IGF-1 Stimulation Research Articles

Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation

ObjectivesInsulin receptor (IR)-mediated signaling is involved in the regulation of pluripotent stem cells; however, its direct effects on regulating the maintenance of pluripotency and lineage development are not fully understood. The main objective of this study is to understand the role of IR signaling in pluripotency and lineage development. MethodsTo explore the role of IR signaling, we generated IR knock-out (IRKO) mouse induced pluripotent stem cells (miPSCs) from E14.5 mouse embryonic fibroblasts (MEFs) of global IRKO mice using a cocktail of four reprogramming factors: Oct4, Sox2, Klf4, cMyc. We performed pluripotency characterization and directed the differentiation of control and IRKO iPSCs into neural progenitors (ectoderm), adipocyte progenitors (mesoderm), and pancreatic beta-like cells (endoderm). We mechanistically confirmed these findings via phosphoproteomics analyses of control and IRKO iPSCs. ResultsInterestingly, expression of pluripotency markers including Klf4, Lin28a, Tbx3, and cMyc were upregulated, while abundance of Oct4 and Nanog were enhanced by 4-fold and 3-fold, respectively, in IRKO iPSCs. Analyses of signaling pathways demonstrated downregulation of phospho-STAT3, p-mTor and p-Erk and an increase in the total mTor and Erk proteins in IRKO iPSCs in the basal unstimulated state. Stimulation with leukemia inhibitory factor (LIF) showed a ∼33% decrease of phospho-ERK in IRKO iPSCs. On the contrary, Erk phosphorylation was increased during in vitro spontaneous differentiation of iPSCs lacking IRs. Lineage-specific directed differentiation of the iPSCs revealed that cells lacking IR showed enhanced expression of neuronal lineage markers (Pax6, Tubb3, Ascl1 and Oligo2) while exhibiting a decrease in adipocyte (Fas, Acc, Pparγ, Fabp4, C/ebpα, and Fsp27) and pancreatic beta cell markers (Ngn3, Isl1, and Sox9). Further molecular characterization by phosphoproteomics confirmed the novel IR-mediated regulation of the global pluripotency network including several key proteins involved in diverse aspects of growth and embryonic development. ConclusionWe report, for the first time to our knowledge, the phosphoproteome of insulin, IGF1, and LIF stimulation in mouse iPSCs to reveal the importance of insulin receptor signaling for the maintenance of pluripotency and lineage determination.

IGF1 suppresses cholesterol accumulation in the liver of growth hormone-deficient mice via the activation of ABCA1.

Recently, several clinical studies have suggested that adult growth hormone (GH) deficiency that also has low concentration of IGF1 is associated with an increased prevalence of fatty liver (FL). ATP-binding cassette transporter A1 (ABCA1) is a pivotal regulator of lipid efflux from cells to apolipoproteins and plays an important role on formation of FL. In this study, we determined the effects of IGF1 on ABCA1 expression in GH-deficient mice to clarify its effects on FL. Western blotting, real-time PCR, and a luciferase assay were employed to examine the effect of IGF1. The binding of FoxO1 to the ABCA1 promoter was assessed by chromatin immunoprecipitation (ChIP) assay. Cholesterol accumulation was analyzed by Oil Red O stain and cholesterol content measurement. We confirmed that IGF1 upregulated the ABCA1 expression. The activity of a reporter construct containing the ABCA1 promoter was induced by IGF1, and this effect was blocked by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). Constitutively active Akt stimulated the ABCA1 promoter activity, and a dominant-negative mutant of Akt or mutagenesis of the FoxO1 response element abolished the effect of IGF1. A ChIP assay indicated that FoxO1 mediated IGF1 transcriptional activity by directly binding to the ABCA1 promoter region. For in vivo experiments, we used an inhibitor for the GH receptor (Pegvisomant) to reduce the IGF1 level. A high-fat diet induced FL in mice (C57BL/6J) given Pegvisomant treatment. IGF1 treatment stimulated ABCA1 expression to improve cholesterol accumulation in these mice. These results show that the PI3K/Akt/FoxO1 pathway contributes to the regulation of ABCA1 expression in response to IGF1 stimulation that suppressed FL in GH-deficient mice.

Proteomic Analysis of Charcoal-Stripped Fetal Bovine Serum Reveals Changes in the Insulin-like Growth Factor Signaling Pathway.

Charcoal-stripped fetal bovine serum (CS-FBS) is commonly used to study androgen responsiveness and androgen metabolism in cultured prostate cancer (CaP) cells. Switching CaP cells from FBS to CS-FBS may reduce the activity of androgen receptor (AR), inhibit cell proliferation, or modulate intracellular androgen metabolism. The removal of proteins by charcoal stripping may cause changes in biological functions and has not yet been investigated. Here we profiled proteins in FBS and CS-FBS using an ion-current-based quantitative platform consisting of reproducible surfactant-aided precipitation/on-pellet digestion, long-column nanoliquid chromatography separation, and ion-current-based analysis. A total of 143 proteins were identified in FBS, among which 14 proteins including insulin-like growth factor 2 (IGF-2) and IGF binding protein (IGFBP)-2 and -6 were reduced in CS-FBS. IGF-1 receptor (IGF1R) and insulin receptor were sensitized to IGFs in CS-FBS. IGF-1 and IGF-2 stimulation fully compensated for the loss of AR activity to maintain cell growth in CS-FBS. Endogenous production of IGF and IGFBPs was verified in CaP cells and clinical CaP specimens. This study provided the most comprehensive protein profiles of FBS and CS-FBS and offered an opportunity to identify new protein regulators and signaling pathways that regulate AR activity, androgen metabolism, and proliferation of CaP cells.

Abstract 1914: Elongation factor 2: A novel target for cancer progression

Abstract Protein translation is one the most energy demanding and highly regulated processes in cells. For this reason, dysregulation of protein synthesis is linked with different types of disease, including cancer. The link between protein synthesis and cancer progression is becoming a central theme among cancer research. Although numerous studies have identified protein synthesis as a process central to cancer progression, further research is required to identify potential novel targets and/or biomarkers. Recently, RACK1 (Receptor for Activated C Kinase 1), a scaffold protein involved in many cell regulatory pathways including cell proliferation and migration, has been found to be part of the ribosomal machinery. Here, RACK1 is able to regulate the synthesis of particular sets of proteins and the translation itself. In fact, RACK1 is located close the mRNA exit channel where is able to recruit kinases, phosphatases and specific mRNA binding proteins. We have identified elongation factor 2 (eEF2) as novel RACK1 interacting protein in our two and three dimensional colon cancer models. Elongation factor 2 is one of the main proteins regulating the elongation phase of protein synthesis, responsible for the translocation of the mRNA along the ribosome. We have confirmed the binding between eEF2 and RACK1 by isolating and purifying a native human form of eEF2 and combining it with a purified human version of RACK1. Interestingly, we have demonstrated that stress conditions and IGF-1 stimulation modulate eEF2 activity in colon cancer cells. Moreover, we have examined the expression of EEF2 and have found that EEF2 gene is dysregulated in our cohort of matched normal and cancer colonic patient tissues. Interestingly, EEF2 expression is upregulated in mucinous cancers (a rare and aggressive type of colorectal cancer) compared to non-mucinous cancers and the expression of EEF2 is as well upregulated in cancers that have recurred within 2 to 3 years after the first surgery. We believe that eEF2 is playing an important role in cancer progression and migration especially by regulating and modulating protein synthesis in certain conditions. A better understanding of the role of this protein will help in the identification of novel targets that can be then translated into therapeutic strategies against colorectal cancer. Citation Format: Beatrice Malacrida, Catríona M. Dowling, Rasmus K. Flygaard, Maeve Kiely, John C. Coffey, Lasse B. Jenner, Patrick A. Kiely. Elongation factor 2: A novel target for cancer progression [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 1914.

IGF1/IGF1R and microRNA let-7e down-regulate each other and modulate proliferation and migration of colorectal cancer cells

ABSTRACT MicroRNA let-7 has been reported to be down-regulated in several human cancers and is now characterized as a tumor suppressor. IGF1R is over-expressed in many cancers and IGF1/IGF1R pathway is attractive target for anticancer therapy. However, the crosstalk between let-7 and IGF1/IGF1R are largely unknown. The present study showed IGF1R were significantly over-expressed in colorectal cancer tissues compared with adjacent normal tissues through immunohistochemical analysis. qRT-PCR results showed that let-7a, let-7b and let-7e were down-regulated in colorectal cancer tissues. Bioinformatics analysis revealed that both IGF1 and IGF1R mRNA are potential targets for let-7 miRNA family. Ectopic transfection of let-7e led to a significant reduction in IGF1R at protein level and their downstream Akt inhibition, as well as a reduction in cell proliferation, migration and invasion in colorectal cancer cells, while inhibition of let-7e enhanced the expression of IGF1R. On the other hand, IGF1 stimulation can significantly down-regulate the expression of let-7e in colorectal cancer cells. Taken together, our findings identify a negative feedback regulation between let-7e and IGF1/IGF1R, and suggest that let-7e could be used in IGF1R-targeted therapeutics in anticancer therapy. Abbreviations: IGF1: insulin-like growth factor 1; IGF1R: IGF1 receptor; miRNA: microRNA; CRC: colorectal cancer; EGFR: epidermal growth factor receptor; HRP: horseradish peroxidase; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; p-Akt: phospho-Akt; PI3K: phosphoinositide 3-kinase; qRT–PCR: quantitative reverse transcription–PCR; IHC: immunohistochemical; siRNA: small interfering RNA; 3′-UTR: 3′-untranslated region

Distinct signalling properties of insulin receptor substrate (IRS)-1 and IRS-2 in mediating insulin/IGF-1 action

Insulin/IGF-1 action is driven by a complex and highly integrated signalling network. Loss-of-function studies indicate that the major insulin/IGF-1 receptor substrate (IRS) proteins, IRS-1 and IRS-2, mediate different biological functions in vitro and in vivo, suggesting specific signalling properties despite their high degree of homology. To identify mechanisms contributing to the differential signalling properties of IRS-1 and IRS-2 in the mediation of insulin/IGF-1 action, we performed comprehensive mass spectrometry (MS)-based phosphoproteomic profiling of brown preadipocytes from wild type, IRS-1−/− and IRS-2−/− mice in the basal and IGF-1-stimulated states. We applied stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of changes in protein phosphorylation. We found ~10% of the 6262 unique phosphorylation sites detected to be regulated by IGF-1. These regulated sites included previously reported substrates of the insulin/IGF-1 signalling pathway, as well as novel substrates including Nuclear Factor I X and Semaphorin-4B. In silico prediction suggests the protein kinase B (PKB), protein kinase C (PKC), and cyclin-dependent kinase (CDK) as the main mediators of these phosphorylation events. Importantly, we found preferential phosphorylation patterns depending on the presence of either IRS-1 or IRS-2, which was associated with specific sets of kinases involved in signal transduction downstream of these substrates such as PDHK1, MAPK3, and PKD1 for IRS-1, and PIN1 and PKC beta for IRS-2. Overall, by generating a comprehensive phosphoproteomic profile from brown preadipocyte cells in response to IGF-1 stimulation, we reveal both common and distinct insulin/IGF-1 signalling events mediated by specific IRS proteins.

Decreased Frequencies of Peripheral Blood CD4+CD25+CD127-Foxp3+ in Patients with Graves' Disease and Graves' Orbitopathy: Enhancing Effect of Insulin Growth Factor-1 on Treg Cells.

Graves' orbitopathy (GO) is characterized by orbital T cell infiltration. We evaluated the regulatory T (Treg) cell fractions induced with IGF-1 in Graves' disease (GD) with and without GO. Peripheral blood mononuclear cells (PBMCs) were obtained from 13 patients with GD without eye manifestations; 10 patients with active GO; and 12 patients with nodular goiter (NG). All the patients from GD, GO, and NG were subclinical hyperthyroid. We analyzed the expression of Treg cell markers (CD4, CD25, CD127-, Foxp3) on T cells and their ability to respond to IGF-1 stimulation. In patients with GD without GO, we found lowered percentages of CD4+ Foxp3+ cells, as compared to nodular goiter 1.77 vs. 5.42% (p=0.0276). Similarly, significantly reduced frequencies of CD4+CD25+CD127-Foxp3+ and CD4+CD25+CD127- cells were observed in GD patients as compared to nodular goiter patients with hyperthyreosis, (0.7 vs. 1.48%) (p=0.0071) and (14.5 vs. 37.2%) (p=0.0051), respectively. In GO with active GO, only the percentage of CD4+CD25+CD127- cells was found to be decreased versus nodular goiter (9.35 vs. 37.2) (p=0.0275). Stimulation of PBMC derived from GO patients with IGF-1 resulted in significant increase of frequency of both CD4+ Foxp3+ and CD4+CD25+CD127- Foxp3 cells. Decreased frequencies of peripheral blood CD4+CD25+CD127-Foxp3+ in patients with GD and GO could be an useful marker of autoimmune process and perhaps a possible target for future therapies. This is the first study demonstrating Treg-enhancing effects of IGF-1. Thus IGF-1 can be accounted for modulating Treg cell-related action in GO.

Induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density

BackgroundIn previous studies it has been shown that bovine granulosa cells (GC) cultured at a high plating density dramatically change their physiological and molecular characteristics, thus resembling an early stage of luteinization. During the present study, these specific effects on the GC transcriptome were comprehensively analysed to clarify the underlying mechanisms.MethodsGC were cultured in serum free medium with FSH and IGF-1 stimulation at different initial plating density. The estradiol and progesterone production was determined by radioimmunoassays and the gene expression profiles were analysed by mRNA microarray analysis after 9 days. The data were statistically analysed and the abundance of selected, differentially expressed transcripts was re-evaluated by qPCR. Bioinformatic pathway analysis of density affected transcripts was done using Ingenuity Pathway Analysis.ResultsThe data showed that at high plating density the expression of 1510 annotated genes, represented by 1575 transcript clusters, showed highly altered expression levels. Nearly two-thirds were up- and one third down-regulated. Within the top up-regulated genes VNN2, RGS2 and PTX3 could be identified, as well as HBA or LOXL2. Down-regulated genes included important key genes of folliculogenesis like CYP19A1 and FSHR. Ingenuity pathway analysis identified “AMPK signaling” as well as “cAMP-mediated signaling” as major pathways affected by the alteration of the expression profile. Main putative upstream regulators were TGFB1 and VEGF, thus indicating a connection with cell differentiation and angiogenesis. A detailed cluster analysis revealed one single cluster that was highly associated with the upstream regulator beta-estradiol. Within this cluster key genes of steroid biosynthesis were not included, but instead, other genes importantly involved in follicular development, like OXT and VEGFA as well as the three most down-regulated genes TXNIP, PAG11 and ARRDC4 were identified.ConclusionsFrom these data we hypothesize that high density conditions induce a stage of differentiation in cultured GC that is similar to early post-LH conditions in vivo. Furthermore we hypothesize that specific cell-cell-interactions led to this differentiation including transformations necessary to promote angiogenesis.

Acute Ethanol Increases IGF-I-Induced Phosphorylation of ERKs by Enhancing Recruitment of p52-Shc to the Grb2/Shc Complex.

Ethanol plays a detrimental role in the development of the brain. Multiple studies have shown that ethanol inhibits insulin-like growth factor I receptor (IGF-IR) function. Because the IGF-IR contributes to brain development by supporting neural growth, survival, and differentiation, we sought to determine the molecular mechanism(s) involved in ethanol's effects on this membrane-associated tyrosine kinase. Using multiple neuronal cell types, we performed Western blot, immunoprecipitation, and GST-pulldowns following acute (1-24 h) or chronic (3 weeks) treatment with ethanol. Surprisingly, exposure of multiple neuronal cell types to acute (up to 24 h) ethanol (50 mM) enhanced IGF-I-induced phosphorylation of extracellular regulated kinases (ERKs), without affecting IGF-IR tyrosine phosphorylation itself, or Akt phosphorylation. This acute increase in ERKs phosphorylation was followed by the expected inhibition of the IGF-IR signaling following 3-week ethanol exposure. We then expressed a GFP-tagged IGF-IR construct in PC12 cells and used them to perform fluorescence recovery after photobleaching (FRAP) analysis. Using these fluorescently labeled cells, we determined that 50 mM ethanol decreased the half-time of the IGF-IR-associated FRAP, which implied that cell membrane-associated signaling events could be affected. Indeed, co-immunoprecipitation and GST-pulldown studies demonstrated that the acute ethanol exposure increased the recruitment of p52-Shc to the Grb2-Shc complex, which is known to engage the Ras-Raf-ERKs pathway following IGF-1 stimulation. These experiments indicate that even a short and low-dose exposure to ethanol may dysregulate function of the receptor, which plays a critical role in brain development. J. Cell. Physiol. 232: 1275-1286, 2017. © 2016 Wiley Periodicals, Inc.

CD99 triggering induces methuosis of Ewing sarcoma cells through IGF-1R/RAS/Rac1 signaling.

CD99 is a cell surface molecule that has emerged as a novel target for Ewing sarcoma (EWS), an aggressive pediatric bone cancer. This report provides the first evidence of methuosis in EWS, a non-apoptotic form of cell death induced by an antibody directed against the CD99 molecule. Upon mAb triggering, CD99 induces an IGF-1R/RAS/Rac1 complex, which is internalized into RAB5-positive endocytic vacuoles. This complex is then dissociated, with the IGF-1R recycling to the cell membrane while CD99 and RAS/Rac1 are sorted into immature LAMP-1-positive vacuoles, whose excessive accumulation provokes methuosis. This process, which is not detected in CD99-expressing normal mesenchymal cells, is inhibited by disruption of the IGF-1R signaling, whereas enhanced by IGF-1 stimulation. Induction of IGF-1R/RAS/Rac1 was also observed in the EWS xenografts that respond to anti-CD99 mAb, further supporting the role of the IGF/RAS/Rac1 axis in the hyperstimulation of macropinocytosis and selective death of EWS cells. Thus, we describe a vulnerability of EWS cells, including those resistant to standard chemotherapy, to a treatment with anti-CD99 mAb, which requires IGF-1R/RAS signaling but bypasses the need for their direct targeting. Overall, we propose CD99 targeting as new opportunity to treat EWS patients resistant to canonical apoptosis-inducing agents.

The Proprotein Convertase Furin Contributes to Rhabdomyosarcoma Malignancy by Promoting Vascularization, Migration and Invasion.

The proprotein convertase (PC) furin cleaves precursor proteins, an important step in the activation of many cancer-associated proteins. Substrates of furin and furin-like PCs play a role in proliferation, metastasis and invasion. Some of them are involved in the progression of the pediatric soft tissue sarcoma rhabdomyosarcoma (RMS). In this study, we show that PCs, and in particular furin, are expressed in RMS cell lines. To investigate the functional role of furin, we generated RMS cell lines with modulated furin activity. Silencing or stable inhibition of furin delayed tumor growth in Rh30 and RD xenografts in vivo, and was correlated with lower microvessel density. Reduced furin activity also decreased migration and invasion abilities in vitro, and inhibition of furin in RMS cells diminished processing of IGF1R, VEGF-C, PDGF-B and MT1-MMP, leading to lower levels of mature proteins. Furthermore, we found that furin activity is required for proper IGF signaling in RMS cells, as furin silencing resulted in reduced phosphorylation of Akt upon IGF1 stimulation. Taken together, our results suggest that furin plays an important role in the malignant phenotype of RMS cells by activating proteins involved in tumor growth and vascularization, metastasis and invasion.

Nuclearly translocated insulin-like growth factor 1 receptor phosphorylates histone H3 at tyrosine 41 and induces SNAI2 expression via Brg1 chromatin remodeling protein.

The insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase that has crucial roles in cell proliferation and protection from apoptosis. It is therefore not surprising that IGF-1R is often found overexpressed in many types of tumors. This has made IGF-1R a prominent target molecule for pharmacological companies to develop new anti-cancer agents. However, several clinical trials during the last 5 years using IGF-1R specific antibodies have shown disappointing results. We have previously shown that upon IGF-1 stimulation, the receptor becomes SUMOylated and translocates into the nucleus of cancer cells to act as a transcription co-factor. Soon after our original study, several others have reported nuclear IGF-1R (nIGF-1R) as well, and some of them have demonstrated a prognostic value of nIGF-1R expression in cancer. In the current study we demonstrate that nIGF-1R binds to and phosphorylates histone H3 at tyrosine 41 (H3Y41) in HeLa cells. Furthermore, our results suggest that phosphorylation of H3Y41 by nIGF-1R, stabilizes the binding of Brg1 chromatin remodeling protein to Histone H3. Our findings suggest that phosphorylated nIGF-1R, rather than total nIGF-1R, plays a superior role in these contexts. We identified SNAI2 oncogene as a target gene for nIGF-1R and its expression was decreased upon mutation of H3Y41 or by Brg1 knockdown. Furthermore, we demonstrate that both IGF-1R and Brg1 binds to the SNAI2 promoter. As SNAI2 protein is implicated in e.g. cancer invasion and metastasis, the nIGF-1R-mediated effects shown in this study may influence such important tumor phenotypic actions.

PTP1B Deficiency Enables the Ability of a High-Fat Diet to Drive the Invasive Character of PTEN-Deficient Prostate Cancers.

Diet affects the risk and progression of prostate cancer, but the interplay between diet and genetic alterations in this disease is not understood. Here we present genetic evidence in the mouse showing that prostate cancer progression driven by loss of the tumor suppressor Pten is mainly unresponsive to a high-fat diet (HFD), but that coordinate loss of the protein tyrosine phosphatase Ptpn1 (encoding PTP1B) enables a highly invasive disease. Prostate cancer in Pten(-/-)Ptpn1(-/-) mice was characterized by increased cell proliferation and Akt activation, interpreted to reflect a heightened sensitivity to IGF-1 stimulation upon HFD feeding. Prostate-specific overexpression of PTP1B was not sufficient to initiate prostate cancer, arguing that it acted as a diet-dependent modifier of prostate cancer development in Pten(-/-) mice. Our findings offer a preclinical rationale to investigate the anticancer effects of PTP1B inhibitors currently being studied clinically for diabetes treatment as a new modality for management of prostate cancer. Cancer Res; 76(11); 3130-5. ©2016 AACR.

Neuroprotection by GH against excitotoxic-induced cell death in retinal ganglion cells

Retinal growth hormone (GH) has been shown to promote cell survival in retinal ganglion cells (RGCs) during developmental waves of apoptosis during chicken embryonic development. The possibility that it might also against excitotoxicity-induced cell death was therefore examined in the present study, which utilized quail-derived QNR/D cells as an in vitro RGC model. QNR/D cell death was induced by glutamate in the presence of BSO (buthionine sulfoxamide) (an enhancer of oxidative stress), but this was significantly reduced (P<0.01) in the presence of exogenous recombinant chicken GH (rcGH). Similarly, QNR/D cells that had been prior transfected with a GH plasmid to overexpress secreted and non-secreted GH. This treatment reduced the number of TUNEL-labeled cells and blocked their release of lactate dehydrogenase (LDH). In a further experiment with dissected neuroretinal explants from ED (embryonic day) 10 embryos, rcGH treatment of the explants also reduced (P<0.01) the number of glutamate-BSO-induced apoptotic cells and blocked the explant release of LDH. This neuroprotective action was likely mediated by increased STAT5 phosphorylation and increased bcl-2 production, as induced by exogenous rcGH treatment and the media from GH-overexpressing QNR/D cells. As rcGH treatment and GH-overexpression cells also increased the content of IGF-1 and IGF-1 mRNA this neuroprotective action of GH is likely to be mediated, at least partially, through an IGF-1 mechanism. This possibility is supported by the fact that the siRNA knockdown of GH or IGF-1 significantly reduced QNR/D cell viability, as did the immunoneutralization of IGF-1. GH is therefore neuroprotective against excitotoxicity-induced RGC cell death by anti-apoptotic actions involving IGF-1 stimulation.

TSH/IGF-1 Receptor Cross Talk in Graves' Ophthalmopathy Pathogenesis.

The TSH receptor (TSHR) is considered the main target of stimulatory autoantibodies in the pathogenesis of Graves' ophthalmopathy (GO); however, it has been suggested that stimulatory IGF-1 receptor (IGF-1R) autoantibodies also play a role. We previously demonstrated that a monoclonal stimulatory TSHR antibody, M22, activates TSHR/IGF-1R cross talk in orbital fibroblasts/preadipocytes obtained from patients with GO (GO fibroblasts [GOFs]). We show that cross talk between TSHR and IGF-1R, not direct IGF-1R activation, is involved in the mediation of GO pathogenesis stimulated by Graves' autoantibodies. Immunoglobulins were purified from the sera of 57 GO patients (GO-Igs) and tested for their ability to activate TSHR and/or IGF-1R directly and TSHR/IGF-1R cross talk in primary cultures of GOFs. Cells were treated with M22 or GO-Igs with or without IGF-1R inhibitory antibodies or linsitinib, an IGF-1R kinase inhibitor. Hyaluronan (hyaluronic acid [HA]) secretion was measured as a major biological response for GOF stimulation. IGF-1R autophosphorylation was used as a measure of direct IGF-1R activation. TSHR activation was determined through cAMP production. A total of 42 out of 57 GO-Ig samples stimulated HA secretion. None of the GO-Ig samples exhibited evidence for IGF-1R autophosphorylation. Both anti-IGF-1R antibodies completely inhibited IGF-1 stimulation of HA secretion. By contrast, only 1 IGF-1R antibody partially blocked HA secretion stimulated by M22 or GO-Igs in a manner similar to linsitinib, whereas the other IGF-1R antibody had no effect on M22 or GO-Ig stimulation. These findings show that the IGF-1R is involved in GO-Igs stimulation of HA secretion without direct activation of IGF-1R. IGF-1R activation by GO-Igs occurs via TSHR/IGF-1R cross talk rather than direct binding to IGF-1R, and this cross talk is important in the pathogenesis of GO.

Pre-stimulation with FGF-2 increases in vitro functional coupling of mesenchymal stem cells with cardiac cells

The functional coupling of transplanted cells with host myocardial cells is a significant challenge in mesenchymal stem cell (MSC) cardiomyoplasty being related to cell survival and therapeutic outcomes. Priming of MSCs with growth factors has been reported to improve their therapeutic efficacy through gap junction-mediated mechanisms. However, the expression pattern of Connexin43 (Cx43) in growth factor-stimulated MSC was not previously addressed. In this study we investigated how the pre-treatment with growth factors modulates MSC ability to integrate into the host tissue after transplantation, with particular focus on the expression of Cx43 and its cellular distribution.Our results showed that stimulation of MSCs with IGF-1, FGF-2, but not TGFβ, increased the level of Cx43 at both mRNA and protein levels. IGF-1 stimulation resulted in a shift of the fibroblast morphology into an epithelial morphology in several well-defined areas of stimulated cells. Confocal microscopy examination revealed that the increase of Cx43 was restricted to the epithelial-like cells and did not occur in other cells. In variance, FGF-2 induced a rod-shape morphology of every single cell, which achieved an extremely low cell index. FGF-2 stimulation also induced a time-dependent increase in Cx43, with a regular distribution pattern in all cells. Dye transfer assay coupled with confocal microscopy and flow cytometry analysis demonstrated functional in vitro cell coupling between FGF-2-stimulated MSCs as well as between FGF-2-stimulated cells and H9c2 cardiomyoblasts, a scenery that mimick MSC transplantation into the myocardium.We conclude that the stimulation of MSCs with FGF-2 prior to transplantation may facilitate their access among the myocardial cells and increase the functional coupling between transplanted and host cells.

Empirical inference of circuitry and plasticity in a kinase signaling network

Our understanding of physiology and disease is hampered by the difficulty of measuring the circuitry and plasticity of signaling networks that regulate cell biology, and how these relate to phenotypes. Here, using mass spectrometry-based phosphoproteomics, we systematically characterized the topology of a network comprising the PI3K/Akt/mTOR and MEK/ERK signaling axes and confirmed its biological relevance by assessing its dynamics upon EGF and IGF1 stimulation. Measuring the activity of this network in models of acquired drug resistance revealed that cells chronically treated with PI3K or mTORC1/2 inhibitors differed in the way their networks were remodeled. Unexpectedly, we also observed a degree of heterogeneity in the network state between cells resistant to the same inhibitor, indicating that even identical and carefully controlled experimental conditions can give rise to the evolution of distinct kinase network statuses. These data suggest that the initial conditions of the system do not necessarily determine the mechanism by which cancer cells become resistant to PI3K/mTOR targeted therapies. The patterns of signaling network activity observed in the resistant cells mirrored the patterns of response to several drug combination treatments, suggesting that the activity of the defined signaling network truly reflected the evolved phenotypic diversity.

PTB-associated splicing factor inhibits IGF-1-induced VEGF upregulation in a mouse model of oxygen-induced retinopathy.

Pathological retinal neovascularization, including retinopathy of prematurity and age-related macular degeneration, is the most common cause of blindness worldwide. Insulin-like growth factor-1 (IGF-1) has a direct mitogenic effect on endothelial cells, which is the basis of angiogenesis. Vascular endothelial growth factor (VEGF) activation in response to IGF-1 is well documented; however, the molecular mechanisms responsible for the termination of IGF-1 signaling are still not completely elucidated. Here, we show that the polypyrimidine tract-binding protein-associated splicing factor (PSF) is a potential negative regulator of VEGF expression induced by IGF stimulation. Functional analysis demonstrated that ectopic expression of PSF inhibits IGF-1-stimulated transcriptional activation and mRNA expression of the VEGF gene, whereas knockdown of PSF increased IGF-1-stimulated responses. PSF recruited Hakai to the VEGF transcription complex, resulting in inhibition of IGF-1-mediated transcription. Transfection with Hakai siRNA reversed the PSF-mediated transcriptional repression of VEGF gene transcription. In summary, these results show that PSF can repress the transcriptional activation of VEGF stimulated by IGF-1 via recruitment of the Hakai complex and delineate a novel regulatory mechanism of IGF-1/VEGF signaling that may have implications in the pathogenesis of neovascularization in ocular diseases.

Phospho-STIM1 is a downstream effector that mediates the signaling triggered by IGF-1 in HEK293 cells

STIM1 is a Ca2+ sensor of the endoplasmic reticulum (ER) that triggers the activation of plasma membrane Ca2+ channels upon depletion of Ca2+ levels within the ER. During thapsigargin-triggered Ca2+ store depletion, ERK1/2 phosphorylates STIM1 at Ser575, Ser608, and Ser621. This phosphorylation plays a role in the regulation of STIM1 dissociation from the microtubule plus-end binding protein EB1, an essential step for STIM1 activation by thapsigargin. However, little is known regarding the physiological role of this phosphorylation. Because IGF-1 triggers the activation of the RAF–MEK–ERK and the phosphoinositide pathways, the role of STIM1 phosphorylation in IGF-1 stimulation was studied. There was found to be phosphorylation of ERK1/2 in both the presence and the absence of extracellular Ca2+, demonstrating that Ca2+ influx is not essential for ERK1/2 activation. In parallel, IGF-1 triggered STIM1 phosphorylation at the aforementioned sites, an effect that was blocked by PD0325901, a MEK1/2 inhibitor used to block ERK1/2 activation. Also, STIM1-GFP was found in clusters upon IGF-1 stimulation, and STIM1-S575A/S608A/S621A-GFP strongly reduced this multimerization. Interestingly, phospho-STIM1 was mainly found in clusters when cells were treated with IGF-1, and IGF-1 triggered the dissociation of STIM1 from EB1, similarly to what has been observed for thapsigargin, suggesting that STIM1 mediates the IGF-1 signaling pathway. A study of IGF-1-stimulated NFAT translocation was therefore performed, finding that STIM1-S575A/S608A/S621A blocked this translocation, as did the fusion protein STIM1-EB1, confirming that both STIM1 phosphorylation and STIM1–EB1 dissociation are required for IGF-1-triggered Ca2+-dependent signaling, and demonstrating that STIM1 phosphorylation plays a role as a downstream effector of the RAF–MEK–ERK pathway and an upstream activator of Ca2+ entry.

The insulin-like growth factor I system: Physiological and pathophysiological implication in cardiovascular diseases associated with metabolic syndrome

Metabolic syndrome is a cluster of risk factors including obesity, dyslipidemia, hypertension, and insulin resistance. A number of theories have been speculated for the pathogenesis of metabolic syndrome including impaired glucose and lipid metabolism, lipotoxicity, oxidative stress, interrupted neurohormonal regulation and compromised intracellular Ca2+ handling. Recent evidence has revealed that adults with severe growth hormone (GH) and insulin-like growth factor I (IGF-1) deficiency such as Laron syndrome display increased risk of stroke and cardiovascular diseases. IGF-1 signaling may regulate contractility, metabolism, hypertrophy, apoptosis, autophagy, stem cell regeneration and senescence in the heart to maintain cardiac homeostasis. An inverse relationship between plasma IGF-1 levels and prevalence of metabolic syndrome as well as associated cardiovascular complications has been identified, suggesting the clinical promises of IGF-1 analogues or IGF-1 receptor activation in the management of metabolic and cardiovascular diseases. However, the underlying pathophysiological mechanisms between IGF-1 and metabolic syndrome are still poorly understood. This mini-review will discuss the role of IGF-1 signaling cascade in the prevalence of metabolic syndrome in particular the susceptibility to overnutrition and sedentary life style-induced obesity, dyslipidemia, insulin resistance and other features of metabolic syndrome. Special attention will be dedicated in IGF-1-associated changes in cardiac responses in various metabolic syndrome components such as insulin resistance, obesity, hypertension and dyslipidemia. The potential risk of IGF-1 and IGF-1R stimulation such as tumorigenesis is discussed. Therapeutic promises of IGF-1 and IGF-1 analogues including mecasermin, mecasermin rinfabate and PEGylated IGF-1 will be discussed.