IR) in biopsies from hyperplastic endometrium and endometrial carcinoma compared with the proliferative endometrium. The receptor was also activated, as judged by increased tyrosine phosphorylation. In addition, in endometrial hyperplasia and carcinoma, the downstream components of the IGF-IR pathway are activated, as reflected in increased Akt phosphorylation. Loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression in endometrial hyperplasia did not correlate with increased activation of IGF-IR. However, the simultaneous loss of PTEN expression and increased IGF-IR activation in hyperplasia was associated with an increased incidence of endometrial carcinoma. These results suggest that up-regulation of IGF-IR and loss of PTEN may be independent events that give rise to complementary activation of the IGF-I pathway and increase the probability of the development of cancer. These studies suggest that increased expression of IGF-IR may be an important contributor to the risk of endometrial hyperplasia and cancer.

Fluid Shear Stress Synergizes with Insulin-like Growth Factor-I (IGF-I) on Osteoblast Proliferation through Integrin-dependent Activation of IGF-I Mitogenic Signaling Pathway

This study tested the hypothesis that shear stress interacts with the insulin-like growth factor-I (IGF-I) pathway to stimulate osteoblast proliferation. Human TE85 osteosarcoma cells were subjected to a steady shear stress of 20 dynes/cm(2) for 30 min followed by 24-h incubation with IGF-I (0-50 ng/ml). IGF-I increased proliferation dose-dependently (1.5-2.5-fold). Shear stress alone increased proliferation by 70%. The combination of shear stress and IGF-I stimulated proliferation (3.5- to 5.5-fold) much greater than the additive effects of each treatment alone, indicating a synergistic interaction. IGF-I dose-dependently increased the phosphorylation level of Erk1/2 by 1.2-5.3-fold and that of IGF-I receptor (IGF-IR) by 2-4-fold. Shear stress alone increased Erk1/2 and IGF-IR phosphorylation by 2-fold each. The combination treatment also resulted in synergistic enhancements in both Erk1/2 and IGF-IR phosphorylation (up to 12- and 8-fold, respectively). Shear stress altered IGF-IR binding only slightly, suggesting that the synergy occurred primarily at the post-ligand binding level. Recent studies have implicated a role for integrin in the regulation of IGF-IR phosphorylation and IGF-I signaling. To test whether the synergy involves integrin-dependent mechanisms, the effect of echistatin (a disintegrin) on proliferation in response to shear stress +/- IGF-I was measured. Echistatin reduced basal proliferation by approximately 60% and the shear stress-induced mitogenic response by approximately 20%. It completely abolished the mitogenic effect of IGF-I and that of the combination treatment. Shear stress also significantly reduced the amounts of co-immunoprecipitated SHP-2 and -1 with IGF-IR, suggesting that the synergy between shear stress and IGF-I in osteoblast proliferation involves integrin-dependent recruitment of SHP-2 and -1 away from IGF-IR.

Role of insulin-like growth factor I signaling in neurodegenerative diseases.

Disturbed trophic support to neurons has long been considered a potential mechanism in neurodegeneration. Recent evidence indicates that intracellular trophic signaling may be compromised in several neurodegenerative diseases. Changes in the levels of insulin-like growth factor I (IGF-I), a trophic hormone with multiple neuroprotective actions, have recently been observed in several human neurodegenerative illnesses. Therefore analysis of IGF-I pathways could help provide greater insight into trophic disturbances to neurons. However, neurodegenerative diseases with similar clinical manifestations show either high or low levels of circulating IGF-I. This apparently puzzling observation can be explained if we consider that IGF-I input to target neurons is disrupted by either lower IGF-I availability or by reduced cell sensitivity to IGF-I. The latter disturbance may be associated with high IGF-I levels. We hypothesize that in the majority of neurodegenerative diseases compromised IGF-I support to neurons emerges as part of the pathological cascade during the degenerative process and contributes to neuronal demise. In addition, loss of IGF-I input to specific neuronal populations might be the cause of a small group of neurodegenerative diseases.

Insulin-like Growth Factor-I Stimulates Dephosphorylation of IκB through the Serine Phosphatase Calcineurin (Protein Phosphatase 2B)

Astrocytes represent the most abundant cell type of the adult nervous system. Under normal conditions, astrocytes participate in neuronal feeding and detoxification. However, following brain injury, local increases in inflammatory cytokines trigger a reactive phenotype in astrocytes during which these cells produce their own inflammatory cytokines and neurotoxic free radicals. Indeed, progression of this inflammatory reaction is responsible for most neurological damage associated with brain trauma. Insulin-like growth factor-I (IGF-I) protects neurons against a variety of brain pathologies associated with glial overproduction of proinflammatory cytokines. Here, we demonstrate that in astrocyte cultures IGF-I regulates NFkappaB, a transcription factor known to play a key role in the inflammatory reaction. IGF-I induces a site-specific dephosphorylation of IkappaBalpha (phospho-Ser(32)) in astrocytes. Moreover, IGF-I-mediated dephosphorylation of IkappaBalpha protects this molecule from tumor necrosis factor alpha (TNFalpha)-stimulated degradation; therefore, IGF-I also inhibits the nuclear translocation of NFkappaB (p65) induced by TNFalpha exposure. Finally, we show that dephosphorylation of IkappaBalpha by IGF-I pathways requires activation of calcineurin. Activation of this phosphatase is independent of phosphatidylinositol 3-kinase and mitogen-activated protein kinase. Thus, these data suggest that the therapeutic benefits associated with IGF-I treatment of brain injury are derived from both its positive effects on neuronal survival and inhibition of the glial inflammatory reaction.

Calvariae from fetal mice with a disrupted Igf1 gene have reduced rates of collagen synthesis but maintain responsiveness to glucocorticoids.

The goals of this study were to examine the role of insulin-like growth factor I (IGF-I) on bone formation and to test the hypothesis that the inhibitory effects of glucocorticoids on bone formation are independent of the IGF-I pathway. In serum-free organ cultures of 18-day fetal mouse calvariae derived from Igf1 null mice (Igf1-/-) and their wild-type (Igf1+/+) and heterozygous (Igf1+/-) littermates, we measured the incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP), percent collagen synthesis (PCS), the incorporation of [3H]thymidine into DNA, and messenger RNA (mRNA) levels of osteoblast markers in the presence or absence of dexamethasone. After 24 h of culture, calvariae of all genotypes had similar levels of PCS. However, after 48-96 h of culture, PCS was significantly lower in Igf1-/- calvariae compared with Igf1+/+ calvariae. Treatment of calvariae with 100 nM of dexamethasone for 48-96 h decreased PCS in all genotypes. After 72 h of culture, [3H]thymidine incorporation was similar in all genotypes and 100 nM dexamethasone caused a significant reduction in [3H]thymidine incorporation in all genotypes. Dexamethasone at 100 nM decreased alpha1(I)-collagen (Colla1) mRNA and increased alkaline phosphatase, bone sialoprotein, and osteopontin mRNA in all genotypes after 72 h of culture. Type I IGF receptor mRNA levels were highest in Igf1-/- calvarial cultures. Dexamethasone at 100 nM increased Igf2 and type I IGF receptor mRNA levels in all genotypes. We conclude that one intact allele for Igf1 is sufficient to maintain normal rates of collagen synthesis in fetal mouse calvarial cultures. Moreover, the inhibitory effects of glucocorticoids on collagen synthesis and cell replication are at least partially independent of the IGF-I pathway in this model.

Insulin-like Growth Factor I-mediated Degradation of Insulin Receptor Substrate-1 Is Inhibited by Epidermal Growth Factor in Prostate Epithelial Cells

We have sought to determine whether insulin-like growth factor I (IGF-I) regulates the levels of insulin receptor substrate-1 (IRS-1) in prostate epithelial cells. Exposure of prostate epithelial cells to IGF-I in the absence of other growth factors leads to a reduction in IRS-1 levels. Ubiquitin content of IRS-1 is increased in the presence of IGF-I, and inhibitors of the proteasome prevented the reduction of IRS-1 levels seen following IGF-I exposure. These results imply that IRS-1 is targeted to the proteasome upon exposure to IGF-I. The addition of epidermal growth factor (EGF) maintained IRS-1 levels even in the presence of IGF-I and inhibits IGF-I-dependent ubiquitination of IRS-1. Thus, these two growth factors, IGF-I and EGF, had antagonistic effects on IRS-1 protein levels in prostate epithelial cells. This regulation of IRS-1 reveals a novel level of cross-talk between the IGF-I and EGF signal pathways, which may have implications in tumors that harbor activating mutations in the EGF receptor.