Kwang Seok Kim and Min-Sun Kim contributed equally to this work.
Regulation of replicative senescence by insulin-like growth factor-binding protein 3 in human umbilical vein endothelial cells
Article first published online: 6 JUN 2007
Volume 6, Issue 4, pages 535–545, August 2007
How to Cite
Kim, K. S., Kim, M.-S., Seu, Y. B., Chung, H. Y., Kim, J. H. and Kim, J.-R. (2007), Regulation of replicative senescence by insulin-like growth factor-binding protein 3 in human umbilical vein endothelial cells. Aging Cell, 6: 535–545. doi: 10.1111/j.1474-9726.2007.00315.x
- Issue published online: 6 JUN 2007
- Article first published online: 6 JUN 2007
- Accepted for publication 11 April 2007
- calorie restriction;
Insulin/insulin-like growth factor (IGF) signaling pathways are among the most conserved processes in aging in organisms ranging from yeast to mammals. Previously, using cDNA microarray technology, we reported that expression of IGF-binding protein 3 (IGFBP3), one of the IGF-binding proteins, was increased with age in human dermal fibroblasts. In this study, the role of IGFBP3 on cellular senescence was studied in human umbilical vein endothelial cells (HUVEC). The expression levels of IGFBP3 mRNA and protein were increased in HUVECs with age. Knockdown of IGFBP3 in old cells with IGFBP3 short hairpin RNA (shRNA) retrovirus resulted in the partial reduction of a variety of senescent phenotypes, such as changes in cell morphology, and decreases in population doubling times and senescence-associated β-galactosidase (SA-β-gal) staining. Down-regulation of IGFBP3 rescued the growth arrest induced by p53 overexpression in young HUVECs. In contrast, up-regulation of IGFBP3 in young cells and prolonged IGFBP3 treatment accelerated cellular senescence, confirmed by cell proliferation and SA-β-gal staining. The FOXO3a (forkhead box O3a) protein level was increased in old IGFBP3 shRNA cells. The treatment of young HUVECs with IGFBP3 repressed the levels of FOXO3a protein. Furthermore, calorie restriction reduced IGFBP3 protein levels, which were found to be increased with age in the rat liver and serum. These results suggest that IGFBP3 might play an important role in the cellular senescence of HUVECs as well as in vivo aging.
Normal somatic cells when cultured in vitro have a limited ability to divide and then enter a state of irreversible proliferative arrest, termed ‘replicative senescence’ (Hayflick & Moorhead, 1961). This process has been proposed to be a typical model for biological aging (Cristofalo et al., 2004) as well as a safeguard against cancer development by suppressing the transformation to immortal cells (Smith & Pereira-Smith, 1996). Like other normal diploid cells, human umbilical vein endothelial cells (HUVEC) enter a state of irreversible growth arrest (Garfinkel et al., 1994; Wagner et al., 2001) following a finite number of cell divisions in vitro. Senescent cells were known to promote aging phenotypes or age-related pathology through not only accumulation of nondividing senescence cells but also secretion of long-range, pleiotropic factors such as degradative enzymes, growth factors and inflammatory cytokines, thereby altering the tissue microenvironment (Campisi, 1998, 2005).
Insulin/insulin-like growth factor (IGF) signaling pathways are major factors that influence lifespan (Hekimi et al., 1998). IGFs act as progression factors, stimulating cell progression from G1 to S phase in the cell cycle in many cell types (Holzenberger et al., 2003). It has been shown that IGF-1 extends the in vitro replicative lifespan of satellite cells by modulating cell cycle regulatory molecules (Florini et al., 1996; Chakravarthy et al., 2000). IGF-1 exerts its pleiotropic effects by binding itself to the type I IGF receptor (IGF-IR) (Chakravarthy et al., 2000), a process that leads to the activation of both the phosphoinositide-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways.
IGF-binding protein 3 (IGFBP3), the most abundant IGFBP in human serum, is synthesized and secreted mainly by hepatic Kupffer cells. IGFBP3 binds over 90% of circulating IGF, resulting in a prolonged half-life of IGF (Baxter & Martin, 1989). IGFBP3 is also produced locally by a variety of normal and tumor cells, suggesting that the cellular microenvironment may directly affect the action of IGF-1 (Clemmons, 1997). IGFBP3 is known to inhibit cell proliferation by interfering with the interaction of IGF-1 and its receptor (Kelley et al., 1996). IGFBP3 can also modulate cell growth and survival independently of IGF action (Firth & Baxter, 2002), presumably via interactions with intracellular proteins such as humanin, a protein known as the Alzheimer's survival protein (Ikonen et al., 2003), importin-β (Schedlich et al., 2000), and nuclear targets such as retinoid X receptor α (Liu et al., 2000). IGFBP3 accumulates in senescent cells and is associated with cell cycle arrest (Yoon et al., 2004; Hampel et al., 2005). A high level of IGFBP3 expression is associated with cellular senescence (Baege et al., 2004). However, the role of IGFBP3 in cellular senescence was not fully understood in primary HUVECs.
The interaction of IGF-1 and its receptor phosphorylates Akt through intracellular signal transduction, and the activated Akt subsequently phosphorylates FOXO (forkhead box O) transcription factors. The phosphorylated FOXO leads to nuclear exclusion and cannot be involved in forkhead transcriptional regulation. FOXO transcription factors have been implicated in regulation of diverse cellular functions including differentiation, metabolism, proliferation and survival (Accili & Arden, 2004; Skurk et al., 2004). The FOXO subfamily of forkhead transcription factors, consisting of three members, FOXO1 (FKHR), FOXO3a (forkhead box O3a; FKHRL-1) and FOXO4 (AFX), are downstream targets of Akt (Brunet et al., 1999). In human endothelial cells, along with cellular senescence, Akt activity is increased; this inhibits FOXO transcription factor activity and increases cellular arrest via the p53/p21 pathway (Miyauchi et al., 2004). Additionally, down-regulation of FOXO3a was known to accelerate cellular senescence in human dermal fibroblasts (HDF) (Kim et al., 2005).
In this study, we investigated the role of IGFBP3 in the regulation of senescence of HUVECs and the relationship between IGFBP3 and FOXO3a activity in HUVECs.
Differential expression of IGFBP3 in young and old endothelial cells
Senescent cells are known to be resistant to mitogen-induced proliferation, express senescence-associated β-galactosidase (SA-β-gal), and have a characteristically enlarged and flattened morphology. In our study, old HUVECs [passage 13, population doubling (PD) > 44] showed senescent phenotypes that distinguished themselves from the early passage cells. To investigate whether IGFs and IGFBP3 are associated with the cellular senescence of HUVECs, we examined the levels of IGFs and IGFBP3 expression in young and old (senescent) HUVECs by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The expression levels of IGFBP3 mRNA and protein were higher in the old cells than in the young cells (Fig. 1A).
Reversal of cellular senescence by IGFBP3 knockdown in senescent HUVECs
To investigate the role of IGFBP3 in cellular senescence, the levels of IGFBP3 mRNA and protein in old cells were down-regulated by gene silencing using IGFBP3 short hairpin RNA (shRNA) retrovirus (Fig. 1B). Repression of IGFBP3 levels in senescent cells showed morphological changes of old cells to resemble young cells (Fig. 1C). SA-β-gal staining and PD time were also decreased in IGFBP3 shRNA cells compared to control cells (Fig. 1D,E). The cell population in G1 phase of the cell cycle was decreased, and the cell population in G2/M and S phases was increased in IGFBP3 shRNA cells compared to old and control cells (Fig. 1F). Additionally, the expression levels of p53 and p21 proteins were also reduced in IGFBP3 shRNA cells (Fig. 1G). To evaluate the effect of IGFBP3 knockdown on reversal of senescence phenotypes, old cells were transduced with p53 shRNA retrovirus as a positive control and senescence phenotypes in p53 shRNA cells were compared to those in IGFBP3 shRNA cells. The percentages of SA-β-gal staining were lower and PD time was shorter in p53 shRNA cells than in IGFBP3 shRNA cells, suggesting that cellular senescence might be reversed less effectively by IGFBP3 knockdown than by p53 knockdown (Fig. 1D,E). As we showed that the FOXO3a protein level was decreased in old HDFs and down-regulation of FOXO3a accelerates cellular senescence in HDFs (Kim et al., 2005), we measured the FOXO3a levels in IGFBP3 shRNA cells. As expected, we found that the FOXO3a level was increased in IGFBP3 shRNA cells (Fig. 1G). However, the FOXO3a level was decreased in p53 shRNA cells (Fig. 1G). These results clearly showed that down-regulation of IGFBP3 in old cells partially reversed senescence phenotypes.
Rescue of p53-induced growth arrest by IGFBP3 knockdown
As the p53 tumor suppressor was known to be the common major effector of the cellular senescence in normal somatic cells (Ferbeyre et al., 2002; Langley et al., 2002; Miyauchi et al., 2004), we tested whether down-regulation of IGFBP3 also rescued the growth arrest induced by p53 overexpression. Following transduction of recombinant p53 adenovirus (Ad-p53) to young HUVECs, IGFBP3 shRNA retrovirus was transduced and then cell proliferation was measured. IGFBP3 knockdown in Ad-p53 cells caused decreases in p53 and p21 protein levels (Fig. 2A). Furthermore, cell proliferation was increased by reduction of IGFBP3 in Ad-p53 cells (Fig. 2B). These results suggested that the p53-dependent pathway might play an important role in IGFBP3-induced cellular senescence in HUVECs.
Acceleration of cellular senescence by IGFBP3 overexpression in young HUVECs
As down-regulation of IGFBP3 levels partially reversed cellular senescence, we examined the effects of IGFBP3 overexpression on senescence in young HUVECs by transduction of IGFBP3 lentivirus. IGFBP3 overexpression was confirmed by Western blotting and resulted in a decrease in the FOXO3a protein level and an increase in the p53 level (Fig. 3A). Up-regulation of IGFBP3 caused a decrease in cell proliferation and an increase in SA-β-gal staining (Fig. 3B,C). To determine whether IGFBP3-induced senescence resulted from the activity of intracellular or extracellular IGFBP3, IGFBP3 up-regulated cells were treated with an IGFBP3 neutralizing antibody (200 ng mL−1) or recombinant human IGF-1 protein (50 ng mL−1) for 4 days. A decrease in cell proliferation by IGFBP3 up-regulation was not recovered by the addition of an IGFBP3 neutralizing antibody or IGF-1 (Fig. 3D), suggesting that IGFBP3-induced senescence resulted from the intracellular action of IGFBP3. Taken together, these results suggest that IGFBP3 might play an important role in the replicative senescence of HUVECs.
Effect of exogenous recombinant human IGFBP3 on cellular senescence in young HUVECs
To further confirm the role of IGFBP3 in cellular senescence, we treated cells with recombinant human IGFBP3 protein (rhIGFBP3) and measured cell proliferation by cell counting. Unlike up-regulation of IGFBP3, we found that IGFBP3 treatment for a few days (3 or 4 days) had no effects on cell proliferation. However, when cells were treated with 100 or 200 ng mL−1 of rhIGFBP3 every 3 days, the cells proliferated slowly during the first 12 days of treatment and showed a significant decrease in cell proliferation around Day 18 (Fig. 4B). Prolonged IGFBP3 treatment also induced up-regulation of p53 and p21 levels (Fig. 4A) and increased SA-β-gal staining (Fig. 4C). As exogenous IGFBP3 can be internalized into cells (Li et al., 1997), prolonged IGFBP3 treatment might result in the accumulation of IGFBP3 in cells and contribute to induction of cellular senescence.
Down-regulation of FOXO3a levels by exogenous IGFBP3
Because FOXO3a levels were increased in IGFBP3 shRNA cells and decreased by IGFBP3 up-regulation, we tested whether exogenous rhIGFBP3 could reduce FOXO3a protein levels in HUVECs. The protein level of FOXO3a in cells treated with rhIGFBP3 was decreased in time- and dose-dependent manners (Fig. 5).
Effects of LY294002 or an IGF-1 receptor antibody on the down-regulation of FOXO3a levels induced by IGFBP3
As IGFBP3 regulates IGF functions by binding to IGFs and because the insulin/IGF signaling pathway was known to regulate members of the FOXO family (Browner et al., 2004), we tested whether the down-regulation of FOXO3a induced by IGFBP3 is mediated through the insulin/IGF signaling pathways. The expression levels of FOXO3a protein in young cells treated with IGFBP3 were measured after pretreatment with LY294002, a PI3-kinase inhibitor, or with an antibody against IGF-1 receptor α (IGF-1Rα) during starvation. IGFBP3-induced FOXO3a down-regulation was not inhibited by pretreatment with LY294002 (Fig. 6A). To validate the activity of LY294002 or IGFBP3, IGF-1 or IGFBP3-induced Akt phosphorylation was examined. As expected, pretreatment with LY294002 inhibited Akt phospholylation induced by IGF-1 or IGFBP3 (Fig. 6A). Additionally, blocking of the binding of IGF-1 to its receptor by an IGF-1Rα antibody had no effect on the down-regulation of FOXO3a induced by IGFBP3 (Fig. 6B). To further characterize the down-regulation of FOXO3a by IGFBP3, cells were transfected with three small interfering RNAs (siRNA) against IGF-1R and the effect of IGFBP3 on the FOXO3a levels was measured. The expression level of FOXO3a was also decreased by IGFBP3 under knockdown of IGF-1Rα/β (Fig. 6C). These results suggest that IGFBP3 might regulate the expression levels of FOXO3a through the IGF-independent pathways.
Repression of IGFBP3 in the livers or serum of old rats by calorie restriction
In an attempt to elucidate the role of IGFBP3 in in vivo aging, we measured the IGFBP3 levels in relation to age in rat liver homogenates by Western blot analysis, as most systemic IGFBP3 is produced in the liver. IGFBP3 levels gradually increased in rat livers as rats aged (Fig. 7A,B). The relative level of IGFBP3 protein in 24-month-old rats was increased about threefold compared to that of 6-month-old counterparts. Caloric restriction was known to be the most consistently effective treatment used to increase mammalian lifespan. Therefore, we measured IGFBP3 levels in the livers of rats that had been under calorie restriction. IGFBP3 levels in the calorie-restricted diet group were slightly increased in the livers of 12-month-old rats, and then gradually decreased through 24 months of age, to levels similar to those of 6-month-old rats. We also measured the IGFBP3 levels in serum of rats with age and found that serum IGFBP3 levels were increased with age. Serum IGFBP3 levels in the calorie-restricted diet group were not increased with age (Fig. 7C,D). These results suggest that IGFBP3 might play an important role in in vivo aging.
Cellular senescence, the limited ability of primary human cells to divide when cultured in vitro, can be used as a model of biological aging. Like any other normal diploid cells, HUVECs have a limited capacity to divide. IGF signaling pathways are one of the major factors influencing lifespan. IGFs regulate cell metabolism, growth, differentiation and survival (Katic & Kahn, 2005). They bind to transmembrane receptors, IGF-1R and IGF-2R. The cellular function of the IGFs is mediated by their receptors. IGF-1 exerts a process that leads to the activation of both the PI3K and MAPK pathways. IGFBPs modulate IGF functions by binding with IGFs. Recent evidence has shown that IGFBPs have dual, IGF-independent functions, including cell survival processes related to cell surface receptors and regulation of transcriptional processes involved in the cell death response and growth inhibition (Rosenzweig, 2004).
The levels of IGFBP3 expression were increased in old HUVECs in vitro (Fig. 1A) and in aged rat livers and serum in vivo (Fig. 7). We confirmed that the down-regulation of IGFBP3 expression in old HUVECs reduces a variety of senescence phenotypes, including morphologic changes of old cells to resemble younger cells, decreases in PD times and SA-β-gal staining, and increases in FOXO3a levels (Fig. 1). In addition, we observed that up-regulation of IGFBP3 levels in young cells decreased cell proliferation and increased SA-β-gal staining (Fig. 3). Prolonged IGFBP3 treatment also decreased cell proliferation and increased p53 and p21 protein levels (Fig. 4). Our results clearly showed that IGFBP3 plays an important role in cellular senescence. Large amounts of IGFBP3 in old cells might block or reduce IGF-1 action through IGF receptors, resulting in attenuation of the mitogenic effect of IGF-1 (Goldstein et al., 1991; Grigoriev et al., 1995). Therefore, the overexpression of IGFBP3 might induce senescence via a concerted antiproliferative effect by interrupting the IGF pathway. Increased IGFBP3 expression may also be associated with increased apoptosis in an IGF-independent manner in cancer cells (Rajah et al., 1997; Hong et al., 2002). While HDFs are more resistant to apoptosis in senescent cells, the senescent HUVECs are prone to apoptotic cell death (Hampel et al., 2004). Resistance to apoptosis in senescent human fibroblasts with high levels of IGFBP3 has been reported to be correlated with the absence of nuclear IGFBP3 (Hampel et al., 2005). Up-regulation of IGFBP3 in senescent fibroblasts has been reported due to an IGFBP3 enhancer element in the 5’ untranslated region of the IGFBP3 gene, which differentially activates IGFBP3 expression in senescent vs. young fibroblasts (Lu et al., 2005). However, further research should be conducted to elucidate the mechanisms by which IGFBP3 expression increases in senescent HUVECs and influences apoptotic cell death.
The p53 pathway functions as a central integration point for various signaling pathways that mediate the response by telomere dysfunction, DNA damage and oxidative stress (Ben-Porath & Weinberg, 2005). The response triggers replicative senescence by activation of p53 in normal cells (Wahl & Carr, 2001). Several reports suggest that the senescence of normal cells can be reversed solely by inactivation of p53 (Itahana et al., 2001; Beausejour et al., 2003; Dirac & Bernards, 2003). We showed that down-regulation of IGFBP3 in HUVECs could reverse the growth arrest induced by overexpression of p53 (Fig. 2), as well as senescence phenotypes caused by replicative senescence (Fig. 1). IGFBP3 was known to be induced by p53 (Buckbinder et al., 1995; Grimberg et al., 2005) and our data also confirmed that IGFBP3 is a direct target of p53 (Fig. 3A), where transduction with p53 adenovirus results in up-regulation of IGFBP3. These results suggested that an increase in IGFBP3 levels in old cells might be attributed to up-regulation of p53 levels during senescence. Interestingly, we found that IGFBP3 shRNA cells showed down-regulation of p53 levels (Fig. 1G) and up-regulation of IGFBP3 and prolonged IGFBP3 treatment enhanced p53 levels, implicating that p53 might be also a target of IGFBP3. Our results suggested that IGFBP3 might play a critical role in modulating senescence through activation of p53. However, further study should be needed to elucidate mechanisms by which IGFBP3 regulates the cellular senescence through the p53 dependent pathway.
We have previously reported that down-regulation of FOXO3a accelerates cellular senescence in human dermal fibroblasts, resulting in phenotypic changes such as increased cell size, generation of reactive oxygen species and expression of the senescence marker, SA-β-gal (Gan et al., 2005; Kim et al., 2005). FOXO proteins are known to be regulated by signal-induced, post-translational modifications. A variety of growth factor signals, such as insulin and IGFs, are known to down-regulate FOXO proteins by nuclear export and proteosomal degradation of FOXO proteins, which is mediated via Akt-dependent FOXO phosphorylation in the nucleus. In contrast, stress signals induce nuclear import and FOXO transcriptional activity, which is mediated via c-Jun N-terminal kinase (JNK)-dependent phosphorylation of cytoplasmic FOXO proteins, and acetylation and deacetylation of nuclear FOXO proteins (Vogt et al., 2005). Given that the binding of IGFBP3 to IGF-1 inhibits the FOXO3a phosphorylation via the PI-3K/Akt signaling pathway, FOXO3a levels should be up-regulated by treatment with rhIGFBP3 and down-regulated by the knockdown of IGFBP3. However, our results showed that knockdown of IGFBP3 in old cells caused an increase in FOXO3a level (Fig. 1G), and treatment of young cells with rhIGFBP3 induced decreases in FOXO3a mRNA and protein levels (Fig. 5). Furthermore, pretreatment with LY294002 or anti-IGF-1R antibody and IGF-1R knockdown did not increase FOXO3a levels, which were down-regulated by rhIGFBP3 treatment (Fig. 6). Taken together, these results suggest that the regulation of FOXO3a levels by IGFBP3 might be mediated via the IGF-independent pathway.
The insulin/IGF signaling pathway is well conserved and is one of the most consistent processes of aging performed in organisms ranging from yeast to mammals (Longo & Finch, 2003; Katic & Kahn, 2005). Calorie restriction is the most reproducible way to extend the lifespan of mammals, and it remains the most robust among all possible aging interventions evaluated to date (Ingram et al., 2006). While the relative level of IGFBP3 protein in 24-month-old rats was threefold higher than those in 6-month-old counterparts, the IGFBP3 level in calorie-restricted 24-month-old rats was similar to that in 6-month-old rats. The level of IGFBP3 protein in serum was also increased in rats with age. However, serum IGFBP3 protein levels in calorie-restricted rats were similar regardless of age. These results suggested that IGFBP3 might play a role also in in vivo aging.
HUVECs and endothelial cell basal medium-2 (EBM-2) containing several growth factors and supplements were purchased from Cambrex Bio Science Inc. (Walkersville, MD, USA). The oligonucleotides for PCR primers of IGFBP3 (forward, gacgccgtgctactcgtt; reverse, cggttcatacccgaggtg) were obtained from Bioneer Inc. (Daejeon, South Korea). The pSM2 retroviral vector encoding IGFBP3 shRNA for knockdown of IGFBP3 was purchased from Open Biosystems Inc. (Huntsville, AL, USA). The pRetroSuper-p53sh vector was kindly gifted from Dr R. Agami (Division of Tumor Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands). The recombinant p53 adenovirus was gladly provided from Dr D. Y. Shin (Dankook University, Cheonan, South Korea). The pcDNA3 vector encoding a full sequence of IGFBP3 gene (pcDNA3/IGFBP3) was kindly donated by Dr Y. M. Oh (Virginia Commonwealth University, Richmond, VA, USA). A rabbit polyclonal antibody against glyceraldehydes 3-phosphate dehydrogenase (GAPDH) was kindly donated by Dr K. S. Kwon (KRIBB, Daejeon, South Korea). Antibodies against FOXO3a, phospho-FOXO3a (pFOXO3a), Akt and phospho-Akt were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). An antibody against human IGF-1R and siRNAs for human IGF-1R were purchased from Santa Cruz Biotech Inc. (Santa Cruz, CA, USA). Nonglycosylated rhIGFBP3 and IGF-I and antibody against IGFBP3 were obtained from R&D System Inc. (Minneapolis, MN, USA). The pLenti6/V5 directional TOPO cloning kit was from Invitrogen Inc. (Carlsbad, CA, USA).
HUVECs in EGM-2 were plated at 2 × 105 cells per 100-mm culture plate and cultured at 37 °C in 5% carbon dioxide (CO2) humidified air. When subcultures reached 80–90% confluence, serial passaging was performed by trypsinization, and the number of PDs was monitored for further experiments. For the experiments, cells were used in either passage 6 (PD < 24) or passage 13 (PD > 44). These are referred to as ‘young’ and ‘old’ cells, respectively. PD was calculated using the geometric equation: PD = log2F/log2I (F, final population number; I, initial population number).
Rat maintenance procedures for specific-pathogen free (SPF) status and dietary composition of chow have been previously reported (Yu et al., 1985). In brief, for the calorie-restricted group, 344 male, SPF Fischer rats were fed a diet of the following composition: 21% soybean protein, 15% sucrose, 43.65% dextrin, 10% corn oil, 0.15% a-methionine, 0.2% choline chloride, 5% salt mix, 2% vitamin mix and 3% Solka-Floc. The ad libitum (AL)-fed group had free access to both food and water. Rats at 6, 12, 18 and 24 months of age were killed by decapitation and the livers were quickly removed and rinsed in ice-cold buffer [100 mm Tris, 1 mm EDTA, 0.2 mm phenylmethylsulfonyl fluoride (PMSF), 1 µm pepstatin, 2 µm leupeptin, 80 mg L−1 trypsin inhibitor and 10 µm N-CBZ-LEU-LEU-LEU-AL pH 7.4]. The tissue was immediately frozen in liquid nitrogen and stored at –80 °C. One gram of liver was homogenized with 2 mL of 50 mm phosphate buffer containing 1 mm EDTA, 0.5 mm PMSF, and 1 µm pepstatin, which was then centrifuged at 900 g at 4 °C for 15 min. The supernatants were centrifuged at 12 000 g at 4 °C for 15 min to yield the mitochondrial fraction and postmitochondrial supernatant fraction. Protein concentration in the supernatants was quantified by the same method as that of cell lysates. To obtain serum samples, rats were decapitated and blood was drawn and allowed to clot at room temperature for 30 min before being centrifuged at 1500 g at 4 °C for 20 min. The supernatant was collected as serum, frozen and stored at –80 °C.
Cell treatment and protein extractions
HUVECs (2 × 105 cells) were seeded in 60 mm dishes and incubated for 24 h in EGM-2. Following overnight serum starvation, cells were treated with 20 µm LY294002 or 100 ng mL−1 of an IGF-1Rα antibody for 1 h prior to treatment with rhIGFBP3 (100 ng mL−1) for 0, 15, 30, 60 or 120 min. For knockdown of IGF-1R, cells were transfected with three siRNAs against IGF-1R with Lipofectamine 2000 (Life Technologies, Inc., Gaithersburg, MD, USA) and incubated for 3 days. The IGF-1R siRNA cells were seeded in 60-mm dishes and treated with rhIGFBP3 (100 ng mL−1) for 60 min. Cells were washed with ice-cold phosphate-buffered saline (PBS), collected by scraping with a rubber policeman, and lysed in 50 µL of ice-cold radioimmunoprecipitation assay (RIPA) buffer [25 mm Tris–HCl pH 7.4, 150 mm KCl, 5 mm EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mm Na3VO4, 5 mm NaF and 1 mm PMSF]. Cell disruption was achieved by vortexing repeatedly for 30-s intervals on ice. The particulate debris was removed by centrifugation at 12 000 g for 10 min. Protein concentration in the supernatants was quantified by the bicinchoninic acid (BCA) method (Pierce Biotechnology Inc., Rockford, IL, USA) using bovine serum albumin as a standard, and the volumes of the supernatants were adjusted for equal protein concentration.
Western blot analysis
Proteins (30 µg) were separated on 10% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. The membranes were incubated overnight at 4 °C with one of the specific antibodies (FOXO3a, pFOXO3a, p-Akt, Akt and IGFBP3). After washing three times in Tween–Tris buffer saline (TTBS), horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit (1 : 5000) antibodies were applied. The proteins were visualized using enhanced chemiluminescence (KPL Inc., Gaithersburg, MD, USA). Some membranes were stripped with a stripping buffer (2% SDS, 100 mmβ-mercaptoethanol and 50 mm Tris–HCl pH 7.0) at 55 °C for 20 min with gentle shaking. The membranes were then reprobed with a GAPDH antibody as a control for protein loading.
Reverse transcription-polymerase chain reaction
Total RNA was extracted from young and old HUVECs using TRI reagent (Molecular Research Center Inc., Cincinnati, OH, USA). RNA was reverse transcribed using 2.5 µm oligo dT primers, 1 mm dNTPs and reverse transcriptase (Promega, Madison, WI, USA), and resulting cDNAs were amplified by a PCR system (Kim et al., 2003). Primers to GAPDH were used to standardize the amounts of RNA in each sample. PCR products were resolved on a 1.5% agarose gel and visualized by ethidium bromide staining.
Preparation of IGFBP3 or p53 shRNA retrovirus and transduction with IGFBP3 or p53 shRNA retrovirus
IGFBP3 or p53 shRNA retrovirus was prepared by transfection of an IGFBP3 or a p53 shRNA retroviral vector into BOSC 23 cells (Pear et al., 1993). After incubation for 3 days, media were collected and centrifuged at 1650 g for 10 min. The viral solution was filtered with 0.45 µm filter membrane and stored at –20 °C. For knockdown of IGFBP3 or p53, old HUVECs (2 × 105) were plated in 60-mm culture plates and incubated overnight. The cells were transduced with IGFBP3 or p53 shRNA retrovirus or empty retrovirus as a control. Following 72-h incubation, IGFBP3 or p53 levels, cell proliferation and SA-β-gal activity were measured.
Preparation of IGFBP3 lentivirus and transduction with IGFBP3 lentivirus
IGFBP3 cDNA was amplified from pcDNA3/IGFBP3 by PCR and cloned into a pLenti6/V5-D-TOPO vector. Nucleotide sequences of IGFBP3 were confirmed by dideoxy sequencing. IGFBP3 lentivirus was prepared according to manufacturer's suggestions. After cells were transduced with IGFBP3 lentivirus or empty lentivirus as a control, IGFBP3 levels and cell proliferation were measured.
Transduction with recombinant p53 adenovirus
Young cells (2 × 105) were plated in 60-mm culture plates and incubated overnight. Cells were transduced with the p53 adenovirus in 2 mL culture media. After 8-h incubation, culture media were exchanged and incubated for the additional 24 h, p53-transduced cells were transfected with IGFBP3 shRNA retrovirus with Lipofectamine (Life Technologies, Inc.). After overnight incubation, fresh culture media were exchanged and the transfected cells were cultured in a CO2 incubator for 2 days.
3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay
Cell proliferation was measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Kim et al., 2005). Cells were seeded on 96-well plates at a density of 3 × 103 cells per well. After treatment, cells were incubated with 1 mg mL−1 MTT solution for 2 h. The medium was aspirated and the formazan product was solubilized with 100 µL dimethyl sulfoxide. Viability was assessed by measuring absorbance at 570 nm with a Bio-Rad microplate reader (Hercules, CA, USA).
SA-β-gal activity assay
SA-β-gal activity for the proportion of senescent HUVECs was measured as described previously (Dimri et al., 1995) with minor modifications. Cells were plated at 1 × 104 in 35-mm culture dishes and cells in subconfluent cultures were washed with PBS and fixed with 3% (v/v) formaldehyde in PBS for 5 min at room temperature. The presence of SA-β-gal activity was determined by incubating cells with a staining solution containing 1 mg mL−1 5-bromo-4-chloro-3-indolyl-β-D-galactoside, 40 mm citric acid-sodium phosphate (pH 6.0), 5 mm potassium ferricyanide, 5 mm potassium ferrocyanide, 150 mm NaCl and 2 mm MgCl2 for 18 h at 37 °C. The percentage of blue cells per 100 cells observed under a light microscope was calculated.
Cell cycle analysis by flow cytometry
Cells were harvested, washed twice with PBS and fixed with 70% ethanol at –20 °C for 1 h. Following the washing of cells with PBS containing 2% FBS and 0.01% CaCl2, RNase (1% w/v) was added and incubated at 37 °C for 30 min. Propidium iodide (50 µg mL−1) was added and cells were incubated for 20 min. The intracellular propidium iodide fluorescence intensity of each population of 10 000 cells was measured in each sample using a Becton-Dickinson FACS Caliber flow cytometer, and the cell cycle was analyzed by CellQuest software (Becton-Dickinson, San Jose, CA, USA).
This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the Aging-associated Vascular Disease Research Center at Yeungnam University [R13-2005-005-01003-0 (2006)] and by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, South Korea (02-PJ10-PG6-AG01-0003). The authors thank the Aging Tissue Bank of Pusan National University and the National Institute of Aging (AG 01188) for supplying rat tissues.
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