Secretion Of IGF-binding Proteins Research Articles

IGFBP3 induced by the TGF-β/EGFRvIII transactivation contributes to the malignant phenotype of glioblastoma

Dual or multi-targets therapy targeting epidermal growth factor receptor variant III (EGFRvIII) and other molecular may relax the constraint for glioblastoma (GBM), putting forward the urgent requirement of finding candidate molecules. Here, the insulin-like growth factor binding protein-3 (IGFBP3) was considered a candidate, whereas the mechanisms of IGFBP3 production remain unclear. We treated GBM cells with exogenous transforming growth factor β (TGF-β) to simulate the microenvironment. We found that TGF-β and EGFRvIII transactivation induced the activation of transcription factor c-Jun, which specifically bound to the promoter region of IGFBP3 through Smad2/3 and ERK1/2 pathways and promoted the production and secretion of IGFBP3. IGFBP3 knockdown inhibited the activation of TGF-β and EGFRvIII signals and the malignant behaviors triggered by them invitro and invivo. Collectively, our results indicated a positive feedback loop of p-EGFRvIII/IGFBP3 under administration of TGF-β, blocking IGFBP3 may be an additional target in EGFRvIII-expressing GBM-selective therapeutic strategy.

Doxorubicin induced ROS-dependent HIF1α activation mediates blockage of IGF1R survival signaling by IGFBP3 promotes cardiac apoptosis.

Doxorubicin (Dox) causes the generation of intracellular reactive oxygen species (ROS) and inactivates insulin-like growth factor 1 (IGF1) signaling, leading to cardiomyocyte apoptosis. IGF-binding protein 3 (IGFBP3) is the most abundant circulating IGF1 carrier protein with high affinity, which has been reported to mediate ROS-induced apoptosis. Hypoxia-inducible factor 1α (HIF1A), an upstream protein of IGFBP3 is regulated by prolyl hydroxylase domain (PHD) through hydroxylation. In this study, we investigated the role of IGFBP3, HIF1A, and PHD in Dox-induced cardiac apoptosis.Cells challenged with 1 μM Dox for 24 h increased ROS generation, augmented intracellular and secreted IGFBP3 levels, and reduced IGF1 signaling. Further, we showed that Dox enhanced the extracellular association of IGF1 with IGFBP3. Moreover, echocardiography parameters, especially ejection fraction (EF) and fractional shortening (FS) were significantly reduced in ventricle tissue of Dox challenged rats. Notably, siRNA approach against IGFBP3 or an anti-IGFBP3 antibody rescued Dox-induced cardiac apoptosis, mitochondrial ROS, and the decrease in the IGF1 signaling activity. Furthermore, silencing HIF1A either using siRNA or inhibitor downregulated intracellular IGFBP3, rescued apoptosis, mitochondrial generation, and reduction in IGF1 signaling. Finally, western blot data revealed that ROS scavenger reversed Dox-induced cardiac apoptosis, increased levels of HIF1A and secreted IGFBP3, and decreased IGF1 survival signaling and PHD expression.These findings suggest that Dox-induced ROS generation suppressed PHD, which might stabilize nuclear HIF1A protein, leading to increased IGFBP3 expression and secretion. This in turn results in enhanced extracellular association of the latter with IGF1 and blocks IGF1 pro-survival signaling and may result in inducing cardiac apoptosis.

Mechanisms linking hypoxia to phosphorylation of insulin-like growth factor binding protein-1 in baboon fetuses with intrauterine growth restriction and in cell culture.

Hypoxia increases fetal hepatic insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation mediated by mechanistic target of rapamycin (mTOR) inhibition. Whether maternal nutrient restriction (MNR) causes fetal hypoxia remains unclear. We used fetal liver from a baboon (Papio sp.) model of intrauterine growth restriction due to MNR (70% global diet of Control) and liver hepatocellular carcinoma (HepG2) cells as a model for human fetal hepatocytes and tested the hypothesis that mTOR-mediated IGFBP-1 hyperphosphorylation in response to hypoxia requires hypoxia-inducible factor-1α (HIF-1α) and regulated in development and DNA-damage responses-1 (REDD-1) signaling. Western blotting (n=6) and immunohistochemistry (n=3) using fetal liver indicated greater expression of HIF-1α, REDD-1 as well as erythropoietin and its receptor, and vascular endothelial growth factor at GD120 (GD185 term) in MNR versus Control. Moreover, treatment of HepG2 cells with hypoxia (1% pO2 ) (n=3) induced REDD-1, inhibited mTOR complex-1 (mTORC1) activity and increased IGFBP-1 secretion/phosphorylation (Ser101/Ser119/Ser169). HIF-1α inhibition by echinomycin or small interfering RNA silencing prevented the hypoxia-mediated inhibition of mTORC1 and induction of IGFBP-1 secretion/phosphorylation. dimethyloxaloylglycine (DMOG) induced HIF-1α and also REDD-1 expression, inhibited mTORC1 and increased IGFBP-1 secretion/phosphorylation. Induction of HIF-1α (DMOG) and REDD-1 by Compound 3 inhibited mTORC1, increased IGFBP-1 secretion/ phosphorylation and protein kinase PKCα expression. Together, our data demonstrate that HIF-1α induction, increased REDD-1 expression and mTORC1 inhibition represent the mechanistic link between hypoxia and increased IGFBP-1 secretion/phosphorylation. We propose that maternal undernutrition limits fetal oxygen delivery, as demonstrated by increased fetal liver expression of hypoxia-responsive proteins in baboon MNR. These findings have important implications for our understanding of the pathophysiology of restricted fetal growth.

Octylphenol and nonylphenol affect decidualization of human endometrial stromal cells

This study investigated the effect of octylphenol (OP) and nonylphenol (NP) on decidualization of endometrial stromal cells. Telomerase-immortalized human endometrial stromal cells were cultured for 12 days and decidualization was induced in vitro. The cells were exposed to OP (5 or 10 μM) or NP (5 μM) during in vitro decidualization. The expression levels of decidualization markers were analyzed using qRT-PCR. The amount of prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1) was measured in medium using ELISA. The expression levels of PRL and IGFBP1 were significantly decreased by treatment with OP or NP. The amounts of PRL and IGFBP1 were also significantly decreased by treatment with OP or NP. The patterns of PRL and IGFBP1 secretion into medium were consistent with their expression patterns. The findings indicate that OP or NP may disrupt the decidualization of endometrial stromal cells and eventually affect the reproductive health of women.

Effects of Xiaotan Tongfu decoction on hepatic metastasis in obesity-associated colorectal cancer.

ObjectiveThis study was performed to assess the influence of obesity on colorectal cancer (CRC) and investigate the efficacy of Xiaotan Tongfu (XTTF) decoction to CRC treatment.MethodsBALB/C mice were used to establish an obesity-associated CRC model by a high-fat diet and tumor implantation. The tumors were harvested from mice inoculated with CT26 cell suspension. Body weight, liver weight, hepatic metastasis, and histological changes were observed. Immunohistochemical analysis and real-time polymerase chain reaction were performed to measure the levels of insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), and IGF binding protein 3 (IGFBP-3).ResultsObesity influenced the secretion of IGFs and aggravated CRC, while XTTF decoction inhibited the process of hepatic metastasis in CRC by upregulating the secretion of IGF-1/IGF-1R and downregulating the secretion of IGFBP-3.ConclusionsThis study provides evidence that XTTF decoction can serve as a candidate curative treatment for CRC.

Proliferation and Molecular Risk Score of Low Risk Myeloma Cells Are Increased in High Risk Microenvironment Via Augmented Bioavailability of Growth Factors

Introduction:Multiple myeloma (MM) cells from patients with smoldering MM (SMM) and low-risk (LR) MM harbor genetic alterations typically seen in patients with high-risk (HR) disease. To test whether the bone marrow (BM) microenvironment plays a role in controlling growth of LR MM cells, we established an experimental model that mimics a HR microenvironment by co-culturing normal mesenchymal stem cells (MSCs) with HR MM cells. We previously have shown that MSC conditioned media (CM) promotes growth of MM cells more effectively than cell-cell contact, as adhesion to MSCs often promotes survival at the expense of proliferation. Therefore, we utilized CM andhypothesized that MSC CM is enriched with bioactive growth factors that facilitate proliferation of LR MM cells.The aim of the study was to test the effect of CM from unprimed and primed MSCs on the survival, growth, and molecular properties of LR MM cells, and identify molecular pathways that mediate these effects.Methods:Primed MSCs were prepared by co-culturing normal MSCs with BM-dependent MM lines for 5 days. MSCs were trypsinized, replated for 40 min followed by serial washing to remove MM cells.Molecularly classified CD138-selected LR MM cells from 8 newly diagnosed patients were treated with 50% primed CM or unprimed CM, or growth media (CONT) for 5 days. Growth and survival of primary MM cells was assessed by MTT assay and detection of annexin V/PI and KI67 by flow cytometry.Microarrays were performed on primed and unprimed MSCs (n=7) and on primary LR MM cells treated with primed and unprimed MSCs CM (n=3). Pathways were analyzed using Ingenuity. Ultra low depth WGS was performed to assess copy number variation. Protein arrays were performed to test levels of secreted factors in CM (n=7).Results:Growth of primary LR MM cells (n=8) was increased by primed CM 5.1±0.05 (p<0.0001) and 7.0±0.6 (p<0.0001) folds compared to unprimed CM or CONT, respectively. In contrast, unprimed MSC CM increased growth in these cells by 1.3 fold. Flow cytometry analyses revealed no differences in proportion of annexin V/PI+ cells. However, the proportion of KI67+ cells was increased from 0.95±0.1% in unprimed CM group to 4.6±1.5% in primed CM group (p<0.04).Primed MSC CM caused MM cell GEP70 score to increase resulting in change from LR to HR in 2 experiments and from an ultra LR score to an intermediate score in another. Pathway analyses on genes differentially expressed between primed CM- and unprimed CM-treated MM cells identified oxidative phosphorylation with mitochondrial dysfunction, cell cycle, mitosis and p53 as the most significantly altered pathways. Top transcription regulators included FOXO3, TP53, E2F4, MYC and E2F1, whereas mir-16-5p and let-7 were the top microRNAs. Top significantly upregulated genes (>2 fold) by primed MSC CM included proliferation-related factors (MKI67, TOP2A, CCNB1, BIRC5 and RRM2), whereas underexpressed genes (< 2 fold) involved regulators of cell dormancy including BCL2 (survival), RICTOR (mTOR), HEY1 (NOTCH), JUN (AP-1) and CXCR4 (adhesion). Four genes we reported to powerfully predict progression of SMM to MM (Khan et al., Haematologica 2015) were highly upregulated in MM cells by primed MSC CM.WGS revealed similar copy number variation in MM cells treated with unprimed and primed CM, suggesting other mechanisms produced the observed gene expression changes.IGF1 is a central MM growth factor and IGF binding proteins (IGFBPs) control its bioavailability. We recently reported that mesenchymal cells are the main source of IGFBPs in BM, with IGFBP2 being the most downregulated gene in MM bone (Schinke et al., CCR 2018). Expression and secretion of IGFBPs (particularly IGFBP2) by MSCs were significantly reduced by priming these cells with MM cells, whereas IGF1 levels remained unchanged. IGFBP2 markedly blocked IGF1-induced MM cell growth (p<0.0003). Addition of IGF1R inhibitor significantly blocked primed CM-induced MM cell growth (p<0.02).Conclusions:MSCs primed by HR MM cells mimic a HR microenvironment, reflected by reduced level of factors that restrain bioavailability of MM growth factors such as IGF1, resulting in shutdown of master regulators of cell dormancy, which then enable a MM cells to proliferate. Such a scenario is particularly applicable in SMM and LR disease where MM cells exhibit a low proliferative index and their expansion is accelerated in distinct HR BM microenvironmental niches such as focal lesions. DisclosuresEpstein:University of Arkansas for Medical Sciences: Employment. Davies:Abbvie: Consultancy; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; MMRF: Honoraria; Janssen: Consultancy, Honoraria; TRM Oncology: Honoraria. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Research Funding.

Chronic endometritis modifies decidualization in human endometrial stromal cells

BackgroundChronic endometritis (CE) is a continuous inflammation of uterine endometrium, and it is usually symptomless. As CE has been thought not to affect the reproductive status and general health of affected women, its significance has not been explored. However, recent studies have shown that CE is related with repeated implantation failures after in vitro fertilization-embryo transfer, unexplained infertility, and recurrent miscarriages. As decidua differentiates to support the implantation process and maintains the pregnancy, we hypothesized that CE may influence the process of decidualization.MethodsSeventeen patients were employed in the experiment involving culture of endometrial stromal cells (ESCs). After obtaining endometrial samples, ESCs were harvested and cultured for 13 days. The concentrations in culture media and the protein expressions in ESCs of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), two well known decidualization markers used in a large number of in vitro models, were analyzed by ELISA and Western blotting, respectively, and the cell numbers were also counted. The mRNA levels of PRL and IGFBP-1 were tested by quantitative real time polymerase chain reaction (RT-PCR). Since sex hormone induce proliferation and differentiation to decidua via binding to the sex hormone receptors (ERα, ERβ, PRA, and PRB), their expression was assessed in another 17 patients’ paraffin-embedded endometrial tissue specimens by immunohistochemistry and semi-quantified by H-score.ResultsIncreased cell numbers and reduced secretion of PRL and IGFBP-1 were detected by ELISA in the ESCs of CE patients after culture for 13 days compared with non-CE patients. The decreased protein expression of IGFBP-1 in ESCs of CE patients was detected by Western blotting. The decreased expression of PRL mRNA and IGFBP-1 mRNA were detected by RT-PCR. Increased expressions of ERα, ERβ, PRA, and PRB were observed in the stromal cells of CE patients in comparison to non-CE patients, whereas increased expressions of ERα and ERβ were detected in the glandular cells of CE.ConclusionOur data suggests that CE modifies decidualization of human ESC through untuning the function of sex steroid hormone receptor.

Suppression of IGF binding protein-3 by palmitate promotes hepatic inflammatory responses.

IGF-binding protein-3 (IGFBP-3) is a liver-derived, anti-inflammatory molecule that is decreased in obesity, a key risk factor for nonalcoholic fatty liver disease (NAFLD). It was not known whether IGFBP-3 levels were altered in NAFLD, whether such alterations could be the result of lipotoxicity, and whether altered IGFBP-3 could affect pathways that are involved in hepatic and systemic inflammation. Serum IGFBP-3 was decreased in patients with NAFLD, whereas liver and circulating IL-8 levels were increased. Palmitate inhibited IGFBP-3 secretion by THP-1 macrophages and enhanced IL-8 expression. Exposure of palmitate-treated THP-1 macrophages to IGFBP-3-deficient conditioned medium led to a 20-fold increase in palmitate-induced IL-8 expression by hepatocytes. Conversely, overexpression of IGFBP-3 suppressed JNK and NF-κB activation and blocked palmitate-induced IL-8 expression in hepatocytes. Silencing IGFBP-3 in Huh7 cells enhanced JNK and NF-κB activity and increased palmitate-induced IL-8 secretion. These data indicate that IGFBP-3 serves as an anti-inflammatory brake in hepatocytes against JNK and NF-κB and limits their activation and downstream production of proinflammatory cytokines. Under lipotoxic conditions, palmitate inhibits hepatic macrophage secretion of IGFBP-3, thereby releasing the brake and enhancing palmitate-induced IL-8 synthesis and secretion.-Min, H.-K., Maruyama, H., Jang, B. K., Shimada, M., Mirshahi, F., Ren, S., Oh, Y., Puri, P., Sanyal, A. J. Suppression of IGF binding protein-3 by palmitate promotes hepatic inflammatory responses.

Branched-chain amino acids regulate insulin-like growth factor-binding protein 1 (IGFBP1) production by decidua and influence trophoblast migration through IGFBP1.

Do branched-chain amino acids (BCAAs) influence the migration of human extravillous trophoblast (EVT) cells through changes in insulin-like growth factor-binding protein 1 (IGFBP1) production in decidual cells? Decidua-derived IGFBP1 had a stimulating effect on migration of EVT. IGFBP1 is abundantly secreted from human decidual cells and influences trophoblast migration in human placenta of early pregnancy. In hepatic cells, the expression of IGFBP1 is influenced by nutritional status and BCAAs regulate IGFBP1 production. This is a laboratory-based study using human decidual cells and trophoblast cells isolated from placental tissue of early pregnancy (n = 50) and grown as primary cultures. Production of IGFBP1 from decidual cells was examined by enzyme-linked immunosorbent assay and immunoblotting after incubation with or without BCAAs. EVT migration was evaluated using the media conditioned by decidual cells. The effect of conditioned media on phosphorylation of focal adhesion kinase (FAK) in EVT was also analyzed by immunoblotting. The same experiments were repeated in the presence of RGD peptide, which inhibits IGFBP1 binding to α5β1 integrin. An EVT migration assay and the immunoblotting of phosphorylated FAK were also conducted with exogenous IGFBP1. The effect of the conditioned media on cytotrophoblast cell number was also assessed using WST-1 in a cell proliferation assay. Deprivation of BCAAs on decidual cells significantly suppressed IGFBP1 secretion (P < 0.05, versus BCAA+). Exogenous IGFBP1-stimulated EVT migration (P < 0.05) and phosphorylation of FAK (P < 0.05), and the RGD peptide inhibited these effects. EVT migration and phosphorylation of FAK were stimulated by the conditioned media, presumably by IGFBP1 in the media. RGD treatment abrogated the stimulating effects of conditioned media. The conditioned media deprived of BCAAs had suppressive effects on EVT migration (P < 0.05, versus BCAA+) and phosphorylation of FAK (P < 0.05, versus BCAA+). The conditioned media did not affect number of cytotrophoblast cells. The conclusions are based on in vitro experiments with human decidual cells and trophoblast cells isolated from placental tissue of early pregnancy, and we were unable to ascertain whether these mechanisms actually operate in vivo. We investigated the effect of decidua-derived IGFBP1 on EVT migration, however, we cannot completely rule out the possibility that endogenous IGF could also influence cell migration. Interruption of the BCAA supply to uterine decidual cells in early pregnancy may suppress EVT migration through reduced IGFBP1 secretion, which may be one of the pathophysiological conditions responsible for pre-eclampsia. None. All funds were obtained through Kyorin University School of Medicine. The authors have no conflict of interest to declare.

Autocrine/Paracrine Insulin-like Growth Factor Binding Protein-3 Acts as Pro-apoptotic Factor for Leydig cells in the Rat Testis

The secretory insulin-like growth factor-binding protein-3 (IGFBP-3) induces apoptosis via both insulin-like growth factor-I (IGF-I)-dependent and -independent mechanisms. Here, we have examined the effects of IGFBP-3 on Leydig cell apoptosis, proliferation and steroidogenesis. Immunohistochemical analysis of testes of rats at different ages revealed that IGFBP-3 is expressed first after 20 days of postnatal life and is present at a high level in the adult testis. In addition, Western blotting showed that the expression of IGFBP-3 in Leydig cells isolated from 60-day-old rats is higher than in 40-day-old animals. The rate of DNA synthesis (as assessed by incorporation of 3H-thymidine in vitro) in Leydig cells from 40-day-old rats is reduced by IGFBP-3, which also blocks the promotion of cell survival by IGF-I. Moreover, IGFBP-3 induces apoptosis in Leydig cells and, at the same time, attenuates the anti-apoptotic action of IGF-I. Furthermore, IGF-I stimulates secretion of IGFBP-3, -4, and -2 by Leydig cells. The pro-inflammatory cytokine tumor necrosis factor- α induces apoptosis in these same cells and increases their secretion of IGFBP-3 and IGFBP-4. These findings provide the first evidence that IGFBP-3 acts as a pro-apoptotic effector of Leydig cells and can also block the positive effect of IGF-I on cell survival. In addition, IGFBPs appear to modulate interactions between IGF-I and pro-inflammatory cytokines in the testis, suggesting possible participation of these proteins in processes such as testicular inflammation and cancer.

Endometrial stromal fibroblasts from women with polycystic ovary syndrome have impaired progesterone-mediated decidualization, aberrant cytokine profiles and promote enhanced immune cell migration in vitro.

Do endometrial stromal fibroblasts (eSF) in women with polycystic ovary syndrome (PCOS) (eSFpcos) exhibit altered estrogen and/or progesterone (P4) responses, which may explain some of the adverse reproductive outcomes and endometrial pathologies in these women? In vitro, eSF from women with PCOS exhibit an aberrant decidualization response and concomitant changes in pro-inflammatory cytokine, chemokine and matrix metalloproteinase (MMP) release and immune cell chemoattraction. In vivo these aberrations may result in suboptimal implantation and predisposition to endometrial cancer. The endometrium in women with PCOS has several abnormalities including progesterone (P4) resistance at the gene expression level, likely contributing to subfertility, pregnancy complications and increased endometrial cancer risk in PCOS women. Prospective, university-based, case-control, in vitro study. Cultures of eSFPCOS (n = 12, Rotterdam and NIH criteria) and eSFControl (Ctrl) (n = 6, regular cycle length, no signs of hyperandrogenism) were treated with vehicle, estradiol (E2, 10 nM) or E2P4 (10 nM/1 μM) for 14 days. Progesterone receptor (PGR) mRNA was assessed with quantitative real-time PCR (qRT-PCR) and eSF decidualization was confirmed by insulin-like growth factor-binding protein-1 (IGFBP-1) transcript and protein expression. Fractalkine (CX3CL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL) 6, 8 and 11, macrophage chemoattractant protein (MCP) 1 and 3, CCL5 (RANTES) and MMPs (MMP1, 2, 3, 7, 9, 10 and 12) were measured in conditioned media by Luminex multiplex assays, and chemotactic activity of the conditioned media was tested in a migration assay using CD14+ monocyte and CD4+ T-cell migration assay. Effects of IL-6 (0.02, 0.2, 2 or 20 ng/ml) or IL-8 (0.04, 0.4, 4, or 40 ng/ml) or combination (0.2 ng/ml IL-6 and 4.0 ng/ml IL-8) on 14-d decidualization were also tested. ANOVA with pre-planned contrasts was used for statistical analysis. Hormonal challenge with E2P4 to induce decidualization revealed two distinct subsets of eSFPCOS. Eight eSFPCOS (dPCOS) and all eSFCtrl (dCtrl) cultures showed a normal decidualization response to E2P4 as determined by morphology and IGFBP-1 secretion. However, 4 eSFPCOS cultures showed blunted decidualization (ndPCOS) in morphological assessment and low IGFBP-1 levels even though all three groups exhibited normal estrogen-mediated increase in PGR expression. Interestingly dPCOS had decreased IL-6 and GM-SCF secretion compared with dCtrl, whereas the ndPCOS cultures showed increased IL-6 and 8, MCP1, RANTES and GM-CSF secretion at base-line and/or in response to E2 or E2P4 compared with dCtrl and/or dPCOS. Furthermore, even though PGR expression was similar in all three groups, P4 inhibition of MMP secretion was attenuated in ndPCOS resulting in higher MMP2 and 3 levels. The conditioned media from ndPCOS had increased chemoattractic activity compared with dCtrl and dPCOS media. Exogenously added IL-6 and/or 8 did not inhibit decidualization in eSFCtrl indicating that high levels of these cytokines in ndPCOS samples were not likely a cause for the aberrant decidualization. This is an in vitro study with a small sample size, utilizing stromal cell cultures from proliferative and secretory phase endometrium. The effect of PCOS on endometrial epithelium, another major histoarchitectural cell compartment of the endometrium, was not evaluated and should be considered in future studies. Furthermore, results obtained should also be confirmed in a larger data set and with mid/late secretory phase in vivo samples and models. The alterations seen in ndPCOS may contribute to endometrial dysfunction, subfertility and pregnancy complications in PCOS women. The results emphasize the importance of understanding immune responses related to the implantation process and normal endometrial homeostasis in women with PCOS. Sigrid Juselius Foundation, Academy of Finland, Finnish Medical Foundation, Orion-Farmos Research Foundation (to T.T.P.), the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) U54HD 055764-07 Specialized Cooperative Centers Program in Reproduction and Infertility Research (to L.C.G.), the NICHD the Ruth L. Kirschstein National Research Service Awards grant 1F32HD074423-03 (to J.C.C.). The authors have no competing interests.

Muscle Mitohormesis Promotes Longevity via Systemic Repression of Insulin Signaling

Mitochondrial dysfunction is usually associated with aging. To systematically characterize the compensatory stress signaling cascades triggered in response to muscle mitochondrial perturbation, we analyzed a Drosophila model of muscle mitochondrial injury. We find that mild muscle mitochondrial distress preserves mitochondrial function, impedes the age-dependent deterioration of muscle function and architecture, and prolongs lifespan. Strikingly, this effect is mediated by at least two prolongevity compensatory signaling modules: one involving a muscle-restricted redox-dependent induction of genes that regulate the mitochondrial unfolded protein response (UPR(mt)) and another involving the transcriptional induction of the Drosophila ortholog of insulin-like growth factor-binding protein 7, which systemically antagonizes insulin signaling and facilitates mitophagy. Given that several secreted IGF-binding proteins (IGFBPs) exist in mammals, our work raises the possibility that muscle mitochondrial injury in humans may similarly result in the secretion of IGFBPs, with important ramifications for diseases associated with aberrant insulin signaling.

Effect of Ghrelin on Hepatic IGF-Binding Protein-1 Production

Ghrelin plays key roles in energy homeostasis by central and peripheral actions that include effects on insulin signalling pathways in liver. Insulin is an important inhibitor of production by hepatocytes of insulin-like growth factor-binding protein-1 (IGFBP-1) which has an endocrine role to inhibit IGF availability. The effects of ghrelin, insulin, an AMPK activator, and an AMPK inhibitor on IGFBP-1 secretion were studied in H4-II-E rat liver cells. Ghrelin (100 nM) blocked the inhibitory effect of a maximally effective concentration of insulin (10 ng/mL) on IGFBP-1 secretion during a 5 h incubation period (P < 0.001) in the absence and presence of an AMPK inhibitor. Ghrelin, alone, had no effect on IGFBP-1 production, but enhanced secretion independently of insulin under conditions of AMPK activation (P < 0.001). In conclusion, IGFBP-1 is identified as a novel target of ghrelin action in liver that may contribute to its metabolic effects in obesity.

Role of Insulin-Like Growth Factor Binding Protein-3 in 1, 25-Dihydroxyvitamin-D3-Induced Breast Cancer Cell Apoptosis

Insulin-like growth factor I (IGF-I) is implicated in breast cancer development and 1, 25-dihydroxyvitamin D3 (1, 25-D3) has been shown to attenuate prosurvival effects of IGF-I on breast cancer cells. In this study the role of IGF binding protein-3 (IGFBP-3) in 1, 25-D3-induced apoptosis was investigated using parental MCF-7 breast cancer cells and MCF-7/VDR cells, which are resistant to the growth inhibitory effects of 1, 25-D3. Treatment with 1, 25-D3 increased IGFBP-3 mRNA expression in both cell lines but increases in intracellular IGFBP-3 protein and its secretion were observed only in MCF-7. 1, 25-D3-induced apoptosis was not associated with activation of any caspase but PARP-1 cleavage was detected in parental cells. IGFBP-3 treatment alone produced cleavage of caspases 7, 8, and 9 and PARP-1 in MCF-7 cells. IGFBP-3 failed to activate caspases in MCF-7/VDR cells; however PARP-1 cleavage was detected. 1, 25-D3 treatment inhibited IGF-I/Akt survival signalling in MCF-7 but not in MCF-7/VDR cells. In contrast, IGFBP-3 treatment was effective in inhibiting IGF-I/Akt pathways in both breast cancer lines. These results suggest a role for IGFBP-3 in 1, 25-D3 apoptotic signalling and that impaired secretion of IGFBP-3 may be involved in acquired resistance to vitamin D in breast cancer.

Metabolism and Cancer: Old and New Players

Metabolism and Cancer: Old and New Players

Seminal plasma enhances and accelerates progesterone-induced decidualisation of human endometrial stromal cells

In preparation for embryo implantation, endometrial stromal cells (ESC) undergo differentiation, termed decidualisation. Enhancing endometrial decidualisation may overcome reduced endometrial receptivity, a major limiting factor in natural and assisted reproduction. To determine whether seminal plasma (SP) influences decidualisation, primary human ESC were treated with progesterone (P4, 50 ng mL(-1)) in the presence or absence of dialysed SP (0.5%) for 24 h or for up to 27 days to investigate immediate early effects or the effects of prolonged exposure, respectively. Combined SP and P4 treatment induced ESC morphological differentiation. Relative to control, P4 alone, and SP alone combined treatment with SP and P4 for 27 days significantly upregulated mRNA levels of the decidua-specific markers prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1). Consistently, PRL protein secretion was significantly increased over the course of 27 days combined SP and P4 treatment relative to control, P4 alone and SP alone. Likewise, IGFBP1 secretion was significantly greater relative to control and P4 alone over the course of 27 days. Thus, SP enhances and accelerates P4-mediated decidualisation of human ESC and may enhance endometrial receptivity.

Effects of Copper on Proliferation and Autocrine Secretion of Insulin-Like Growth Factor-1 (IGF-1) and IGF-Binding Protein-3 (IGFBP-3) in Chondrocytes from Newborn Pigs In Vitro

Chondrocytes from the lateral trochlear ridge of the distal femur taken from 1-day-old piglets were cultured in medium supplemented with 0, 7.8, 15.6, 31.2, and 62.5μmol/L copper. Insulin-like growth factor-1 (IGF-1) and IGF-binding protein 3 (IGFBP-3) levels in culture medium were determined by radioimmunoassay. DNA synthesis in chondrocytes was measured by tritiated thymidine ((3)H-TdR) incorporation. Proliferation-promoting activity and incorporation of (3)H-TdR in chondrocytes were increased in all culture media supplemented with copper and 15% fetal calf serum (FCS). The contents of IGF-1 and IGFBP-3 were also enhanced significantly in culture media containing 15% FCS and supplemented with copper at 15.6, 31.2, and 62.5μmol/L. The optimal copper concentration for promoting chondrocyte proliferation and autocrine secretion of IGF-1 and IGFBP-3 was 31.2μmol/L.

Acute Effects of Blood Transfusion on Growth Hormone and Insulin-Like Growth Factor-1 Levels in Children with Thalassemia

Background/Aim: Children with β-thalassemia have chronic anemia and growth retardation. Impaired growth hormone (GH)-insulin-like growth factor-1 (IGF-1) axis function has been demonstrated. Growth improvement has been demonstrated following optimal blood transfusion. Whether correction of anemia by blood transfusion augments GH-IGF-1 axis function has not been established. Methods: Twenty children with thalassemia aged 11.7 years (5.3–16.3 years) were recruited. GH provocative tests were performed twice, before and 1 week after blood transfusion. IGF-1, IGF-binding protein-3 (IGFBP-3) and hematocrit were measured. Results: Median IGF-1 and IGFBP-3 concentrations were significantly increased at 1 week following transfusion: pre- versus posttransfused concentrations: 86.4 versus 143.5 ng/ml (p < 0.001) and 2.95 versus 3.75 mg/l (p < 0.001), respectively. However, median peak GH levels and areas under the curve of GH during GH testing between pre- and posttransfusion periods were not different. The pretransfused hematocrit level was correlated with pretransfused IGF-1 (r = 0.662, p < 0.001) and IGFBP-3 (r = 0.691, p < 0.001) levels. Conclusions: In thalassemics, correction of anemia by blood transfusion rapidly enhanced GH-mediated IGF-1 and IGFBP-3 secretion. This suggests thatanemia may be one of the factors causing partial GH insensitivity.

Progesterone decreases bone morphogenetic protein (BMP) 7 expression and BMP7 inhibits decidualization and proliferation in endometrial stromal cells

Regulation of decidualization is decisive for proper implantation and the establishment of pregnancy. Recent studies have suggested that several bone morphogenetic proteins (BMPs) play physiological roles in reproduction. In the present study, we examined the expression of BMP7 in the endometrium and the effect of BMP7 on decidualization and proliferation of endometrial stromal cells (ESC). The gene expression of BMP7 in endometrial tissues collected from women with regular menstrual cycles was determined and the effect of ovarian steroid hormones on BMP7 gene expression was investigated in cultured ESC. The effect of BMP7 on the decidualization of ESC was determined by measuring the gene expression and protein secretion of insulin-like growth factor binding protein 1 (IGFBP1), a marker of decidualization. The effect of BMP7 on the proliferation of ESC was examined by the bromodeoxyuridine (BrdU) incorporation assay. The gene expression of BMP7 in endometrial tissues was low at and after the mid-secretory phase of the menstrual cycle. Progesterone suppressed the gene expression of BMP7 in cultured ESC. Treatment with progesterone and estradiol for 12 days achieved decidualization of ESC, increasing the gene expression and protein secretion of IGFBP1. Addition of BMP7 protein to the culture almost completely inhibited these increases. BMP7 suppressed BrdU incorporation in ESC, which indicated an antiproliferative effect of BMP7 on ESC. Progesterone-induced suppression of BMP7 and BMP7-induced inhibition of decidualization and proliferation of ESC suggest an elaborate regulatory mechanism for decidualization through BMP7 in the endometrium.

NOX4 mediates hypoxia-induced proliferation of human pulmonary artery smooth muscle cells: the role of autocrine production of transforming growth factor-β1 and insulin-like growth factor binding protein-3

Persistent hypoxia can cause pulmonary arterial hypertension that may be associated with significant remodeling of the pulmonary arteries, including smooth muscle cell proliferation and hypertrophy. We previously demonstrated that the NADPH oxidase homolog NOX4 mediates human pulmonary artery smooth muscle cell (HPASMC) proliferation by transforming growth factor-beta1 (TGF-beta1). We now show that hypoxia increases HPASMC proliferation in vitro, accompanied by increased reactive oxygen species generation and NOX4 gene expression, and is inhibited by antioxidants, the flavoenzyme inhibitor diphenyleneiodonium (DPI), and NOX4 gene silencing. HPASMC proliferation and NOX4 expression are also observed when media from hypoxic HPASMC are added to HPASMC grown in normoxic conditions, suggesting autocrine stimulation. TGF-beta1 and insulin-like growth factor binding protein-3 (IGFBP-3) are both increased in the media of hypoxic HPASMC, and increased IGFBP-3 gene expression is noted in hypoxic HPASMC. Treatment with anti-TGF-beta1 antibody attenuates NOX4 and IGFBP-3 gene expression, accumulation of IGFBP-3 protein in media, and proliferation. Inhibition of IGFBP-3 expression with small interfering RNA (siRNA) decreases NOX4 gene expression and hypoxic proliferation. Conversely, NOX4 silencing does not decrease hypoxic IGFBP-3 gene expression or secreted protein. Smad inhibition does not but the phosphatidylinositol 3-kinase (PI3K) signaling pathway inhibitor LY-294002 does inhibit NOX4 and IGFBP-3 gene expression, IGFBP-3 secretion, and cellular proliferation resulting from hypoxia. Immunoblots from hypoxic HPASMC reveal increased TGF-beta1-mediated phosphorylation of the serine/threonine kinase (Akt), consistent with hypoxia-induced activation of PI3K/Akt signaling pathways to promote proliferation. We conclude that hypoxic HPASMC produce TGF-beta1 that acts in an autocrine fashion to induce IGFBP-3 through PI3K/Akt. IGFBP-3 increases NOX4 gene expression, resulting in HPASMC proliferation. These observations add to our understanding hypoxic pulmonary vascular remodeling.