Age-Related Insulin-Like Growth Factor Binding Protein-4 Overexpression Inhibits Osteogenic Differentiation of Rat Mesenchymal Stem Cells.

BACKGROUND/AIMS: Insulin-like growth factor binding proteins (IGFBP) play important roles in bone metabolism. IGFBP4 is involved in senescent-associated phenomena in mesenchymal stem cells (MSCs). The goal of the present study was to determine whether age-related IGFBP4 overexpression is associated with the impaired osteogenic differentiation potential of aged bone marrow derived MSCs. METHODS: MSCs were isolated from Sprague-Dawley rats aged 3-26 months. The bone morphogenetic protein (BMP)-2-induced osteogenic differentiation of rat MSCs was assessed by analyzing the expression levels of osteoblast marker genes [runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteocalcin (OC)], ALP activity and calcification. RESULTS: Our study showed that IGFBP4 mRNA and protein expression increased with age in parallel with impaired osteogenic differentiation of MSCs cultured in BMP2-containing osteogenic medium, as evidenced by the downregulation of osteoblast marker genes, and decreased ALP activity and calcium deposits. IGFBP4 overexpression impaired BMP2-induced osteogenic differentiation potential of young MSCs, whereas IGFBP4 knockdown restored the osteogenic potency of aged MSCs. Moreover, IGFBP4 knockdown stimulated the activation of Erk and Smad by increasing phosphorylation. CONCLUSION: Collectively, our results demonstrate that IGFBP4 overexpression plays a role in the impairment of MSC differentiation potential via the Erk and Smad pathways, suggesting potential targets to improve MSC function for cell therapy applications.

IGFBP-4 and -5 are expressed in first-trimester villi and differentially regulate the migration of HTR-8/SVneo cells.

BACKGROUND: Adverse gestational outcomes such as preeclampsia (PE) and intrauterine growth restriction (IUGR) are associated with placental insufficiency. Normal placental development relies on the insulin-like growth factors -I and -II (IGF-I and -II), in part to stimulate trophoblast proliferation and extravillous trophoblast (EVT) migration. The insulin-like growth factor binding proteins (IGFBPs) modulate the bioavailability of IGFs in various ways, including sequestration, potentiation, and/or increase in half-life. The roles of IGFBP-4 and -5 in the placenta are unknown, despite consistent associations between pregnancy complications and the levels of two IGFBP-4 and/or -5 proteases, pregnancy-associated plasma protein -A and -A2 (PAPP-A and PAPP-A2). The primary objective of this study was to elucidate the effects of IGFBP-4 and -5 on IGF-I and IGF-II in a model of EVT migration. A related objective was to determine the timing and location of IGFBP-4 and -5 expression in the placental villi. METHODS: We used wound healing assays to examine the effects of IGFBP-4 and -5 on the migration of HTR-8/SVneo cells following 4 hours of serum starvation and 24 hours of treatment. Localization of IGFBP-4, -5 and PAPP-A2 was assessed by immunohistochemical staining of first trimester placental sections. RESULTS: 2 nM IGF-I and -II each increased HTR-8/SVneo cell migration with IGF-I increasing migration significantly more than IGF-II. IGFBP-4 and -5 showed different levels of inhibition against IGF-I. 20 nM IGFBP-4 completely blocked the effects of 2 nM IGF-I, while 20 nM IGFBP-5 significantly reduced the effects of 2 nM IGF-I, but not to control levels. Either 20 nM IGFBP-4 or 20 nM IGFBP-5 completely blocked the effects of 2 nM IGF-II. Immunohistochemistry revealed co-localization of IGFBP-4, IGFBP-5 and PAPP-A2 in the syncytiotrophoblast layer of first trimester placental villi as early as 5 weeks of gestational age. CONCLUSIONS: IGFBP-4 and -5 show different levels of inhibition on the migration-stimulating effects of IGF-I and IGF-II, suggesting different roles for PAPP-A and PAPP-A2. Moreover, co-localization of the pappalysins and their substrates within placental villi suggests undescribed roles of these molecules in early placental development.

Temporal expression patterns of insulin-like growth factor binding protein-4 in the embryonic and postnatal rat brain.

BACKGROUND: IGFBP-4 has been considered as a factor involving in development of the central nervous system (CNS), but its role needs to be further clarified. In present study, the localization of IGFBP-4 expression in the embryonic forebrain, midbrain and hindbrain was determined using immunohistochemistry, and the levels of IGFBP-4 protein and mRNA were semi-quantified using RT-PCR and Western blot in the embryonic (forebrain, midbrain and hindbrain) and postnatal brain (cerebral cortex, cerebellum and midbrain). RESULTS: A clear immunoreactivity of IGFBP-4 covered almost the entire embryonic brain (forebrain, midbrain, hindbrain) from E10.5 to E18.5, except for the area near the ventricle from E14.5. The change of IGFBP-4 mRNA level was regularly from E10.5 to E18.5: its expression peaked at E13.5 and E14.5, followed by gradual decreasing from E15.5. The expression of IGFBP-4 protein was similar to that of mRNA in embryonic stage. After birth, the pattern of IGFBP-4 expression was shown to be rather divergent in different brain areas. In the cerebral cortex, the IGFBP-4 mRNA increased gradually after birth (P0), while the protein showed little changes from P0 to P28, but decreased significantly at P70. In the cerebellum, the IGFBP-4 mRNA decreased gradually from P0, reached the lowest level at P21, and then increased again. However, its protein level gradually increased from P0 to P70. In the midbrain, the IGFBP-4 mRNA first decreased and reached its lowest level at P28 before it increased, while the protein remained constant from P0 to P70. At P7, P14, P21, P28 and P70, the levels of IGFBP-4 mRNA in the cerebral cortex were significantly higher than that in the cerebellum or in the midbrain. Differently, the protein levels in the cerebellum were significantly higher than that either in the cerebral cortex or in the midbrain at P14, P21, P28 and P70. CONCLUSIONS: The temporal expression pattern of IGFBP-4 in the embryonic brain from E10.5 to E18.5 was consistent with the course of neurogenesis in the ventricular zone, suggesting an important role of IGFBP-4 in regulating differentiation of neural stem cells. A strikingly higher abundance of the IGFBP-4 protein observed in the cerebellum from P14 to P70 suggests that IGFBP-4 may participate in the maintenance of cerebellar plasticity.

TCDD induces the expression of insulin-like growth factor binding protein 4 in 5L rat hepatoma cells: a cautionary tale of the use of this cell line in studies on dioxin toxicity.

Previous quantitative proteomic studies on the actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 5L rat hepatoma cells, a cell model frequently used for investigating the mechanisms of TCDD toxicity, had indicated that dioxin exposure reduced the abundance of numerous proteins which are regulated at the level of protein synthesis initiation. In the present study, we have analysed the mechanism mediating this inhibition. TCDD treatment of the cells largely prevented the activation of eukaryotic translation initiation factor 4E-binding protein 1, a regulator of translation initiation and substrate of the mammalian target of rapamycin (mTOR). By "working upwards" from mTOR, we observed that TCDD inhibited endogenous and IGF-I-induced AKT and ERK activation by interfering with tyrosine phosphorylation of insulin receptor substrate 1. This inhibition was mediated by a TCDD-induced secreted factor which was identified as insulin-like growth factor binding protein 4 (IGFBP-4). The induction of IGFBP-4 protein was dependent on a functional aryl hydrocarbon receptor and was preceded by a rapid increase in the level of IGFBP-4 mRNA indicating that IGFBP-4 is a previously unknown transcriptional target of TCDD in 5L cells. IGFBP-4 was not induced by TCDD in the parental cell line of 5L cells, Fao, and in various closely related rat hepatoma cell lines as well as in other unrelated cell types. Analysis of 5L cell chromosomes by multicolour spectral karyotyping (SKY) revealed that the cells carry several hitherto uncharacterised chromosomal translocations. The observations suggest that in 5L cells the Igfbp-4 gene may have got under the control of a promoter containing dioxin responsive element(s) leading to the induction of IGFBP-4 by TCDD. These findings emphasise a particular caution when interpreting and extrapolating results on the action mechanisms of TCDD obtained in studies using 5L cells as a model system.

Intramammary infusion of Panax ginseng extract in the bovine mammary gland at cessation of milking modifies components of the insulin-like growth factor system during involution.

The objective of this study was to evaluate the effects of a single intramammary infusion of Panax ginseng extract (GS) on insulin-like growth factors (IGF) in bovine mammary gland during early involution. Eight mammary quarters from six nonpregnant cows in late lactation were infused with 10 mL of ginseng extract solution (3 mg/mL), six quarters were treated with 10 mL of placebo (vehicle alone) and six quarters were maintained as uninoculated controls. Milking was interrupted after infusion. Concentrations of IGF1 in mammary secretions were higher in GS-treated quarters than in placebo and uninoculated control quarters at 24, 48 and 72 h post-treatment (p<0.05). Treatment with GS did not affect mammary secretion of IGF2 (p=0.942). At 7 d of post-lactational involution, a decrease of immunostained area and mRNA expression for IGF1 was observed in mammary tissue of GS-treated quarters compared with placebo-treated quarters and uninoculated controls (p<0.05). The IGF2 immunostained area and mRNA expression for this growth factor were not affected by GS treatment (p=0.216 and p=0.785, respectively). An increase in protein levels and mRNA expression in mammary tissue of IGFBP3, IGFBP4 and IGFBP5 was observed in GS-treated quarters compared with placebo-treated quarters and uninoculated controls (p<0.05). These results provide evidence that intramammary inoculation of GS extract at cessation of milking may promote early mammary involution through the inhibition of IGF1 local production and bioavailability.

An in situ hybridization study of the insulin-like growth factor system in developing condylar cartilage of the fetal mouse mandible.

The objective of this study was to investigate the involvement of the insulin-like growth factor (IGF) system in the developing mandibular condylar cartilage and temporomandibular joint (TMJ). Fetal mice at embryonic day (E) 13.0-18.5 were used for in situ hybridization studies using [35S]-labeled RNA probes for IGF-I, IGF-II, IGF-I receptor (-IR), and IGF binding proteins (-BPs). At E13.0, IGF-I and IGF-II mRNA were expressed in the mesenchyme around the mandibular bone, but IGF-IR mRNA was not expressed within the bone. At E14.0, IGF-I and IGF-II mRNA were expressed in the outer layer of the condylar anlage, and IGF-IR mRNA was first detected within the condylar anlage, suggesting that the presence of IGF-IR mRNA in an IGF-rich environment triggers the initial formation of the condylar cartilage. IGFBP-4 mRNA was expressed in the anlagen of the articular disc and lower joint cavity from E15.0 to 18.5. When the upper joint cavity was formed at E18.5, IGFBP-4 mRNA expression was reduced in the fibrous mesenchymal tissue facing the upper joint cavity. Enhanced IGFBP-2 mRNA expression was first recognized in the anlagen of both the articular disc and lower joint cavity at E16.0 and continued expression in these tissues as well as in the fibrous mesenchymal tissue facing the upper joint cavity was observed at E18.5. IGFBP-5 mRNA was continuously expressed in the outer layer of the perichondrium/fibrous cell layer in the developing mandibular condyle. These findings suggest that the IGF system is involved in the formation of the condylar cartilage as well as in the TMJ.

Expression of a protease-resistant insulin-like growth factor-binding protein-4 inhibits tumour growth in a murine model of breast cancer.

BACKGROUND: Insulin-like growth factor 1 (IGF1) promotes breast cancer and disease progression. Bioavailability of IGF1 is modulated by IGF-binding proteins (IGFBPs). IGFBP4 inhibits IGF1 activity but cleavage by pregnancy-associated plasma protein-A (PAPP-A) protease releases active IGF1. METHODS: Expression of IGF pathway components and PAPP-A was assessed by western blot or RT-PCR. IGFBP4 (dBP4) resistant to PAPP-A cleavage, but retaining IGF-binding capacity, was used to block IGF activity in vivo. 4T1.2 mouse mammary adenocarcinoma cells transfected with empty vector, vector expressing wild-type IGFBP4 or vector expressing dBP4 were implanted in the mammary fat pad of BALB/c mice and tumour growth was assessed. Tumour angiogenesis and endothelial cell apoptosis were assessed by immunohistochemistry. RESULTS: 4T1.2 cells expressed the IGF1R receptor and IGFBP4. PAPP-A was expressed within mammary tumours but not by 4T1.2 cells. Proliferation and vascular endothelial growth factor (VEGF) production by 4T1.2 cells was increased by IGF1(E3R) (recombinant IGF1 resistant to binding by IGFBPs) but not by wild-type IGF1. IGF1-stimulated microvascular endothelial cell proliferation was blocked by recombinant IGFBP4. 4T1.2 tumours expressing dBP4 grew significantly more slowly than controls or tumours expressing wild-type IGFBP4. Inhibition of tumour growth by dBP4 was accompanied by the increased endothelial cell apoptosis. CONCLUSION: Protease-resistant IGFBP4 blocks IGF activity, tumour growth and angiogenesis.

Insulin-like growth factor binding proteins IGFBP3, IGFBP4, and IGFBP5 predict endocrine responsiveness in patients with ovarian cancer.

PURPOSE: This study sought to explore the predictive value of the insulin-like growth factor (IGF) binding proteins (IGFBP) as markers of response in ovarian cancer patients treated with the aromatase inhibitor letrozole. EXPERIMENTAL DESIGN: IGFBP mRNA expression in cell lines was measured by quantitative reverse transcription-PCR and IGFBP protein expression measured in sections from primary tumors of patients treated with letrozole by semiquantitative immunohistochemistry. RESULTS: Quantitative reverse transcription-PCR analysis showed that IGFBP3 and IGFBP5 were down-regulated and IGFBP4 was up-regulated by 17beta-estradiol (E(2)) in an estrogen receptor (ER)-positive ovarian cancer cell line. Expressions of IGFBP1, IGFBP2, and IGFBP6 were unaffected by E(2). The E(2) modulation of these genes was reversed by tamoxifen. Using ERalpha-specific (propyl pyrazole triol) and ERbeta-specific (diarylpropionitrile) agonists, the gene expression modulations produced by E(2) could be replicated by propyl pyrazole triol but not by diarylpropionitrile. For ovarian cancer patients being treated with letrozole, we tested the predictive value of the IGFBPs in paraffin-fixed sections from their primary tumors by semiquantitative immunohistochemistry. Using serum CA125 as an indicator of progression/response, significant differences in expression levels of IGFBPs were observed between tumors from CA125 responding/stable patients compared with tumors from progressing patients. Mean immunoscores for IGFBP3 and IGFBP5 were significantly lower, and mean expression of IGFBP4 was significantly higher in tumors from patients demonstrating CA125 response or stabilization compared with CA125 progression. CONCLUSION: These results indicate that expression levels of certain IGFBP family members in ovarian cancers are estrogen regulated and can, thus, help identify patients who could benefit from endocrine therapy.

Insulin-like growth factor binding protein-4 (IGFBP-4) is a novel anti-angiogenic and anti-tumorigenic mediator secreted by dibutyryl cyclic AMP (dB-cAMP)-differentiated glioblastoma cells.

cAMP has been shown to reverse the transformed phenotype of various cancer cells. Human glioblastoma U87MG cells exposed to 500 microM dB-cAMP for 6 days showed reduced proliferation, attenuated invasiveness, and inability to induce angiogenic responses in human brain endothelial cells (HBECs) grown in Matrigeltrade mark. VEGF was the principal mediator of angiogenic actions of U87MG conditioned media (CM), since VEGF neutralizing antibody completely inhibited U87MG-induced angiogenic responses and no detectable levels of IGF, bFGF, and PlGF were found in U87MG CM. VEGF release was induced ( approximately 20%) in dB-cAMP-treated U87MG cells, suggesting a simultaneous induction of anti-angiogenic mediators. Down-stream effectors of dB-cAMP actions in U87MG were investigated by microarray gene expression analysis. Detected increases in differentiation genes, staniocalcin-1 and Wnt-5a, and angiogenesis-related genes, PAI-1, SPARC, IGFBP-4, IGFBP-7, PAPP-A, and PRSS-11 in dB-cAMP-treated U87MG cells were validated by real-time PCR, Western blot, and/or ELISA. A subsequent series of experiments identified IGFBP-4 as the principal anti-angiogenic mediator secreted by glioblastoma cells in response to dB-cAMP. Human recombinant IGFBP-4 inhibited the angiogenic response of HBEC induced by U87MG CM, whereas anti-human IGFBP-4 antibody restored the pro-angiogenic activity of dB-cAMP-treated U87MG CM. Since neither U87MG nor HBEC cells secreted detectable levels of IGF-I, and there are no known cellular IGFBP-4 receptors, the anti-angiogenic effect of IGFBP-4 was likely IGF-I-independent and indirect. IGFBP-4 also antagonized angiogenic effects of VEGF(165), PlGF, and bFGF, and reduced U87MG colony formation in soft-agar. IGFBP-4 is a novel dB-cAMP-induced anti-angiogenic and anti-tumorigenic mediator that may be a promising candidate for glioblastoma therapy.

Neuromuscular stimulation causes muscle phenotype-dependent changes in the expression of the IGFs and their binding proteins in developing slow and fast muscle of chick embryos.

Insulin-like growth factor (IGF) -1 and -2 and binding protein (IGFBP-2, -4, and -5) expression can be affected by several environmental factors, but the impact of movement on the IGF axis during late embryogenesis has yet to be fully characterized. Movement was promoted in chick embryos during mid-embryogenesis using 4-aminopyridine (4-AP). The results indicate an increase in IGF-1 (P < 0.01) and a decrease in IGFBP-2 (P < 0.05) mRNA expression in slow muscle of the stimulated group compared with the control group. In fast muscle, there was a decrease in IGF-2 (P < 0.01) on embryonic day (ED) 16 and an increase in IGFBP-2 (P < 0.01) and IGFBP-4 (P < 0.05) and in IGFBP-5 (P < 0.05) expression on ED18 in the stimulated group compared with the control group. These results indicate that neuromuscular stimulation with 4-AP influences IGF axis gene expression in a muscle fiber type-dependent manner. Consequences of the changes in the IGF system for each muscle are discussed.

Insulin-like growth factor-binding protein (IGFBP)-1, -2, -3, -4, -5, and -6 and IGFBP-related protein 1 during rainbow trout postvitellogenesis and oocyte maturation: molecular characterization, expression profiles, and hormonal regulation.

In the present study we report the full coding sequence of rainbow trout IGF-binding protein-1 (IGFBP1), -2, -3, -5, and -6 and IGFBP-related protein-1 (IGFBP-rP1) mRNAs as well as the partial coding sequence of IGFBP-4 mRNA. To our knowledge, this is the first report of IGFBP4, IGFBP6, and IGFBP-rP1 in a nonmammalian species. The tissue distribution of all mRNAs was studied, and the ovarian expression profiles of IGFBP2 to -6 and IGFBP-rP1 between late vitellogenesis and oocyte maturation were characterized. In addition, in vitro hormonal regulation by the maturation-inducing steroid 17,20beta-dihydroxy-4-pregnen-3-one (17,20betaP), gonadotropin, and estradiol were studied. We observed that besides IGFBP1, which was only found in liver, IGFBP2 to -6 and IGFPB-rP1 were expressed in the preovulatory ovary. IGFBP3 was also detected in liver, trunk, kidney, skin, and gills, whereas IGFBP2 to -6 and IGFBP-rP1 exhibited a wider tissue distribution. In the preovulatory ovary, IGFBP3 was strongly down-regulated during the postvitellogenesis period, whereas IGFBP5 exhibited a limited up-regulation. In addition, IGFBP6 and IGFBP-rP1 were up-regulated during oocyte maturation. Hormonal regulation data indicated that all ovarian IGFBPs and IGFBP-rP1 transcripts are regulated under gonadotropic stimulation at a concentration that induced 100% oocyte maturation. In addition, IGFBP2 to -5 transcripts are regulated by 17,20betaP and estradiol. Together, our observations strongly suggest that during final oocyte maturation, a down-regulation of IGFBP3, -4, and -5 occurs in the oocyte in response to gonadotropic and 17,20betaP (IGFBP3 and -5) stimulation, whereas an up-regulation of IGFBP2 and -6 occurs in follicular layers or extrafollicular tissue in response to gonadotropic stimulation.

Myocardial insulin-like growth factor-I gene expression during recovery from heart failure after combined left ventricular assist device and clenbuterol therapy.

BACKGROUND: Patients who undergo mechanical support with a left ventricular assist device (LVAD) exhibit reverse remodeling and in some cases recover from heart failure. We have developed a combination therapy using LVAD support combined with pharmacological therapy to maximize reverse remodeling, followed by the beta2 adrenergic agonist clenbuterol. We recently found that clenbuterol induces insulin-like growth factor I (IGF-I) in cardiac myocytes in vitro. The purpose of this study is to examine IGF-I expression in recovery patients after combination therapy. METHODS AND RESULTS: Myocardial mRNA levels were determined by real-time quantitative polymerase chain reaction in 12 recovery patients (at LVAD implantation, explantation, and 1 year after explantation). IGF-I mRNA was elevated at the time of LVAD explantation relative to donors, with 2 groups distinguishable: Those with low IGF-I mRNA at implantation who showed significant increase during recovery and those with high IGF-I mRNA at implantation who remained high. Levels returned to normal by 1 year after explantation. Microarray analysis of implantation and explantation samples of recovery patients further revealed elevated IGF-II and IGF binding proteins IGFBP4 and IGFBP6. IGF-I levels correlated with stromal cell-derived factor mRNA measured both in LVAD patients and in a wider cohort of heart failure patients. CONCLUSIONS: The data suggest involvement of elevated myocardial IGF-I mRNA in recovery. IGF-I may act to limit atrophy and apoptosis during reverse remodeling and to promote repair and regeneration in concert with stromal cell derived factor.

Oxygen-dependent modulation of insulin-like growth factor binding protein biosynthesis in primary cultures of rat hepatocytes.

Higher levels of IGF-binding protein 1 (IGFBP-1) mRNA are expressed in the less aerobic perivenous zone of the liver. Because gradients in oxygen tension (pO(2)) may contribute to zonated gene expression, the influence of arterial and venous pO(2) on IGFBP-1 biosynthesis was studied in primary cultures of rat hepatocytes. Maximal IGFBP-1 mRNA and protein levels were observed under venous pO(2), whereas less than 30% of maximal levels were observed under arterial pO(2). In contrast, the expression of IGFBP-4 was greatest under arterial pO(2), indicating that this effect of hypoxia on IGFBP-1 gene expression is specific. The response to hypoxia appears to involve reactive oxygen species, because treatment with H(2)O(2) results in a dose-dependent decrease of IGFBP-1 mRNA levels under venous pO(2), whereas IGFBP-1 mRNA expression under arterial pO(2) was not affected. Inhibition of the hypoxia-dependent IGFBP-1 mRNA induction by actinomycin D indicates that this effect is mediated at the level of gene transcription, and inhibition of IGFBP-1 mRNA by the iron chelator desferrioxamine under both venous and arterial pO(2) suggested the involvement of hypoxia-inducible transcription factors (HIF). Transfection experiments demonstrated that especially HIF-3alpha and HIF-2alpha, and to a lesser extent HIF-1alpha, contribute to the induction of IGFBP-1 mRNA expression in isolated hepatocytes, whereas experiments with vectors for the HIF prolyl hydroxylases (PHD) indicated a major role of PHD-2 in destabilization of HIFs, attenuating the induction of IGFBP-1 under venous pO(2). Reporter gene studies indicate that hypoxia stimulates IGFBP-1 expression through a putative HIF response element located approximately 250 bp upstream from the transcription initiation site. Together, these results support the concept that iron, radical oxygen species, and the HIF-2 and -3 as well as the PHD pathways play important roles in mediating effects of hypoxia on IGFBP-1 gene expression in the liver.

Steroid-modulated proliferation of human endometrial carcinoma cell lines: any role for insulin-like growth factor signaling?

OBJECTIVES: Estrogen-stimulated proliferation of the normal and malignant human endometrium is balanced by the differentiating properties of progesterone. This study evaluated the role of insulin-like growth factor (IGF) signaling in steroid-induced modulation of endometrial cancer cell proliferation. METHODS: We used the human endometrial, estrogen-responsive ECC-1 and progesterone-responsive PRAB-36 cell lines. Proliferation studies with IGFs in combination with either estrogen or progesterone were conducted. Furthermore, the mRNA and protein expression of insulin-like growth factor-binding proteins (IGFBPs) was evaluated. RESULTS: Using the ECC-1 cell line, we observed that estrogen-induced proliferation is modulated via the IGF-receptor signaling pathway, and that IGF-1-induced stimulation of proliferation does not influence estrogen receptor signaling. Furthermore, expression of the main modulators of IGF action, the IGFBPs, was found to be regulated by estrogen and progesterone in both cell lines. IGFBP-4 was up-regulated by estrogen in the ECC-1 cell line, and IGFBP-3 and IGFBP-6 were down-regulated by progesterone in the PRAB-36 cell line. CONCLUSION: Estrogen-induced stimulation of proliferation of ECC-1 endometrial cancer cells is partly achieved via IGF signaling. Furthermore, the IGFBPs are regulated by estrogens as well as progestagens and could potentially play a role in the modulation of endometrial cancer cell proliferation.

Pregnancy-associated plasma protein-A (PAPP-A) expression and insulin-like growth factor binding protein-4 protease activity in normal and malignant ovarian surface epithelial cells.

Pregnancy-Associated Plasma Protein-A (PAPP-A) proteolyses insulin-like growth factor binding protein-4 (IGFBP-4), thereby regulating local IGF availability. Reduced PAPP-A mRNA expression has been reported in ovarian cancer specimens compared to normal ovarian surface epithelial cells (OSE). To characterize PAPP-A expression and proteolytic activity in OSE, we developed a lifespan-extended human cell model using a temperature-sensitive mutant of the SV40 large T antigen (SV40LT). These OSE(tsT) cells proliferate at 34 degrees C (i.e., when SV40LT-positive), but not at 39 degrees C, a temperature at which the SV40LT is unstable (SV40LT-negative). Proteolysis of radiolabeled IGFBP-4 in conditioned media from OSE(tsT) lines was IGF-dependent and blocked by anti-PAPP-A antisera. Temperature shifts that eliminated stable SV40LT induced a 7-fold increase in PAPP-A mRNA and a 4-fold increase in protein. The converse experiment (shifting to SV40LT-positive conditions) resulted in decreased levels of PAPP-A mRNA but little change in PAPP-A protein. Nevertheless, there was a marked reduction in IGF-BP-4 proteolytic activity in medium of SV40LT-positive OSE-(tsT) cells. This decreased PAPP-A activity coincided with a nearly 20-fold increase in mRNA encoding a physiological inhibitor of PAPP-A, the precursor form of eosinophil Major Basic Protein (proMBP), and 4- to 5-fold increases in proMBP protein. Primary cultures of unmodified OSE expressed high levels of PAPP-A and undetectable proMBP, and therefore produced abundant IGFBP-4 protease activity. Short-term ovarian tumor cell cultures expressed variable levels of PAPP-A and high levels of proMBP, and consequently secreted little or no IGFBP-4 protease activity. The concurrent regulation of PAPP-A and its inhibitor, proMBP, suggests that IGFBP-4 proteolysis and local regulation of IGF availability may be altered in malignant ovarian epithelial cells.

Up date on IGFBP-4: regulation of IGFBP-4 levels and functions, in vitro and in vivo.

Of the six known high affinity insulin-like growth factor binding-proteins (IGFBPs), IGFBP-4 appears to be unique in that it is the only IGFBP that functions mostly like a traditional binding protein. In this regard, none of the IGF independent effects that have been ascribed for other IGFBPs have been described for IGFBP-4. However, recent in vitro and in vivo studies, in particular the recent identification of pregnancy-associated plasma protein-A as a major IGFBP-4 protease, are consistent with the idea that IGFBP-4 is an extremely important component of IGF system in several tissues including gonads and bone. In this review, we have provided an update on IGFBP-4 research and we have summarized our current understanding of the regulation of levels and actions of IGFBP-4 and proteolytic fragments both in vitro and in vivo.

Expression of insulin-like growth factor I (IGF-I) gene and of genes for IGF-binding proteins 1, 2, 3, 4 (IGFBP-1-IGFBP-4) in non-neoplastic human thyroid cells and in certain human thyroid cancers. Effect of exogenous IGF-I on this expression.

AIM: The aim of this study was to examine the expression of the IGF-I gene and of genes for IGFBP-1, -2, -3, and -4 in cells from nodular goiters (NG), and from different human thyroid carcinomas (papillary--PTC, anaplastic--ATC, and medullary--MTC), cultured in monolayers. The influence, exerted by exogenous IGF-I on the expression of these genes, was also investigated. METHODS: Thyroid tissue specimens were obtained from 65 patients during subtotal or total thyroidectomies. After approximately 2-3 weeks of culture, thyroid cells were incubated for 24 hours with IGF-I in concentrations of: 0, 1, 10 and 100 ng/ml. The total mRNA was isolated according to the method described by Chomczynski and Sacchi with our own modifications. Afterwards, mRNA encoding IGF-I, IGFBP-1-IGFBP-4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were amplified, using the reverse transcription-polymerase chain reaction (RT-PCR); GAPDH gene served as a control gene. PCR products were electrophoresed and then submitted to densitometric analysis. RESULTS AND CONCLUSIONS: Our study has shown that in carcinoma cells (ATC, PTC, MTC), IGF-I reveals a stimulatory influence on the expression of its own gene, that effect being most distinctive in ATC cells. These facts indicate an important role of IGF-I in the pathogenesis and invasiveness of the analyzed malignant neoplasms.

Insulin-like growth factor binding protein-1-6 expression in activated microglia.

In the CNS insulin-like growth factor-1 (IGF-1) enhances survival of neurons, promotes myelin synthesis and acts as a mitogen for microglia. The effects of IGF-1 are regulated by a family of 6 IGF binding proteins (IGFBPs). We investigated mRNA expression patterns of IGFBPs in primary rat microglia under basal conditions and after activation with lipopolysaccharide (LPS). Under basal conditions, microglia expressed IGFBP-2 to -6, whereas, IGFBP-1 could not be detected. Following 2 h treatment with LPS mRNA levels for IGFBP-4 and -6 displayed a down regulation, and IGFBP-5 became undetectable. Levels of IGFBP-2 and -3 remained unaltered. Expression patterns of IGFBPs might play an important role in regulating the autocrine/paracrine IGF-1 actions on microglia under inflammatory conditions.

The influence of undernutrition during gestation on skeletal muscle cellularity and on the expression of genes that control muscle growth.

We examined the effects of two levels of gestational undernutrition (50 % and 40 % of ad libitum) on postnatal growth rate, skeletal muscle cellularity and the expression of genes that control muscle growth, in the offspring at weaning. The results showed that the rat pups born to mothers fed the 50 % diet during gestation and a control diet during lactation had an increased postnatal growth rate compared with the pups fed the more restricted diet (40 % of ad libitum). Surprisingly, the growth rate of the control group (ad libitum) was intermediate between the 50 % and 40 % groups. The restricted diets did not alter the number of muscle fibres in the semitendinosus muscle of the offspring but the number of muscle nuclei was reduced by 16 % in the 40 % group compared with the control group. In the 50 % group, the lightest pups at birth (L) had elevated muscle insulin-like growth factor (IGF)-1, IGF binding protein (BP)-5 and proliferating cell nuclear antigen (PCNA) mRNA compared with the L pups from both the control and 40 % groups. The heaviest pups at birth (H) in the 50 % group had increased levels of IGFBP-4, PCNA and M-cadherin mRNA compared with both the control and 40 % groups. Levels of IGF-1 receptor, myostatin and MyoD mRNA did not correlate with postnatal growth. Both H and L pups from the 40 % group had reduced muscle IGF-1 mRNA but all other transcripts examined were similar to control levels. The results suggest that the increased postnatal growth rate, which accompanied milder fetal undernutrition (50 %), may be due to a more active local muscle IGF system and increased muscle-cell proliferation.

Retinoic acid increases insulin-like growth factor-binding protein-4 expression in cultured rat hepatocytes.

Hepatic insulin-like growth factor binding protein (IGFBP) expression is controlled by diverse factors including thyroid hormone, which enhances IGFBP-4 production in hepatocytes. In the present work, we have investigated whether hepatic IGFBP-4 expression is regulated by retinoic acid (RA), which acts via nuclear receptors belonging to the steroid/thyroid hormone receptor superfamily. Primary cultures of adult rat hepatocytes were incubated with two natural stereoisomers of RA, all-trans RA and 9-cis RA (atRA and 9cRA), and with the synthetic RA receptor (RAR)-selective agonist TTNPB. IGFBP-4 mRNA abundance was measured by Northern blot and protein production was evaluated by Ligand blot on hepatocyte-conditioned culture media. Our results indicate that atRA, 9cRA, and TTNPB increase IGFBP-4 expression by cultured hepatocytes, both at the mRNA and protein level. The RARs play a definite role in this regulation, which is independent from ongoing protein synthesis but dependent on active transcription. AtRA and thyroid hormone act synergistically in increasing hepatic IGFBP-4 expression. Our data establish a role for hormonal factors such as thyronines and retinoids in regulating the hepatic IGF system directly at the IGFBP-4 level.