Abstract
The biological actions of insulin-like growth factor-1 (IGF-1) are critical from embryonic development through adulthood. IGF-1 exerts its actions almost exclusively through the IGF-1 receptor (IGF-1 R), perpetuating a receptor-tyrosine kinase signaling cascade to mediate the many pleiotropic effects of IGF-1, which include cell proliferation, differentiation and survival. Signals activated by the IGF- 1/IGF-1R system include phosphatidylinositol 3’OH-kinase (PI3-kinase) and mitogen-activated protein (MAP) kinase (ERK1/2) pathways, as well as protein kinase C (PKC) in some cells.|Following vascular interventional procedures for symptomatic occlusive arterial disease, symptomatic “restenosis” of the vessel occurs in 25-55% of patients. This arterial lumen renarrowing occurs as a result of vascular smooth muscle cell (VSMC) accumulation in the vessel intima forming a ‘neointima’, a process now believed to be due to an imbalance between cell proliferation and apoptosis. Although several factors have been implicated, IGF-1 may play an especially pivotal role. Indeed, a local IGF-1 /IGF-1 R autocrine/paracrine loop in VSMCs has been recently shown to be required for VSMC growth in vivo. In acromegalics, high levels of IGF-1 are thought to be the main contributor to the accelerated atherosclerosis seen in these patients. Moreover, IGF-1 mRNA, protein and the IGF-1 receptor are elevated in both human and animal models of atherosclerotic and post-angioplasty restenotic vascular lesions. Evidence that c-myc is involved in this process and, that IGF-1 potently inhibits c-myc-induced death, suggests that maintaining VSMC viability during cell injury may have dire consequences for the genesis of occlusive vascular diseases. However, despite our understanding of IGF-1 signals mediating growth, those signals regulating VSMC survival are mostly unknown.|In this study, we examined the pathways of staurosporine (STAU)-induced death and c-/nyc-induced death in human and rat VSMCs, respectively, and the regulation of these apoptotic pathways by IGF-1. Scanning and transmission electron microscopic studies, as well as DNA fragmentation and annexin V assays, showed that morphological and biochemical apoptosis can be induced in human VSMCs (hVSMCs) using STAU (a broad-spectrum protein kinase inhibitor). However, there was a lack of intense ‘blebbing’ in these cells relative to that observed in rat VSMCs (rVSMCs) undergoing myc-induced apoptosis. IGF-1 profoundly inhibited both STAU-induced hVSMC death and c-myc-induced apoptosis of rVSMCs. STAU also produced a 20-fold decrease in PKC-e protein over 24 h, which was partially prevented by IGF-1. Cytosolic and particulate/membrane translocation studies (for PKC activation) showed that IGF-1 activates PKC-e in hVSMCs, but not PKC-a or PKC-8. In fact, pretreatment of hVMSCs with STAU resulted in a slight increase in PKC-e activation over that with IGF-1 alone.|In regards to apoptosis, inhibition of PI3-kinase by wortmannin and LY294002 showed that PI3-kinase (and the downstream survival-related enzyme, Akt/PKB) were required for greater than 60% of the survival effects of IGF-1 in human and rat VSMCs. Moreover, exposure of cells to the MEK inhibitors PD098059 or U0126 showed that IGF-1 also required the MEK-ERK1/2 MAP kinase pathway for preventing STAU- and c-myc-induced human and rat VSMC apoptosis. In addition, IGF-1 produced a strong activation of the PI3-kinase downstream survival enzyme Akt/PKB in hVSMCs. Interestingly, Akt activation peaked at 2 h but returned to near baseline at 6 h using IGF-1 alone, while in the presence of the death stimulus STAU, IGF-1 produced a sustained and greater than 10-fold increase in Akt/PKB activation at 6 h. Sustained Akt/PKB activation for at least 12 h also occurred by IGF-1 during myc-induced apoptosis inhibition. Indeed, IGF-1 sustained the activation of Akt for the duration of apoptosis inhibition. Use of phosphorothioate antisense oligonucleotides, designed to hybridize to PKC-e mRNA, leading to its degradation and thereby specifically inhibiting PKC-e expression, reversed the survival effects of IGF-1 on hVSMCs during STAU-induced apoptosis by about 50%.|Further stress/death pathway studies led to the findings that both STAU and c- Myc, during their respective apoptosis induction programs, induce a strong and sustained activation of the stress kinase family member, JNK/SAPK. This activity was strongly inhibited by IGF-1 at 1 h during either death stimulus, whereas at 2 h inhibition was lost and at 4 and 6 h, the presence of IGF-1 led to a significant augmentation of JNK activity in hVSMCs. Studies on the phosphorylation status of c-Jun, the downstream transcriptional-regulatory target of JNK/SAPK, showed that both apoptosis and activating c-Jun phosphorylations paralleled increases in JNK activity during both apoptotic stimuli. As with JNK, c-Jun phosphorylation at 1 h was only transiently inhibited by IGF-1. Use of selective enzyme inhibitors showed that the transient inhibition of JNK activity and c-Jun phosphorylation (at 1 h) by IGF-1 was dependent on PI3-kinase and ERK1/2 MAP kinase pathways, but not on PKC.|Importantly, studies on MAP kinases revealed that IGF-1 produced a relatively small, but sustained activation of ERK1/2 for up to 2 h in human VSMCs.|To explore the link between the survival abilities of PI3-kinase, Akt/PKB and PKC-e, constitutively active and dominant negative expression vectors for Akt/PKB, PDK1, and PKC-e and PKC-^ were transiently transfected into cells and apoptosis assessed in the absence or presence of IGF-1. These data showed not only that Akt/PKB was important and itself sufficient to inhibit c-myc-induced apoptosis, but also that active PKC-e could partially prevent this form of death. Further, dominant negative PDK1 partially reversed the ability of IGF-1 to prevent myc-induced death. The VSMC survival functions of both PKC-e and PDK1, especially in response to IGF-1, were previously unknown. Taken together, these data suggest that a preferential activation by IGF-1 of all the characterized survival substrates, including Akt/PKB, PKC-e and ERK1/2, occurred when apoptotic stimuli were present. This appears to be highly responsive and benefits the cell by allowing its survival during stress-inducing, death-promoting stimuli. Moreover, multiple pathways previously uncharacterized in human and rat VSMCs, such as ERK1/2, PDK1, Akt and PKC-e, create this environment in response to IGF-1, some in a sustained fashion (ERKs, Akt), while PKC-e does so within about 30 minutes or less. As a consequence of these studies, it is hoped that understanding this signaling tapestry will culminate in a greater diversity of cellular targets for therapeutic intervention. Ultimately, inhibition of gene expression, which is/are regulated by these IGF-1-induced signaling cascades, will likely provide the best targets for disease modification.