The matrix metalloproteinase-9 regulates the insulin-like growth factor-triggered autocrine response in DU-145 carcinoma cells

The androgen-independent human prostate adenocarcinoma cell line DU-145 proliferates in serum-free medium and produces insulin-like growth factors (IGF)-I, IGF-II, and the IGF type-1 receptor (IGF-1R). They also secrete three IGF-binding proteins (IGFBP), IGFBP-2, -3, and -4. Of these, immunoblot analysis revealed selective proteolysis of IGFBP-3, yielding fragments of 31 and 19 kDa. By using an anti-IGF-I-specific monoclonal antibody (mAb), we detect surface receptor-bound IGF-I on serum-starved DU-145 cells, which activates IGF-1R and triggers a mitogenic signal. Incubation of DU-145 cells with blocking anti-IGF-I, anti-IGF-II, or anti-IGF-I plus anti-IGF-II mAb does not, however, inhibit serum-free growth of DU-145. Conversely, anti-IGF-1R mAb and IGFBP-3 inhibit DNA synthesis. IGFBP-3 also modifies the DU-145 cell cycle, decreases p34(cdc2) levels, and IGF-1R autophosphorylation. The antiproliferative IGFBP-3 activity is not IGF-independent, since des-(1-3)IGF-I, which does not bind to IGFBP-3, reverses its inhibitory effect. DU-145 also secretes the matrix metalloproteinase (MMP)-9, which can be detected in both a soluble and a membrane-bound form. Matrix metalloproteinase inhibitors, but not serpins, abrogate DNA synthesis in DU-145 associated with the blocking of IGFBP-3 proteolysis. Overexpression of an antisense cDNA for MMP-9 inhibits 80% of DU-145 cell proliferation that can be reversed by IGF-I in a dose-dependent manner. Inhibition of MMP-9 expression is also associated with a decrease in IGFBP-3 proteolysis and with reduced signaling through the IGF-1R. Our data indicate an IGF autocrine loop operating in DU-145 cells, specifically modulated by IGFBP-3, whose activity may in turn be regulated by IGFBP-3 proteases such as MMP-9.     

IGFBP-3 Inhibits the Proliferation of Neural Progenitor Cells

Insulin like growth factor-1 (IGF-1) plays an important role in the proliferation and differentiation of neural progenitor cells. The effects of IGF-1 can be regulated by insulin like growth factor binding protein-3 (IGFBP-3) which can either inhibit or stimulate the proliferation of cells depending on the expression of proteases that can release IGF-1 from IGF1-IGFBP3 complex. Although IGF-1 is essential for the development of brain, both IGFBP-3 and IGF-1 are elevated in the brains of children younger than 6 months of age. Likewise, IGFBP-3 is also upregulated following cerebral ischemia and hypoxia. However, the role of IGFBP-3 in neurogenesis is not clear. Using an in vitro culture system of rat neural progenitor cells, we demonstrate that IGFBP-3 specifically regulates the IGF-1 mediated neural progenitor cell proliferation via down regulation of phopho-Akt, and cyclin D1. In addition, IGFBP-3 also decreased the content of nestin in the neural progenitor cells indicating its potential role in neurogenesis.     

EGCG inhibits activation of the insulin-like growth factor-1 receptor in human colon cancer cells

The IGF/IGF-1R system, which includes the IGF, IGF-1R, and IGFBPs proteins, plays an important role in the development and growth of colorectal cancer. We previously reported that in the HT29 human colon cancer cell line EGCG, the major biologically active component of green tea, inhibits activation of the RTKs EGFR, HER2, and HER3, and that this is associated with inhibition of multiple downstream signaling pathways. Since IGF-1R is also a RTK, in this study we examined the effects of EGCG on the activity of IGF/IGF-1R system in human colon cancer cells. We found that the colon cancer cell lines Caco2, HT29, SW837, and SW480 express high levels of the IGF-1R receptor, and that both SW837 and SW480 cells display constitutive activation of this receptor. Treatment of SW837 cells with 20 microg/ml of EGCG (the IC50 concentration for growth inhibition) caused within 6 h a decrease in the phosphorylated (i.e., activated) form of the IGF-1R protein. At 12 h, there was a decrease in the levels of both IGF-1 protein and mRNA and within 3-6 h there was an increase in the levels of both IGFBP-3 protein and mRNA. The increased expression of the latter protein was sustained for at least 48 h. When SW837 cells were treated with EGCG for a longer time, i.e., 96 h, a very low concentration (1.0 microg/ml) of EGCG also caused inhibition of activation of IGF-1R, a decrease in the IGF-1 protein, and an increase in the IGFBP-3 protein. EGCG also caused a decrease in the levels of mRNAs that encode MMPs-7 and -9, proteins that proteolyze IGFBP-3. In addition, treatment with EGCG caused a transient increase in the expression of TGF-beta2, an inducer of IGFBP-3 expression. These findings expand the roles of EGCG as an inhibitor of critical RTKs involved in cell proliferation, providing further evidence that EGCG and related compounds may be useful in the chemoprevention or treatment of colorectal cancer.     

Expression profile of IGF system during lung injury and recovery in rats exposed to hyperoxia: a possible role of IGF-1 in alveolar epithelial cell proliferation and differentiation

Although several studies have shown that an induction of insulin-like growth factor (IGF) components occurs during hyperoxia-mediated lung injury, the role of these components in tissue repair is not well known. The present study aimed to elucidate the role of IGF system components in normal tissue remodeling. We used a rat model of lung injury and remodeling by exposing rats to > 95% oxygen for 48 h and allowing them to recover in room air for up to 7 days. The mRNA expression of IGF-I, IGF-II, and IGF-1 receptor (IGF-1R) increased during injury. However, the protein levels of these components remained elevated until day 3 of the recovery and were highly abundant in alveolar type II cells. Among IGF binding proteins (IGFBPs), IGFBP-5 mRNA expression increased during injury and at all the recovery time points. IGFBP-2 and -3 mRNA were also elevated during injury phase. In an in vitro model of cell differentiation, the expression of IGF-I and IGF-II increased during trans-differentiation of alveolar epithelial type II cells into type-I like cells. The addition of anti-IGF-1R and anti-IGF-I antibodies inhibited the cell proliferation and trans-differentiation to some extent, as evident by cell morphology and the expression of type I and type II cell markers. These findings demonstrate that the IGF signaling pathway plays a critical role in proliferation and differentiation of alveolar epithelium during tissue remodeling.     

Contrasting effects of IGF binding protein-3 expression in mammary tumor cells and the tumor microenvironment

IGFBP-3 has both stimulatory and inhibitory effects on cancer progression. The growth of EO771 mammary carcinoma cells as syngeneic tumors in C57BL/6 mice is reduced in Igfbp3-null (BP3KO) mice, suggesting that systemic IGFBP-3 enhances tumor progression. In this study we assessed the growth of EO771 cells expressing human IGFBP-3 in BP3KO mice. Cells expressing hIGFBP-3 showed decreased proliferation in vitro and increased levels of IGF-1 receptor (IGF1R) protein but not mRNA, consistent with sequestration of endogenous IGF by IGFBP-3. The growth rate of these cells was restored by exposure to IGF-1 or analogues with reduced affinity for IGFBP-3 (long Arg³-IGF-1) or IGF1R (Leu²⁴-IGF-1). In EO771 cells implanted orthotopically into mice, hIGFBP-3 expression by the cells inhibited tumor establishment in BP3KO but not wild-type mice. For tumors that successfully established, final weight was not affected significantly by hIGFBP-3 expression. However, final tumor weight was inversely related to intratumoral T cell counts, and sera from BP3KO mice with tumors showed low-titer immunoreactivity against IGFBP-3. The contrasting effects on tumor establishment and progression of IGFBP-3 expressed by mammary carcinoma cells, compared to systemic stromal and circulating IGFBP-3, highlights the complexity of growth regulation by IGFBP-3 in mammary tumors.     

Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells

The insulin-like growth factor type 1 receptor (IGF-1R) is part of the receptor tyrosine kinase superfamily. The activation of IGF-1R regulates several key signaling pathways responsible for maintaining cellular homeostasis, including survival, growth, and proliferation. In addition to mediating signal transduction at the plasma membrane, in serum-based models, IGF-1R undergoes SUMOylation by SUMO 1 and translocates to the nucleus in response to IGF-1. In corneal epithelial cells grown in serum-free culture, however, IGF-1R has been shown to accumulate in the nucleus independent of IGF-1. In this study, we report that the insulin-like growth factor binding protein-3 (IGFBP-3) mediates nuclear translocation of IGF-1R in response to growth factor withdrawal. This occurs via SUMOylation by SUMO 2/3. Further, IGF-1R and IGFBP-3 undergo reciprocal regulation independent of PI3k/Akt signaling. Thus, under healthy growth conditions, IGFBP-3 functions as a gatekeeper to arrest the cell cycle in G0/G1, but does not alter mitochondrial respiration in cultured cells. When stressed, IGFBP-3 functions as a caretaker to maintain levels of IGF-1R in the nucleus. These results demonstrate mutual regulation between IGF-1R and IGFBP-3 to maintain cell survival under stress. This is the first study to show a direct relationship between IGF-1R and IGFBP-3 in the maintenance of corneal epithelial homeostasis.     

Development of IGF Signaling Antibody Arrays for the Identification of Hepatocellular Carcinoma Biomarkers

Purpose

Our objective was to develop a system to simultaneously and quantitatively measure the expression levels of the insulin-like growth factor (IGF) family proteins in numerous samples and to apply this approach to profile the IGF family proteins levels in cancer and adjacent tissues from patients with hepatocellular carcinoma (HCC).

Experimental Design

Antibodies against ten IGF family proteins (IGF-1, IGF-1R, IGF-2, IGF-2R, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, and Insulin) were immobilized on the surface of a glass slide in an array format to create an IGF signaling antibody array. Tissue lysates prepared from patient''s liver cancer tissues and adjacent tissues were then applied to the arrays. The proteins captured by antibodies on the arrays were then incubated with a cocktail of biotinylated detection antibodies and visualized with a fluorescence detection system. By comparison with standard protein amount, the exact protein concentrations in the samples can be determined. The expression levels of the ten IGF family proteins in 25 pairs of HCC and adjacent tissues were quantitatively measured using this novel antibody array technology. The differential expression levels between cancer tissues and adjacent tissues were statistically analyzed.

Results

A novel IGF signaling antibody array was developed which allows the researcher to simultaneously detect ten proteins involved in IGF signal pathway with high sensitivity and specificity. Using this approach, we found that the levels of IGF-2R and IGFBP-2 in HCC tissues were higher than those in adjacent tissues.

Conclusion

Our IGF signaling antibody array which can detect the expression of ten IGF family members with high sensitivity and specificity will undoubtedly prove a powerful tool for drug and biomarker discovery.     

Modulation of insulin-like growth factor actions in L6A1 myoblasts by insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5: A dual role for IGFBP-5

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. J. Cell. Physiol. 177:47–57, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of insulin-like growth factor 1 binding protein 3 levels by epidermal growth factor and retinoic acid in cervical epithelial cells

Insulin-like growth factors (IGFs) are important regulators of epithelial cell growth. The mitogenic activity of these factors is influenced by the levels of extracellular IGF binding proteins, including insulin-like growth factor binding protein 3 (IGFBP-3). In the present report we study the effects of epidermal growth factor (EGF) and all-trans-retinoic acid (RA) on IGFBP-3 RNA and protein levels in human papillomavirus-immortalized cervical epithelial cells. Treatment of ECE16-1 cells with 3–20 ng/ml EGF causes a marked reduction in IGFBP-3 levels. In contrast, 1 μM RA increases IGFBP-3 mRNA and protein levels in the presence or absence of 20 ng/ml EGF. The response is concentration dependent with a half-maximal increase observed at 1 nM RA. RA is able to reverse the EGF suppression when added simultaneously or 3 days after initiation of EGF treatment. Conversely, when cells are treated with RA, IGFBP-3 levels increase within 24 h and subsequent addition of EGF is without effect. Thus, the RA-dependent increase in IGFBP-3 levels is dominant over the EGF suppression. The increased IGFBP-3 levels are correlated with RA suppression of proliferation. Similar RA effects on IGFBP-3 mRNA levels were observed in other cervical epithelial cell lines (i.e., ECE16-D1, ECE16-D2, and CaSki). These results suggest that RA may act to inhibit cervical cell growth by increasing IGFBP-3 levels and reducing the extracellular concentration of free insulin-like growth factor I (IGFI) and/or, alternatively, IGFBP-3 may inhibit cell growth by direct effects on the cell, independent of IGFI. © 1994 Wiley-Liss, Inc.     

Fibroblast growth factor-2 over-rides insulin-like growth factor-I induced proliferation and cell survival in human neuroblastoma cells

The insulin-like growth factor (IGF) system is a key regulator of cell growth, survival and differentiation, and these functions are co-modulated by other growth factors including fibroblast growth factor-2 (FGF-2). To investigate IGF/FGF interactions in neuronal cells, we employed neuroblastoma cells (SK-N-MC). In serum free conditions proliferation of the SK-N-MC cells was promoted by IGF-I (25 ng/ml), but blunted by FGF-2 (50 ng/ml). IGF-I-induced proliferation was abolished in the presence of FGF-2 even when IGF-I was used at 100 ng/ml. In addition to our previously described FGF-2 induced proteolytic cleavage of IGFBP-2, we found that FGF-2 increased IGFBP-6 levels in conditioned medium (CM) without affecting IGFBP-6 mRNA abundance. Modulation of IGFBP-2 and -6 levels were not significant mechanisms involved in the blockade of IGF-I action since the potent IGF-I analogues [QAYL]IGF-I and des(1-3)IGF-I (minimal IGFBP affinity) were unable to overcome FGF-2 inhibition of cell proliferation. FGF-2 treated cells showed morphological differentiation expressing the TUJ1 neuronal marker while cells treated with IGF-I alone showed no morphological change. When IGF-I was combined with FGF-2, however, cell morphology was indistinguishable from that seen with FGF-2 alone. FGF-2 inhibited proliferation and enhanced differentiation was also associated with a 70% increase in cell death. Although IGF-I alone was potently anti-apoptotic (60% decreased), IGF-I was unable to prevent apoptosis when administrated in combination with FGF-2. Gene-array analysis confirmed FGF-2 activation of the intrinsic and extrinsic apoptotic pathways and blockade of IGF anti-apoptotic signaling. FGF-2, directly and indirectly, overcomes the proliferative and anti-apoptotic activity of IGF-I by complex mechanisms, including enhancement of differentiation and apoptotic pathways, and inhibition of IGF-I induced anti-apoptotic signalling. Modulation of IGF binding protein abundance by FGF-2 does not play a significant role in inhibition of IGF-I induced mitogenesis.     

Estrogen utilization of IGF-1-R and EGF-R to signal in breast cancer cells

As breast cancer cells develop secondary resistance to estrogen deprivation therapy, they increase their utilization of non-genomic signaling pathways. Our prior work demonstrated that estradiol causes an association of ERα with Shc, Src and the IGF-1-R. In cells developing resistance to estrogen deprivation (surrogate for aromatase inhibition) and to the anti-estrogens tamoxifen, 4-OH-tamoxifen, and fulvestrant, an increased association of ERα with c-Src and the EGF-R occurs. At the same time, there is a translocation of ERα out of the nucleus and into the cytoplasm and cell membrane. Blockade of c-Src with the Src kinase inhibitor, PP-2 causes relocation of ERα into the nucleus. While these changes are not identical in response to each anti-estrogen, ERα binding to the EGF-R is increased in response to 4-OH-tamoxifen when compared with tamoxifen. The changes in EGF-R interactions with ERα impart an enhanced sensitivity of tamoxifen-resistant cells to the inhibitory properties of the specific EGF-R tyrosine kinase inhibitor, AG 1478. However, with long term exposure of tamoxifen-resistant cells to AG 1478, the cells begin to re-grow but can now be inhibited by the IGF-R tyrosine kinase inhibitor, AG 1024. These data suggest that the IGF-R system becomes the predominant signaling mechanism as an adaptive response to the EGF-R inhibitor. Taken together, this information suggests that both the EGF-R and IGF-R pathways can mediate ERα signaling.To further examine the effects of fulvestrant on ERα function, we examined the acute effects of fulvestrant, on non-genomic functionality. Fulvestrant enhanced ERα association with the membrane IGF-1-receptor (IGF-1-R). Using siRNA or expression vectors to knock-down or knock-in selective proteins, we further demonstrated that the ERα/IGF-1-R association is Src-dependent. Fulvestrant rapidly induced IGF-1-R and MAPK phosphorylation. The Src inhibitor PP2 and IGF-1-R inhibitor AG1024 greatly blocked fulvestrant-induced ERα/IGF-1-R interaction leading to a further depletion of total cellular ERα induced by fulvestrant and further enhanced fulvestrant-induced cell growth arrest. More dramatic was the translocation of ERα to the plasma membrane in combination with the IGF-1-R as shown by confocal microscopy. Taken in aggregate, these studies suggest that secondary resistance to hormonal therapy results in usage of both IGF-R and EGF-R for non-genomic signaling.     

Expression of mRNAs encoding insulin-like growth factor (IGF) ligands, IGF receptors and IGF binding proteins during follicular growth and atresia in the ovine ovary throughout the oestrous cycle

The components of the insulin-like growth factor (IGF) system appear to be involved in the regulation of ovarian follicular growth and atresia in sheep. However, previous studies have only investigated a select few components of the system. The aim of the present study was to investigate the expression of mRNA encoding all of the components of the sheep IGF system among follicles of varying size and health status throughout the oestrous cycle using sheep-specific ribonucleotide probes and in situ hybridisation. For all IGF components, gene expression was unaffected by stage of oestrous cycle. IGF-I mRNA expression in all classes of follicle was generally low throughout the oestrous cycle, while IGFBP-1 mRNA expression could not be demonstrated at all. In contrast, there was relatively intense follicular expression of mRNAs encoding all remaining IGF system components. For IGF-II, both IGF receptors and IGFBP-2, -3, -4, -5, and -6, gene expression decreased as follicles increased in diameter (P < 0.01). IGF-II, type I IGF-R and IGFBP-2, -3, -4, and -6 mRNA expression significantly decreased as follicles progressed from healthy to atretic status (P < 0.01), whereas gene expression for type II IGF-R and IGFBP-5 was greater in atretic follicles (P < 0.01). This study demonstrates the spatial patterns of follicular gene expression for all of the IGF system components in cycling sheep for the first time. These results further highlight the potential functional role of IGF-II, in contrast to IGF-I, in the autocrine and/or paracrine regulation of follicle growth in sheep.     

IGF-1 receptor contributes to the malignant phenotype in human and canine osteosarcoma

To further define the role of insulin-like growth factor-1 (IGF-1) and its receptor (IGF-1R) in osteosarcoma (OS), human OS cell lines with low (SAOS-2) and high (SAOS-LM2) metastatic potential and three canine OS-derived cell lines were studied. Cell lines were evaluated for: IGF-1R expression; expression of IGF binding proteins (IGFBPs); effect of IGF-1 on tumor cell growth, invasion, expression of urokinase plasminogen activator (uPA), and soluble uPA receptor (suPAR), and; ectopic and orthotopic tumorigenicity of the canine OS cells in athymic mice. All cell lines exhibited steady-state mRNA expression of IGF-1R. The SAOS-2 and SAOS-LM2 cells expressed 9,138 and 10,234 cell-associated binding sites, respectively. Canine OS cells expressed from 1,728 to 3,883 binding sites. Two IGF-1-treated cell lines displayed enhanced proliferation. Two cell lines formed colonies in semisolid media, and IGF-1 increased colony number. Matrigel invasion was enhanced in one cell line following IGF-1 treatment. uPA and suPAR were unchanged in SAOS-2 and SAOS-LM2 cells following IGF-1 treatment, but the highly metastatic OS line SAOS-LM2 expressed five times more suPAR and displayed enhanced invasion compared to the parental, low metastatic SAOS-2. IGFBP-5 was detected in four of five cell lines, and IGFBP-3 was detected in two canine OS cell lines. Two canine OS lines were tumorigenic, and one metastasized spontaneously. In conclusion, OS cells express IGF-1R, which can contribute to their growth and invasion. There is suggestive evidence that increasing receptor number may contribute to in vivo tumorigenesis. Additional studies are needed to determine how IGF-1/IGF-1R interactions contribute to the malignant phenotype of OS.     

Duplication and diversification of the hypoxia-inducible IGFBP-1 gene in zebrafish

Background

Gene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenability to genetic and experimental manipulation and because it possess a large number of duplicated genes.

Methodology/Principal Findings

We report the identification and characterization of two hypoxia-inducible genes in zebrafish that are co-ortholgs of human IGF binding protein-1 (IGFBP-1). IGFBP-1 is a secreted protein that binds to IGF and modulates IGF actions in somatic growth, development, and aging. Like their human and mouse counterparts, in adult zebrafish igfbp-1a and igfbp-1b are exclusively expressed in the liver. During embryogenesis, the two genes are expressed in overlapping spatial domains but with distinct temporal patterns. While zebrafish IGFBP-1a mRNA was easily detected throughout embryogenesis, IGFBP-1b mRNA was detectable only in advanced stages. Hypoxia induces igfbp-1a expression in early embryogenesis, but induces the igfbp-1b expression later in embryogenesis. Both IGFBP-1a and -b are capable of IGF binding, but IGFBP-1b has much lower affinities for IGF-I and -II because of greater dissociation rates. Overexpression of IGFBP-1a and -1b in zebrafish embryos caused significant decreases in growth and developmental rates. When tested in cultured zebrafish embryonic cells, IGFBP-1a and -1b both inhibited IGF-1-induced cell proliferation but the activity of IGFBP-1b was significantly weaker.

Conclusions/Significance

These results indicate subfunction partitioning of the duplicated IGFBP-1 genes at the levels of gene expression, physiological regulation, protein structure, and biological actions. The duplicated IGFBP-1 may provide additional flexibility in fine-tuning IGF signaling activities under hypoxia and other catabolic conditions.     

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.