Insulin-like Growth Factor (IGF)-I Regulates IGF-binding Protein-5 Gene Expression through the Phosphatidylinositol 3-Kinase, Protein Kinase B/Akt, and p70 S6 Kinase Signaling Pathway*

Expression of the insulin-like growth factor-binding protein 5 (IGFBP-5) gene in vascular smooth muscle cells is up-regulated by IGF-I through an IGF-I receptor-mediated mechanism. In this study, we studied the possible involvement of the mitogen-activated protein kinase (MAPK) and PI 3-kinase signaling pathways in mediating IGF-I-regulated IGFBP-5 gene expression. The addition of Des(1–3)IGF-I, an IGF analog with reduced affinity to IGFBPs, resulted in a transient activation of p44 and p42 MAPK. Inhibition of the MAPK activation by PD98059, however, did not affect IGF-I-stimulated IGFBP-5 expression. Des(1–3)IGF-I treatment also strongly activated PI 3-kinase. This activation was probably mediated through IRS-1, because IGF-I stimulation resulted in a significant increase in IRS-1- but not IRS-2-associated PI 3-kinase activity. This activation occurred within 5 min and was sustained at high levels for over 6 h. Likewise, Des(1–3)IGF-I caused a long lasting activation of PKB/Akt and p70s6k. When LY294002 and wortmannin, two specific inhibitors of PI 3-kinase, were added with Des(1–3)IGF-I, the IGF-I-regulated IGFBP-5 expression was negated. The addition of rapamycin, which inhibits IGF-I-induced p70s6k activation, significantly inhibited IGF-I-regulated IGFBP-5 gene expression. These results suggest that the action of IGF-I on IGFBP-5 gene expression requires the activation of the PI 3-kinase-PKB/Akt-p70s6k pathway but not the MAPK pathway in vascular smooth muscle cells.

Abnormal vascular smooth muscle cell (VSMC) 1 proliferation and directed migration from the media into the intima play major roles in the pathogenesis of atherosclerotic lesions, the formation of restenosis after angioplasty, and the accelerated arteriopathy after cardiac transplantation (1). These cellular events are regulated by a number of peptide growth factors including insulin-like growth factor-I (IGF-I). Studies have shown that IGF-I is a mitogen and strong chemoattractant for cultured VSMCs (2)(3)(4). The growth and migration-promoting actions of IGF-I are mediated through the IGF-IR, a tyrosine kinase receptor that is linked to the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathways (3). The bioactivity of IGF-I is further modulated by several high affinity binding proteins (IGFBPs) present in the local vasculature. These IGFBPs control the distribution of IGF-I between extracellular environment and cell surface binding sites and can alter IGF bioactivity by modulating its interaction with the receptor (4). Previous studies have shown that VSMCs secrete several IGFBPs, including IGFBP-5 (5)(6)(7)(8)(9)(10)(11). In cultured porcine VSMCs, expression of the IGFBP-5 gene is up-regulated by IGF-I (11). The increased IGFBP-5 protein, in turn, modulates the IGF-I action by a feedback mechanism in these cells (12).
The regulatory effect of IGF-I on IGFBP-5 gene expression in VSMCs is mediated through the IGF-IR (11). However, how the activation of the IGF-IR leads to the elevation of IGFBP-5 gene expression is not well understood. In this study, we investigated the effect of IGF-I on MAPK and PI 3-kinase activation and the role of these signaling pathways in mediating IGF-Istimulated IGFBP-5 gene expression in cultured VSMCs. IGF-I exposure resulted in the activation of both MAPK and PI 3-kinase signaling cascades in these cells. When the activation of PI 3-kinase or p70 s6k was suppressed using specific inhibitors, the IGF-I-regulated IGFBP-5 gene expression was negated. The addition of PD98059, which inhibits IGF-I-induced MAPK activation, had no such effect. These results suggest that the action of IGF-I on IGFBP-5 gene expression requires the activation of the PI 3-kinase-PKB/Akt-p70 s6k pathway but not the MAPK pathway in VSMCs.

EXPERIMENTAL PROCEDURES
Materials-All chemicals and reagents were purchased from Sigma unless noted otherwise. PI 3-kinase and 4G10 anti-phosphotyrosine antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The IRS-1 and IRS-2 antibodies were a gift from Dr. Ildiko Szantos-Denes (Harvard Medical School, Boston). Phosphospecific (activated) and control antibodies for MAPK, PKB/Akt, and p70 s6k as well as mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitor PD98059 were purchased from New England Biolabs (Beverly, MA). Horseradish peroxidase-linked anti-rabbit antibody, rainbow molecular weight markers, [ 32 P]ATP, and [ 32 P]dCTP were from NEN Life Science Products. Human IGF-I and Des(1-3)IGF-I were obtained from GroPep (Adelaide, Australia). LY294002 was purchased from BIOMOL (Plymouth Meeting, PA). Fetal bovine serum, Dulbecco's minimum essential medium with high glucose, and penicillin-streptomycin were purchased from Life Technologies, Inc. Trypsin was obtained from Roche Molecular Biochemicals.
Cell Culture-Porcine VSMCs were isolated from thoracic aorta of newborn piglets (11). The cells were grown in 10-cm dishes (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) in Dulbecco's minimum essential medium supplemented with 4 mM glutamine, penicillin (100 units/ml), streptomycin (100 g/ml), and 10% fetal bovine serum. The medium was changed every fourth day until the cells became confluent. Prior to stimulation experiments, medium was changed to serum-free Dulbecco's minimum essential medium for 18 -24 h. This serum-free Dulbecco's minimum essential medium was then replaced with serum-free Dulbecco's minimum essential medium plus indicated growth factors for various times.
Western Immunoblotting Analysis-For Western immunoblotting analysis, cells were lysed with lysis buffer (50 mM Tris, 0.2 M NaCl, 0.5% IGEPAL CA-630, 0.1% bovine serum albumin, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml aprotinin). Equal amounts of lysates were separated by 12.5% SDS-polyacrylamide gel electrophoresis and transferred to Immobilon P membrane (0.45-m pore size, Millipore, Bedford, MA). The membranes were blocked in 3% bovine serum albumin (Fisher) in Tris-buffered saline-Tween 20 (TBST). For anti-PKB/Akt and p70 s6k blotting, membranes were incubated with a 1:1000 dilution of the primary antibodies in TBST-bovine serum albumin buffer and then washed three times with TBST. All other blots were incubated with a 1:5000 dilution of the indicated antibody in blocking buffer for 2 h at room temperature. Blots were then washed with TBST and incubated with a 1:3000 dilution of horseradish peroxidase-linked anti-rabbit or 1:8000 dilution anti-mouse secondary antibodies in blocking buffer for 2-3 h, followed by further washing. Enhanced chemiluminescence was performed according to the manufacturer's instructions (Amersham Pharmacia Biotech). Densitometry was performed by scanning the autoradiographs (ScanJet IIcx; Hewlett-Packard) and then analyzed using Scion Image software (Frederick, MD).
PI 3-Kinase Assay-After growth factor treatment, cell cultures were washed, lysed (1% IGEPAL, 137 mM NaCl, I mM MgCl 2 , 150 M vanadate, 1 g/ml leupeptin, 1 g/ml aprotinin), and incubated with primary antibody overnight followed by further incubation with protein A-Sepharose for 2 h. After three washes, the PI 3-kinase assay was performed as described previously (13). Briefly, samples were resuspended in 30 l of PI 3-kinase buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 0.5 mM EGTA), and 20 g of phosphatidylinositol were added. After 5 min at room temperature, 10 Ci of [ 32 P]ATP was added. After 10 min at room temperature, the reaction was stopped with 80 l of 1 M HCl, and lipids were extracted with 160 l of MeOH/chloroform (1:1). Samples were spotted on 1% potassium oxalate-treated TLC plates (Analtech, Newark, DE) and developed in CHCl 3 /MeOH/H 2 O/NH 4 OH (64:47: 11.3:2). The highest migrating spots on the TLC plate, representing phosphatidylinositol phosphate, were quantitated as described above.
RNA Isolation and Northern Blot Analysis-Total RNA was isolated from cell cultures using TriReagent following the manufacturer's instructions (Molecular Research Center, Inc., Cincinnati, OH) and was quantified by measuring UV absorption at OD 260 nm. RNA samples were size-fractionated on a 1% agarose gel, blotted, and fixed onto a Hybond-N membrane (Amersham Pharmacia Biotech). They were hybridized with [ 32 P]dCTP-labeled porcine IGFBP-5 cDNAs (14). cDNA probes for rat glyceraldehyde-3-phosphate dehydrogenase and bovine IGFBP-2 were used to determine that the same amounts of RNA were loaded. Densitometry was performed as described above.
Statistical Analysis-Values are means Ϯ S.E. Differences among groups were analyzed by one-way analysis of variance followed by Fisher's protected least significance difference test using Statview (Abacus Concepts, Inc., Berkeley, CA).

Des(1-3)IGF-I Stimulation Results in a Transient
Activation of the MAPK, but Inhibition of MAPK Does Not Affect IGF-Idependent IGFBP-5 Gene Expression-To determine the signaling mechanism(s) that IGF-I utilizes to regulate IGFBP-5 gene expression, the effect of IGF-I on MAPK activation was investigated using a phosphospecific MAPK antibody that only detects the doubly phosphorylated (at Thr 202 and Tyr 204 residues) ERK1 and ERK2. Activation of MAPK occurs through phosphorylation of Thr 202 and Tyr 204 by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (15). Since it is known that IGFBP-5 can modulate IGF-I activities in VSMCs (12), Des(1-3)IGF-I, an IGF-I analog that binds to IGF-IR with normal affinity but has significantly reduced affinity for IGFBPs (16), was used to activate the IGF-IR. In cultured porcine VSMCs, Des(1-3)IGF-I induced a concentration-and time-dependent increase in the phosphorylation/acti-vation of ERK1 and ERK2 (Fig. 1). ERK1 and ERK2 phosphorylation was observed at 5 min, and the maximum phosphorylation occurred within 10 min (Fig. 1A). This effect disappeared rapidly thereafter and returned to the basal levels within 30 min. The minimum effective concentration of Des(1-3)IGF-I was 5 ng/ml. This is followed by a concentration-dependent increase, with the maximum phosphorylation response seen at approximately 20 ng/ml of Des(1-3)IGF-I (Fig.  1B). PD98059, which noncompetitively blocks the activation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase by Raf-1 without affecting other known serine/threonine and tyrosine kinases (17), was used to block the activation of MAPK by Des(1-3)IGF-I. Pretreatment with PD98059 at 20 M for 2 h inhibited the Des(1-3)IGF-I-stimulated ERK1 and ERK2 phosphorylation by approximately 90% ( Fig. 2A). This inhibition was specific, because LY294002, wortmannin, and rapamycin had no such effect. Inhibition of MAPK activation by PD98059 at the concentrations of 20 -40 M had little effect in changing IGF-I-stimulated IGFBP-5 gene expression (Fig. 2, B and C). PD98059 at the same concentrations, however, significantly inhibited IGF-I-induced VSMC migration (data not shown). These results indicate that MAPK activation is not required for IGF-I-induced IGFBP-5 gene expression in porcine VSMCs.

Des(1-3)IGF-I Stimulation Results in a Sustained Activation of PI 3-Kinase and PKB/Akt, and Inhibition of PI 3-Kinase
Activation Abolishes IGF-I-dependent IGFBP-5 Gene Expression-Since activation of the MAPK signaling cascade is not required for IGF-I-dependent IGFBP-5 expression, we next tested whether this action of IGF-I is mediated through the PI 3-kinase signaling pathway. For this, the effect of Des(1-3)IGF-I in activating PI 3-kinase was first examined. Serumstarved or Des(1-3)IGF-I-treated cells were immunoprecipitated using an anti-phosphotyrosine antibody (4G10), and the PI 3-kinase activity was analyzed by an in vitro kinase assay. As shown in Fig. 3, Des(1-3)IGF-I treatment resulted in a significant increase in the phosphotyrosine-associated PI 3-kinase activity. Binding and phosphorylation of a member of the IRS proteins is an early and essential step for the signal trans- duction initiated by the IGF-IR (18,19). IRS proteins then serve as a multiple docking protein by binding to numerous Src homology 2 domain-containing proteins, including the p85 regulatory subunit of PI 3-kinase. To determine whether IRS-1, IRS-2, or both are involved in IGF-I signaling in VSMCs, cell lysates were immunoprecipitated with either an anti-IRS-1 or anti-IRS-2 antibody, and the associated PI 3-kinase activity was determined. Des(1-3)IGF-I resulted in a significant increase of IRS-1-associated PI 3-kinase activity (Fig. 3). In con-trast, there was no change in the IRS-2-associated PI 3-kinase activity after Des(1-3)IGF-I stimulation. These results suggest that IGF-I primarily utilizes IRS-1 to activate PI 3-kinase and its downstream signaling molecules in porcine VSMCs. The Des(1-3)IGF-I-induced increase in IRS-I-associated PI 3-kinase activity was dose-dependent at concentrations ranging from 1 to 50 ng/ml (Fig. 4B). At the maximum concentration (50 ng/ml), Des(1-3)IGF-I induced an over 100-fold increase. This activation occurred within 5 min of Des(1-3)IGF-I stimulation and was sustained at high levels for 6 h (Fig. 4A). The activity began to decline after 6 h but remained significantly higher than the basal level even after 24 h. To further examine the effect of Des(1-3)IGF-I in activating the PI 3-kinase signaling cascade, the phosphorylation of PKB/Akt, a downstream target of PI 3-kinase, was examined using a phosphospecific and control PKB/Akt antibody. Consistent with the PI 3-kinase assay results, the Des(1-3)IGF-I-induced PKB/Akt phosphorylation was observed within 5 min and lasted for several hours (Fig. 5A). Des(1-3)IGF-I induced the phosphorylation of PKB/ Akt in a concentration-dependent manner in concentrations ranging from 1 to 20 ng/ml (Fig. 5B). The maximum phosphorylation response was seen at 20 ng/ml Des(1-3)IGF-I. To determine whether the IGF-I-stimulated IGFBP-5 gene expression is mediated through the PI 3-kinase signaling pathway, two structurally distinct PI 3-kinase inhibitors, wortmannin and LY294002, were used to block PI 3-kinase activation. The effectiveness of these inhibitors was examined by directly monitoring the Des(1-3)IGF-I-induced changes in PI 3-kinase activity as well as the phosphorylation state of PKB/Akt. As shown in Fig. 6A, pretreatment of porcine VSMCs with wortmannin (20 M) completely abolished the Des(1-3)IGF-I-induced PI 3-kinase activation. LY294002 (10 M) also significantly inhibited the activation. PD98059 (40 M) had no such inhibitory effect (Fig. 6A). Likewise, both wortmannin and LY294002 but not PD98059 or rapamycin blocked the IGF-Istimulated PKB/Akt phosphorylation (Fig. 6B). When co-incubated with Des(1-3)IGF-I (100 ng/ml), LY294002 (10 M) completely inhibited the Des(1-3)IGF-I-stimulated IGFBP-5 gene expression (Fig. 7). Similarly, wortmannin at the concentration of 20 M blocked the effect of Des(1-3)IGF-I in up-regulating IGFBP-5 gene expression (Fig. 7). These results indicate that PI 3-kinase activation is required for IGF-I-induced IGFBP-5 gene expression in porcine VSMCs.

Des(1-3)IGF-I Stimulation Results in a Sustained
Activation of p70 s6k , and Inhibition of p70 s6k Inhibits IGF-I-dependent IGFBP-5 Gene Expression-We next tested whether p70 ribosomal protein S6 kinase (p70 s6k ) is required for this action of IGF-I in cultured porcine VSMCs. p70 s6k is a serine/threonine kinase downstream of PKB/Akt in the PI 3-kinase pathway. Activation of p70 s6k necessitates its phosphorylation at Ser 411 , Thr 421 , and Ser 424 residues, which lie within the pseudosubstrate region (20,21). Phosphorylation of these residues is thought to lead to activation of p70 s6k via relief of pseudosubstrate repression. The possible effect of IGF-I in activating p70 s6k in cultured porcine VSMCs was examined by Western immunoblot analysis using antibodies specific for Ser 411 as well as Thr 421 /Ser 424 -phosphorylated p70 s6k , respectively. As shown in Fig. 8, Des(1-3)IGF-I stimulation resulted in a concentration-and time-dependent increase in the phosphorylation of p70 s6k at Ser 411 and Thr 421 /Ser 424 residues. Phosphorylation of p70 s6k was observed at 5 min after IGF-I treatment. The maximum phosphorylation occurred at 10 -30 min (Fig. 8A), and this high level was sustained for 6 h. The phosphorylation levels gradually decreased thereafter. As shown in Fig. 8B, Des(1-3)IGF-I was effective at concentrations as low as 1 ng/ ml. This was followed by a concentration-dependent increase with the maximum phosphorylation response seen at a concentration of 10 -20 ng/ml.
To determine whether p70 s6k is required for IGF-I action in porcine VSMCs, the immunosuppressant rapamycin was used to inhibit p70 s6k activation. p70 s6k phosphorylation induced by Des(1-3)IGF-I (20 ng/ml) was effectively inhibited by 20 ng/ml rapamycin as well as LY294002 and wortmannin (20 M) (Fig.  9). In contrast, PD98059 (40 M) had no such effect. Preincubation of cells with rapamycin (20 ng/ml) inhibited the effect of Des(1-3)IGF-I on IGFBP-5 expression (Fig. 10). These experiments were repeated three times, and an average of 46 Ϯ 11% inhibition was obtained (p Ͻ 0.05). These results indicate that p70 s6k is involved in IGF-I-regulated IGFBP-5 gene expression in VSMCs. To ascertain that the change was not due to any toxic effect of these compounds used, we examined the effects of these compounds on the forskolin-induced IGFBP-5 gene expression. Our previous study indicated that forskolin treatment increases IGFBP-5 gene expression through a cAMPmediated mechanism (14). As shown in Fig. 10C, forskolin (10 M) treatment resulted in a 12-fold increase in IGFBP-5 mRNA abundance. This effect was additive with IGF-I, suggesting that they act through different mechanisms. When LY294002 (20 M), rapamycin (20 ng/ml), or PD98059 (40 M) was added with forskolin, it did not affect the forskolin induction of IGFBP-5 gene expression (Fig. 10C).

DISCUSSION
It has been shown that IGF-I stimulates the expression of IGFBP-5 in VSMCs, and this up-regulation of IGFBP-5 by its own ligand is mediated through the IGF-IR (11). How the activation of the IGF-IR leads to the elevation of IGFBP-5 gene expression is poorly defined. In this study, we present evidence that IGF-I stimulation caused a strong and sustained activation of the PI 3-kinase and its downstream signaling molecules, PKB/Akt and p70 s6k , in primary porcine VSMCs. We found that inhibition of PI 3-kinase activation by LY294002 or wortmannin abolished the IGF-I-stimulated IGFBP-5 gene expression. Likewise, inhibition of p70 s6k also inhibited the IGF-Istimulated IGFBP-5 gene expression. These results suggest that the action of IGF-I on IGFBP-5 gene expression requires the activation of the PI-3-kinase-PKB/Akt and p70 s6k pathway in cultured porcine VSMCs. Studies using "model" systems (i.e. various tumor or transformed cell lines) have revealed that a complex network of signaling events are activated upon ligand occupancy of the IGF-IR (18). Two homologous adapter proteins, IRS-1 and IRS-2, have been identified as major substrates of the activated IGF-IR (19). In this study, we found that IGF-I stimulation resulted in a significant increase in IRS-1-but not IRS-2associated PI 3-kinase activity. These results strongly suggest (but do not definitively prove) that IGF-I activates the PI 3-kinase signaling pathway primarily through IRS-1. IRS-1 has over 20 potential tyrosine phosphorylation sites, which serve as docking sites for numerous Src homology 2 domain-containing proteins, including the p85 subunit of PI 3-kinase (19). Like other cell types, activation of IRS-1 in cultured porcine VSMCs results in the activation of the PI 3-kinase pathways. In these primary cells, Des(1-3)IGF-I is a potent activator of PI 3-kinase. This activation is strong and sustained for more than 6 h. In this study, we have used two potent and specific inhibitors of PI 3-kinase, LY294002 and wortmannin, to determine the possible involvement of PI 3-kinase in mediating the effect of IGF-I in regulating IGFBP-5 gene expression. The use of these two structurally distinct compounds has established the important role of PI 3-kinase in transmitting the antiapoptotic and differentiation signals of IGF-I (22)(23)(24). Recent studies have shown that PI 3-kinase is required for the effect of insulin on IGFBP-1 gene expression in rat hepatocytes using these compounds (25). In this study, we have found that IGF-I stimula- tion of IGFBP-5 gene expression was blocked by LY294002 and wortmannin at concentrations that effectively inhibited IGF-Istimulated PI 3-kinase activity. Thus, activation of PI 3-kinase is required for the action of IGF-I on IGFBP-5 gene expression in VSMCs. This finding is contrary to a recent report indicating that the up-regulation of the IGFBP-5 gene by IGF-I/insulin was rapamycin-sensitive but was not inhibited by LY294002 or wortmannin in mouse C2 myoblast cells (26). In that particular study, however, high concentrations of insulin instead of IGF-I were used for the gene expression experiments. In such a setting, one would expect that the insulin receptor would also be activated in addition to the IGF-IR. Therefore, differences in ligands used, experiment conditions, cell type, or species may account for the different findings.
Treatment of porcine VSMCs with Des(1-3)IGF-I also has a profound and long lasting effect in activating PKB/Akt and p70 s6k in cultured porcine VSMCs. PKB/Akt is a serine-threonine kinase immediately downstream of PI 3-kinase (27). PKB/ Akt has been shown to be a major mediator for IGF-I actions in promoting neuronal survival (22,23). A recent study by Cichy et al. (28) has shown that PKB/Akt mediates the inhibitory effect of insulin on IGFBP-1 gene expression in hepatocytes. More recent studies from the same group have identified FKHR, a human homologue of the forkhead/winged helix transcription factor initially discovered in Caenorhabditis elegans, as the target of the insulin receptor-PKB/Akt signaling pathway (29). Phosphorylation of FKHR by PKB/Akt at serine 256 disrupts the inhibitory effect of insulin on IGFBP-1 gene transcription (29). Acting further downstream in this pathway is p70 s6k . IGF-I-induced cell cycle progression and protein synthesis have been reported to be inhibited by inhibition of p70 s6k (27). However, other cellular responses mediated by PI 3-kinase, including inhibition of apoptosis, activation of glycogen synthesis, and glucose transport are independent of p70 s6k (30,31). In this study, we have found that rapamycin (20 ng/ml) effectively inhibited Des(1-3)IGF-I-induced p70 s6k phosphorylation without affecting MAPK, PI 3-kinase, and PKB/Akt activity or phosphorylation states in porcine VSMCs. Inhibition of p70 s6k activation by rapamycin significantly inhibited the IGF-I-stimulated IGFBP-5 gene expression, suggesting that the action of IGF-I in up-regulating IGFBP-5 gene is mediated through the PI 3-kinase, PKB/Akt, and p70 s6k signaling pathway in porcine VSMCs. The downstream events of the PI 3-kinase-PKB/Akt-p70 s6k pathway that are involved in IGF-I regulation of IGFBP-5 gene expression are not clear at present. p70 s6k phosphorylates and activates several nuclear proteins involved in transcriptional regulation of specific genes (32). One of the nuclear proteins phosphorylated by p70 s6k is the transcription factor cAMP-responsive element modulator (33), which constitutes a target for mitogenic signaling through p70 s6k . It has also been reported that rapamycin treatment leads to a reduction in the phosphorylation of retinoblastoma tumor suppressor (Rb) in human and rat VSMCs (34). The relevance of any of these nuclear proteins to IGFBP-5 gene transcription, however, has yet to be established. It is of interest to note that while inhibition of PI 3-kinase activity by either LY294002 or wortmannin completely abolished IGF-I-induced IGFBP-5 gene expression, rapamycin was only partially effective in inhibiting IGF-I-induced IGFBP-5 expression. Since rapamycin at the concentrations used completely suppressed p70 s6k , it is possible that another p70 s6k -independent signaling pathway(s) is also involved in this action of IGF-I. Recently we have shown that several PKC isoforms, including PKC-␤I, -⑀, -, and -, are expressed in porcine VSMCs and are under the regulation of IGF-I (13). Down-regulation or inhibition of PKC activity by high doses of phorbol 12-myristate 13-acetate or a specific PKC inhibitor (GF109203X) abolished IGF-I-induced IGFBP-5 gene expression in these cells. Several studies indicate that several PKC isoforms, including PKC-and PKC-⑀, act downstream of the PI 3-kinase (35)(36)(37). Therefore, some of these PKC isoforms may be essential signaling intermediates for IGF-I acting downstream of PI 3-kinase in the IGF signaling network in VSMCs (13).
In addition to the PI 3-kinase pathway, we consistently observed a time-and concentration-dependent phosphorylation of ERK1 and -2 induced by Des(1-3)IGF-I treatment in these cells. Although several previous studies reported that IGF-I did not activate MAPK in cultured human and bovine VSMCs (2,38,39), other studies reported an increase in MAPK activity after IGF-I treatment in rat and porcine VSMCs (3, 40 -42). These conflicting data cannot simply be attributed to species differences, because IGF-I is a strong activator of MAPK in many human and bovine cell types (24,43). Our results with porcine VSMCs are not necessarily in contradiction with previous studies using human and bovine VSMCs. Those studies focused on the comparison of PDGF-BB and IGF-I. Indeed, we found that IGF-I is a weaker activator of the MAPK pathway in comparison with PDGF-BB in this cell type; PDGF-BB at a concentration of 5 ng/ml results in a greater response than that of 100 ng/ml IGF-I (data not shown). Likewise, Thommes et al. (40) also reported that IGF-I was only 30% as effective as PDGF in inducing MAPK activation in rat VSMCs. Furthermore, our results indicated that the IGF-I-induced MAPK activation is a transient event, characterized by a rapid rise and decline within minutes and is only observed when high doses of IGF-I are added. These variations may explain the previous negative findings.
Activation of the MAPK signaling pathway by IGF-I has been shown in a variety of cell types (18). The importance of MAPK in cell proliferation and gene expression is generally acknowledged. Several targets of this pathway have been defined, including transcription factors such as Elk-1 or SAP-1 (44). This provides a common route by which signals from various growth factors and hormones converge at a major regulatory element in the promoters of c-fos and other coregulated genes, the serum response element. Several lines of evidence suggested that the up-regulating of the IGFBP-5 gene by IGF-I observed in porcine VSMCs may not act though this pathway. In these primary cells, IGF-I up-regulates IGFBP-5 gene expression in a time frame consistent with that of an intermediate effect rather than an immediate early response. No consensus serum response element is present in the promoter region of the IGFBP-5 gene. In addition, cycloheximide abrogates IGF-I-induced IGFBP-5 gene expression, indicating a requirement for the synthesis of an intermediate protein(s) (11). Furthermore, IGF-I stimulation does not change the c-fos levels in rat VSMCs (40). In this study, we further demonstrated that specific inhibition of the MAPK activation did not significantly decrease the IGF-I-dependent IGFBP-5 gene expression. Since PD98059 at the given concentration almost completely suppressed IGF-I-induced MAPK activation and inhibited IGF-Idirected VSMC migration, we conclude that MAPK activation is not critical for IGF-I-induced IGFBP-5 gene expression.
In addition to the IGFBP-5 gene, IGF-I has been shown to regulate transcription of a number of genes in a variety of cell types, but a consensus IGF-responsive element has yet to be established. In VSMCs, IGF-I has been shown to regulate expression of the elastin gene (45)(46)(47). This regulation has been attributed to several GC-rich sequences in these genes that are capable of binding to IGF-I-regulated nuclear proteins isolated from rat VSMCs (45,48). Two of the proteins have been shown to be transcription factors Sp1 and Sp3 (46,47). The IGFBP-5 promoter contains several GC-rich regions superficially resembling the Sp1 element. In particular, the DNA sequence 5Ј-CCCCACCCCCACCC-3Ј at positions Ϫ147 to Ϫ134 has this potential. Although this highly conserved sequence contains two overlapping AP-2 elements 5Ј-CCCCACCC-3Ј and is capable of binding to AP-2 in vitro, it does not appear to mediate the AP-2 regulation in vivo (14). This region contains sequences identical to the Rb control element 5Ј-CCACCC-3Ј. The Rb control element motif has been identified as a Sp1-binding sequence responsible for Rb-induced trans-activation (49). Previous studies by Jensen et al. (46) suggested that IGF-I may disrupt Sp1 binding to the GC-rich domain of the elastin gene by affecting the phosphorylation state of Rb in rat VSMCs. In preliminary studies, we have found that IGF-I stimulation resulted in Rb phosphorylation in cultured porcine VSMCs. 2 This regulation of Rb phosphorylation state by IGF-I may require p70 s6k activation, because it has been reported that rapamycin treatment leads to a reduction in the phosphorylation of Rb in human and rat VSMCs (34). Further experiments are needed to determine whether this Rb control element motif and/or another cis sequence(s) is responsible for the IGF-Istimulated IGFBP-5 gene expression in VSMCs.