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Purpose: We have previously identified insulin-like growth factor 2 (IGF2) and insulin-like growth factor 1 receptor (IGF1R) as essential proteins for tip cell maintenance and sprouting angiogenesis. In this study, we aim to identify other IGF family members involved in endothelial sprouting angiogenesis., Methods: Effects on sprouting were analyzed in human umbilical vein endothelial cells (HUVECs) using the spheroid-based sprouting model, and were quantified as mean number of sprouts per spheroid and average sprout length. RNA silencing technology was used to knockdown gene expression. Recombinant forms of the ligands (IGF1 and IGF2, insulin) and the IGF-binding proteins (IGFBP) 3 and 4 were used to induce excess effects. Effects on the tip cell phenotype were analyzed by measuring the fraction of CD34 + tip cells using flow cytometry and immunohistochemistry in a 3D angiogenesis model. Experiments were performed in the presence and absence of serum., Results: Knockdown of IGF2 inhibited sprouting in HUVECs, in particular when cultured in the absence of serum, suggesting that components in serum influence the signaling of IGF2 in angiogenesis in vitro. We then determined the effects of IGFBP3 and IGFBP4, which are both present in serum, on IGF2-IGF1R signaling in sprouting angiogenesis in the absence of serum: knockdown of IGFBP3 significantly reduced sprouting angiogenesis, whereas knockdown of IGFBP4 resulted in increased sprouting angiogenesis in both flow cytometry analysis and immunohistochemical analysis of the 3D angiogenesis model. Other IGF family members except INSR did not affect IGF2-IGF1R signaling., Conclusions: Serum components and IGF binding proteins regulate IGF2 effects on sprouting angiogenesis. Whereas IGFBP3 acts as co-factor for IGF2-IGF1R binding, IGFBP4 inhibits IGF2 signaling. more...

Genomic sequencing of hepatocellular carcinoma (HCC) uncovers a paucity of actionable mutations, underscoring the necessity to exploit epigenetic vulnerabilities for therapeutics. In HCC, EZH2-mediated H3K27me3 represents a major oncogenic chromatin modification, but how it modulates the therapeutic vulnerability of signaling pathways remains unknown. Here, we show EZH2 acts antagonistically to AKT signaling in maintaining H3K27 methylome through epigenetic silencing of IGFBP4. ChIP-seq revealed enrichment of Ezh2/H3K27me3 at silenced loci in HBx-transgenic mouse-derived HCCs, including Igfbp4 whose down-regulation significantly correlated with EZH2 overexpression and poor survivals of HCC patients. Functional characterizations demonstrated potent growth- and invasion-suppressive functions of IGFBP4, which was associated with transcriptomic alterations leading to deregulation of multiple signaling pathways. Mechanistically, IGFBP4 stimulated AKT/EZH2 phosphorylation to abrogate H3K27me3-mediated silencing, forming a reciprocal feedback loop that suppressed core transcription factor networks (FOXA1/HNF1A/HNF4A/KLF9/NR1H4) for normal liver homeostasis. Consequently, the in vivo tumorigenicity of IGFBP4-silenced HCC cells was vulnerable to pharmacological inhibition of EZH2, but not AKT. Our study unveils chromatin regulation of a novel liver tumor suppressor IGFBP4, which constitutes an AKT-EZH2 reciprocal loop in driving H3K27me3-mediated epigenetic reprogramming. Defining the aberrant chromatin landscape of HCC sheds light into the mechanistic basis of effective EZH2-targeted inhibition. more...

Insulin-like growth factor binding protein 4 (IGFBP-4) and cyclooxygenase2 (COX-2) are associated with tumor inflammatory microenvironment which is involved in the progression of tumor. However, it is unclear that the roles of IGFBP-4 in lung cancer and the effects of IGFBP-4 on COX-2 expression. In this study, we showed that IGFBP-4 could decrease COX-2 production in lung cancer A549 cells. IGFBP-4 expression was significantly lower but COX-2 expression was higher in lung cancer tissues compared to matched adjacent normal tissues. In addition, IGFBP-4 could inhibit lung cancer cell proliferation, migration and invasion, and suppress the phosphorylation of PI3 K/AKT, ERK, and CREB. These results indicate that IGFBP-4 has potent antitumor effects in non-small cell lung cancer cells., (© 2017 International Federation for Cell Biology.) more...

Giant cell tumor (GCT) of bone is a generally benign tumor with a locally aggressive behavior. Histologically, GCTs consist of multinucleated giant cells, mononuclear histiocytes and the neoplastic fibroblast-like stromal cells (GCTSC). Growing evidence exists that GCTSCs develop from mesenchymal stem cells (MSCs), but little is known about the underlying molecular mechanisms. In previous studies we observed inactivation of the ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene in primary GCTSC due to strong DNA hypermethylation, indicating that epigenetic silencing might be involved in neoplastic transformation of MSCs. Here we investigated further candidate genes and identified strong hypermethylation of the insulin-like growth factor binding protein 4 (IGFBP4) promoter, resulting in IGFBP4 downregulation in GCTs compared to MSCs. Overexpression of UCHL1 and IGFBP4 by stable transfection of GCTSC did not influence cell viability, proliferation, migration and chemosensitivity compared to parental cells. However, colony-formation was significantly decreased suggesting that rescue of UCHL1 and IFGBP4 suppresses clonogenicity of GCT stromal cells. The observation of reduced expression of the stem-cell-specific transcription factors OCT4 and SOX2 in these cell lines further supported our findings. Epigenetic silencing of UCHL1 and IGFBP4 in GCTs might thus be a crucial event during the malignant transformation of MSCs in the context of GCT development and represent promising targets for the development of new diagnostic and therapeutic strategies., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.) more...

Background: Fetal growth restriction (FGR) is a leading cause of perinatal mortality and morbidity. Animal studies suggest dysregulation of IGF-binding protein (IGFBP)-4 is significant in the development of FGR, although human data are lacking. We postulated that IGFBP-4 is expressed at the maternal fetal interface and plays a role in regulating IGF bioavailability. Thus, maternal serum levels of IGFBP-4 may be associated with complications of abnormal placental growth and development including FGR., Methods: Circulating levels of IGFBP-4 and its protease, pregnancy-associated plasma protein-A (PAPP-A), were examined in healthy pregnancies. Their expression in villi and bed as possible sources of the circulating products were examined by immunohistochemistry. From the large Ottawa and Kingston (OaK) Birth Cohort, a nested case-control study was conducted to examine circulating levels of IGBP-4, PAPP-A, IGF-I, and IGF-II by Western blot in early gestation in 36 women who went on to develop FGR and 36 controls having normal-weight babies., Results: IGFBP-4 was elevated in early pregnancy compared with nonpregnant women and women in later pregnancy, consistent with the presence of abundant extravillous trophoblasts and decidual cells that highly expressed IGFBP-4. High expression of PAPP-A was observed in extravillous trophoblasts and decidual cells in early pregnancy but hardly detectable in the circulation at this time, suggesting maternal circulating PAPP-A originates more likely from syncytiotrophoblasts. Increased IGFBP-4 in the maternal circulation in early pregnancy was associated with the development of FGR [0.48 (0.28-0.74) in control vs. 1.22 (0.66-1.65) in FGR; odds ratio = 22 (95% confidence interval = 2.7-181)]. No difference was observed in circulating PAPP-A, IGF-I and IGF-II in the FGR vs. control group., Conclusion: Our findings support the role of IGFBP-4 in regulating IGF bioavailability and provide new clues for the prevention and treatment of FGR, raising the possibility of clinical use of IGFBP-4 as an early biomarker for this condition. more...

The insulin-like growth factor (IGF) signaling pathway is a conserved pathway that regulates animal development, growth, metabolism, reproduction, and aging. The biological actions of IGFs are modulated by IGF-binding proteins (IGFBPs). Although the structure and function of fish IGFBP-1, -2, -3, and -5 have been elucidated, there is currently no report on the full-length structure of a fish IGFBP-4 nor its biological action. In this study, we cloned and characterized the IGFBP-4 gene from fugu. Sequence comparison, phylogenetic, and synteny analyses indicate that its chromosomal location, gene, and protein structure are similar to its mammalian orthologs. Fugu IGFBP-4 mRNA was easily detectable in all adult tissues examined with the exception of spleen. Older animals tended to have higher levels of IGFBP-4 mRNA in the muscle and eyes compared with younger animals. Starvation resulted in significant increases in IGFBP-4 mRNA abundance in the muscle, liver, gallbladder, and brain. Overexpression of fugu and human IGFBP-4 in zebrafish embryos caused a significant decrease in body size and somite number, suggesting that fugu IGFBP-4 inhibits growth and development, possibly by binding to IGFs and inhibiting their binding to the IGF receptors. These results provide new information about the structural and functional conservation, expression patterns, and physiological regulation of the IGFBP-4 gene in a teleost fish. more...

IGFs are required for normal prenatal and postnatal growth. Although actions of IGFs can be modulated by a family of IGF-binding proteins (IGFBPs) in vitro, these studies have identified a complicated pattern of stimulatory and inhibitory IGFBP effects, so that understanding relevant aspects of IGFBP action in vivo has been limited. Here we have produced a null mutation of one specific IGFBP, IGFBP-4, which is coexpressed with IGF-II early in development. Surprisingly, mutation of IGFBP-4, believed from in vitro studies to be exclusively inhibitory, leads to a prenatal growth deficit that is apparent from the time that the IGF-II growth deficit first arises, which strongly suggests that IGFBP-4 is required for optimal IGF-II-promoted growth during fetal development. Mice encoding a mutant IGFBP-4 protease (pregnancy-associated plasma protein-A), which facilitates IGF-II release from an inactive IGF-II/IGFBP-4 complex in vitro, are even smaller than IGFBP-4 mutant mice. However, the more modest IGFBP-4 growth deficit is completely restored in double IGFBP-4/pregnancy-associated plasma protein-A-deficient mice. Taken together these results indicate not only that IGFBP-4 functions as a local reservoir to optimize IGF-II actions needed for normal embryogenesis, but also establish that IGFBP-4 proteolysis is required to activate most, if not all, IGF-II mediated growth-promoting activity. more...

The biological activity of IGF-I and -II is controlled by six binding proteins (IGFBPs), preventing the IGFs from interacting with the IGF receptor. Proteolytic cleavage of IGFBPs is one mechanism by which IGF can be released to bind the receptor. The IGFBPs are usually studied individually, although the presence of more than one of the IGFBPs in most tissues suggests a cooperative function. Thus, the IGFBPs are part of regulatory networks with proteolytic enzymes in one end and the IGF receptor in the other end. We have established a model system that allows analysis of the dynamics between IGF, IGFBP-4 and -5, the IGF receptor, and the proteolytic enzyme PAPP-A, which specifically cleaves both IGFBP-4 and -5. We demonstrate different mechanisms of IGF release from IGFBP-4 and -5: cooperative binding to IGF is observed for the proteolytic fragments of IGFBP-5, but not fragments of IGFBP-4. Furthermore, we find that PAPP-A-mediated IGF-dependent cleavage of IGFBP-4 is inhibited by IGFBP-5, which sequesters IGF from IGFBP-4, and that cleavage of both IGFBP-4 and -5 is required for the release of bioactive IGF. Finally, we show that cell surface-localized proteolysis of IGFBP-4 represents the final regulatory step of efficient IGF delivery to the receptor. Our data define a regulatory system in which molar ratios between the IGFBPs and IGF and between the different IGFBPs, sequential proteolytic cleavage of the IGFBPs, and surface association of the activating proteinase are key elements in the regulation of IGF receptor stimulation. more...

Insulin-like growth factor binding protein-4 (IGFBP-4) is an important member of the insulin-like growth factor (IGF) system. The IGFBP-4 has three domains of which the N-terminal sequence is important for the binding of IGF. It acts as a transport protein for IGF-I and IGF-II and modulates their biological effects. There is increasing evidence that IGFBP-4 inhibits IGF-induced cellular growth both in vitro and in vivo. IGFBP-4 can also mediate its actions through a mechanism independent of IGFs. IGFBP-4 level and expression in various tissues are influenced by IGFBP protease, nutrition, several growth factors and hormones. Overexpression of IGFBP-4 in transgenic animal models causes reduced growth of organs containing smooth muscle. Most cancers express IGFBP-4 at levels which correlate with their state of differentiation. However, the effects of IGFBP-4 on tumor growth are uncertain. In vitro studies have shown that overexpression of IGFBP-4 inhibit the growth of some colon cancer cells. Overexpression of IGFBP-4 in vivo has been reported to decrease the growth of prostate cancer. The effect of altered expression of IGFBP-4 in vivo in colon and other cancers needs to be explored as locally available IGFs appear to stimulate mitogenesis. more...

We have previously demonstrated that triiodothyronine (T(3)) stimulates hepatic IGFBP-4 expression in rats. Since there is evidence that some of the genes whose expression is regulated by T(3) are also sensitive to 3,5-diiodothyronine (T(2)), we used the adult rat hepatocyte model in primary cultures directly exposed to T(2) to evaluate insulin-like growth factor binding protein-4 (IGFBP-4) expression by Northern and Ligand blot analyses in this study. Our results demonstrate that T(2), like T(3), is able to enhance IGFBP-4 mRNA and protein after 12-24 h of incubation. The potency of the two iodothyronines is comparable as judged by dose-dependence experiments. The T(2)-induced IGFBP-4 increase is independent from ongoing protein synthesis but dependent on active transcription. Since T(3) and T(2) do not affect IGF-I production, it appears that the iodothyronines affect the hepatic IGF system at the IGFBP level. Our data, demonstrating that T(2) mimics the stimulatory effect of T(3) on IGFBP-4 expression by rat hepatocytes, allow us to include IGFBP-4 gene among the genes regulated by the two iodothyronines. more...

Effects of insulin-like growth factor-binding protein-4 (IGFBP-4) on proliferation, colony formation, and cell migration were assessed in IGF-sensitive and -insensitive colorectal cancer cell lines. In IGF-insensitive Isreco-1 cells, overexpression of IGFBP-4 reduced colony formation but not cell proliferation and migration, whereas exogenous IGF-II had no effect. In IGF-dependent LS1034 cells, IGFBP-4 inhibited all parameters of growth tested, whereas IGF-II partially restored reduced proliferation and cell migration only. In Isreco-2 cells, which lack endogenous IGF expression but are IGF sensitive, colony formation was also reduced by IGFBP-4. Therefore, specific parameters of malignant progression of colon carcinoma cells are distinctly affected by IGF-dependent and IGF-independent effects of IGFBP-4. more...

Insulin-like growth factor binding protein 4 (IGFBP-4) is abundantly expressed in bone and is generally believed to function as an inhibitor of IGF action. To investigate the function of locally produced IGFBP-4 in bone in vivo, we targeted expression of IGFBP-4 to osteoblasts using a human osteocalcin promoter to direct transgene expression. IGFBP-4 protein levels in calvaria of transgenic (OC-BP4) mice as measured by Western ligand blot were increased 25-fold over the endogenous level. Interestingly, levels of IGFBP-5 were decreased in the OC-BP4 mice, possibly because of a compensatory alteration in IGF-1 action. Morphometric measurements showed a decrease in femoral length and total bone volume in transgenic animals compared with the controls. Quantitative histomorphometry at the distal femur disclosed a striking reduction in bone turnover in the OC-BP4 mice. Osteoblast number/bone length and bone formation rate/bone surface in OC-BP4 mice were approximately one-half that seen in control mice. At birth, OC-BP4 mice were of normal size and weight but exhibited striking postnatal growth retardation. Organ allometry (mg/g body weight) analysis revealed that, whereas most organs exhibited a proportional reduction in weight, calvarial and femoral wet weights were disproportionally small (approximately 70% and 80% of control, respectively). In conclusion, paracrine overexpression of IGFBP-4 in the bone microenvironment markedly reduced cancellous bone formation and turnover and severely impaired overall postnatal skeletal and somatic growth. We attribute these effects to the sequestration of IGF-1 by IGFBP-4 and consequent impairment of IGF-1 action in skeletal tissue. more...

Recent studies support a critical role for the paracrine IGF/IGF-binding protein system in the regulation of vascular smooth muscle cell growth. In this study we have explored the hypothesis that the abundance of individual IGF-binding proteins in smooth muscle is subject to regulation during postnatal life and in response to injury. IGF-binding protein-2 was the predominant binding protein secreted by neonatal rat vascular smooth muscle cells, whereas IGF-binding protein-4 was most prevalent in adult vascular smooth muscle cells coincident with increased IGF-binding protein-4 protease activity. After arterial injury, IGF-binding protein-4 mRNA increased, associated with greater IGF-binding protein-4 proteolytic activity, resulting in stable steady state levels of the IGF-binding protein-4 protein. Expression of pregnancy-associated plasma protein A mRNA, recently identified as an IGF-binding protein-4 protease, was expressed at higher levels in adult than neonatal vascular smooth muscle cell lines, but did not change significantly after arterial injury. The peak of immunoreactive pregnancy-associated plasma protein A from hydrophobic interaction chromatography fractions of smooth muscle cell-conditioned medium coincided, but did not fully overlap, with the fractions containing maximal IGF-binding protein-4 protease activity. In conclusion, our data point to a developmental switch from IGF-binding protein-2 to IGF-binding protein-4 in vascular smooth muscle cells postnatally. Moreover, IGF-binding protein-4 expression is coregulated with IGF-binding protein-4 protease activity, suggesting that biosynthesis and degradation of this binding protein are coordinated events important for regulating biological activity of IGF-I. more...

Insulin-like growth factor-binding proteins (IGFBPs) both stimulate and inhibit IGF activity, and in the M12 prostate cancer cell line, overexpression of IGFBP-4 was shown to delay tumorigenesis while decreasing the production of IGFBP-2. We have performed the reverse experiment, inhibition of IGFBP-4 expression with antisense complementary DNA, in two prostate tumor cell lines, ALVA-31 and M12. Expression of antisense messenger RNA transcripts was verified by RNase protection assays, and inhibition of mature IGFBP-4 in cell medium was demonstrated by Western blotting. Both transfected lines (ALVA-31asBP4 and M12asBP4) proliferated more slowly in monolayer culture than parental controls. Colony formation in soft agar was strongly inhibited in both cases, and the rate of tumor formation and growth in male athymic nude mice injected with M12asBP4 was markedly reduced relative to that in mice receiving M12 control cells. Apoptosis induced by the topoisomerase inhibitor etoposide was also enhanced in transfected cells. The effects on colony formation in soft agar and tumor formation in mice were maintained for the duration of the experiments, in contrast to the delayed growth observed in the previous study of IGFBP-4 overexpression. A significant difference was found in the patterns of IGFBP expression; production of both messenger RNA and protein for IGFBP-3 and IGFBP-6 was greatly increased in the M12asBP4 and ALVA31asBP4 cell lines. Up-regulation of these binding proteins has been observed in association with actions of 1,25-dihydroxyvitamin D(3) in prostate cancer cells, and the data suggest a role for IGFBP-3 and IGFBP-6 in the suppression of prostate tumor cell growth. more...

Decreased bone formation plays an important role in the development of lytic lesions during the late stage of multiple myeloma (MM). Release of insulin-like growth factor binding protein-4 (IGFBP4) by tumour cells adjacent to bone may inhibit IGF-I-stimulated osteoblast growth and contribute to decreased bone formation. The present study demonstrates that the human MM cell line, ARH-77, expresses IGFBP4 and, to a lesser extent, IGFBP6 mRNA and protein. IGFBP4 expression in myeloma cells may be modulated by cytokines released by stromal cells and T cells in the microenvironment. We tested the effect of recombinant interferon-gamma (INF) on IGFBP4 expression in ARH-77. INF increased IGFBP4 mRNA and protein levels at 12 h, with a decline to baseline by 24 h. In contrast, IGFBP4 was not regulated in response to IL-6, TNF-alpha, PDGF BB, bFGF, TGF-beta or the cAMP agonist, forskolin. In other systems. IGFBP4 may also be regulated post-transcriptionally by a protease that is activated by IGF-I or -II. Conditioned medium from ARH-77 cultures incubated with IGF-I or -II for up to 24 h failed to demonstrate proteolytic activity. Proteolysis was also not observed when conditioned medium containing exogenous rhIGFBP4 was incubated with IGF-I or -II under cell-free conditions. To determine if human myeloma tumours also express IGFBP4, total RNA was isolated from four tumour biopsies. All samples expressed detectable levels of IGFBP4 mRNA. These findings indicate that interferon-gamma may indirectly modulate bone formation via the the release of tumour-derived IGFBP4. suggesting that the immune system may influence bone turnover in MM. Failure of myeloma cells to release protease activity may promote IGFBP4 accumulation in the microenvironment during tumour growth. more...

We have previously shown that the insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of skeletal muscle cells in culture, and that these actions in L6A1 muscle cells may be modulated by three secreted IGF binding proteins (IGFBPs), IGFBP-4, -5, and -6. Since we found that the temporal expression pattern of IGFBP-4 and IGFBP-5 differed dramatically during the transition from proliferating myoblasts to differentiated myotubes, we undertook the current study to examine the effects of purified IGFBP-4 and IGFBP-5 on IGF-stimulated actions in L6A1 muscle cells. As has been shown for other cell types, we found that IGFBP-4 had only inhibitory actions, inhibiting IGF-I and IGF-II-stimulated proliferation and differentiation. In contrast, IGFBP-5 exhibited both inhibitory and stimulatory actions. When added in the presence of 30 ng/ml IGF-I, IGFBP-5 (250 ng/ml) inhibited all markers of the early proliferative response: the tyrosine phosphorylation of the cytoplasmic signaling molecules IRS-1 and Shc, the activation of the MAP kinases, ERK1 and 2, the elevation of c-fos mRNA, the early inhibition of the elevation in myogenin mRNA, and the increase in cell number. In contrast, IGFBP-5 stimulated all aspects of the myogenic response to IGF-I: the later rise in myogenin mRNA, the elevation of creatine kinase activity, and the fusion of myoblasts into myotubes. This dual response to IGFBP-5 was greatest when it was added at a molar ratio of IGFBP-5 to IGF-I of 2:1. In contrast, when IGFBP-5 was added in the presence of IGF-II, it inhibited both proliferation and differentiation. Neither IGFBP had any effect when added in the presence of R3 IGF-I, an analog with substantially reduced affinity for IGFBPs. Our results suggest that the role of IGFBP-4 is mainly to sequester excess IGFs, and thus inhibit all actions. IGFBP-5, however, is capable of eliciting a dual response, possibly due to its unique ability to associate with the cell membrane. more...

Using a recently developed human osteoblastic cell line (hFOB/ER9) with high levels (approximately 4,000 per nucleus) of estrogen receptors and the characteristic phenotype of the mature osteoblast, we tested the hypothesis that estrogen decreases bone formation by inhibiting the action of the insulin-like growth factor (IGF) paracrine/autocrine system. IGF-II, the predominant IGF produced by osteoblastic cells, was measurable in hFOB/ER9-conditioned medium (approximately 10 ng/mL) and its level did not change significantly after treatment with 17 beta-estradiol (E2) or anti-estrogens. Treatment with E2 at 0.1-100 nM decreased [3H]thymidine uptake to 53% of control (p < 0.001) in a dose-dependent fashion. The predominant IGF-binding proteins (IGFBPs) produced by hFOB/ER9 and by normal trabecular osteoblasts are IGFBP-3 and IGFBP-4, of which IGFBP-4 is consistently inhibitory of IGF action. Treatment with E2 at 0.01-10 nM for 48 h increased IGFBP-4 mRNA to 346% +/- 90% (mean +/- SE) of control (p < 0.05) and IGFBP-4 protein to 278% +/- 75% of control (p < 0.01) in a dose-dependent fashion but did not alter IGFBP-3 mRNA or protein. E2 treatment also attenuated IGF-dependent, IGFBP-4 specific proteolysis to approximately 50% of control. ICI 182,780, a pure anti-estrogen, completely blocked E2-mediated decreases in cell proliferation and increases in levels of IGFBP-4 mRNA and protein. Treatment of the hFOB/ER9 cells with recombinant human IGFBP-4 (200 ng/mL) decreased cell proliferation to 55% of control (p < 0.01). Thus, E2 acts on osteoblastic cells to increase availability of inhibitory IGFBP-4, by both increasing its production and decreasing its degradation, which may oppose the mitogenic effect of the IGFs on osteoblastic cells. This action may mediate, at least in part, the decreases in bone formation that are observed after estrogen treatment in vivo. more...

Insulin-like growth factor (IGF)-I and IGF-II, and their binding proteins (IGFBPs) have been demonstrated to play important roles in follicular development as intraovarian regulators. Previous studies have demonstrated that the follicular fluid of atretic follicles contains high levels of IGFBP-2 and IGFBP-4, which are known to inhibit the action of IGFs. In this study, we identified IGFBP-4 protease activity in the follicular fluid of developing but not atretic follicles. To elucidate the regulation mechanism of IGFBP-4 proteolytic activity in the ovary, cultured luteinized granulosa cells (GCs) were incubated with various hormones, and proteolyzed IGFBP-4 in the medium was analyzed. IGFBP-4 proteolytic activity was increased when GCs were incubated with IGFs, estradiol or follicle-stimulating hormone (FSH) but not with testosterone. We also showed that IGFBP-4 inhibited IGF-1-induced estradiol release by GCs while proteolyzed IGFBP-4 did not. These results suggest that human luteinized GCs produce IGFBP-4 protease, and that FSH and IGFs may stimulate folliculogenesis by modulating IGFBP-4 degradation in the ovary. more...