Tumor-derived insulin-like growth factor-binding protein-1 contributes to resistance of hepatocellular carcinoma to tyrosine kinase inhibitors.

BACKGROUND: Antiangiogenic tyrosine kinase inhibitors (TKIs) provide one of the few therapeutic options for effective treatment of hepatocellular carcinoma (HCC). However, patients with HCC often develop resistance toward antiangiogenic TKIs, and the underlying mechanisms are not understood. The aim of this study was to determine the mechanisms underlying antiangiogenic TKI resistance in HCC. METHODS: We used an unbiased proteomic approach to define proteins that were responsible for the resistance to antiangiogenic TKIs in HCC patients. We evaluated the prognosis, therapeutic response, and serum insulin-like growth factor-binding protein-1 (IGFBP-1) levels of 31 lenvatinib-treated HCC patients. Based on the array of results, a retrospective clinical study and preclinical experiments using mouse and human hepatoma cells were conducted. Additionally, in vivo genetic and pharmacological gain- and loss-of-function experiments were performed. RESULTS: In the patient cohort, IGFBP-1 was identified as the signaling molecule with the highest expression that was inversely associated with overall survival. Mechanistically, antiangiogenic TKI treatment markedly elevated tumor IGFBP-1 levels via the hypoxia-hypoxia inducible factor signaling. IGFBP-1 stimulated angiogenesis through activation of the integrin α5ß1-focal adhesion kinase pathway. Consequently, loss of IGFBP-1 and integrin α5ß1 by genetic and pharmacological approaches re-sensitized HCC to lenvatinib treatment. CONCLUSIONS: Together, our data shed light on mechanisms underlying acquired resistance of HCC to antiangiogenic TKIs. Antiangiogenic TKIs induced an increase of tumor IGFBP-1, which promoted angiogenesis through activating the IGFBP-1-integrin α5ß1 pathway. These data bolster the application of a new therapeutic concept by combining antiangiogenic TKIs with IGFBP-1 inhibitors.

Insulin-like growth factor binding protein 1 ameliorates lipid accumulation and inflammation in nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Insulin-like growth factor binding protein 1 (IGFBP1) is recently proved to be associated with glucose regulation and insulin resistance. However, little is known about its direct impact on nonalcoholic fatty liver disease (NAFLD). This study aims to investigate the effect and potential mechanism of IGFBP1 in NAFLD. METHODS: We first measured the expression level of IGFBP1 in NAFLD patients, mice, and cells. Then in in vivo study, C57BL/6 mice were fed with a methionine/choline-deficient (MCD) diet for 4 weeks to establish the model of NAFLD. And for the last 2 weeks, the mice were injected intraperitoneally with vehicle or recombinant mouse IGFBP1 0.015 mg/kg/d. The L02 cells were treated with free fatty acids (FFA) or palmitate acids (PA) and recombinant IGFBP1 for 48 h. Integrin-linked kinase (ILK) inhibitor and small interfering RNA were used to explore the potential interactions between IGFBP1 and integrin ß1 (ITGB1). RESULTS: The expression of IGFBP1 was increased in NAFLD patients, mice, and cells. IGFBP1 treatment significantly ameliorated lipid accumulation and hepatic injury in MCD-fed mice. IGFBP1 downregulated hepatic lipogenesis and upregulated lipid ß-oxidation. In addition, IGFBP1 attenuated the nuclear factor-kappa B (NF-κB) and extracellular regulated protein kinases (ERK) signaling pathways. In vitro, we proved that IGFBP1 relieved FFA-induced lipid accumulation via interacting with ITGB1 and alleviated inflammation by inhibiting NF-κB and ERK signaling pathways. CONCLUSIONS: IGFBP1 treatment significantly ameliorated hepatic steatosis by interacting with ITGB1 and suppressed inflammation by inhibiting NF-κB and ERK signaling pathways. Therefore, IGFBP1 might be a potential therapeutic target for NAFLD.

Inflammation-Associated Cytokines IGFBP1 and RANTES Impair the Megakaryocytic Potential of HSCs in PT Patients after Allo-HSCT.

Prolonged isolated thrombocytopenia (PT) is a severe complication in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Whether the megakaryoctic potential of hematopoietic stem cells (HSCs) in bone marrow is intact and what factors drive the pathological process of PT remain elusive. A retrospective study in patients (n = 285) receiving HSCT revealed that the occurrence of PT was approximately 8% and the number of platelets and megakaryocytes in PT patients is much lower compared with control subjects. To test whether the deficiency of thrombopoiesis was caused by the activities of HSCs, the megakaryocytic differentiation potential of HSCs before or after transplantation was assessed. Interestingly, a substantial decrease of megakaryocytic differentiation was observed 2 weeks after transplantation of HSCs in all of the allo-HSCT recipients. However, 4 weeks after transplantation, the ability of HSCs to generate CD41+CD42b+ megakaryocytes in successful platelet engraftment patients recovered to the same level as those of HSCs before implantation. In contrast, HSCs derived from PT patients throughout the postimplantation period exhibited poor survival and failed to differentiate properly. A protein array analysis demonstrated that multiple inflammation-associated cytokines were elevated in allo-HSCT recipients with PT. Among them, insulin-like growth factor-binding protein 1 and regulated on activation, normal T cell expressed and secreted were found to significantly suppress the proliferation and megakaryocytic differentiation of HSCs in vitro. Our results suggested that the occurrence of PT may be attributed, at least partially, to the damage to HSC function caused by inflammation-associated cytokines after HSCT. These findings shed light on the mechanism underlying HSC megakaryocytic differentiation in PT patients and may provide potential new strategies for treating PT patients after HSCT.

Insulin-Like Growth Factor Binding Protein 1 Could Improve Glucose Regulation and Insulin Sensitivity Through Its RGD Domain.

Low circulating levels of insulin-like growth factor binding protein 1 (IGFBP-1) are associated with insulin resistance and predict the development of type 2 diabetes. IGFBP-1 can affect cellular functions independently of IGF binding through an Arg-Gly-Asp (RGD) integrin-binding motif. Whether causal mechanisms underlie the favorable association of high IGFBP-1 levels with insulin sensitivity and whether these could be exploited therapeutically remain unexplored. We used recombinant IGFBP-1 and a synthetic RGD-containing hexapeptide in complementary in vitro signaling assays and in vivo metabolic profiling in obese mice to investigate the effects of IGFBP-1 and its RGD domain on insulin sensitivity, insulin secretion, and whole-body glucose regulation. The RGD integrin-binding domain of IGFBP-1, through integrin engagement, focal adhesion kinase, and integrin-linked kinase, enhanced insulin sensitivity and insulin secretion in C2C12 myotubes and INS-1 832/13 pancreatic ß-cells. Both acute administration and chronic infusion of an RGD synthetic peptide to obese C57BL/6 mice improved glucose clearance and insulin sensitivity. These favorable effects on metabolic homeostasis suggest that the RGD integrin-binding domain of IGFBP-1 may be a promising candidate for therapeutic development in the field of insulin resistance.

Assessment of T-2 toxin effect and its metabolite HT-2 toxin combined with insulin-like growth factor I, leptin and ghrelin on progesterone secretion by rabbit ovarian fragments.

Assessment of A-trichothecene mycotoxins (T-2 and HT-2 toxins) effect combined with growth factor IGF-I, and the metabolic hormones leptin and ghrelin on progesterone secretion by rabbit ovarian fragments was studied. Rabbit ovarian fragments were incubated without (control group) or with T-2/HT-2 toxin, or their combinations with insulin-like growth factor I (IGF-I), leptin or ghrelin at various concentrations for 24 h. Secretion of progesterone was determined by ELISA. First, T-2 toxin and HT-2 toxins at all doses used (0.01, 0.1, 1, 10, and 100 ng mL(-1)) were not shown to be potential regulators of progesterone secretion in rabbit ovarian fragments. Second, T-2 toxin but not HT-2 toxin combined with IGF-I was shown to be potential regulator of progesterone secretion in rabbit ovarian fragments. T-2 toxin at all doses used (0.01; 0.1; 1; 10; and 100 ng mL(-1)) combined with IGF-I (at dose 100 ng mL(-1)) significantly (P < 0.05) decreased progesterone secretion by rabbit ovarian fragments. Third, T-2 toxin and HT-2 toxin at all doses used in the study (0.01, 0.1, 1, 10, and 100 ng mL(-1)) combined with leptin (at dose 1000 ng mL(-1)) were not shown to be potential regulators of progesterone secretion in rabbit ovarian fragments. Furthermore, T-2 toxin and HT-2 toxin at all doses used in the study (0.01, 0.1, 1, 10, and 100 ng mL(-1)) combined with ghrelin (500 ng mL(-1)) were not shown to be potential regulators of progesterone secretion in rabbit ovarian fragments. Results in this study showed that trichothecene as T-2 toxin combined with IGF-I but not HT-2 toxin was able to decrease progesterone secretion in rabbit ovarian fragments in vitro. Experimental results of T-2 and HT-2 toxins combined with leptin and ghrelin did not confirm ability to modulate progesterone secretion by ovarian fragments in rabbits.

Effects of IGFBP-1 and IGFBP-2 and their fragments on migration and IGF-induced proliferation of human dermal fibroblasts.

OBJECTIVE: A family of six insulin-like growth factor (IGF) binding-proteins (IGFBP) bind to IGF-I and IGF-II with high affinity and modulate their activity. We have recently shown that a neutrophil-derived protease activity cleaved IGFBP-1, -2 and -4. IGFBP-1 and IGFBP-2 have a C-terminal Arg-Gly-Asp (RGD) sequence, and IGFBP-1 has been shown by others to stimulate migration through binding of its RGD sequence to α5ß1 integrin. The aim of this study was to determine the effect of this IGFBP protease on IGF-induced proliferation and the effect of IGFBP-1 and IGFBP-2 and their proteolytic fragments on migration in normal and high glucose of human dermal fibroblasts (HDF). DESIGN: We investigated the effect of intact or cleaved IGFBP-1 and -2 on proliferation in cultured HDFs and on HDF migration in normal and high glucose. RESULTS: Both IGFBP-1 and IGFBP-2 and their proteolytic fragments stimulated HDF migration and the stimulatory effect was abolished by pre-treating cells with a α5ß1 integrin antibody. High glucose impaired migration of HDFs; however, the addition of IGFBP-1, IGFBP-2 or fragments increased migration to levels observed in normoglycemia. IGFBP-2 inhibited IGF-II induced proliferation; however, the inhibitory effect was reduced after being cleaved. Intact native IGFBP-1 showed either potentiating or inhibitory effects on IGF-I induced proliferation depending on the confluence of cells, and proteolysis of IGFBP-1 did not change these effects. IGFBP-1 was found to increase phosphorylation of FAK and ERK1/2 and this effect was inhibited by the monoclonal integrin a5ß1 ab. CONCLUSIONS: IGFBP-1 and -2 and their proteolytic fragments may improve tissue repair under inflammatory conditions, through effects on proliferation and migration of HDFs in normal and high glucose.

Insulin-like growth factor binding protein-1 inhibits cancer cell invasion and is associated with poor prognosis in hepatocellular carcinoma.

Insulin-like growth factor binding protein-1 (IGFBP-1) plays an important role in the development and progression of cancer. However, the expression of IGFBP-1 remains equivocal, and little is known about its clinicopathological significance and prognostic value in hepatocellular carcinoma (HCC). In this study, we evaluated the expression of IGFBP-1 in 90 paired HCC tissues and adjacent non-cancerous liver tissues and analyzed its clinical and prognostic significance. The results showed that IGFBP-1 was detected in cytoplasm as well as cell nucleus, and down-regulated in HCC tissues compared to the adjacent non-cancerous liver tissues. The decreased expression of IGFBP-1 was correlated with tumor differentiation, liver cirrhosis, microvascular invasion or metastasis, TNM stage and poor survival. Moreover, low levels of IGFBP-1 may be an independent prognostic indicator for the survival of patients with HCC. We also evaluated its function by adding recombinant IGFBP-1 to the cultured HCC cell lines HepG2 and MHCC97-H. The result of the invasion chamber assay showed that IGFBP-1 could inhibit the invasion of HepG2 and MHCC97-H. MMP-9 secretion by these cells was significantly decreased when the cells were treated with IGFBP-1. Our results suggest that IGFBP-1 inhibits the invasion and metastasis of HCC cells and that IGFBP-1 may be useful as a valuable marker for the prognosis of patients with HCC.

Low-dose insulin-like growth factor binding proteins 1 and 2 and angiopoietin-like protein 3 coordinately stimulate ex vivo expansion of human umbilical cord blood hematopoietic stem cells as assayed in NOD/SCID gamma null mice.

INTRODUCTION: Insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs) and angiopoietin-like proteins (ANGPTLs) can enhance the ex vivo expansion of hematopoietic stem cells (HSCs) when used with a standard cytokine cocktail of stem cell factor (SCF), thrombopoietin (TPO) and FLT3 ligand (FL). In order to determine the optimal dose and combination of IGFs, IGFBPs and ANGPTLs, serial dilution and full permutation of IGFBP1, IGFBP2, IGF2 and ANGPTL3 were applied on a cryopreserved umbilical cord blood mononuclear cell (UCB-MNC) ex vivo expansion system. METHODS: In this system, 4 × 105 cells/ml of UCB-MNCs were inoculated in serum-free Stemspan® medium (Stemcell technologies, vancouver, BC, Canada) supplied with standard basal cytokine combination of 100 ng/ml SCF, 50 ng/ml FL and 100 ng/ml TPO and supported by a bone marrow mesenchymal stromal cell layer. RESULTS: Paradoxically, experiment results showed that the highest expansion of CD34+CD38-CD90+ primitive progenitor was stimulated by cytokine combination of SCF + TPO + FL + IGFBP1 + IGFBP2 + ANGPTL3 at a low dose of 15 ng/ml IGFBP1 and 20 ng/ml IGFBP2 and ANGPTL3. This ex vivo expansion was further validated in 8-week-old to 10-week-old nonobese diabetic/severe combined immunodeficiency interleukin 2 gamma chain null (NOD/SCID-IL2Rγ-/-) mice. Limiting dilution assay showed excellent correlation between the HSC ex vivo surface marker of CD34+CD38-CD90+ and the in vivo competitive repopulating unit (CRU) functional assay. CONCLUSION: IGFBP1, IGFBP2, IGF2 and ANGPTL3 can stimulate the expansion of CD34+CD38-CD90+ primitive progenitor at low dose. The optimal combination comprises IGFBP1, IGFBP2 and ANGPTL3 together with the standard cytokine cocktail of SCF, FL and TPO. The CD34+CD38-CD90+ phenotype can serve as a surrogate ex vivo surface marker for HSCs due to consistency with the in vivo CRU functional assay.

Cross-talk between integrin receptor and insulin-like growth factor receptor in regulation of collagen biosynthesis in cultured fibroblasts.

PURPOSE: Cellular processes are regulated by signals generated by adhesion receptors and growth factor receptors. IGFbinding protein 1 (IGFBP-1) is a molecule which may affect the both signaling pathways through inactivation of IGF-I (ligand for IGF-IR) and binding to RGD region of integrin receptors. Whether this phenomenon is important in communication between insulin-like growth factor receptor (IGF-IR) and ß1-integrin receptor in regulation of prolidase activity and collagen biosynthesis is the aim of this study. MATERIAL AND METHOD: We studied the effects of IGFBP-1, IGF-I, thrombin (integrin activator), echistatin (disintegrin), phosphatidylinositol 3-kinase inhibitor (LY-294002) and ERK 1/2 inhibitors (PD98059 and UO126) on prolidase activity, collagen biosynthesis and expression of proteins participating in pathways generated by these receptors. RESULTS: Stimulation of ß1-integrin and IGF-I receptors by standard ligands was proved to up-regulate collagen synthesis in cultured fibroblasts. IGFBP-1, similarly as echistatin and studied inhibitors, contributed to down-regulation of ERK1/2, Akt, mTOR expression and up-regulation of NFκB. It was accompanied by parallel decrease in prolidase activity and collagen biosynthesis. CONCLUSION: The data suggest that "cross talk" between IGF-I receptor and integrin receptor may play important role in regulation of prolidase activity and collagen biosynthesis.

Sex steroids, GHRH, somatostatin, IGF-I, and IGFBP-1 modulate ghrelin's dose-dependent drive of pulsatile GH secretion in healthy older men.

CONTEXT: Ghrelin is a potent endogenous stimulator of GH secretion. However, clinical factors that regulate ghrelin dose-responsiveness are incompletely defined. OBJECTIVE: The aim of the study was to test the multipathway hypothesis that testosterone (T) and estradiol, GHRH, and somatostatin (SS) jointly modulate ghrelin's action. DESIGN/PARTICIPANTS/SETTING: Healthy older men (n = 21) participated in a double-blind, prospectively randomized, placebo (Pl)-controlled study in a Clinical Translational Research Center. INTERVENTIONS: To create a range of sex-steroid milieus, men received leuprolide + Pl (n = 10) or leuprolide + T addback (n = 11). Sixteen to 21 d later, subjects received three separate randomly ordered overnight constant i.v. infusions of saline, GHRH, and SS. Interactions between the peptide clamp and ghrelin were tested by superimposed injections of four randomly ordered bolus i.v. doses of ghrelin (0.03, 0.135, 0.60, and 2.7 µg/kg). GH was measured every 10 min, and GH responses were assessed by nonlinear dose-response analysis. Linear associations were assessed by stepwise regression. OUTCOME MEASURES/RESULTS: The descending numerical order of ghrelin efficacy (maximal GH secretory-burst mass; micrograms/liter) was 107 (GHRH + Pl), 104 (GHRH + T), 73 (saline + T), 73 (SS + T), 60 (saline + Pl), and 52 (SS + Pl) [means], wherein SS + T exceeded SS + Pl. GHRH and IGF binding protein-1 augmented, whereas IGF-I attenuated ghrelin potency. Age and IGF-I decreased ghrelin/GHRH synergy. Ghrelin sensitivity was independent of interventions. CONCLUSIONS: These studies introduce composite regulatory effects of sex hormones, GHRH, SS, IGF binding protein-1, and IGF-I on ghrelin dose-responsiveness, suggesting multipathway modulation of GH-secretagogue action.

Complex regulation of GH autofeedback under dual-peptide drive: studies under a pharmacological GH and sex steroid clamp.

To test the postulate that sex difference, sex steroids, and peptidyl secretagogues control GH autofeedback, 11 healthy postmenopausal women and 14 older men were each given 1) a single iv pulse of GH to enforce negative feedback and 2) continuous iv infusion of saline vs. combined GHRH/GHRP-2 to drive feedback escape during pharmacological estradiol (E(2); women) or testosterone (T; men) supplementation vs. placebo in a double-blind, prospectively randomized crossover design. By three-way ANCOVA, sex difference, sex hormone treatment, peptide stimulation, and placebo/saline responses (covariate) controlled total (integrated) GH recovery during feedback (each P < 0.001). Both sex steroid milieu (P = 0.019) and dual-peptide stimulation (P < 0.001) determined nadir (maximally feedback-suppressed) GH concentrations. E(2)/T exposure elevated nadir GH concentrations during saline infusion (P = 0.003), whereas dual-peptide infusion did so independently of T/E(2) and sex difference (P = 0.001). All three of sex difference (P = 0.001), sex steroid treatment (P = 0.005), and double-peptide stimulation (P < 0.001) augmented recovery of peak (maximally feedback-escaped) GH concentrations. Peak GH responses to dual-peptidyl agonists were greater in women than in men (P = 0.016). E(2)/T augmented peak GH recovery during saline infusion (P < 0.001). Approximate entropy analysis corroborated independent effects of sex steroid treatment (P = 0.012) and peptide infusion (P < 0.001) on GH regularity. In summary, sex difference, sex steroid supplementation, and combined peptide drive influence nadir, peak, and entropic measurements of GH release under controlled negative feedback. To the degree that the pharmacological sex steroid, GH, and dual-peptide clamps provide prephysiological regulatory insights, these outcomes suggest major determinants of pulsatile GH secretion in the feedback domain.

Polymerization of insulin-like growth factor-binding protein-1 (IGFBP-1) potentiates IGF-I actions in placenta.

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1), the main secretory protein of decidua that binds to IGFs and has been shown to inhibit or stimulate IGFs' bioactivities. Polymerization, one of the posttranslational modifications of IGFBP-1, has been shown to lead to loss of inhibiting effect of IGFBP-1 on IGF-I actions. The current studies were undertaken to elucidate the effects of steroid hormones on IGFBP-1 polymerization in trophoblast cell cultures. Placental tissues were obtained during legal, elective procedures of termination of pregnancy performed between 7 and 10 weeks of gestation, and primary trophoblast cells were separated. IGFBP-1 polymerization was analyzed by SDS-PAGE and immunoblotting. IGFBP-1 was polymerized when IGFBP-1 was added to trophoblast cell cultures. Polymerization of IGFBP-1 was inhibited by the addition of anti-tissue transglutaminase antibody into the culture media. There was an increase in the intensity of polymerized IGFBP-1 bands with the addition of medroxyprogesterone acetate (MPA), while no such difference was observed upon treatment with estradiol. MPA also increased the expression of tissue transglutaminase on trophoblast cell membranes. IGF-I stimulated trophoblast cell migration, while IGFBP-1 inhibited this IGF-I-induced trophoblast response. Addition of MPA attenuated the inhibitory effects of IGFBP-1 on IGF-I-induced trophoblast cell migration. IGFBP-1 was polymerized by tissue transglutaminase on the cell surface of trophoblasts, and MPA increased tissue transglutaminase expression on the cell surface and facilitated IGFBP-1 polymerization. These results suggest that progesterone might facilitate polymerization of decidua-secreted IGFBP-1 and increase IGF-I actions at feto-maternal interface, thereby stimulating trophoblast invasion of maternal uterus.

Insulin-like growth factor binding protein-1 activates integrin-mediated intracellular signaling and migration in oligodendrocytes.

In multiple sclerosis (MS), oligodendrocytes in lesions are lost, leaving damaged tissue virtually devoid of these myelin-producing cells. Our group has recently demonstrated enhanced expression of insulin-like growth factor (IGF) binding protein-1 (IGFBP-1) in oligodendrocytes (CNPase(+)) localized adjacent to MS lesions. In the present study, we demonstrate IGF-1-independent actions of IGFBP-1 on OLN-93 oligodendroglial cells, including activation of kinases ERK1/2, focal adhesion kinase and p21-activated kinase as well as small monomeric GTPases Rac and Ral. Activation of these intracellular signaling components was inhibited by GRGDS peptide, indicating signaling through integrin receptors. While both IGF-1 and IGFBP-1 demonstrated rapid induction of actin polymerization, IGFBP-1 proved to be a more potent inducer of migration than IGF-1, inducing a threefold increased migration rate. Furthermore, through integrin receptor signaling IGFBP-1 induced rapid transient translocalization of intracellular Rac toward punctuated structures followed by translocation of Rac to the plasma membrane. Our results suggest that up-regulation of IGFBP-1 in oligodendrocytes in MS may serve two functions: (i) regulate IGF-1 actions, (ii) exert IGF-independent effects through its RGD sequence.

The effects of hepatocyte growth factor and insulin-like growth factor-1 on the myogenic differentiation of satellite cells in human urethral rhabdosphincter.

AIMS: To examine the effects of hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) on the myogenic differentiation of human urethral rhabdosphincter (RS) satellite cells. METHODS: Human RS was obtained from patients undergoing radical prostatectomy for prostate cancer. Selectively cultured RS satellite cells, transfected with temperature sensitive simian virus-40 T antigen (ts-SV40 Tag) to extend their lifespan, were cultured at 33 degrees C, and then incubated at 39 degrees C to induce myogenic differentiation. Varying concentrations of HGF and IGF-1 were added to the differentiation medium. Inactivation of SV40 Tag and evaluations of myogenic differentiation were examined by immunocytochemistry, western blotting and real-time RT-PCR. RESULTS: At 39 degrees C, ts-SV40 Tag-transfected RS satellite cells slowly proliferated, fused to form multinucleated myotubes, and expressed myosin heavy chain (MHC) in association with the temperature-dependent inactivation of SV40 Tag. IGF-1 significantly accelerated the MHC expression in a dose-dependent fashion through activation of the PI3-kinase pathway. Conversely, HGF did not promote MHC expression due a reduction in phosphorylation of both the MAP-kinase and the PI3-kinase pathways, a pattern of response different than the response of proliferating RS satellite cells to HGF. CONCLUSIONS: HGF did not induce myogenic differentiation of RS satellite cells. Conversely, IGF-1 promoted myogenic differentiation of RS satellite cells via the PI3-K pathway likewise proliferating RS satellite cells. These findings may be useful for developing novel techniques for regenerating the human RS to treat urinary incontinence.

[Influence of insulin-like growth factor 1 on PECAM-1 and ICAM-1 adhesion molecules expression in tumor necrosis factor alpha and hyperglycemia induced umbilical vein endothelial cells]. / Wplyw insulinopodobnego czynnika wzrostu 1 na ekspresji czasteczek adhezyjnych PECAM-1 i ICAM-1 na powierzchni komórek sródblonka naczyn zylnych pepowiny indukowanych czynnikiem martwicy nowotworu alpha oraz hiperglikemia.

UNLABELLED: Activation, proliferation and regeneration of endothelium form developmental background of many pathological states. In those processes adhesion molecules and growth factors are crucial. Insulin-like growth factor 1 (IGF-1) is an important regulator of vascular function, capable of expressing pleiotropic actions. Interactions between endothelial cells and growth factors are complex and environment dependent. Cytokines, including tumor necrosis factor alpha (TNF-alpha) activate endothelium cells during inflammatory process. Endothelial dysfunction, with enhanced adhesion molecules expression, can be caused also by hyperglycemia. Aim of the study was to assess the influence of IGF-1 on platelet/endothelial cell adhesion molecule 1 (PECAM-1; CD31) and intercellular adhesion molecule 1 (ICAM-1; CD54) expression on human umbilical vein endothelial cells (HUVECs) stimulated by hyperglycemia and TNF-alpha. MATERIAL AND METHODS: Material for the study was obtained from fresh umbilical cord. Cell cultures were incubated with IGF-1 concentrations of 50 ng/ml and 200 ng/ml, glucose concentrations of 11.1 mmol/l and 22.2 mmol/l, TNF-alpha concentrations of 10 ng/ml and 20 ng/ml, as well as combinations of above. Ratio of PECAM-1 and ICAM-1 expression revealing cells was obtained using flow cytometry method. In statistic analysis ANOVA test and Duncan's new multiple range test were used. RESULTS: It was demonstrated, that IGF-1 concentrations of 50 ng/ml, 200 ng/ml enhance PECAM-1 and ICAM-1 endothelial cells expression (respectively to 57.2 +/- 4.8%, 76.1 +/- 1.5% and 91.7 +/- 5.0%, 86.8 +/- 4.7%) compared to control group (respectively: 20.9 +/- 0.5% and 26.6 +/- 0.5%). Expression under influence of 10 ng/ml and 20 ng/ml TNF-alpha increases up to respectively: 78.4 +/- 0.9% and 86.5 +/- 0.7% for CD31 and respectively: 65.1 +/- 3.8% and 70.8 +/- 1.4% for CD54. Glucose concentration of 22.2 mmol/l increases CD31 expression to 44.6 +/- 5.2% and CD54 expression to 47.3 +/- 4.8%. Combination of influence of IGF-1 and hyperglycemia enhances CD31 expression from 82.2 +/- 0.6% up to 85.3 +/- 0.7%, whereas CD54 expression from 89.3 +/- 3.5% up to 94.4 +/- 0.5%, depending of concentrations. Combination of 20 ng/ml TNF-alpha with 50 ng/ml IGF-1 enhances expression of CD54 to 96.8 +/- 1.2%, with 200 ng/ml IGF-1 enhances CD31 and CD54 expression respectively to 90.5 +/- 2.3% and 96.6 +/- 0.6%. CONCLUSIONS: 1. It was concluded, that IGF-1, hyperglycemia and TNF-alpha enhance PECAM-1 and ICAM-1 adhesion molecules expression on HUVEC cells, activating the endothelium. 2. Combination of IGF-1, TNF-alpha and hyperglycemia influence synergistically enhances PECAM-1 and ICAM-1 expression on endothelial cells surface.

Insulin-like growth factor binding protein-1 induces decidualization of human endometrial stromal cells via alpha5beta1 integrin.

Progesterone is known to induce decidualization of human endometrial stromal cells in vitro. Decidualized stromal cells produce insulin-like growth factor binding protein-1 (IGFBP-1) as well as prolactin (PRL). In this study, we tested the possibility that IGFBP-1 directly stimulates endometrial stromal cell decidualization. Endometrial stromal cells were obtained from normal menstruating patients with uterine myoma at hysterectomy. Stromal cells were cultured for up to 4 weeks with estradiol (E(2)) and/or medroxy progesterone acetate (MPA) in the presence or the absence of IGFBP-1 and, LR(3)-IGF-I (an IGF-I analogue) that binds to the IGF-I receptor but has reduced affinity for IGFBPs. Decidualization of endometrial stromal cells was evaluated by morphological changes and PRL release into culture media. The binding of IGFBP-1 to endometrial cells was analysed using a biosensor. MPA and E(2) induced decidualization of stromal cells, while LR(3)-IGF-I inhibited decidualization by MPA and E(2) as well as PRL and IGFBP-1 secretion into medium. IGFBP-1 induced decidualization of stromal cells in the absence of MPA and E(2) in the medium. IGFBP-1-induced decidualization was not inhibited by the addition of LR(3)IGF-1 but was inhibited by the addition of an RGD peptide, however, the RGD peptide had no effect on decidualization when added alone. The binding analysis showed that IGFBP-1 bound to the surface of endometrial stromal cells and an anti-alpha5beta1 integrin antibody inhibited its binding. These results suggest that IGFBP-1 produced by endometrium can mediate progesterone-induced decidualization possibly by interacting with alpha5beta1 integrin on the surface of endometrial stromal cells.

Insulin-like growth factor-binding protein 2 secreted by a tumorigenic cell line supports ex vivo expansion of mouse hematopoietic stem cells.

Successful hematopoietic stem cell (HSC) transplantation is often limited by the numbers of HSCs, and robust methods to expand HSCs ex vivo are needed. We previously showed that angiopoietin-like proteins (Angptls), a group of growth factors isolated from a fetal liver HSC-supportive cell population, improved ex vivo expansion of HSCs. Here, we demonstrate that insulin-like growth factor-binding protein 2 (IGFBP2), secreted by a tumorigenic cell line, also enhanced ex vivo expansion of mouse HSCs. On the basis of these findings, we established a completely defined, serum-free culture system for mouse HSCs, containing SCF, thrombopoietin, fibroblast growth factor 1, Angptl3, and IGFBP2. As measured by competitive repopulation analyses, there was a 48-fold increase in numbers of long-term repopulating mouse HSCs after 21 days of culture. This is the first demonstration that IGFBP2 stimulates expansion or proliferation of murine stem cells. Our finding also suggests that certain cancer cells synthesize proteins that can stimulate HSC expansion. Disclosure of potential conflicts of interest is found at the end of this article.

The divergent effect of insulin-like growth factor binding protein (IGFBP) - 1 on IGF-induced Steroidogenesis in bovine adrenocortical cells is not due to its phosphorylation status.

In previous studies we have shown that insulin-like growth factor (IGF)-II stimulates basal as well as ACTH-induced cortisol secretion from bovine adrenocortical cells more potently than IGF-I. The steroidogenic effect of both, IGF-I and IGF-II, is mediated through the IGF-I receptor and modified by locally produced IGF binding proteins. Further studies demonstrate that bovine adrenocortical cells do synthesize IGFBP-1 to -4 and that their secretion is regulated differentially by IGFs and ACTH. Since IGFBP-1 selectively is induced 3- to 4- fold by ACTH, the aim of the following studies was to evaluate the effect of IGFBP-1 on IGF-induced cortisol secretion from bovine adrenocortical cells in primary culture. Coincubation of bovine adrenocortical cells with purified IGFBP-1 (30 nM) induced a time--and dose-dependent potentiating effect (38+/-2%) on IGF-I-stimulated (6.5 nM) cortisol-secretion, wheras the IGF-II induced steroidogenic effect was inhibited (40+/-3%) by IGFBP-1. Similar results were found when bovine adrenocortical cells were preincubated with IGFBP-1 before stimulation experiments with IGFs were performed. In order to evaluate the influence of different phosphorylation states of IGFBP-1 on the steroidogenic effect of IGF-I and IGF-II and on the affinity to IGFs, a highly phosphorylated (pIGFBP-1) and a dephosphorylated isoform (dIGFBP-1) of IGFBP-1 have been separated by anion exchange chromatography for further incubation experiments and binding studies. No different effects on IGF-I or IGF-II-induced steroidogenesis were observed when a highly phosphorylated IGFBP-1 isoform was used, compared to the dephosphorylated IGFBP-1 isoforms. In binding studies IGFBP-1 did show high affinity binding for IGF-I with a calculated association constant (K (a)) of 2,17 x 10 (9) M (-1). Similar association constants were calculated for dIGFBP-1 and pIGFBP-1 (1,93 x 10 (9) M (-1) and 2,67 x 10 (9) M (-1) respectively) and no difference in binding capacity was observed when IGF-II was used as ligand. IN CONCLUSION, these results demonstrate that in bovine adrenocortical cells IGFBP-1 time- and dose-dependently inhibits the steroidogenic effect of IGF-II and stimulates the effect of IGF-I. The observed modulatory effect of IGFBP-1 is independent of the mode of incubation and its phosphorylation status. The previously observed stronger steroidogenic potency of IGF-II in bovine adrenocortical cells therefore can not be explained by an interaction with IGFBP-1. The mechanisms by which IGFBP-1 divergently regulates the steroidogenic effect of IGF-I and IGF-II remain unclear at present and further investigation is necessary to elucidate the mechanisms by which IGFBP-1 modulates IGF action in the adult adrenal gland.

Human chorionic gonadotropin and decidualization in vitro inhibits cytochalasin-D-induced apoptosis in cultured endometrial stromal fibroblasts.

Endometrial apoptosis increases from the proliferative phase through the secretory phase and peaks at menses. However, with the onset of pregnancy, the corpus luteum is rescued and stromal cells, instead of undergoing apoptosis, reorganize the cytoskeleton and then begin to differentiate. We hypothesized that in the presence of hormones (estradiol-17beta and medroxyprogesterone acetate), chorionic gonadotropin (hCG) as an early embryonic signal, and induction of decidualization by dibutyryl-cAMP (dbcAMP), endometrial stromal cells are rescued by the regulation of proteins that inhibit apoptosis. The percentage of cells stained with annexin V, an early apoptotic marker, increased dramatically after cytoskeletal disruption with cytochalasin D compared with non-cytochalasin-D-treated controls (P < 0.05). However, treatment of cells with hCG or dbcAMP in the presence of hormones significantly (P < 0.05) decreased the percentage of annexin-V-stained cells compared with cells treated with cytochalasin D alone. This inhibition was further confirmed by immunodetection of cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The inhibition of apoptosis by hCG and dbcAMP was via the intrinsic pathway because the cytochalasin-D-treated cells stained intensely for Bax, whereas the cells treated with hormones, hCG, or dbcAMP stained predominantly for Bcl-2. Treatment of cytochalasin-D-treated cells with hormones and dbcAMP resulted in an increase in the secretion of IGF-binding protein-1 (IGFBP-1) and prolactin. Treatment of cytochalasin-D-treated cells with recombinant IGFBP-1 and prolactin also inhibited apoptosis. These data suggest that under in vitro conditions, both hCG and the induction of decidualization play a direct role in preventing uterine stromal cells from undergoing apoptosis. Furthermore, this inhibition of apoptosis may be mediated in part by IGFBP-1 and prolactin and the alteration in the expression of Bcl-2 and Bax.

IGFBP-1 inhibits EGF mitogenic activity in cultured endometrial stromal cells.

The properties of the insulin-like growth factor-binding proteins (IGFBP-1 to 6) are not limited to modulation of IGF actions. IGFBP-1, which shares an Arg-Gly-Asp (RGD) motif in its C-terminal domain, modulates cell motility by binding to integrin alpha5beta1. The cross-talks between integrins and growth factor receptor signalling pathways are extensively documented, particularly in the case of the epidermal growth factor receptor (EGFR). However, whether IGFBP-1 can modulate growth factor signalling through its interaction with integrin alpha5beta1 has not yet been studied. As EGF is involved in the decidualisation of endometrial stromal cells (ESCs) and as decidualised ESCs are a source of IGFBP-1, we investigated if IGFBP-1 can modulate EGF effects on ESCs. RGD- and IGF-independent inhibition of EGF mitogenic activity and EGFR signalling by IGFBP-1 were demonstrated in ESC primary cultures, A431, cells and in mouse fibroblasts lacking IGF receptors.