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.

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.

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.

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.

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.

Thyroid hormone and retinoic acid induce the synthesis of insulin-like growth factor-binding protein-4 in mouse osteoblastic cells.

Thyroid hormone (T3) is a known regulator of the transcription rate of specific genes. By subtractive hybridization of T2-treated osteoblastic cells, differentially expressed messenger RNAs (mRNAs) were enriched in the form of double stranded complementary DNA (cDNA) fragments. Sequencing of a differentially expressed cDNA that detects a 2.6-kilobase mRNA in Northern blots revealed to homology in the EMBL-Genebank data bases. A mouse genomic library was screened, and the isolated genomic DNA was identified as part of the insulin-like growth factor-binding protein-4 (IGFBP-4) gene including the 3'-untranslated region to which the cloned cDNA fragment was mapped by sequencing. We observed an up-regulation of the 2.6-kilobase IGFBP-4 mRNA transcript in the presence of T3 or retinoic acid. The induction of the IGFBP-4 transcript persisted up to 48 h. This response was inhibited by cycloheximide as well as actinomycin D. Long term induction studies revealed that the T3 effect is present during the complete culture period, with a constant rise in IGFBP-4 mRNA levels until 14 days. Under these culture conditions, the DNA content of MC3T3-E1 cells were significantly reduced by T3 and retinoic acid, indicating the repressive effect of both hormones on cell growth. Western immunoblots showed that the transcriptional induction is consequently transduced to increased IGFBP-4 levels in the conditioned medium of T3-treated cells. Our data show that thyroid hormone and retinoic acid stimulate transcription of IGFBP-4 mRNA in osteoblasts, resulting in increased IGFBP-4 secretion into the medium. IGFBP-4, a known inhibitor of cellular proliferation, might contribute to the antiproliferative effect of T3 and retinoic acid on osteoblasts.

Testosterone and insulin-like growth factor (IGF) I interact in controlling IGF-binding protein production in androgen-responsive foreskin fibroblasts.

The growth of the male external genitalia is primarily regulated by androgens. However, human genital fibroblast growth is also stimulated by insulin-like growth factor (IGF) I. In this study, we report that IGF-binding protein (IGFBP) production in human foreskin fibroblasts is regulated by androgens and IGF-I. Human foreskin fibroblasts secrete IGFBP-3, IGFBP-4, and IGFBP-5. IGF-I increased the abundance of both intact IGFBP-3 and -5 in the culture medium. Testosterone increased IGFBP-3, and the combination of IGF-I and testosterone had an additive effect. Following its secretion, IGFBP-5 was degraded, but the effect of IGF-I on IGFBP-5 peptide abundance in conditioned media did not seem to be due to inhibition of proteolysis. Testosterone had no effect on IGFBP-5 degradation. Intact IGFBP-4 was decreased by IGF-I, and the combination resulted in a similar reduction. The mechanism seemed to be decreased synthesis, since IGFBP-4 messenger RNA was also decreased. The increase in IGFBP-5 synthesis was associated with an increase in the abundance of intact IGFBP-5 in the extracellular matrix. The combination of testosterone and IGF-I resulted in a synergistic stimulation of total protein synthesis by the fibroblast cultures, suggesting that a maximum anabolic response requires both hormones. These observations suggest that combined exposure to androgen and IGF-I altered the abundance of some forms of IGFBPs and that the IGFBPs that are regulated may play a role in modulating the effects of IGF-I on the anabolic response.

Localization and regulation of pregnancy-associated plasma protein a expression in healing human skin.

Pregnancy-associated plasma protein A (PAPP-A) is an IGF-binding protein-4 (IGFBP-4) metalloproteinase that cleaves inhibitory IGFBP-4 to amplify local IGF-I bioavailability in vitro. Thus it has functional implications in injury/repair responses. In this study we determined PAPP-A expression in healing human skin. Wounds were induced with a scalpel on the forearms of three normal subjects and were allowed to heal by first intention. Biopsies obtained on d 0, 2, 8, and 14 were processed for immunohistochemical detection of PAPP-A, IGF-I, and IGFBP-4. In uninjured skin (d 0), strong staining for PAPP-A was present in the epidermis, sweat and sebaceous gland epithelial cells, hair follicles, and blood vessels; no PAPP-A was detected in dermal fibroblasts or with mature collagen bundles. IGF-I localized strongly to epithelial cells of skin glands was weak to moderate in epidermis and blood vessels, and was absent in dermal cells. Weak focal staining for IGFBP-4 was found within uninjured epidermis. During wound healing, PAPP-A expression was induced in dermal granulation tissue within and adjacent to the injury. PAPP-A was present in dermis on d 2 and was increased in intensity and extent on d 8 and 14. PAPP-A expression also increased in the epidermis. PAPP-A expression in cells of granulation tissue colocalized with alpha-smooth actin staining of myofibroblasts and new blood vessels as well as with CD68 staining of macrophages and was associated with the compact, newly synthesized collagen of the healing wound. IGF-I staining was enhanced in the epidermis localized to the area of the incision and in granulation tissue associated with lymphoid cells. IGFBP-4 staining of the epidermis remained unchanged during wound healing, but was induced in the fibroblastic cells of granulation tissue over time. These data demonstrate localized and regulated expression of PAPP-A in human skin and suggest that PAPP-A may play an important role in an integrated IGF system in wound healing and tissue remodeling in vivo.

Recombinant synthesis of insulin-like growth factor-binding protein-4 (IGFBP-4): Development, validation, and application of a radioimmunoassay for IGFBP-4 in human serum and other biological fluids.

Insulin-like growth factor-binding protein-4 (IGFBP-4), like the five other IGFBPs present in human serum, acts as a transport protein for insulin-like growth factor I (IGF-I) and IGF-II and modulates their biological effects. To investigate the role of IGFBP-4 in the physiology of the IGF system, we developed a sensitive RIA for IGFBP-4 employing, as antigen, tracer, and standard, recombinant human IGFBP-4 (rhIGFBP-4) expressed in Escherichia coli as a fusion protein with glutathione S-transferase and affinity purified with glutathione-derivatized resin. Antibody against the rhIGFBP-4 fusion protein was raised in guinea pigs; tracer and standard were provided by the rhIGFBP-4 moiety that had been cleaved from the rhIGFBP-4 fusion protein and repurified by reverse phase high pressure liquid chromatography. We report that both IGFBP-4 purified from PC3 human prostate cell-conditioned medium and rhIGFBP-4 bound IGF and migrated in electrophoresis gels in an identical manner; that in gel permeation chromatography, rhIGFBP-4 coeluted with the IGFBP-4 present in human serum; and that both are equally immunoreactive with the IGFBP-4 antiserum. Employing this IGFBP-4 RIA, we determined that no IGFBP other than IGFBP-4 reacted with the IGFBP-4 antiserum, and that recovery of IGFBP-4 from serum samples exceeded 90% when exogenous IGFBP-4 was added and was unaffected by the addition of IGFs or by repeated freezing and thawing of the sample. We employed this IGFBP-4 RIA to demonstrate an increase in IGFBP-4 in TE85 human osteosarcoma cell-conditioned medium after treatment with dibutyryl cAMP, PTH, and 1,25-dihydroxyvitamin D3, agents known to increase the IGFBP-4 messenger ribonucleic acid level. Application of this RIA to the measurement of IGFBP-4 in human serum revealed that the circulating level of IGFBP-4 in 41 individuals in the 61-87 yr age group (546 +/- 135 microgram/L) was 35% higher than that in 24 individuals in the 23-40 yr age group (404 +/- 156 microgram/L). The mean circulating level of PTH was also 20% higher in the 61-87 yr group compared to that in the 23-40 yr group (P < 0.01). In addition, serum IGFBP-4 amounts showed a significant positive correlation with age (r = 0.54; P < 0.001) and serum PTH (r = 0.26; P < 0.01). These data validate this IGFBP-4 RIA and illustrate its utility in illuminating the physiological mechanisms that regulate IGFBP-4 in vivo and influence its effects on the IGFs in both normal and abnormal pathology and in aging.

Interferon-gamma and activin A promote insulin-like growth factor-binding protein-2 and -4 accumulation by human luteinizing granulosa cells, and interferon-gamma promotes their apoptosis.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) antagonize IGF and gonadotropin actions on granulosa cells. Human atretic follicles express IGFBP-2 in granulosa cells more strongly and contain higher levels of IGFBP-2 and IGFBP-4 than healthy follicles. We studied the effects of interferon-gamma (IFN gamma) and activin A, which decrease progesterone accumulation, on granulosa cell IGFBP production and apoptosis. Conditioned media from luteinizing granulosa cells cultured with IFN gamma or activin A and/or LH were subjected to ligand blotting; northern blots of total ribonucleic acid (RNA) from these cells were probed for IGFBP-2 and -4. Apoptosis was measured by in situ DNA end labeling. LH decreased medium IGFBP-2 to 21% of the control value. Although IFN gamma did not alter basal medium IGFBP-2, in the presence of LH it increased IGFBP-2 3.4-fold, with parallel changes in messenger RNA levels. Activin A also tended to increase medium IGFBP-2 in LH-treated cultures. In conditioned medium, IGFBP-4 was consistently decreased by LH, whereas both IFN gamma and activin A increased IGFBP-4 and decreased IGFBP-4 protease activity. Both LH and IFN gamma modestly stimulated IGFBP-4 messenger RNA levels. Follistatin antagonized the action of activin A, but not that of IFN gamma. IFN gamma, but not activin A, increased granulosa cell apoptosis. In conclusion, IFN gamma produced by activated lymphocytes may decrease endogenous IGF activity through stimulation of IGFBPs and may promote apoptosis of granulosa-lutein cells in vivo and, thus, luteal regression. Activin A similarly promotes IGFBP accumulation, but it does not promote apoptosis.

Regulation of IGF-I, IGFBP-4 and IGFBP-5 gene expression by loading in mouse skeletal muscle.

Gene expression of IGF-I, IGFBP-4 and IGFBP-5 was studied in hindhimb skeletal muscle of mice, which were either overloaded or unloaded for 8 days. Overloading induced a 15% hypertrophy in soleus muscle associated with a 60% increase of IGF-I transcript levels and a doubling of IGFBP-4 mRNA levels. IGFBP-5 mRNA levels were decreased to one third of the control value. Changes in IGFBPs mRNA always preceded changes in IGF-I gene expression. Unloading by hindlimb suspension resulted in atrophy of soleus muscle (20%) and phenotype change towards the fast type associated with a transient decrease of IGF-I mRNA (30%) and a sustained increase (x2) of IGFBP-5 transcript. These alterations in IGFBPs expression, in unloaded or overloaded soleus, suggest that they may play a role in skeletal muscle adaptation to changes in loading.

Developmental changes in the expression patterns of IGFs, type 1 IGF receptor and IGF-binding proteins-2 and -4 in perinatal rat lung.

The insulin-like growth factors (IGF-I and IGF-II), their receptors and binding proteins (IGFBPs) are endogenously expressed in a number of tissues including the lung during fetal and neonatal development. This endogenous autocrine/paracrine IGF 'system', together with endocrine sources, contributes to the regulation of lung cell proliferation. We investigated the expression of the mRNAs encoding IGF-I, IGF-II, the type 1 IGF receptor (IGF-T1R) and two IGF-binding proteins (IGFBP-2 and IGFBP-4) in rat lung during the perinatum. These were compared in lung with surfactant apoprotein A (Sp-A) mRNA levels. mRNA in extracts of fetal tissues collected between day 17 of gestation (17f) and day 9 after birth (9d) was estimated by Northern blot or RNase protection analysis. At day 20 of gestation IGF-I, IGF-T1R and IGFBP-4 mRNA levels were higher in lung than liver (all P < 0.01), whereas IGF-II and IGFBP-2 mRNA levels were higher in liver than lung (each P < 0.02). The expression of IGF-1, IGFBP-2 and IGFBP-4 in lung was high before birth (days 17-20f) but decreased to low levels at days 21f, 22f or at birth (1d) but increased in the neonatal lung. IGF-II expression in lung was high at 17f but decreased before birth and remained low after birth. The IGF-T1R was expressed at moderate levels before birth, decrease before birth but peaked at days 2-5 after birth. The decrease in expression of these growth regulators before birth expression of these growth regulators before birth was matched by an increased in Sp-A expression which was clearly seen at day 20f, peaked at 1d and then was clearly seen at day 20f, peaked at 1d and then was maintained at high levels after birth. Primary cell cultures of 18f lung epithelia express IGFBP-2 while fibroblasts from the same animals express only IGFBP-4. Cells grown from 22f lung tissue express IGFBP-2 and IGFBP-4 at lower levels, behaving in vitro as they do in vivo. The contrasting levels of expression of different components of the IGF system in the fetal lung and liver indicate organ-specific regulation. IGFBP-2 and IGFBP-4 expression in different cell types within lung but with similar temporal changes suggests cell-specific regulation, perhaps by a common agent. The patterns by a common agent. The patterns of expression of IGF-I, IGF-T1R, IGFBP-2 and IGFBP-4, but not IGF-II, in developing lung correspond to previously described phasic changes in lung cell proliferation rates. The nadir in expression of these four major components of the lung IGF system occurs in the saccular phase when the lung begins to differentiate, probably under the influence of certain endocrine agents.

Increased expression of type 1 insulin-like growth factor receptor messenger RNA in rat hippocampal formation is associated with aging and behavioral impairment.

Insulin-like growth factor messenger RNAs are expressed in adult rat brain. However, little is known about the effects of aging on the expression of the insulin-like growth factors, their receptors, and their binding proteins in different regions of rat brain. The goal of the current study was to assess whether there is altered expression of the insulin-like growth factor system during normal aging in the hippocampal formation, a region particularly vulnerable to the aging process. A spatial learning task in the Morris water maze was used to assess the cognitive status of young (7-8-month-old) and aged (28-29-month-old) male Long-Evans rats. Sites of expression and abundance of insulin-like growth factor-I, type 1 insulin-like growth factor receptor, and insulin-like growth factor binding protein-4 messenger RNAs were then examined by in situ hybridization histochemistry and solution or northern blot hybridization assays. In situ hybridization histochemistry revealed no qualitative differences in the regional distribution of insulin-like growth factor-I, type 1 receptor, and insulin-like growth factor binding protein-4 messenger RNAs within the hippocampal formation of young and aged rats. However, quantitative analysis of messenger RNA abundance in hippocampal tissue homogenates showed a significant age-related increase in type 1 receptor messenger RNA (n = 25; t = -2.5; P < 0.02). Furthermore, linear regression analysis indicated that type 1 receptor messenger RNA abundance was significantly correlated with spatial learning impairment in the water maze (r = 0.44; P < 0.03) such that greater behavioral impairment was associated with higher type 1 receptor messenger RNA levels in the hippocampal formation. Neither insulin-like growth factor-I nor insulin-like growth factor binding protein-4 messenger RNA abundance was related to age or behavior. However, linear regression revealed a negative correlation between insulin-like growth factor-I messenger RNA abundance and type 1 receptor messenger RNA abundance in aged hippocampus (r = -0.72, P < 0.01). These data indicate that increased hippocampal expression of type 1 receptor messenger RNA is associated with aging and cognitive decline. The correlation between type 1 receptor and insulin-like growth factor-I messenger RNA abundance in the hippocampal formation of aged rats suggests that insulin-like growth factor availability may influence type 1 receptor expression. However, because no overall age difference was found in the amount of insulin-like growth factor-I messenger RNA in the hippocampal formation, decreased insulin-like growth factor from other sources such as the cerebrospinal fluid and the peripheral circulation may be involved in up-regulating type 1 receptor messenger RNA. Alternatively, type 1 receptor messenger RNA regulation may be part of a trophic response to the degenerative and regenerative events that occur within the hippocampal formation during aging.

Modulation of insulin-like growth factor (IGF) and IGF binding protein biosynthesis by hypoxia in cultured vascular endothelial cells.

Endothelial cells (EC) are hypoxia-tolerant and their capacity to proliferate in low oxygen tension is essential to maintain vascular endothelium integrity. The present study addresses whether hypoxia alters the expression of insulin-like growth factor (IGF) and IGF binding protein (IGFBP) genes in bovine aortic EC (BAEC) and bovine pulmonary artery EC (BPAEC). EC were cultured in normoxic (21%) conditions and exposed to 0% oxygen for 24, 48, or 72 h; some cells were reoxygenated by exposure to 21% oxygen for 24 or 48 h following hypoxia. IGF-I peptide and mRNA levels were very low in both cell types, and decreased further with exposure to hypoxia. Ligand blotting showed that both cell types synthesized 24 kDa (IGFBP-4), 30 kDa (IGFBP-5 and/or IGFBP-6), 43 kDa and 48 kDa IGFBPs (IGFBP-3 glycosylation variants). IGFBP-4 was the predominant IGFBP expressed by both cell types and did not change with exposure to hypoxia. Hypoxia caused a significant increase in IGFBP-3 secretion in BPAEC but not in BAEC. IGFBP-3 stable mRNA levels in BPAEC were increased correspondingly. IGFBP-5 was expressed only in BAEC and decreased with exposure to hypoxia. IGFBP-6 mRNA expression was low and increased in both cell types with exposure to hypoxia. These results demonstrate that EC IGFBP baseline expression as well as its expression in hypoxia vary in different vascular beds and suggest that the IGFBPs may be the dominant paracrine regulators of proliferation of EC as well as maintenance of endothelium integrity during hypoxia.

Regulation of IGF binding protein synthesis by a bovine mammary epithelial cell line.

IGF-I has been proposed as a key regulator of mammary epithelial cell (MEC) growth and differentiation. As IGF-I bioactivity is modulated by specific, high-affinity binding proteins (IGFBP), the forms of IGFBP that are secreted by the bovine MEC line, MAC-T, were identified. Media conditioned by MAC-T cells contained four forms of IGFBP that were identified, by western blotting with specific antibodies, as IGFBP-2, -3, -4 and -6. The amounts of IGFBP-3 in conditioned media were relatively low under basal conditions when analyzed by ligand blotting with 125I-IGF-II, but were increased dramatically relative to serum-free controls by exposure to IGF-I (100 ng/ml) or IGF-II (100 ng/ml) for 24 h. These increases in IGFBP-3 protein corresponded with dose-dependent increases in IGFBP-3 mRNA, with IGF-II eliciting a smaller response than was elicited by IGF-I at each concentration. Leu-IGF-I, which has reduced affinity for the IGF-I receptor but normal affinity for IGFBPs, failed to increase IGFBP-3 protein and mRNA levels, whereas B-chain IGF-I (normal affinity for the receptor but reduced affinity for IGFBPs) elicited the response, thus implying an IGF-I receptor-mediated event. Time-course studies indicated that IGFBP-3 mRNA was increased fourfold by 3 h of IGF-I treatment, with maximal increases of eightfold above serum-free controls observed between 8 and 13 h of treatment. By 24 h of treatment, IGFBP-3 mRNA levels had declined and were approximately threefold above controls in cells exposed to IGF-I. Amounts of messenger RNA of IGFBP-6 and IGFBP-2 were not increased by IGF treatment. However, retinoic acid (10(-6) M) stimulated both IGFBP-2 and IGFBP-6 protein and mRNA levels, but it decreased IGFBP-3 mRNA levels relative to controls. The combination of retinoic acid plus IGF-I had no additional effect on IGFBP-6 or -2 above that observed with retinoic acid alone, whereas IGF-I together with retinoic acid attenuated the decrease in IGFBP-3 observed with retinoic acid alone. Protein kinase A-mediated pathways were also shown to alter IGFBP synthesis. Forskolin, which increases cAMP, increased IGFBP-3 protein and mRNA levels. The combination of IGF-I plus forskolin resulted in greater increases in both protein and mRNA than were observed with either treatment alone. In contrast, forskolin decreased IGFBP-6 mRNA relative to controls, but had no effect on IGFBP-2. The decrease in IGFBP-6 was less marked when cells were treated with a combination of IGF-I and forskolin. Forskolin had no effect on IGFBP-2 mRNA levels. In summary, the ability of IGF-I specifically to regulate IGFBP-3 synthesis represents a mechanism whereby IGF-I may regulate its own bioactivity. In addition, the differential regulation of IGFBP-2, -3 and -6 by retinoic acid (which inhibits proliferation) and IGF-I (which stimulates proliferation) suggests that these forms of IGFBP have different roles in regulating mammary epithelial cell physiology.

Transforming growth factor-alpha stimulates insulin-like growth factor binding protein-4 (IGFBP-4) expression and blocks follicle-stimulating hormone regulation of IGFBP-4 production in rat granulosa cells.

The ability of TGF-alpha to regulate insulin-like growth factor binding protein-4 (IGFBP-4), was investigated. Primary cultures of rat granulosa cells (GC) were grown in serum-free medium with rat (r) TGF-alpha and/or rFSH, and secreted IGFBP-4 protein and its steady state mRNA levels were measured by Western immunoblotting and Northern blotting, respectively. Control (untreated) cells secreted IGFBP-4 spontaneously, and the levels were increased by rTGF-alpha in a dose- and time-dependent manner. rTGF-alpha abolished FSH-induced IGFBP-4 protease activity and suppressed FSH-dependent effects on IGFBP-4 production. IGFBP-4 mRNA levels were decreased and increased by FSH and TGF-alpha, respectively, and TGF-alpha blocked the FSH effects. These results demonstrate that TGF-alpha is a potent stimulator of IGFBP-4 expression in rat GC and can overcome the regulatory effects of FSH on IGFBP-4 production. The consequence of these TGF-alpha effects is a marked, sustained increase in the levels of IGFBP-4 in the microenvironment.