Absence Of Insulin-like Growth Factor-1 Research Articles

APELIN AS A POTENTIAL DOWNSTREAM MEDIATOR OF THE IGF-1 CARDIOPROTECTIVE PATHWAY IN THE HYPERTROPHIED MYOCARDIUM OF SPONTANEOUSLY HYPERTENSIVE RATS

Objective: Hypertension is a complex cardiovascular disease and contributes to worldwide morbidity and mortality associated to the development of hypertensive cardiac hypertrophy and the progression of heart failure. Hypertensive myocardium is characterized by mitochondrial dysfunction and redox disbalance. On the other hand, aerobic exercise upregulates several humoral factors such as insulin like growth factor-1 (IGF-1) and apelin, involved in cardioprotective effects. Particularly, whether apelin mediates IGF-1 cardioprotective effects by an autocrine paracrine mechanism is not clear yet. Objective. To determine the acute effects of IGF-1/apelin on oxidative stress and mitochondrial status, as well as the possible crosstalk between the signaling pathways triggered by them in spontaneously hypertensive rats (SHR) myocardium. Design and method: Adult SHR isolated hearts or cardiomyocytes were perfused during 10 min in the presence or absence of IGF-1 or apelin, and the specific antagonist of their receptors (AG1024 and ML221 for IGF1R and APJ, respectively). We explored reactive oxygen species (ROS) production, SOD activity, APJ cellular distribution, mitochondrial membrane potential (↖ ⇘) and permeability transition pore opening through the calcium retention capacity (CRC). Results: IGF-1 exerted an antioxidant effect evidenced by an increase in SOD activity (U/mg, IGF-1: 59.8±5.2, vs Control: 41.6±3) and decrease in ROS production (%, IGF-1: 66±12, vs Control: 100±8). In mitochondria, IGF-1 prevented loss induced by H2O2 (IGF-1: 0.934±0.016, vs Control: 0.827±0.027) and increased CRC (IGF-1: 251±34, vs Control: 135±23). Interestingly, IGF-1 upregulated APJ expression (IGF-1: 175.8±27.7 vs Control: 100±16 % mRNA) while favoring its translocation to the sarcolemma (Pearson coefficient: IGF-1: 0.880±0.011 vs Control: 0.758±0.031) in isolated cardiomyocytes. Moreover, exogenous apelin was able to emulate IGF-1 antioxidant and mitochondrial actions (SOD: 64±5.3 U/mg; ROS: 64± 7 %; ↖ ⇘ loss induced by H2O2: 0.981±0.025 and CRC 264±31). These effects were prevented by inhibition of APJ (Apelin+ML221: SOD: 33±4.4, CRC: 84±17, 0.808±0.042) but not by AG1024 (Apelin+AG: 0.985±0.068, CRC: 245±47). Conclusions: In conclusion, our data suggest that IGF-1 by sequentially activating its own receptor and the apelin/APJ pathway is responsible for cardioprotection. Therefore, apelin emerges as a downstream effector of the cardiac IGF-1 signaling pathway.

Effects of gonadotropins, 11-ketotestosterone, and insulin-like growth factor-1 on target gene expression and growth of previtellogenic oocytes from shortfinned eels, Anguilla australis, in vitro.

Pituitary gonadotropins, metabolic hormones, and sex steroids are known factors affecting the advanced stages of ovarian development in teleost fish. However, the effects of these hormones and of the interactions between them on the growth of previtellogenic ovarian follicles are not known. In order to address this void in understanding, previtellogenic ovarian fragments from eel, Anguilla australis, were incubated in vitro with recombinant Japanese eel follicle-stimulating hormone (rec-Fsh), human chorionic gonadotropin (hCG), or 11-ketotestosterone (11-KT) in the presence or absence of recombinant human insulin-like growth factor-1 (IGF1). The results of long-term in vitro culture (21days) demonstrated that rec-Fsh and 11-KT, rather than hCG, caused significant increases in the diameter of previtellogenic oocytes. Meanwhile, only 11-KT induced a significant increase in lipid accumulation. Moreover, a greater effect on oocyte growth was observed when IGF1 supplementation was combined with 11-KT rather than with rec-Fsh or hCG. For short-term culture (24h), treatment with 11-KT in the presence or absence of IGF1 had no significant effects on mRNA levels of target genes (lhr, cyp19, cyp11b, lpl, and ldr) except for upregulation of fshr. There were no significant effects of rec-Fsh on expression of any target gene, whereas hCG downregulated the expression of these genes. There was no evidence for any interaction between the gonadotropins and IGF1 that resulted in growth of previtellogenic oocytes. Taken together, these results suggest that hormones from both the reproductive and the metabolic axes regulate the growth of previtellogenic oocytes in Anguilla australis.

Abstract 5839: Growth hormone receptor (GHR) expression confers resistance to ruxolitinib in endocrine-resistant breast cancer cells

Abstract More than 30% of patients with early-stage estrogen receptor-positive breast cancer (ER+) treated with adjuvant endocrine therapy will relapse within fifteen years and all patients with metastatic breast cancer expressing ER eventually acquire resistance to antiestrogen therapy. The insulin-like growth factor 1 (IGF-1) and its receptor (IGF-1R) are involved in the development of resistance to endocrine therapies such as tamoxifen. The IGF-1/growth hormone (GH) signaling axis has been implicated as a mitogenic pathway in the development and progression of breast carcinogenesis. Preclinical data demonstrate that blockade of the IGF-1R inhibits breast cancer growth, progression, and drug resistance. Unfortunately, IGF-1R targeted therapies have failed to show a benefit in prolonging either disease-free or overall survival in clinical trials. IGF-1R inhibition results in upregulation of GH due to disruption of a negative feedback pathway. We propose GH is able to stimulate mitogenic pathways independently of IGF-IR and the GHR is a potential therapeutic target in endocrine-resistant breast cancers. Tamoxifen-resistant (TamR) and long-term estrogen deprived (LTED) cells were derived from parental MCF-7L and T47D estrogen receptor-positive breast cancer cells. The endocrine-resistant cell lines express increased levels of growth hormone receptor (GHR) mRNA compared to the parental cell lines from which they were derived when measured by RT-PCR. TamR and LTED cell lines have reduced IGF-1R expression, but continue to phosphorylate insulin receptor substrate 1(IRS-1) downstream of IGF-1R. In these endocrine-resistant cells, GH treatment activates protein kinase B (Akt), phosphoinositide 3-kinase (PI3K), and mitogen activated protein kinase (MAPK) pathways in the absence of IGF-1. To determine if GHR signals through janus kinase-2 (JAK-2) in these cells, we treated MCF-7L and T47D TamR cells with 500 nM GH, which resulted in JAK-2 phosphorylation. To examine growth effects, we treated cells with the JAK-2 inhibitor ruxolitinib. Both MCF-7L and T47D endocrine-resistant cell lines treated with ruxolitinib have increased IC50 values compared to their parental counterparts. The MCF-7L parental cells had an IC50 value of 6.7 µM compared to 20 µM and 21.7 µM in TamR and LTED cells, respectively. The T47D parental cells had an IC50 value of 28.6 µM whereas the IC50 of T47D TamR cells was 45 µM. These data indicate endocrine-resistant breast cancer cell lines with elevated GHR expression have decreased sensitivity to JAK-2 inhibition. Based on these results, GHR and its downstream signaling should be considered as a target in the development of novel therapeutics to treat breast cancers that are resistant to traditional endocrine therapies. Citation Format: Anja N. Holtz, Douglas Yee, Heather Beckwith. Growth hormone receptor (GHR) expression confers resistance to ruxolitinib in endocrine-resistant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5839.

IgA Enhances IGF-1 Mitogenic Activity Via Receptor Modulation in Glomerular Mesangial Cells: Implications for IgA-Induced Nephropathy

Background/Aim: Glomerulonephritis due to mesangial proliferation is responsible for renal dysfunction in IgA nephropathy (IgAN), however molecular mechanisms of pathogenesis are not well known. We examined the effect of IgA on Insulin-like Growth Factor-1 (IGF-1) activity, a potent mitogen with vital role in growth and development of children, and IGF-1 receptor (IGF-1R) in cultures of glomerular mesangial cells (GMC). Methods: GMC were isolated from rat kidneys using sieving and enzymatic digestion of tissue homogenates, and cultured in RPMI 1640 medium. GMC cultures were treated with IgA (0-10 µg/ml) in the presence or absence of IGF-1 and fetal bovine serum (FBS), and BrdU incorporation was measured. IGF-1 levels were assayed along with real-time PCR quantification of IGF-1R mRNA. Results: Treatment of GMC with IgA (5 -10 µg/ml) significantly (p < 0.01) increased the BrdU incorporation in the presence or absence of FBS or IGF-1. IgA-mediated effects were more pronounced in IGF-1 treated cells that were significantly (p < 0.01) blocked by pretreatment of cells with IGF-1 receptor antibody or genistein. IgA significantly increased the levels of IGF-1 in culture supernatants and GMC homogenates. IGF-1R mRNA was significantly (p < 0.01) increased in IgA treated cells particularly by co-treatment with IGF-1. Conclusion: These findings show that IgA enhances the IGF-1 activity in GMC via stimulation of IGF-1R gene transcription and suggest a role for IGF-1 in pathogenesis of IgAN.

Growth hormone (GH)-transgenic insulin-like growth factor 1 (IGF1)-deficient mice allow dissociation of excess GH and IGF1 effects on glomerular and tubular growth.

Growth hormone (GH)‐transgenic mice with permanently elevated systemic levels of GH and insulin‐like growth factor 1 (IGF1) reproducibly develop renal and glomerular hypertrophy and subsequent progressive glomerulosclerosis, finally leading to terminal renal failure. To dissociate IGF1‐dependent and ‐independent effects of GH excess on renal growth and lesion development in vivo, the kidneys of 75 days old IGF1‐deficient (I −/−) and of IGF1‐deficient GH‐transgenic mice (I −/− /G), as well as of GH‐transgenic (G) and nontransgenic wild‐type control mice (I +/+) were examined by quantitative stereological and functional analyses. Both G and I −/− /G mice developed glomerular hypertrophy, hyperplasia of glomerular mesangial and endothelial cells, podocyte hypertrophy and foot process effacement, albuminuria, and glomerulosclerosis. However, I −/− /G mice exhibited less severe glomerular alterations, as compared to G mice. Compared to I +/+ mice, G mice exhibited renal hypertrophy with a significant increase in the number without a change in the size of proximal tubular epithelial (PTE) cells. In contrast, I −/− /G mice did not display significant PTE cell hyperplasia, as compared to I −/− mice. These findings indicate that GH excess stimulates glomerular growth and induces lesions progressing to glomerulosclerosis in the absence of IGF1. In contrast, IGF1 represents an important mediator of GH‐dependent proximal tubular growth in GH‐transgenic mice.

Insulin-like growth factor 1 increases apical fibronectin in blastocysts to increase blastocyst attachment to endometrial epithelial cells in vitro.

Does insulin-like growth factor 1 (IGF1) increase adhesion competency of blastocysts to increase attachment to uterine epithelial cells in vitro? IGF1 increases apical fibronectin on blastocysts to increase attachment and invasion in an in vitro model of implantation. Fibronectin integrin interactions are important in attachment of blastocysts to uterine epithelial cells at implantation. Mouse blastocysts (hatched or near completion of hatching) were cultured in serum starved (SS) medium with varying treatments for 24, 48 or 72 h. Treatments included 10 ng/ml IGF1 in the presence or absence of the PI3 kinase inhibitor LY294002, an IGF1 receptor (IGF1R) neutralizing antibody or fibronectin. Effects of treatments on blastocysts were measured by attachment of blastocysts to Ishikawa cells, blastocyst outgrowth and fibronectin and focal adhesion kinase (FAK) localization and expression. Blastocysts were randomly allocated into control and treatment groups and experiments were repeated a minimum of three times with varying numbers of blastocysts used in each experiment. FAK and integrin protein expression on Ishikawa cells was quantified in the presence or absence of IGF1. Fibronectin expression and localization in blastocysts was studied using immunofluorescence and confocal microscopy. Global surface expression of integrin αvβ3, β3 and β1 was measured in Ishikawa cells using flow cytometry. Expression levels of phosphorylated FAK and total FAK were measured in Ishikawa cells and blastocysts by western blot and image J analysis. Blastocyst outgrowth was quantified using image J analysis. The presence of IGF1 significantly increased mouse blastocyst attachment to Ishikawa cells compared with SS conditions (P < 0.01). IGF1 treatment resulted in distinct apical fibronectin staining on blastocysts, which was reduced by the PI3 kinase inhibitor LY294002. This coincided with a significant increase in blastocyst outgrowth in the presence of IGF1 (P < 0.01) or fibronectin (P < 0.001), which was abolished by LY294002 (P < 0.001). Apical expression of integrin αvβ3, β3 and β1 in Ishikawa cells was unaltered by IGF1. However, IGF1 increased phosphorylated FAK (P < 0.05) and total FAK expression in Ishikawa cells. FAK signalling is linked to integrin activation and can affect the integrins' ability to bind and recognize extracellular matrix proteins such as fibronectin. Treatment of blastocysts with IGF1 before co-culture with Ishikawa cells increased their attachment (P < 0.05). This effect was abolished in the presence of LY294002 (P < 0.001) or an IGF1R neutralizing antibody (P < 0.05). This study uses an in vitro model of attachment that uses mouse blastocysts and human endometrial cells. This involves a species crossover and although this use has been well documented as a model for attachment (as human embryo numbers are limited) the results should be interpreted carefully. This study presents mechanisms by which IGF1 improves attachment of blastocysts to Ishikawa cells and documents for the first time how IGF1 can increase adhesion competency in blastocysts. Failure of the blastocyst to implant is the major cause of human assisted reproductive technology (ART) failure. As growth factors are absent during embryo culture, their addition to embryo culture medium is a potential avenue to improve IVF success. In particular, IGF1 could prove to be a potential treatment for blastocysts before transfer to the uterus in an ART setting.

Effects of insulin-like growth factor-1 and dexamethasone on cytokine-challenged cartilage: relevance to post-traumatic osteoarthritis

Interleukin-1 is one of the inflammatory cytokines elevated after traumatic joint injury that plays a critical role in mediating cartilage tissue degradation, suppressing matrix biosynthesis, and inducing chondrocyte apoptosis, events associated with progression to post-traumatic osteoarthritis (PTOA). We studied the combined use of insulin-like growth factor-1 (IGF-1) and dexamethasone (Dex) to block these multiple degradative effects of cytokine challenge to articular cartilage. Young bovine and adult human articular cartilage explants were treated with IL-1α in the presence or absence of IGF-1, Dex, or their combination. Loss of sulfated glycosaminoglycans (sGAG) and collagen were evaluated by the DMMB and hydroxyproline assays, respectively. Matrix biosynthesis was measured via radiolabel incorporation, chondrocyte gene expression by qRT-PCR, and cell viability by fluorescence staining. In young bovine cartilage, the combination of IGF-1 and Dex significantly inhibited the loss of sGAG and collagen, rescued the suppression of matrix biosynthesis, and inhibited the loss of chondrocyte viability caused by IL-1α treatment. In adult human cartilage, only IGF-1 rescued matrix biosynthesis and only Dex inhibited sGAG loss and improved cell viability. Thus, the combination of IGF-1 + Dex together showed combined beneficial effects in human cartilage. Our findings suggest that the combination of IGF-1 and Dex has greater beneficial effects than either molecule alone in preventing cytokine-mediated cartilage degradation in adult human and young bovine cartilage. Our results support the use of such a combined approach as a potential treatment relevant to early cartilage degradative changes associated with joint injury.

The Effects of Insulin-like Growth Factor 1 and Growth Hormone on Human Meibomian Gland Epithelial Cells

A phase 1 study has reported that dry eye disease is the most common adverse effect of human exposure to the antibody figitumumab, an anticancer drug that prevents insulin-like growth factor 1 (IGF-1) from binding to its receptor. We hypothesized that the mechanism underlying this effect is the inhibition of IGF-1 action in epithelial cells of the meibomian gland. To test the hypothesis that IGF-1 stimulates meibomian gland function in vitro and to examine whether growth hormone, a closely related hormone of IGF-1, has the same effect. DESIGN, SETTING, AND MATERIAL: Immortalized human meibomian gland epithelial cells were cultured in the presence or the absence of IGF-1, growth hormone, and an IGF-1 receptor-blocking antibody. Signaling pathways, cell proliferation, neutral lipid staining, and a key protein involved in lipid biogenesis were evaluated. Application of IGF-1 and growth hormone to human meibomian gland epithelial cells. Immunoblotting, cell counting, and neutral lipid staining. RESULTS Insulin-like growth factor 1 activated the phosphoinositol 3-kinase/Akt and forkhead box O1 pathways (showing a dose-dependent effect on immunoblotting), stimulated cellular proliferation (about 1.8-fold increase in cell number), increased sterol regulatory element-binding protein 1 expression (about 3-fold increase on immunoblotting), and promoted lipid accumulation in human meibomian gland epithelial cells (about 2-fold increase in lipid staining). These IGF-1 actions, which may be blocked by cotreatment with the anti-IGF-1 antibody, were accompanied by inconsistent effects on extracellular signal-regulated kinase phosphorylation. We were not able to demonstrate activation of Akt, forkhead box O1, extracellular signal-regulated kinase, Janus kinase 2, or signal transducers and activators of transcription 5, induced cell proliferation, or lipid accumulation in these cells by growth hormone application. Our results support the hypothesis that IGF-1 acts on human meibomian gland epithelial cells and may explain why treatment with figitumumab, the IGF-1 inhibitor, causes dry eye disease. Ophthalmic care for dry eye disease may be needed when patients with cancer undergo treatment with drugs that inhibit IGF-1 action.

Insulin-like growth factor-1 prevents dorsal root ganglion neuronal tyrosine kinase receptor expression alterations induced by dideoxycytidine in vitro.

Dideoxycytidine (zalcitabine, ddC) produces neurotoxic effects. It is particularly important to understand the toxic effects of ddC on different subpopulations of dorsal root ganglion (DRG) neurons which express distinct tyrosine kinase receptor (Trk) and to find therapeutic factors for prevention and therapy for ddC-induced peripheral sensory neuropathy. Insulin-like growth factor-1 (IGF-1) has been shown to have neurotrophic effects on DRG sensory neurons. However, little is known about the effects of ddC on distinct Trk (TrkA, TrkB, and TrkC) expression in DRG neurons and the neuroprotective effects of IGF-1 on ddC-induced neurotoxicity. Here, we have tested the extent to which the expression of TrkA, TrkB, and TrkC receptors in primary cultured DRG neurons is affected by ddC in the presence or absence of IGF-1. In this experiment, we found that exposure of 5, 25, and 50μmol/L ddC caused a dose-dependent decrease of the mRNA, protein, and the proportion of TrkA-, TrkB-, and TrkC-expressing neurons. IGF-1 (20nmol/L) could partially reverse the decrease of TrkA and TrkB, but not TrkC, expression with ddC exposure. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (10μmol/L) blocked the effects of IGF-1. These results suggested that the subpopulations of DRG neurons which express distinct TrkA, TrkB, and TrkC receptors were affected by ddC exposure. IGF-1 might relieve the ddC-induced toxicity of TrkA- and TrkB-, but not TrkC-expressing DRG neurons. These data offer new clues for a better understanding of the association of ddC with distinct Trk receptor expression and provide new evidence of the potential therapeutic role of IGF-1 on ddC-induced neurotoxicity.

Elevated nuclear Foxo1 suppresses excitability of skeletal muscle fibers

Forkhead box O 1 (Foxo1) controls the expression of proteins that carry out processes leading to skeletal muscle atrophy, making Foxo1 of therapeutic interest in conditions of muscle wasting. The transcription of Foxo1-regulated proteins is dependent on the translocation of Foxo1 to the nucleus, which can be repressed by insulin-like growth factor-1 (IGF-1) treatment. The role of Foxo1 in muscle atrophy has been explored at length, but whether Foxo1 nuclear activity affects skeletal muscle excitation-contraction (EC) coupling has not yet been examined. Here, we use cultured adult mouse skeletal muscle fibers to investigate the effects of Foxo1 overexpression on EC coupling. Fibers expressing Foxo1-green fluorescent protein (GFP) exhibit an inability to contract, impaired propagation of action potentials, and ablation of calcium transients in response to electrical stimulation compared with fibers expressing GFP alone. Evaluation of the transverse (T)-tubule system morphology, the membranous system involved in the radial propagation of the action potential, revealed an intact T-tubule network in fibers overexpressing Foxo1-GFP. Interestingly, long-term IGF-1 treatment of Foxo1-GFP fibers, which maintains Foxo1-GFP outside the nucleus, prevented the loss of normal calcium transients, indicating that Foxo1 translocation and the atrogenes it regulates affect the expression of proteins involved in the generation and/or propagation of action potentials. A reduction in the sodium channel Nav1.4 expression in fibers overexpressing Foxo1-GFP was also observed in the absence of IGF-1. We conclude that increased nuclear activity of Foxo1 prevents the normal muscle responses to electrical stimulation and that this indicates a novel capability of Foxo1 to disable the functional activity of skeletal muscle.

Promotive effect of insulin‐like growth factor‐1 for epithelial sealing to titanium implants

Improvement of oral epithelial adhesion to titanium (Ti) may significantly enhance the efficacy of dental implants. Here, we investigated whether insulin-like growth factor-1 (IGF-1) improved the sealing of the peri-implant epithelium (PIE) around the implant. Right maxillary first molars were extracted from rats and replaced with experimental implants. After 4 weeks of IGF-1 treatment, the implant-PIE interface exhibited a band of immunoreactive laminin-332 (Ln-5), similar to the tooth-junctional epithelium interface, that was partially absent in the untreated group. Immunoelectron microscopy showed a characteristic Ln-5-positive band including hemidesmosomes at both the apical and upper portions of the implant-PIE interface in the IGF-1-treated group. We also investigated the effects of IGF-1/PI3K inhibitors on the dynamics of rat oral epithelial cells (OECs) grown on Ti plates. In OECs cultured with IGF-1, adhesion protein expression increased, cell adherence to Ti plates was higher, and proliferation was faster, whereas migration and apoptosis were induced in the absence of IGF-1 or in the presence of both IGF-1 and a PI3K inhibitor. These data suggest that PI3K mediates the promotive effects of IGF-1, and that IGF-1 is effective at enhancing epithelial integration around Ti implants.

Oxidative stress due to radiation in CD34+ Hematopoietic progenitor cells: protection by IGF-1

Radiation exerts direct as well as indirect effects on DNA through the generation of reactive oxygen species (ROS). Irradiated hematopoietic progenitor cells (HPCs) experience DNA strand breaks, favoring genetic instability, due to ROS generation. Our aim was to study the effect of a range of radiation doses in HPCs and the possible protective mechanisms activated by insulin-like growth factor-1 (IGF-1). ROS generation was evaluated, in the presence or absence of IGF-1 in liquid cultures of human HPCs-CD34+ irradiated with 1-, 2- and 5-Gy X-rays, using a flow cytometry assay. Manganese superoxide dismutase (MnSOD) expression was studied by western blot analysis and visualized by an immunofluorescence assay. Apoptosis was estimated using the following assays: Annexin-V assay, DNA degradation assay, BCL-2/BAX mRNA and protein levels and caspase-9 protein immunofluorescence visualization. Viability and clonogenic potential were studied in irradiated HPCs. The generation of superoxide anion radicals at an early and a late time point was increased, while the hydrogen peroxide generation at a late time point was stable. IGF-1 presence further enhanced the radiation-induced increase of MnSOD at 24 h post irradiation. IGF-1 inhibited the mitochondria-mediated pathway of apoptosis by regulating the m-RNA and protein expression of BAX, BCL-2 and the BCL-2/BAX ratio and by decreasing caspase-9 protein expression. IGF-1 presence in culture media of irradiated cells restored the clonogenic capacity and the viability of HPCs as well. In conclusion, IGF-1 protects HPCs-CD34+ from radiation effects, by eliminating the oxidative microenvironment through the enhancement of MnSOD activation and by regulating the mitochondria-mediated pathway of apoptosis.

Bilayered constructs aimed at osteochondral strategies: The influence of medium supplements in the osteogenic and chondrogenic differentiation of amniotic fluid-derived stem cells

The development of osteochondral tissue engineered interfaces would be a novel treatment for traumatic injuries and aging associated diseases that affect joints. This study reports the development of a bilayered scaffold, which consists of both bone and cartilage regions. On the other hand, amniotic fluid-derived stem cells (AFSCs) could be differentiated into either osteogenic or chondrogenic cells, respectively. In this study we have developed a bilayered scaffolding system, which includes a starch/polycaprolactone (SPCL) scaffold for osteogenesis and an agarose hydrogel for chondrogenesis. AFSC-seeded scaffolds were cultured for 1 or 2weeks in an osteochondral-defined culture medium containing both osteogenic and chondrogenic differentiation factors. Additionally, the effect of the presence or absence of insulin-like growth factor-1 (IGF-1) in the culture medium was assessed. Cell viability and phenotypic expression were assessed within the constructs in order to determine the influence of the osteochondral differentiation medium. The results indicated that, after osteogenic differentiation, AFSCs that had been seeded onto SPCL scaffolds did not require osteochondral medium to maintain their phenotype, and they produced a protein-rich, mineralized extracellular matrix (ECM) for up to 2weeks. However, AFSCs differentiated into chondrocyte-like cells appeared to require osteochondral medium, but not IGF-1, to synthesize ECM proteins and maintain the chondrogenic phenotype. Thus, although IGF-1 was not essential for creating osteochondral constructs with AFSCs in this study, the osteochondral supplements used appear to be important to generate cartilage in long-term tissue engineering approaches for osteochondral interfaces. In addition, constructs generated from agarose–SPCL bilayered scaffolds containing pre-differentiated AFSCs may be useful for potential applications in regeneration strategies for damaged or diseased joints.

193 INSULIN-LIKE GROWTH FACTOR-1 EXERTS A THERMOPROTECTIVE ROLE ON MITOCHONDRIAL FUNCTION OF BOVINE OOCYTES EXPOSED TO HEAT SHOCK

The series of events associated with oocyte maturation are susceptible to disruption by elevated temperature. These events are regulated by a variety of growth factors, such as insulin-like growth factor-1 (IGF-1). Exposure of bovine oocytes to heat shock compromises oocyte competence and triggers apoptosis. It has been shown that cellular stresses often alter mitochondrial function and activate the mitochondrial apoptotic cascade. Therefore, the objective of this study was to determine the effect of heat shock on bovine oocyte mitochondrial activity and the role of IGF-1 in this context. Slaughterhouse derived cumulus–oocyte complexes (COC) were subjected to control (38.5°C for 22 h) and heat shock (41°C for 14 h, followed by 38.5°C for 8 h) treatments in the presence of 0 or 100 ng mL–1 of IGF-1 during in vitro maturation (IVM). After 22 h, IVM COC were mechanically denuded and subjected to MitoTracker Red CMX-Ros assay (Invitrogen M-7512) to localize and quantify active mitochondria. Denuded oocytes were incubated in TCM-199-HEPES containing 10 μg mL–1 of polyvinyl alcohol and 50 nM MitoTracker at 37°C for 15 min. Oocytes were evaluated under fluorescence microscope and digital images were obtained and stored as TIFF files. Mitochondrial activity from each oocyte was quantified using the software Image J 1.43. This experiment was replicated 6 times using 97 to 204 COC/treatment. Data were analyzed by least-squares analysis of variance using the general linear model procedure of SAS. In the absence of IGF-1, heat shock reduced (P &lt; 0.001) mitochondrial activity from 64.31 ± 1.91 to 56.74 ± 1.26 arbitrary units for control and heat shock groups, respectively. Addition of IGF-1 to maturation medium did not affect mitochondrial activity in the control group (66.25 ± 1.56). However, IGF-1 improved (temperature × IGF-1; P &lt; 0.001) mitochondrial activity of bovine oocytes subjected to heat shock (70.32 ± 1.32). In conclusion, heat shock reduced bovine oocyte mitochondrial activity, suggesting activation of mitochondrial apoptotic cascade. Moreover, IGF-1 exerted a thermoprotective role, reducing the mitochondrial damage caused by elevated temperature.

Growth Factor-Dependent Trafficking of Cerebellar NMDA Receptors via Protein Kinase B/Akt Phosphorylation of NR2C

NMDA receptor subunit composition varies throughout the brain, providing molecular diversity in NMDA receptor function. The NR2 subunits (NR2A-D) in large part dictate the distinct functional properties of NMDA receptors and differentially regulate receptor trafficking. Although the NR2C subunit is highly enriched in cerebellar granule cells and plays a unique role in cerebellar function, little is known about NR2C-specific regulation of NMDA receptors. Here, we demonstrate that PKB/Akt directly phosphorylates NR2C on serine 1096 (S1096). In addition, we identify 14-3-3epsilon as an NR2C interactor, whose binding is dependent on S1096 phosphorylation. Both growth factor stimulation and NMDA receptor activity lead to a robust increase in both phosphorylation of NR2C on S1096 and surface expression of cerebellar NMDA receptors. Finally, we find that NR2C expression, unlike NR2A and NR2B, supports neuronal survival. Thus, our data provide a direct mechanistic link between growth factor stimulation and regulation of cerebellar NMDA receptors.

Chronic-alcohol exposure alters IGF1 signaling in H9c2 cells via changes in PKC delta

Previously, we have demonstrated that chronic-alcohol exposure alters insulin-like growth factor 1 (IGF1) signaling in adult rat heart cells. This report examines the effects of alcohol in vitro on the expression of protein kinase C (PKC) alpha, delta, and epsilon using the embryonic heart cell line, H9c2, and how this may be linked to changes in IGF1 signal transduction. Western blot analyses of H9c2 protein preparations demonstrate that there are significant increases in the total protein levels of PKC delta and epsilon after 4 days exposure to alcohol, and similar increases were found after 2 and 6 days exposure. In addition, there was a significant increase in PKC delta and epsilon in the membranal fractions and a decrease in the cytosolic fractions. No change was found in the expression or activity levels for PKC alpha. Chronic-alcohol exposure (100 mM, 4 days) increased the basal tyrosine kinase activity of the IGF1 receptor (IGF1R), and altered its rate of activation. Chronic-alcohol exposure also reduced the rate of Erk1/Erk2 activation by IGF1. Chronic alcohol blocked the proliferative effects of IGF1 on cell growth and reduced cell viability both in the presence and absence of IGF1, and this alcohol-induced reduction in cell viability was blocked using siRNA to inhibit PKC delta. In addition, a reduction in the amount of myosin light chain 2 was found in the alcohol-exposed cells. In conclusion, chronic alcohol alters PKC delta and epsilon expression and activity, and suppresses the IGF1 signaling pathway in embryonic heart cell culture. Blockage of PKC delta expression using siRNA inhibits the suppressive effects of alcohol on cell viability.

Cumulative Activation of Akt and Consequent Inhibition of Glycogen Synthase Kinase-3 by Brain-Derived Neurotrophic Factor and Insulin-Like Growth Factor-1 in Cultured Hippocampal Neurons

Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) seem to play key roles in mediating neuronal plasticity in the hippocampus. In the current studies, we have used cultured hippocampal neurons to study possible interactions between the two growth factors in modulating neuronal signaling pathways. BDNF and IGF-1 were found to each effectively activate the neuroprotective Akt pathway, with the magnitude of activation being at least additive when cultures were simultaneously treated with supramaximal concentrations of peptides. Likewise, a cumulative inhibitory Akt-dependent phosphorylation of proapoptotic glycogen synthase kinase-3 was observed. Immunofluorescent studies demonstrated that a single population of neurons responded to BDNF and IGF-1. In contrast, the magnitude of BDNF-stimulated extracellular signal-regulated kinase (ERK) activation was found to be much greater than that of IGF-1-stimulated ERK, such that the difference in magnitude stimulated by BDNF in the presence and absence of IGF-1 did not reach statistical significance. Consistent with the observed agonist-stimulated activation of Akt, BDNF and IGF-1 were both found to act as neurotrophins, enhancing neuronal survival under low-insulin culture conditions. Maximal survival was achieved when both growth factors were present. These findings provide insight into the significance of multiple growth factors stimulating activation of ERK and Akt in the central nervous system. In some cases, the magnitude of activation required to elicit biological responses may be achieved only with a combination of compounds.

Identification of calcium-regulated heat-stable protein of 24 kDa (CRHSP24) as a physiological substrate for PKB and RSK using KESTREL

A substrate for PKBalpha (protein kinase Balpha) was detected in liver extracts, and was purified and identified as CRHSP24 (calcium-regulated heat-stable protein of apparent molecular mass 24 kDa). PKBalpha, as well as SGK1 (serum- and glucocorticoid-induced protein kinase 1) and RSK (p90 ribosomal S6 kinase), phosphorylated CRHSP24 stoichiometrically at Ser52 in vitro and its brain-specific isoform PIPPin at the equivalent residue (Ser58). CRHSP24 became phosphorylated at Ser52 when HEK-293 (human embryonic kidney) cells were stimulated with IGF-1 (insulin-like growth factor-1) and this was prevented by inhibitors of PI3K (phosphoinositide 3-kinase), but not by rapamycin [an inhibitor of mTOR (mammalian target of rapamycin)] or PD 184352, an inhibitor of the classical MAPK (mitogen-activated protein kinase) cascade and hence the activation of RSK. IGF-1 induced a similar phosphorylation of CRHSP24 in ES (embryonic stem) cells from wild-type mice or mice that express the PDK1 (3-phosphoinositide-dependent kinase 1) mutant (PDK1[L155E]) that activates PKBalpha normally, but cannot activate SGK. CRHSP24 also became phosphorylated at Ser52 in response to EGF (epidermal growth factor) and this was prevented by blocking activation of both the classical MAPK cascade and the activation of PKBalpha, but not if just one of these pathways was inhibited. DYRK2 (dual-specificity tyrosine-phosphorylated and -regulated protein kinase 2) phosphorylated CRHSP24 at Ser30, Ser32 and Ser41 in vitro, and Ser41 was identified as a site phosphorylated in cells. These and other results demonstrate that CRHSP24 is phosphorylated at Ser52 by PKBalpha in response to IGF-1, at Ser52 by PKBalpha and RSK in response to EGF, and at Ser41 in the absence of IGF-1/EGF by a DYRK isoform or another proline-directed protein kinase(s).

Insulin-like growth factor-binding protein 6 inhibits survival and differentiation of rat oligodendrocyte precursor cells.

Insulin-like growth factor 1 (IGF-1) is a growth and survival factor for oligodendrocyte lineage cells and promotes myelination. We demonstrate that IGF-binding protein 6 (IGFBP-6) is expressed and localized to the Golgi complex in rat oligodendrocyte precursor (O2A) cells. IGFBP-6 mRNA showed a developmentally regulated expression pattern, displaying a transient decrease during early development, and enhanced levels upon cell maturation. IGFBP-6 mRNA expression could be reduced by addition of basic fibroblast growth factor and progesterone while estrogen increased IGFBP-6 mRNA. IGF-1, platelet-derived growth factor, and insulin had no effect. When added exogenously, IGFBP-6 reduced O2A cell survival in the absence of IGF-1 and inhibited IGF-1-stimulated survival in a partially IGF-1-dependent and partially IGF-1-independent fashion. In addition, IGFBP-6 reduced the IGF-stimulated expression of two myelin proteins, CNPase and MAG. Taken together, the data show that IGFBP-6 is a new negative effector of oligodendrocyte survival and differentiation.

Rapid Identification of Small Binding Motifs with High-Throughput Phage Display: Discovery of Peptidic Antagonists of IGF-1 Function

A panel of 22 naı̈ve peptide libraries was constructed in a polyvalent phage display format and sorted against insulin-like growth factor-1 (IGF-1). The libraries were pooled to achieve a total diversity of 4.4 × 10 11. After three rounds of selection, the majority of the phage clones bound specifically to IGF-1, with a disulfide-constrained CX 9C scaffold dominating the selection. Four monovalently displayed sub-libraries were designed on the basis of these conserved motifs. Sub-library maturation in a monovalent format yielded an antagonistic peptide that inhibited the interactions between IGF-1 and two cell-surface receptors and those between IGF-1 and two soluble IGF binding proteins with micromolar potency. NMR analysis revealed that the peptide is highly structured in the absence of IGF-1, and peptides that preorganize the binding elements were selected during the sorting.