The caspase-8 homologue FLICE-inhibitory protein (FLIP) functions as a caspase-8 dominant negative, blocking apoptosis induced by the oligomerization of the adapter protein FADD/MORT-1. FLIP expression correlates with resistance to apoptosis induced by various members of the tumor necrosis factor family such as TRAIL. Furthermore, forced expression of FLIP renders cells resistant to Fas-mediated apoptosis. Although FLIP expression is regulated primarily by MEK1 activity in activated T cells, the oncogenic signaling pathways that regulate FLIP expression in tumor cells are largely unknown. In this report, we examined the roles of the MAP kinase and phosphatidylinositol (PI) 3-kinase signaling pathways in the regulation of FLIP expression in tumor cells. We observed that the MEK1 inhibitor PD98059 reduced FLIP levels in only 2 of 11 tumor cell lines tested. In contrast, disruption of the PI 3-kinase pathway with the specific inhibitor LY294002 reduced Akt (protein kinase B) phosphorylation and the levels of FLIP protein and mRNA in all cell lines evaluated. The introduction of a dominant negative Akt adenoviral construct also consistently reduced FLIP expression as well as the phosphorylation of the Akt target glycogen synthase kinase-3. In addition, infection of the same cell lines with a constitutively active Akt adenovirus increased FLIP expression and the phosphorylation of GSK-3. These data add FLIP to the growing list of apoptosis inhibitors in which expression or function is regulated by the PI 3-kinase-Akt pathway.The p55 TNF 1 receptor, Fas, LARD (DR3), and the TRAIL receptors DR4 and DR5 are members of the TNF receptor family that contain a unique cytoplasmic sequence termed a death domain (DD). Upon engagement by ligand or antibody cross-linking, these receptors trimerize and recruit an adapter protein called FADD (Fas-associated death domain) (1-3), which interacts through its own DD with those of the receptors or other adapters (e.g. TRADD). The resulting multimolecular complex, referred to as a death-inducing signaling complex or DISC, recruits an inactive procaspase called FLICE or caspase-8, which is then cleaved to an active form, initiating a cascade of proteolytic events resulting in the cleavage of numerous proteins essential for DNA repair and the maintenance of cell viability (4). It has been shown that FADD multimerization is sufficient to kill some cells even in the absence of TNF family ligands or receptor cross-linking (5). For example, the apoptosis induced by certain chemotherapeutic agents is thought to rely on ligand-independent FADD multimerization, since it can be blocked by a dominant negative FADD (5). Collectively, these data suggest that FADD plays a critical role in apoptosis induced by the cross-linking of DD-containing membrane receptors of the TNF receptor family (e.g. Fas) as well as that resulting from exposure to cytotoxic drugs. Many tumors express Fas (6) and TRAIL receptors (7, 8) yet are resistant to apoptosis induced by exposure to Fas ligand, TRAIL, or agonist anti-...

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Abstract-Fas is constitutively expressed on endothelial cells, but in contrast to smooth muscle and other cell types, endothelial cells are highly resistant to Fas-mediated apoptosis. In this study, we examined the role of the serine/threonine kinase Akt/PKB in controlling the sensitivity of endothelial cells to Fas-mediated apoptosis. Serum deprivation inhibited expression of the caspase-8 inhibitor FLICE-inhibitory protein (FLIP), which functions downstream from Fas. FLIP expression levels were restored when serum-depleted cells were treated with vascular endothelial growth factor. Treatment with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin or LY294002 or infection of the adenoviral construct expressing dominant-negative Akt (Adeno-dnAkt) also inhibited the expression of FLIP in endothelial cells, whereas the MEK inhibitor PD98059 had no effect. Conversely, adenovirusmediated transfection of a constitutively-active Akt gene abolished the wortmannin-and LY294002-mediated downregulation of FLIP. Suppression of PI 3-kinase signaling sensitized endothelial cells to Fas-mediated apoptosis.Under conditions of suppressed PI 3-kinase signaling, restoration of FLIP expression reversed the induced sensitivity of endothelial cells to Fas-mediated apoptosis. These data suggest that inhibition of Fas-mediated apoptosis, via promotion of FLIP expression, is a mechanism through which Akt signaling can promote endothelial cell survival.

Abstract-Fas ligand (FasL) is a death factor that induces apoptosis in cells bearing its receptor, Fas. Fas and FasL have been detected in the vessel wall, and it has been proposed that Fas-mediated apoptosis has a role in physiological and pathological cell turnover in the vasculature. Here, we evaluated the expression of Fas in the presence and absence of cytokines on both endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). We also examined the sensitivity of ECs and VSMCs to Fas

Glycogen synthase kinase-3␤ (GSK3␤) plays important roles in metabolism, embryonic development, and tumorigenesis. Here we investigated the role of GSK3␤ signaling in vascular biology by examining its function in endothelial cells (ECs). In EC, the regulatory phosphorylation of GSK3␤ was found to be under the control of phosphoinositide 3-kinase-, MAPK-, and protein kinase A-dependent signaling pathways. The transduction of a nonphosphorylatable constitutively active mutant of GSK␤ promoted apoptosis under the conditions of prolonged serum deprivation or the disruption of cellmatrix attachments. Conversely, the transduction of catalytically inactive GSK3␤ promoted EC survival under the conditions of cellular stress. Under normal cell culture conditions, the activation of GSK3␤ signaling inhibited the migration of EC to vascular endothelial growth factor or basic fibroblast growth factor. Angiogenesis was inhibited by GSK3␤ activation in an in vivo Matrigel plug assay, whereas the inhibition of GSK3␤ signaling enhanced capillary formation. These data suggest that GSK3␤ functions at the nodal point of converging signaling pathways in EC to regulate vessel growth through its control of vascular cell migration and survival.

Interleukin-1 induced a time-dependent release of high levels of nitric oxide from rat vascular smooth muscle cells up to 96 hours. A time-dependent release of lactate dehydrogenase was also induced by Interleukin-1 from 72 to 96 hours after its stimulation. In situ nick end-labeling assay revealed that incubation for 48 hours with interleukin-1 induced a positive staining of fragmented nuclei. However, NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, inhibited both lactate dehydrogenase release and DNA fragmentation induced by interleukin-1. Furthermore, sodium nitroprusside, a nitric oxide donor, also induced lactate dehydrogenase release and DNA fragmentation. Fluorescent staining of DNA revealed patches of irregularly dispersed, brightly staining, and condensed chromatin in rat vascular smooth muscle cells treated with sodium nitroprusside. Flow cytometric analysis with monoclonal antibody against human Fas revealed that expression of Fas was upregulated by sodium nitroprusside in human vascular smooth muscle cells. Methylene blue, an inhibitor of soluble guanylate cyclase, did not affect sodium nitroprusside-induced upregulation of Fas. Furthermore, 8-bromo-guanosine 3':5'-cyclic monophosphate, an analogue of cGMP, did not upregulate Fas expression. These findings indicate that nitric oxide released from vascular smooth muscle cells may induce apoptosis in vascular smooth muscle cells themselves and also induced upregulation of Fas via a cGMP-independent mechanism. Thus, nitric oxide could trigger the remodeling of atherosclerotic plaques.

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