hsa_circ_0058122 knockdown prevents steroid-induced osteonecrosis of the femoral head by inhibiting human umbilical vein endothelial cells apoptosis via the miR-7974/IGFBP5 axis.

BACKGROUND: Steroid-induced osteonecrosis of femoral head (SONFH) is a serious complication of glucocorticoid overused. Recent evidence has demonstrated that circRNAs exert key pathophysiological roles in a variety of disease processes. However, the role of circRNA in SONFH remains largely unknown. The current study sought to evaluate how hsa_circ_0058122 affects SONFH in dexamethasone (DEX) treated human umbilical vein endothelial cells (HUVECs) model. METHODS: RT-PCR was used to demonstrate the hsa_circ_0058122 expression level in Dex-treated HUVECs cells. The effects of hsa_circ_0058122 on HUVECs apoptosis were evaluated via overexpression plasmid and siRNA. Using dual-luciferase and fluorescence in situ hybridization assays, we demonstrated that hsa_circ_0058122 binds to miR-7974 thereby facilitating HUVECs apoptosis. Bioinformatics analysis and western blot were performed to confirm target genes of hsa-miR-7974. RESULTS: In our previous work, we revealed the top 20 elevated circRNAs in SONFH patients were hsa_circ_0010027, hsa_circ_0058115, hsa_circ_0010026, hsa_circ_0058839, hsa_circ_0056886, hsa_circ_0056885, hsa_circ_0058146, hsa_circ_0058105, hsa_circ_0058112, hsa_circ_0058143, hsa_circ_0058102, hsa_circ_0058090, hsa_circ_0075353, hsa_circ_0058126, hsa_circ_0058130, hsa_circ_0058140, hsa_circ_0058122, hsa_circ_0058123, hsa_circ_0058103, and hsa_circ_0058121. Among these, hsa_circ_0058122 was finally selected for further investigation. We found hsa_circ_0058122 expression was markedly elevated in Dex-treated HUVECs cells, and the Dex-mediated HUVEC apoptosis was impaired in hsa_circ_0058122-silenced cells and increased in hsa_circ_0058122-overexpressing cells. hsa_circ_0058122 competitively binds to hsa-miR-7974, which in turn interacts with insulin-like growth factor binding protein 5 (IGFBP5). CONCLUSIONS: hsa_circ_0058122/miR-7974/IGFBP5 was proposed to be a key regulatory pathway for SONFH. DEX treatment upregulated hsa_circ_0058122 expression in HUVECs, which sponged miR-7974, thereby increasing IGFBP5 expression, the hsa_circ_0058122/miR-7974/IGFBP5 axis contributed to the Dex-mediated apoptosis. These findings may identify novel targets for SONFH molecular therapy.

Homocysteine downregulates cardiac homeobox transcription factor NKX2.5 via IGFBP5.

Homocysteine (Hcy) is an independent risk factor of congenital heart disease (CHD), but its exact underlying mechanism is unclear. In this study, we collected amniotic fluid (AF) supernatant samples from pregnant women carrying CHD-affected (n = 16) or normal (n = 16) fetuses. We found that Hcy concentrations were higher in the AF of the CHD group when compared with normal pregnancies. Also, Western blot showed that NK2 homeobox 5 (NKX2.5) was decreased and insulin-like growth factor binding protein 5 (IGFBP5) was increased in the AF of the CHD group. In the H9C2 cell culture experiment, 500 µmol/L Hcy downregulated NKX2.5 and upregulated IGFBP5. Real-time PCR and Western blot showed that NKX2.5 expression was reduced in H9C2 cells treated with IGFBP5. Luciferase reporter gene demonstrated that IGFBP5 decreased the transcription of the NKX2.5 promoter. Chromatin immunoprecipitation and electrophoretic mobility shift assay suggested that IGFBP5 binds to the NKX2.5 promoter region. Thus, the data indicated that one of the possible mechanisms by which Hcy is involved in CHD may be that Hcy inhibits NKX2.5 expression partly through IGFBP5.NEW & NOTEWORTHY We found that Hcy and IGFBP5 were increased, whereas NKX2.5 was decreased, in AF of CHD. Meanwhile, Hcy could upregulate IGFBP5 but downregulate NKX2.5, and IGFBP5 inhibited NKX2.5 expression in vitro. Moreover, IGFBP5 can bind to the NKX2.5 promoter region and reduce NKX2.5 transcriptional activity.

Local application of IGFBP5 protein enhanced periodontal tissue regeneration via increasing the migration, cell proliferation and osteo/dentinogenic differentiation of mesenchymal stem cells in an inflammatory niche.

BACKGROUND: Periodontitis is a widespread infectious disease ultimately resulting in tooth loss. The number of mesenchymal stem cells (MSCs) in patients with periodontitis is decreased, and MSC functions are impaired. Rescuing the impaired function of MSCs in periodontitis is the key for treatment, especially in a manner independent of exogenous MSCs. Our previous study found that overexpressed insulin-like growth factor binding protein 5 (IGFBP5) could promote exogenous MSC-mediated periodontal tissue regeneration. Here, we investigate the role of IGFBP5 protein in MSCs and periodontal tissue regeneration independent of exogenous MSCs in an inflammatory niche. METHODS: TNFα was used to mimic the inflammatory niche. Lentiviral IGFBP5 shRNA was used to silence IGFBP5 and recombinant human IGFBP5 protein (rhIGFBP5) was used to stimulate the periodontal ligament stem cells (PDLSCs) and bone marrow stem cells (BMSCs). The effects of IGFBP5 on PDLSCs were evaluated using the scratch-simulated wound migration, Transwell chemotaxis, alkaline phosphatase (ALP) activity, Alizarin red staining, Cell Counting Kit-8, Western blot, Real-time PCR, Co-IP and ChIP assays. The swine model of periodontitis was used to investigate the functions of IGFBP5 for periodontal regeneration and its anti-inflammation effect. RESULTS: We discovered that 0.5 ng/ml rhIGFBP5 protein enhanced the migration, chemotaxis, osteo/dentinogenic differentiation and cell proliferation of MSCs under the inflammatory condition. Moreover, 0.5 ng/ml rhIGFBP5 application could rescue the impaired functions of IGFBP5-silenced-MSCs in the inflammatory niche. Furthermore, local injection of rhIGFBP5 could promote periodontal tissue regeneration and relieve the local inflammation in a minipig model of periodontitis. Mechanistically, we found that BCOR negatively regulated the expression of IGFBP5 in MSCs. BCOR formed a protein complex with histone demethylase KDM6B and raised histone K27 methylation in the IGFBP5 promoter. CONCLUSIONS: This study revealed that rhIGFBP5 could activate the functions of MSCs in an inflammatory niche, provided insight into the mechanism underlying the activated capacities of MSCs, and identified IGFBP5 as a potential cytokine for improving tissue regeneration and periodontitis treatment independent of exogenous MSCs and its potential application in dental clinic.

Human Adipocytes Induce Inflammation and Atrophy in Muscle Cells During Obesity.

Inflammation and lipid accumulation are hallmarks of muscular pathologies resulting from metabolic diseases such as obesity and type 2 diabetes. During obesity, the hypertrophy of visceral adipose tissue (VAT) contributes to muscle dysfunction, particularly through the dysregulated production of adipokines. We have investigated the cross talk between human adipocytes and skeletal muscle cells to identify mechanisms linking adiposity and muscular dysfunctions. First, we demonstrated that the secretome of obese adipocytes decreased the expression of contractile proteins in myotubes, consequently inducing atrophy. Using a three-dimensional coculture of human myotubes and VAT adipocytes, we showed the decreased expression of genes corresponding to skeletal muscle contractility complex and myogenesis. We demonstrated an increased secretion by cocultured cells of cytokines and chemokines with interleukin (IL)-6 and IL-1ß as key contributors. Moreover, we gathered evidence showing that obese subcutaneous adipocytes were less potent than VAT adipocytes in inducing these myotube dysfunctions. Interestingly, the atrophy induced by visceral adipocytes was corrected by IGF-II/insulin growth factor binding protein-5. Finally, we observed that the skeletal muscle of obese mice displayed decreased expression of muscular markers in correlation with VAT hypertrophy and abnormal distribution of the muscle fiber size. In summary, we show the negative impact of obese adipocytes on muscle phenotype, which could contribute to muscle wasting associated with metabolic disorders.

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.

IGFBP-5 enhances epithelial cell adhesion and protects epithelial cells from TGFß1-induced mesenchymal invasion.

TGFß1 is a major fibrotic factor and its actions involve induction of epithelial cell death, together with the stimulation and transdifferentiation of fibroblasts into collagen- and fibronectin-secreting myofibroblasts. These actions of TGFß1 are also consistent with a pro-metastatic role, by aiding epithelial cell escape through mesenchymal tissues. Recently IGFBP-5 has been described as a pro-fibrotic (pro-metastatic?) agent and the aim of this study was to compare and contrast the actions of IGFBP-5 with TGFß1. We used NMuMG cells and cloned stable epithelial and mesenchymal lines from the parent cells. TGFß1 induced apoptosis and/or EMT in the epithelial cells, whereas it enhanced mesenchymal cell survival and migration. IGFBP-5, in contrast, enhanced both cell-cell and cell-ECM adhesion and also improved wound closure in epithelial cells whereas, in mesenchymal cells, IGFBP-5 decreased adhesion and migration. Furthermore, IGFBP-5 was able to antagonise the actions of TGFß1. In a co-culture model simulating epithelial-mesenchymal boundaries, IGFBP-5 was able to antagonise the disruptive transgressions induced by TGFß1. Overall, these findings suggest that IGFBP-5 is important in maintaining epithelial-mesenchymal boundaries and thus may limit metastasis and fibrosis by inducing an orderly repair mechanism, very distinct from the fibrotic disruption induced by TGFß1. A role for IGFBP-5 in the inhibition of metastasis is supported by immunohistochemical studies of breast cancer microarrays, where we show that elevated IGFBP-5 expression is associated with increased disease-free survival.

Cell motility in models of wounded human skin is improved by Gap27 despite raised glucose, insulin and IGFBP-5.

Reducing Cx43 expression stimulates skin wound healing. This is mimicked in models when Cx43 function is blocked by the connexin mimetic peptide Gap27. IGF-I also stimulates wound healing with IGFBP-5 attenuating its actions. Further, the IGF-I to IGFBP-5 ratio is altered in diabetic skin, where wound closure is impaired. We investigated whether Gap27 remains effective in augmenting scrape-wound closure in human skin wound models simulating diabetes-induced changes, using culture conditions with raised glucose, insulin and IGFBP-5. Gap27 increased scrape-wound closure in normal glucose and insulin (NGI) and to a lesser extent in high glucose and insulin (HGI). IGF-I enhanced scrape-wound closure in keratinocytes whereas IGFBP-5 inhibited this response. Gap27 overcame the inhibitory effects of IGFBP-5 on IGF-I activity. Connexin-mediated communication (CMC) was reduced in HGI, despite raised Cx43, and Gap27 significantly decreased CMC in NGI and HGI. IGF-I and IGFBP-5 did not affect CMC. IGF-I increased keratinocyte proliferation in NGI, and Gap27 increased proliferation in NGI to a greater extent than in HGI. We conclude that IGF-I and Gap27 stimulate scrape-wound closure by independent mechanisms with Gap27 inhibiting Cx43 function. Gap27 can enhance wound closure in diabetic conditions, irrespective of the IGF-I:IGFBP-5 balance.

Insulin-like growth factor-binding protein-5 (IGFBP-5) inhibits TNF-α-induced NF-κB activity by binding to TNFR1.

IGFBP-5 is known to be involved in various cell phenomena such as proliferation, differentiation, and apoptosis. However, the exact mechanisms by which IGFBP-5 exerts its functions are unclear. In this study, we demonstrate for the first time that IGFBP-5 is a TNFR1-interacting protein. We found that ectopic expression of IGFBP-5 induced TNFR1 gene expression, and that IGFBP-5 interacted with TNFR1 in both an in vivo and an in vitro system. Secreted IGFBP-5 interacted with GST-TNFR1 and this interaction was blocked by TNF-α, demonstrating that IGFBP-5 might be a TNFR1 ligand. Furthermore, conditioned media containing secreted IGFBP-5 inhibited PMA-induced NF-κB activity and IL-6 expression in U-937 cells. Coimmunoprecipitation assays of TNFR1 and IGFBP-5 wild-type and truncation mutants revealed that IGFBP-5 interacts with TNFR1 through its N- and L-domains. However, only the interaction between the L-domain of IGFBP-5 and TNFR1 was blocked by TNF-α in a dose-dependent manner, suggesting that the L-domain of IGFBP-5 can function as a TNFR1 ligand. Competition between the L-domain of IGFBP-5 and TNF-α resulted in inhibition of TNF-α-induced NF-κΒ activity. Taken together, our results suggest that the L-domain of IGFBP-5 is a novel TNFR1 ligand that functions as a competitive TNF-α inhibitor.

Effects of IGF-binding protein 5 in dysregulating the shape of human hair.

The hair follicle has a unique dynamic property to cyclically regenerate throughout life. Despite significant progress in hair structure and hair shape determination using animal models, the mechanisms controlling the architecture and the shape of the human hair remain largely unexplored. In this study, comparison of the genetic expression of several human genes, especially those involved in growth, development, and differentiation, between Caucasian curly hair and naturally straight hair was performed. Thereafter, analyses using human recombinant and lentiviral vector technologies were conducted to further dissect and elucidate a molecular mechanism that regulates hair growth and development, particularly in controlling the shape of the hair shaft. Overexpression of IGF-binding protein 5 (IGFBP-5) in the human hair xenografts obtained from straight- and curly-haired individuals was found to result in the decreased expression of several extracellular matrix proteins and disassembly of adhesional junctions, resulting in twisted hair shafts as well as an unusual deposition of hair cuticle that may be derived from the disturbance of normal proliferation and differentiation. This study provides evidence that IGFBP-5 has an effect on human hair shape, and that lentiviral transduction regimen can be used for functional analysis of genes involved in human hair morphogenesis.

Fibrotic response in fibroblasts from congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are characterized by a generalized underglycosylation of proteins. CDG is associated with multiple symptoms such as psychomotor retardation, hypotonia, hormonal disturbances, liver fibrosis and coagulopathies. The molecular basis of these symptoms is poorly understood considering the large extent of affected glycoproteins. To better understand the cellular responses to protein underglycosylation in CDG, we have investigated the differences in gene expression between healthy control and CDG fibroblasts by transcriptome comparison. This analysis revealed a strong induction of several genes encoding components of the extracellular matrix, such as collagens, COMP, IGFBP5 and biglycan. The extent of this response was confirmed at the protein level by showing increased production of collagen type-I for example. This fibrotic response of CDG fibroblasts was not paralleled by a differentiation to myofibroblasts and by increased TGF-ß signalling. We could show that the addition of recombinant IGFBP5, one of the induced proteins in CDG, to healthy control fibroblasts increased the production of collagen type-I to levels similar to those found in CDG fibroblasts. The fibrotic response identified in CDG fibroblasts may account for the elevated tissue fibrosis, which is often encountered in CDG patients.

Insulin-like growth factor-binding protein-5-induced laminin gamma1 transcription requires filamin A.

Insulin-like growth factor-binding protein-5 (IGFBP-5) has IGF-1-independent intranuclear effects that are poorly defined. Treatment of cells with IGFBP-5 induces migration, prevents apoptosis, and leads to increased laminin subunit transcription. Similarly, filamin A (FLNa), an actin-binding protein that participates in cell attachment, plays important additional roles in signal transduction and modulation of transcriptional responses. In this report, we show that IGFBP-5 leads to dephosphorylation of FLNa with subsequent FLNa cleavage. Following cleavage, there is enhanced recruitment of Smad3/4 to a C-terminal FLNa fragment with nuclear translocation and subsequent binding to the promoter region of the laminin gamma1 (lamc1) gene. FLNa knockdown prevents IGFBP-5-mediated increases in lamc1 transcription. These data indicate that IGFBP-5 induces formation of a FLNa-based nuclear shuttle that recruits transcription factors and regulates transcription of IGFBP-5 target genes. These studies provide new insights into the mechanisms whereby IGFBP-5 and FLNa exert intranuclear effects.

The pro-fibrotic factor IGFBP-5 induces lung fibroblast and mononuclear cell migration.

We have previously shown that insulin-like growth factor-binding protein-5 (IGFBP-5) is overexpressed in fibrotic lung tissues and that it induces production of extracellular matrix components such as collagen and fibronectin both in vitro and in vivo. We recently observed mononuclear cell infiltration in lung tissues of mice expressing IGFBP-5. We therefore examined the role of IGFBP-5 on the migration of immune cells. Migration assays demonstrated that IGFBP-5 induced migration of peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. Preferential migration of monocytes/macrophages, natural killer cells, and T cells was observed. Moreover, the CD4/CD8 ratio of migrating cells was significantly higher in vitro and in vivo in response to IGFBP-5. IGFBP-5 resulted in preferential migration of activated CD4(+) T cells and monocytes. Interestingly, IGFBP-5 also induced migration of primary human lung fibroblasts. Exogenous administration of IGFBP-5 induced activation of mitogen-activated protein kinase (MAPK) signaling cascade but not PI3K in PBMCs. IGFBP-5-induced migration was blocked by the MEK1/2 inhibitor U0126, suggesting that IGFBP-5-induced migration occurs via MAPK activation. Furthermore, monocytes treated with recombinant IGFBP-5 expressed the mesenchymal markers alpha-smooth muscle actin and fibronectin in vitro and in vivo, suggesting that IGFBP-5 can induce the transformation of monocytes into mesenchymal cells. Collectively, our results suggest that IGFBP-5 induces cell migration via MAPK-dependent and IGF-I-independent mechanisms.

Vitronectin modulates human mesenchymal stem cell response to insulin-like growth factor-I and transforming growth factor beta 1 in a serum-free environment.

The use of animal sera for the culture of therapeutically important cells impedes the clinical use of the cells. We sought to characterize the functional response of human mesenchymal stem cells (hMSCs) to specific proteins known to exist in bone tissue with a view to eliminating the requirement of animal sera. Insulin-like growth factor-I (IGF-I), via IGF binding protein-3 or -5 (IGFBP-3 or -5) and transforming growth factor-beta 1 (TGF-beta(1)) are known to associate with the extracellular matrix (ECM) protein vitronectin (VN) and elicit functional responses in a range of cell types in vitro. We found that specific combinations of VN, IGFBP-3 or -5, and IGF-I or TGF-beta(1) could stimulate initial functional responses in hMSCs and that IGF-I or TGF-beta(1) induced hMSC aggregation, but VN concentration modulated this effect. We speculated that the aggregation effect may be due to endogenous protease activity, although we found that neither IGF-I nor TGF-beta(1) affected the functional expression of matrix metalloprotease-2 or -9, two common proteases expressed by hMSCs. In summary, combinations of the ECM and growth factors described herein may form the basis of defined cell culture media supplements, although the effect of endogenous protease expression on the function of such proteins requires investigation.

Insulin-like growth factor (IGF) binding protein-5 blocks skeletal muscle differentiation by inhibiting IGF actions.

Signaling through the IGF-I receptor by locally produced IGF-I or -II is critical for normal skeletal muscle development and repair after injury. In most tissues, IGF action is modulated by IGF binding proteins (IGFBPs). IGFBP-5 is produced by muscle cells, and previous studies have suggested that when overexpressed it may either facilitate or inhibit IGF actions, and thus potentially enhance or diminish IGF-mediated myoblast differentiation or survival. To resolve these contradictory observations and discern the mechanisms of action of IGFBP-5, we studied its effects in cultured muscle cells. Purified wild-type (WT) mouse IGFBP-5 or a variant with diminished extracellular matrix binding (C domain mutant) each prevented differentiation at final concentrations as low as 3.5 nm, whereas analogs with reduced IGF binding (N domain mutant) were ineffective even at 100 nm. None of the IGFBP-5 variants altered cell number. An IGF-I analog (R(3)IGF-I) with diminished affinity for IGFBPs promoted full muscle differentiation in the presence of IGFBP-5(WT), showing that IGFBP-5 interferes with IGF-dependent signaling pathways in myoblasts. When IGFBP-5(WT) or variants were overexpressed by adenovirus-mediated gene transfer, concentrations in muscle culture medium reached 500 nm, and differentiation was inhibited, even by IGFBP-5(N). As 200 nm of purified IGFBP-5(N) prevented activation of the IGF-I receptor by 10 nm IGF-II as effectively as 2 nm of IGFBP-5(WT), our results not only demonstrate that IGFBP-5 variants with reduced IGF binding affinity impair muscle differentiation by blocking IGF actions, but underscore the need for caution when labeling effects of IGFBPs as IGF independent because even low-affinity analogs may potently inhibit IGF-I or -II if present at high enough concentrations in biological fluids.

Involvement of ligand occupancy in Insulin-like growth factor-I (IGF-I) induced cell growth in osteoblast like MC3T3-E1 cells.

Growth factors and matrix proteins regulate the proliferation and differentiation of osteoblasts. The insulin-like growth factor (IGF) system comprises IGF-I, IGF-II, and six high-affinity IGF-binding proteins (IGFBPs). IGFs stimulate cell growth in many types of tissue; IGF-binding proteins regulate cellular actions and can affect cell growth. IGF-I is involved in differentiation, proliferation, and matrix formation in osteoblasts; IGFBP-5 is associated with the extracellular matrix (ECM) and can potentiate the actions of IGF-I. We investigated the effect of ECM proteins on the responses of MC3T3-E1 osteoblast cells to IGF-I and IGFBP-5. In addition, because extracellular signal-regulated kinases 1 and 2 (Erk 1/2) affect cell growth, we evaluated the effects of IGFBP-5 on Erk 1/2 phosphorylation in MC3T3-E1 cells. IGF-I caused an increase in IGFBP-5 expression in cultured MC3T3-E1 cells, and IGF-I plus IGFBP-5 significantly increased cell growth. Likewise, the addition of IGF-I and IGFBP-5 to cultured MC3T3-E1 cells increased the synthesis of the ECM proteins osteopontin (OPN) and thrombospondin-1 (TSP-1), which can bind to alphaVbeta3 integrin receptors on the cell surface. By contrast, the addition of an antibody against ECM proteins inhibited the effects of OPN and TSP-1 on IGFBP-5 expression. The stimulatory effect of IGFBP-5 was mediated via Erk 1/2 activation. These data suggest that IGFBP-5 regulates Erk 1/2 phosphorylation in cultured MC3T3-E1 cells via ECM proteins that may ultimately stimulate the growth of osteoblasts. We determined whether occupation of the alphaVbeta3 integrin receptor affects IGF-I receptor (IGF-IR)-mediated signaling and function in MC3T3-E1 osteoblast cells. Occupation of the alphaVbeta3 integrin receptor with ECM proteins induced IGF-I-stimulated IGF-IR phosphorylation. Conversely, in the presence of the alphaVbeta3-specific disintegrin echistatin, IGF-I-stimulated IGF-IR activation was inhibited. IGF-I-stimulated IGF-IR phosphorylation was accompanied by IRS-1 phosphorylation and MAPK activation. However, these effects were attenuated by echistatin. Thus, occupancy of the alphaVbeta3 disintegrin receptor modulates IGF-I-induced IGF-IR activation and IGF-IR-mediated function in MC 3T3-E1 osteoblasts.

Insulin-like growth factor binding protein (IGFBP)-mediated hair cell survival on the mouse utricle exposed to neomycin: the roles of IGFBP-4 and IGFBP-5.

CONCLUSION: This study suggests for the first time that 1) IGF-I, IGFBP-4, and -5 alone and IGF-I+IGFBP-5 mixture stimulated hair cell survival and prevented neomycin-induced hair cell loss in the sensory epithelial culture of mouse utricles, 2) When administered together, IGFBP-4 diminished the effect of IGF-I, 3) In P3-5 mice utricle, IGF-I, IGFBP-4, and IGFBP-5 are expressed in the cytoplasm of hair cells. And Insulin/IGF-I Receptor is expressed in the nucleus of hair cells. OBJECTIVES: Several growth factors have been demonstrated to protect auditory sensory cells in vitro and in vivo from aminoglycoside toxicity. IGF-I is one of the most well-known mitogenic and protective substance working in the inner ear. However, there are no reports available regarding the function of IGFBPs in the inner ear. In the present study, the effects of IGFBP-4 and -5 on hair cell survival were investigated in mouse utriclular organ cultures. MATERIALS AND METHODS: The amount of cellular damage and cell viability in vestibular organs were assessed by counting hair cells stained with a rhodamine-phalloidin probe. The expressions of IGFBP-4, IGFBP-5, IGF-IR, and IGF-I were localized by immunohistochemistry. RESULTS: When treated with IGF-I, IGFBP-4, or IGFBP-5 for 24 h, explant culture showed hair cell survival rates of 136+/-18%, 140+/-15%, and 133+/-6%, respectively, compared to controls. Neomycin (1 mM) induced hair cell loss resulted in 45+/-17% of hair cell survival. However, pre-treatment of IGF-I, IGFBP-4, or -5 before neomycin insult showed survival rates of 113+/-14%, 98+/-8%, and 73+/-24%, respectively. Similar to IGF-I, IGFBP-4 and IGFBP-5 were significantly protective. IGFBP-4 and -5 immunoreactivities were observed in the cytoplasm of normal explanted vestibular hair cells as well as in the P3 mouse utricular hair cells in vivo.

Induction of cellular senescence by insulin-like growth factor binding protein-5 through a p53-dependent mechanism.

The insulin-like growth factor (IGF) signaling pathway plays a crucial role in the regulation of cell growth, differentiation, apoptosis, and aging. IGF-binding proteins (IGFBPs) are important members of the IGF axis. IGFBP-5 is up-regulated during cellular senescence in human dermal fibroblasts and endothelial cells, but the function of IGFBP-5 in cellular senescence is unknown. Here we show that IGFBP-5 plays important roles in the regulation of cellular senescence. Knockdown of IGFBP-5 in old human umbilical endothelial cells (HUVECs) with IGFBP-5 micro-RNA lentivirus caused partial reduction of a variety of senescent phenotypes, such as changes in cell morphology, increases in cell proliferation, and decreases in senescence-associated beta-galactosidase (SA-beta-gal) staining. In addition, treatment with IGFBP-5 protein or up-regulation of IGFBP-5 in young cells accelerates cellular senescence, as confirmed by cell proliferation and SA-beta-gal staining. Premature senescence induced by IGFBP-5 up-regulation in young cells was rescued by knockdown of p53, but not by knockdown of p16. Furthermore, atherosclerotic arteries exhibited strong IGFBP-5-positive staining along intimal plaques. These results suggest that IGFBP-5 plays a role in the regulation of cellular senescence via a p53-dependent pathway and in aging-associated vascular diseases.

Preparation of cultured skin for transplantation using insulin-like growth factor I in conjunction with insulin-like growth factor binding protein 5, epidermal growth factor, and vitronectin.

BACKGROUND: Cultured skin for transplantation is routinely prepared by growing patient keratinocytes in the presence of semidefined sources of growth factors including serum and feeder cells, but these materials require substantial risk remediation and can contribute to transplant rejection. METHODS: We have therefore investigated the potential of a novel combination of recombinant and purified growth factors to replace serum and feeder cells in cultures of human keratinocytes suitable for clinical application. Our technique was investigated with respect to culture establishment, serial propagation, colony-forming efficiency, immunocytochemistry, epidermal reconstruction, and suitability to support transplantation by aerosolization. RESULTS: We demonstrate that insulin-like growth factor (IGF)-I--used in conjunction with epidermal growth factor (EGF), insulin-like growth factor binding protein (IGFBP)-5 and vitronectin--supports growth in the absence of serum. Moreover, a threefold greater number of cells are generated within 7 days compared to those grown under current best practice conditions using serum (P<0.05). The resulting test cultures are suitable for epidermal reconstruction and support the option for delivery in the form of an aerosolized cell suspension. Serial propagation, with the view to producing confluent sheets for extensive injuries, was achieved but with less consistency and this result correlated with a significant decline in colony-forming efficiency compared to controls. CONCLUSIONS: IGF-I used in conjunction with IGFBP-5, EGF, and vitronectin provides a superior alternative to serum for the rapid expansion and transplantation of cultured keratinocytes within the first week of treatment. Nevertheless, further optimization is required with respect to elimination of feeder cells and serial expansion of cultures for treatment of extensive injuries.

Rat glomerular mesangial cells require laminin-9 to migrate in response to insulin-like growth factor binding protein-5.

Temporal and spatial differences in extracellular matrix play critical roles in cell proliferation, differentiation and migration. Different migratory stimuli use different substrates and receptors to achieve cell migration. To understand the mechanism of insulin-like growth factor binding protein-5 (IGFBP-5)-induced migration in mesangial cells, the roles of integrins and substrates were examined. IGFBP-5 induced an increase in mRNA expression for laminin (LN) chains lama4, lamb2, and lamc1, suggesting that LN-9 might be required for migration. Antibodies to the LNalpha(4) and LNbeta(2) chains, but not LNbeta(1), blocked IGFBP-5-induced migration. Anti-sense morpholino oligonucleotide inhibition of expression of LNalpha(4) substantially reduced expression of LN-8/9 (alpha(4)beta(1)gamma(1)/alpha(4)beta(2)gamma(1), 411/421) and prevented IGFBP-5-induced migration. Anti-sense inhibition of lamb2 reduced expression of LN-9. Absence of LN-9 prevented IGFBP-5-induced migration, which was not preserved by continued expression of LN-8. The requirement for LN-9 was further supported by studies of T98G cells, which express predominantly LN-8. IGFBP-5 had little effect on migration in these cells, but increased migration when T98G cells were plated on LN-8/9. IGFBP-5-mediated mesangial cell migration was inhibited by antibodies that block attachment to alpha(6)beta(1)-integrins but was unaffected by antibodies and disintegrins that block binding to other integrins. Furthermore, in cells with anti-sense inhibited expression of LN-9, integrin alpha(6)beta(1) was no longer detected on the cell surface. These studies suggest the specificity of mechanisms of migration induced by specific stimuli and for the first time demonstrate a unique function for LN-9 in mediating IGFBP-5-induced migration.

SOM230 inhibits insulin-like growth factor-I action in mammary gland development by pituitary independent mechanism: mediated through somatostatin subtype receptor 3?

Somatostatin analogs (SAs) treat acromegaly by lowering pituitary GH secretion, which, in turn, lowers systemic IGF-I. The profound systemic effect is often greater than expected in the face of only partial GH suppression. Here we report that the SA SOM230 can also act by a nonpituitary-mediated inhibition of IGF-I action. SOM230 inhibited mammary development in intact and hypophysectomized female rats, a process requiring IGF-I. IGF-I overcame this inhibition. SOM230 also inhibited other actions of IGF-I (inhibition of apoptosis, phosphorylation of insulin receptor substrate-1, and cell division). SOM230 did not reduce IGF-I mRNA abundance in mammary gland but did stimulate IGF binding protein 5 (IGFBP5). IGFBP5 was 3.75 times higher in mammary epithelium of SOM230 than in placebo animals (P < 0.001). Administration of IGFBP-5 also inhibited GH-induced mammary development (P < 0.001). Measurement of sstr(1-5) (somatostatin subtype receptor) by real-time RT-PCR revealed that the mammary glands had an abundance of sstr(3) and lower amounts of sstr(4) and sstr(5) but no sstr(1) or sstr(2.) That mammary development was also inhibited to a lesser degree than SOM230 by octreotide, whose main action is through sstr(2), strongly suggests that sstr(3) is at least in part mediating the effects of the SAs. We conclude that 1) SAs inhibit IGF-I action in the mammary gland through a novel nonpituitary mechanism; 2) IGFBP-5, here shown to inhibit pubertal mammary development, might mediate the effect; and 3) Measurement of available sstr receptors in the mammary gland suggests that sstr(3) mediates the SA activity, but sstr(5) is also a possible mediator.