Regulation of insulin-like growth factors I and II and their binding proteins in human bone marrow stromal cells by dexamethasone

Glucocorticoids inhibit the proliferation, but induce the differentiation, of bone marrow stromal cells into osteoblast-like cells. The mechanisms, however, are still conjectural. Since insulin-like growth factors (IGFs) have profound effects on osteoblast growth and differentiation, it is possible that glucocorticoids exert their effects on bone marrow stromal cells in part via regulation of IGFs. Therefore, we analyzed the effects of dexamethasone (Dex) on the expression of IGF I and IGF II in cultured preosteoblastic normal human bone marrow stromal cells (HBMSC). Whereas Dex decreased the concentration of IGF I in the conditioned medium since early in the treatment, the concentration of IGF II was increased progressively as culture period lengthened. As the activities of IGF I and IGF II are regulated by the IGF binding proteins (IGFBPs), we analyzed the effects of Dex on the expression of IGFBPs. Dex increased IGFBP-2 in a time-dependent manner. The increase in IGFBP-2, however, was only to the same extent as that of IGF II at most, depending on the length of treatment. Therefore, the increase in IGFBP-2 would dampen, but not eliminate, the increased IGF II activities. By contrast, Dex decreased IGFBP-3 levels, the latter increasing the bioavailability of IGF II. Although IGFBP-4 mRNA levels were stimulated by Dex, IGFBP-4 concentration in the conditioned medium was unchanged as measured by RIA. IGFBP-5 and IGFBP-6 mRNA levels were decreased by Dex in a time-dependent fashion. IGFBP-5 protein level was also decreased 1–4 days after Dex treatment. IGFBP-1 mRNA was not detectable in HBMSC. These accumulated data indicate that Dex regulates IGF I and IGF II and their binding proteins differentially in normal human bone marrow stromal cells. The progressive increase in IGF II may contribute to Dex-induced cell differentiation. J. Cell. Biochem. 71:449–458, 1998. © 1998 Wiley-Liss, Inc.     

Involvement of growth hormone (GH) and insulin-like growth factor (IGF) system in ovarian folliculogenesis

During the last decade, involvement of growth hormone (GH), insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in ovarian folliculogenesis has been extensively studied. This review provides an update on the GH, IGF system and their role in ovarian follicular development. In vitro studies and knockout experiments demonstrated an important role of GH in preantral follicle growth and differentiation through their binding with GH receptors, which are located both in the oocyte and follicular somatic tissues. Furthermore, GH stimulates the development of small antral follicles to gonadotrophin-dependent stages, as well as maturation of oocytes. With regard to the IGF system, IGF-I has no effects on primordial follicle development, but both IGF-I and IGF-II stimulate growth of secondary follicles. Depending on the species studies and method used, these proteins have been detected in oocytes and/or somatic cells. In antral follicles, these IGFs stimulate granulosa cell proliferation and steroidogenesis in most mammals. The bioavailability of IGFs is regulated by a family of intrafollicular expressed IGF binding proteins (IGFBPs). Facilitation of IGF can be increased through the activity of specific IGFBP proteases, which degrade the IGF/IGFBP complex, resulting in the production of IGFBP fragments and release of attached IGF.     

Structure and function of bone marrow environment

Bone marrow and spleen are the major hematopoietic tissue in adult mice. However, little is known about the specific mechanism regulating hematopoiesis within these tissues. Since Dexter et al. first described conditions to maintain bone marrow hematopoiesis, long term bone marrow culture (LTBMC) has been developed in order to analyze the mechanism of the maintenance of proliferation and differentiation of hematopoietic stem cells in vitro. Furthermore, several stromal cell lines which are able to support the growth and differentiation of hematopoietic lineage, has been established from LTBMC. Although it is well known that bone marrow stromal cell lines are able to produce colony stimulating factors, it has been suggested that the stromal cell factors which involve membrane bound moieties must have a key role in the regulation of hematopoiesis. We expect that monoclonal antibodies to the surface of bone marrow stromal cells could detect such a critical stroma-associated protein that bounds the cell surface of the bone marrow stroma.     

A role of EphB4 receptor and its ligand,ephrin-B2, in erythropoiesis

Erythropoiesis is regulated not only by erythropoietin but also by microenvironments which are composed of transmembrane molecules. We have previously shown that a receptor tyrosine kinase EphB4 is predominantly expressed on human erythroid progenitors in bone marrow. EphB4 is expressed in approximately 45% of hematopoietic progenitor cells, which are CD34-positive and c-Kit-positive in human umbilical cord blood (hUCB). The transmembrane ligand for EphB4 or ephrin-B2 is expressed on bone marrow stromal cells and arterial endothelial cells. When such EphB4-positive hematopoietic progenitor cells were co-cultured with stromal cells which express ephrin-B2, they were immediately detached from stromal cells and differentiated to mature erythroid cells. At that time, expression of EphB4 immediately down-regulated. In contrast, on ephrin-B2 non-expressing stromal cells, they remained EphB4-positive cells and the generated number of mature erythroid cells was less than that on ephrin-B2 expressing stromal cells. Additionally, ephrin-B2 expression on endothelial cells up-regulated under hypoxic condition. Taken together, we propose that one of the molecular cues that regulate erythropoiesis is ephrin-B2 on stromal cells.     

Bone marrow stromal cells selectively stimulate the rapid expansion of lineage-restricted myeloid progenitors

Bone marrow stromal cells serve hematopoietic microenvironments where different blood cells are controlled in their growth and differentiation. To characterize functions of stromal cells, 33 bone marrow stromal cells including preadipocytes, endothelial cells, and fibroblasts were established from transgenic mice harboring temperature-sensitive SV40 T-antigen gene and their selective stimulatory abilities to support large colony formation of lineage-specific hematopoietic progenitor cells (erythroid, monocyte/macrophage, granulocyte, and monocyte-granulocyte) were examined. Among established stromal cells, 27 clones showed erythropoietic stimulatory activity in the presence of erythropoietin. On myeloid progenitors, the stromal cells showed lineage-restricted stimulatory activity and a reciprocal relationship was observed between granulocyte formation and macrophage formation, but these activities were not dependent on the amount of produced colony-stimulating factors (CSFs). Our present study with many stromal cells established from bone marrow indicated that each stromal cell in the bone marrow may provide the preferable microenvironment for a rapid expansion of the lineage-restricted progenitor cells in combination with CSFs. © 1995 Wiley-Liss, Inc.     

Induction of circulating neonatal stem cell populations

Hematopoietic cell differentiation and growth are regulated by paracrine molecules that include insulin and insulin-like growth factors (IGFs). IGF-I and -II stimulation of erythropoiesis in cultures of adult bone marrow and peripheral blood cells and murine fetal liver cells has been previously reported. In order to investigate whether these paracrines also influence differentiation and proliferation of human neonatal progenitor cells, we assessed their effects in cultures of umbilical cord blood and adult blood and marrow cells, using a serum-substituted system. IGF-I stimulated colony-forming unit-erythroid (CFU-E)-derived colony formation by adult cells by up to 265%, while IGF-II augmented colony formation by up to 100% in the presence of erythropoietin. Stimulation occurred in a saturable fashion over concentrations of 0 to 200 ng/ml. Similar results were obtained in subcultures of adult-circulating progenitors. Moreover, a subpopulation of erythropoietin-independent adult CFU-E was stimulated to proliferate by IGF-I but not by IGF-II. In contrast to these effects in adult marrow culture, IGF-II exerted a greater stimulatory effect on neonatal CFU-E proliferation than did IGF-I in erythropoietin-containing cultures. Additionally, IGF-II stimulated proliferation of erythropoietin-independent neonatal CFU-Es in a concentration-dependent fashion. Together, the data are consistent with the hypothesis that somatomedins are involved in developmental regulation of erythropoiesis.     

Insulin-like growth factor-binding protein-3 activates a phosphotyrosine phosphatase. Effects on the insulin-like growth factor signaling pathway

The proliferative action of insulin-like growth factors (IGF-I and -II) is mediated via the type I IGF receptor (IGF-IR) and is modulated by their association with high affinity binding proteins, IGFBP-1 to -6. We recently found that, in addition to its ability to bind IGFs, IGFBP-3 also inhibits IGF-IR activation independently of IGF binding and without interacting directly with IGF-IR. Here, we show that IGFBP-3 is capable of blocking the signal triggered by IGFs. Breast carcinoma-derived cells (MCF-7) were stimulated by des(1-3)IGF-I or [Gln(3),Ala(4),Tyr(15),Leu(16)]IGF-I, two IGF analogues with intact affinity for IGF-IR, but with weak or virtually no affinity for IGFBPs, then incubated with IGFBP-3. The activated IGF-IR was desensitized through reversal of its autophosphorylation, following which both phosphatidylinositol 3-kinase and p42(MAPK) activities were depressed. Direct measurement of phosphotyrosine phosphatase activity and reconstitution experiments using tyrosine-phosphorylated insulin receptor substrate-1 (IRS-1) indicated that IGFBP-3 activated a phosphotyrosine phosphatase (PTPase). This action appeared to be peculiar to IGFBP-3 among the IGFBPs, since neither IGFBP-1 nor IGFBP-5 (structurally the closest to IGFBP-3), had any such effect. Several cell lines derived from normal or tumor cells responsive to IGF-I were used to show that IGFBP-3-stimulated PTPase is cell type-specific. Although the precise nature of the phosphatase remains to be determined, the results of this study demonstrate that IGFBP-3 stimulates a phosphotyrosine phosphatase activity that down-regulates the IGF-I signaling pathway, suggesting a major role for IGFBP-3 in regulating cell proliferation.     

Insulin-like growth factor-binding protein-5 inhibits osteoblast differentiation and skeletal growth by blocking insulin-like growth factor actions

Signaling through the IGF-I receptor by locally synthesized IGF-I or IGF-II is critical for normal skeletal development and for bone remodeling and repair throughout the lifespan. In most tissues, IGF actions are modulated by IGF-binding proteins (IGFBPs). IGFBP-5 is the most abundant IGFBP in bone, and previous studies have suggested that it may either enhance or inhibit osteoblast differentiation in culture and may facilitate or block bone growth in vivo. To resolve these contradictory observations and discern the mechanisms of action of IGFBP-5 in bone, we studied its effects in differentiating osteoblasts and in primary bone cultures. Purified wild-type (WT) mouse IGFBP-5 or a recombinant adenovirus expressing IGFBP-5WT each prevented osteogenic differentiation induced by the cytokine bone morphogenetic protein (BMP)-2 at its earliest stages without interfering with BMP-mediated signaling, whereas an analog with reduced IGF binding (N domain mutant) was ineffective. When added at later phases of bone cell maturation, IGFBP-5WT but not IGFBP-5N blocked mineralization, prevented longitudinal growth of mouse metatarsal bones in short-term primary culture, and inhibited their endochondral ossification. Because an IGF-I variant (R3IGF-I) with diminished affinity for IGFBPs promoted full osteogenic differentiation in the presence of IGFBP-5WT, our results show that IGFBP-5 interferes with IGF action in osteoblasts and provides a framework for discerning mechanisms of collaboration between signal transduction pathways activated by BMPs and IGFs in bone.     

Cell-specific localization of insulin-like growth factor binding protein mRNAs in rat liver

The liver is a major source of circulating insulin-like growth factor I (IGF-I), and it also synthesizes several classes of IGF binding proteins (IGFBPs). Synthesis of IGF-I and IGFBPs is regulated by hormones, growth factors, and cytokines. They are nutritionally regulated and expressed in developmentally specific patterns. To gain insight into cellular regulatory mechanisms that determine hepatic synthesis of IGF-I and IGFBPs and to identify potential target cells for IGF-I within the liver, we studied the cellular sites of synthesis of IGF-I, IGF receptor, growth hormone (GH) receptor, and IGFBPs in freshly isolated rat hepatocytes, endothelial cells, and Kupffer cells. We also localized cellular sites of IGFBP synthesis by in situ hybridization histochemistry. Western ligand and immunoblot analyses were used to determine IGFBP secretion by isolated cells. Two IGF-I mRNA subtypes with different 5' ends (class 1 and class 2) were detected in all isolated liver cell preparations. Type 1 IGF receptor mRNA was detected in endothelial cells, indicating that these cells are a local target for IGF actions in liver. GH receptor was expressed in all cell preparations, consistent with GH regulation of IGF-I and IGFBP synthesis in multiple liver cell types. The IGFBPs expressed striking cell-specific expression. IGFBP-1 was synthesized only in hepatocytes, and IGFBP-3 was expressed in Kupffer and endothelial cells. IGFBP-4 was expressed at high levels in hepatocytes and at low levels in Kupffer and endothelial cells. Cell-specific expression of distinct IGFBPs in the liver provides the potential for cell-specific regulation of hepatic and endocrine actions of IGF-I.     

Kupffer cells support extramedullary erythropoiesis induced by nitrogen-containing bisphosphonate in splenectomized mice

Our previous study indicated that injecting nitrogen-containing bisphosphonate (NBP) induced the site of erythropoiesis to shift from the bone marrow (BM) to the spleen. This was due to the depletion of BM-resident macrophages, which support erythropoiesis. In this study, we examined NBP treatment-induced extramedullary hematopoiesis in splenectomized mice, focusing on hepatic hematopoiesis. NBP-treated mice did not display anemia or significant change in erythropoietin production, while megakaryopoiesis and erythropoiesis were constantly observed in the liver. Erythroblastic islands were detected in the sinusoidal lumen. Kupffer cells expressed VCAM-1 following NBP treatment, which is an important factor for erythroblast differentiation. Cl₂MBP-liposome treatment depleted the erythroblastic islands, and decreased the number of hematopoietic cells in the liver, as determined by colony forming assays. Together, these results indicate that Kupffer cells support erythropoiesis, acting as stromal cells in the liver, and that they might act as a niche for hematopoietic precursor cells in an emergency.     

A new candidate for the regulation of erythropoiesis. Insulin-like growth factor I

The effect of pure human insulin-like growth factor I (IGF I) on the colony formation of late stage erythroid precursor cells (CFU-e) from fetal mouse liver and adult bone marrow was studied in a serum-free culture system. We found that IGF I in physiological concentrations stimulated erythroid colony formation. The combined effect of IGF I and erythropoietin was smaller than the sum of their single effects. The number of colonies induced by IGF I was linearly dependent on the number of plated cells. Our results indicate that IGF I is the first clearly defined mitogen that stimulates the late stages of erythroid differentiation independently of erythropoietin.     

Endogenous IGF-I regulates IGF binding protein production in human intestinal smooth muscle cells

Human intestinal smooth muscle in culture produces insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3, IGFBP-4, and IGFBP-5, which modulate the effects of IGF-I. This study examined the regulation of IGFBP production by endogenous IGF-I. R3-IGF-I, an agonist unaffected by IGFBPs, elicited concentration-dependent increase in growth, measured by [(3)H]thymidine incorporation, and production of IGFBP-3, IGFBP-4, and IGFBP-5, measured by Western blot. Antagonists of the IGF-I receptor, IGF-I Analog or monoclonal antibody 1H7, elicited concentration-dependent inhibition of growth and decrease in IGFBP-3, IGFBP-4, and IGFBP-5 production, implying that endogenous IGF-I stimulated growth and IGFBP production. R3-IGF-I-induced increase in IGFBP-3, IGFBP-4, and IGFBP-5 production was partially inhibited by a mitogen-activated protein (MAP) kinase or a phosphatidylinositol-3-kinase (PI 3-kinase) inhibitor and abolished by the combination. We conclude that endogenous IGF-I stimulates growth and IGFBP-3, IGFBP-4, and IGFBP-5 production in human intestinal smooth muscle cells. Regulation of IGFBP production by IGF-I is mediated by activation of distinct MAP kinase and PI 3-kinase pathways, the same pathways through which IGF-I stimulates growth.     

Role of insulin-like growth factor binding protein-3 (IGFBP-3) in the differentiation of primary human adult skeletal myoblasts

Although muscle satellite cells were identified almost 40 years ago, little is known about the induction of their proliferation and differentiation in response to physiological/pathological stimuli or to growth factors/cytokines. In order to investigate the role of the insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system in adult human myoblast differentiation we have developed a primary human skeletal muscle cell model. We show that under low serum media (LSM) differentiating conditions, the cells secrete IGF binding proteins-2, -3, -4 and -5. Intact IGFBP-5 was detected at days 1 and 2 but by day 7 in LSM it was removed by proteolysis. IGFBP-4 levels were also decreased at day 7 in the presence of IGF-I, potentially by proteolysis. In contrast, we observed that IGFBP-3 initially decreased on transfer of cells into LSM but then increased with myotube formation. Treatment with 20 ng/ml tumour necrosis factor-alpha (TNFalpha), which inhibits myoblast differentiation, blocked IGFBP-3 production and secretion whereas 30 ng/ml IGF-I, which stimulates myoblast differentiation, increased IGFBP-3 secretion. The TNFalpha-induced decrease in IGFBP-3 production and inhibition of differentiation could not be rescued by addition of IGF-I. LongR(3)IGF-I, which does not bind to the IGFBPs, had a similar effect on differentiation and IGFBP-3 secretion as IGF-I, both with and without TNFalpha, confirming that increased IGFBP-3 is not purely due to increased stability conferred by binding to IGF-I. Furthermore reduction of IGFBP-3 secretion using antisense oligonucleotides led to an inhibition of differentiation. Taken together these data indicate that IGFBP-3 supports myoblast differentiation.     

Insulin-like Growth Factor (IGF) I Down-Regulates Type 1 IGF Receptor (IGF 1R) and Reduces the IGF I Response in A549 Non-Small-Cell Lung Cancer and Saos-2/B-10 Osteoblastic Osteosarcoma Cells

The insulin-like growth factor type 1 receptor (IGF 1R) mediates the acute metabolic effects of IGF I as well as IGF I-stimulated cell proliferation and protection from apoptosis. IGF binding proteins (IGFBPs) can modulate these responses. We, therefore, investigated whether intrinsic IGFBPs interfere with IGF I-induced regulation of IGF 1R expression and with the biological response to IGF I in two human tumor cell lines, the non-small-cell lung cancer cell line A549 and the osteoblastic osteosarcoma cell line Saos-2/B-10. We compared the growth rates, IGFBP production, IGF I binding characteristics, IGF 1R protein and mRNA levels, and the acute IGF I response (stimulation of glycogen synthesis) after pretreatment of the cells in serum-free medium with or without added IGF I or medium supplemented with 5% fetal calf serum (FCS). In contrast to A549 cells, which produce IGF I and significant amounts of IGFBPs, survival and proliferation of Saos-2/B-10 cells, which do not produce IGF I or significant amounts of IGFBPs, depended on the addition of exogenous IGF I. IGF I increased the concentration of IGFBP-2 and -3 and decreased the concentration of IGFBP-4 in the medium of A549 cells. As compared to FCS, IGF I pretreatment in both cell lines decreased the number of specific IGF I binding sites, down-regulated total and membrane IGF 1R protein, and largely reduced or abolished the acute IGF I response without affecting IGF 1R mRNA levels. The data suggest that the IGF 1R protein of the two cell lines is translationally and/or posttranslationally down-regulated by its ligand in the presence and in the absence of locally produced IGFBPs and that the cell lines have retained this negative feedback to counteract IGF I stimulation.     

Insulin-like growth factor-binding protein-3 binds fibrinogen and fibrin.

Following tissue injury, a fibrin network formed at the wound site serves as a scaffold supporting the early migration of stromal cells needed for wound healing. Growth factors such as insulin-like growth factor-I (IGF-I) concentrate in wounds to stimulate stromal cell function and proliferation. The ability of IGF-binding proteins (IGFBPs) such as IGFBP-3 to reduce the rate of IGF-I clearance from wounds suggests that IGFBP-3 might bind directly to fibrinogen/fibrin. Studies presented here show that IGFBP-3 does indeed bind to fibrinogen and fibrin immobilized on immunocapture plates, with K(d) values = 0.67 and 0.70 nM, respectively, and competitive binding studies suggest that the IGFBP-3 heparin binding domain may participate in this binding. IGF-I does not compete for IGFBP-3 binding; instead, IGF-I binds immobilized IGFBP-3.fibrinogen and IGFBP-3.fibrin complexes with affinity similar to that of IGF-I for the type I IGF receptor. In the presence of plasminogen, most IGFBP-3 binds directly to fibrinogen, although 35-40% of the IGFBP-3 binds to fibrinogen-bound plasminogen. IGFBP-3 also binds specifically to native fibrin clots, and addition of exogenous IGFBP-3 increases IGF-I binding. These studies suggest that IGF-I can concentrate at wound sites by binding to fibrin-immobilized IGFBP-3, and that the lower IGF affinity of fibrin-bound IGFBP-3 allows IGF-I release to type I IGF receptors of stromal cells migrating into the fibrin clot.     

Chronic inflammation inhibits GH secretion and alters the serum insulin-like growth factor system in rats

Adjuvant-induced arthritis in rats is associated with growth failure, hypermetabolism and accelerated protein breakdown. The aim of this work was to study the effects of adjuvant-induced arthritis on GH and insulin-like growth factor-I (IGF-I). Arthritis was induced by an intradermal injection of complete Freund's adjuvant and rats were killed 18 and 22 days later. IGF-I and GH levels were measured by radioimmunoassay. Pituitary GH mRNA was analyzed by northern blot and IGF binding proteins (IGFBPs) by western blot. Arthritic rats showed a decrease in both serum and hepatic concentrations of IGF-I. On the contrary, arthritis increased the circulating IGFBPs. The serum concentration of IGF-I in the arthritic rats was negatively correlated with the body weight loss observed in these animals. Arthritis decreased the serum concentration of GH and this decrease seems to be due to an inhibition of GH synthesis, since pituitary GH mRNA content was decreased in arthritic rats (p<0.01). These data suggest that the decrease in body weight gain in arthritic rats may be, at least in part, secondary to the decrease in GH and IGF-I secretion. Furthermore, the increased serum IGFBPs may also be involved in the disease process.     

Regulation of vascular smooth muscle cell responses to insulin-like growth factor (IGF)-I by local IGF-binding proteins

Insulin-like growth factor (IGF)-I is a pleiotropic hormone that regulates vascular smooth muscle cell (VSMC) migration, proliferation, apoptosis, and differentiation. These actions are mediated by the IGF-I receptor. How activation of the same receptor by the same ligand leads to these diverse cellular responses is not well understood. Here we describe a novel mechanism specifying VSMC responses to IGF-I stimulation, distinctive for the pivotal roles of local IGF-binding proteins (IGFBPs). The role of local IGFBPs was indicated by comparing the activities of IGF-I and des-1-3-IGF-I, an IGF-I analog with reduced binding affinity to IGFBPs. Compared with IGF-I, des-1-3-IGF-I was more potent in stimulating DNA synthesis but much less potent in inducing directed migration of VSMCs. When the effects of individual IGFBPs were tested, IGFBP-2 and IGFBP-4 were found to inhibit IGF-I-stimulated DNA synthesis and migration. IGFBP-5 had an inhibitory effect on IGF-I-stimulated DNA synthesis, but it strongly potentiated IGF-I-induced VSMC migration. By using a non-IGF-binding IGFBP-5 mutant and an IGF-I-neutralizing antibody, it was demonstrated that IGFBP-5 also stimulates VSMC migration in an IGF-independent manner. This effect of IGFBP-5 was inhibited by soluble heparin and by treating cells with heparinase. Mutation of the heparin-binding motif of IGFBP-5 reduced its migration promoting activity. These findings suggest that local IGFBPs are important determinants of cellular responses to IGF-I stimulation, and a key player in this paradigm is IGFBP-5. IGFBP-5 not only modulates IGF-I actions, but it also stimulates cell migration by interacting with cell-surface heparan sulfate proteoglycans.