Mutations in PTEN occur in 60 -80% of prostate cancers and lead to a constitutive activation of the phosphatidylinositol 3-kinase pathway and a resultant loss of activity of the FOXO family of forkhead transcription factors FKHRL1 and FKHR. To provide insight into the role of PTEN mutations in prostate cancer, we used microarrays to identify genes regulated by FKHRL1 and FKHR in LAPC4 prostate carcinoma cells. These studies revealed that adenoviral overexpression of FKHRL1 and FKHR in the LAPC4 prostate cancer cell line resulted in apoptosis and induced the expression of many genes that affect cellular proliferation or survival. The expression of one of these FOXO-regulated genes, TRAIL, a pro-apoptotic member of the tumor necrosis factor family, was decreased in human metastatic prostate tumors. The altered expression of TRAIL in these tumors correlated directly with decreased PTEN expression and the resultant loss of FKHRL1 and FKHR activity. Analysis of the effects of FOXO proteins on the TRAIL promoter localized the FKHRL1 responsive element of the TRAIL promoter to nucleotides ؊138 to ؊121 and demonstrated that TRAIL is a direct target of FKHRL1. These findings suggest that the decreased activity of FKHRL1 and FKHR in prostate cancers resulting from loss of PTEN leads to a decrease in TRAIL expression that may contribute to increased survival of the tumor cells.The forkhead transcription factors are DNA-binding proteins characterized by the presence of a conserved 110-amino acid winged helix DNA binding domain (1). They play important roles in embryogenesis, tumorigenesis, and maintenance of differentiation status. In Caenorhabditis elegans the forkhead transcription factor DAF-16 is under control of the insulin receptor/PI 3-kinase 1 pathway (2). The human orthologs of DAF-16 include FKHRL1, FKHR, and AFX and belong to the FOXO subfamily of forkhead transcription factors. The PI 3-kinase pathway, via activation of its downstream kinase Akt, phosphorylates each of the FOXO proteins at three different Ser/Thr residues (3). These phosphorylated FOXO proteins interact with 14-3-3 proteins and are subsequently sequestered in the cytoplasm where they are inactive. Inhibition of the PI 3-kinase pathway by PTEN overexpression or pharmacologic means leads to dephosphorylation and nuclear translocation of active FKHRL1, FKHR, and AFX, which in turn leads to cell cycle arrest and apoptosis (4). Conversely, loss of PTEN activity results in increased Akt activity leading to inhibition of FOXO protein activity through their phosphorylation and cytoplasmic sequestration. In addition, the expression of dominant negative FKHRL1 results in the inhibition of apoptosis, demonstrating that FOXO transcriptional activity controls cellular proliferation and apoptosis downstream of PTEN (5).The PTEN gene was initially identified by its frequent loss in glioblastomas (6), but subsequent studies have shown that PTEN is commonly mutated in prostate cancer (7-12). In prostate tumors, the loss of PTEN occurs late in the tumorigenic pro...

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Oncostatin M is a member of the IL-6 family of cytokines that is primarily known for its effects on cell growth. Endothelial cells have an abundance of receptors for oncostatin M, and may be its primary target. We determined if oncostatin M induces a key endothelial cell function, initiation of the inflammatory response. We found that subcutaneous in-

Brown recluse spider (Loxosceles reclusa) venom induces severe dermonecrotic lesions. The mechanism for this is unknown but presents an interesting paradox: necrosis is completely dependent on the victim's neutrophils, yet neutrophils are not activated by the venom. We show Loxosceles venom is a potent, but disjointed, endothelial cell agonist. It weakly induced E-selectin expression, but not intercellular adhesion molecule-i or IL-6 expression, yet significantly stimulated release of IL-8 and large amounts of GM-CSF by 4 h. In contrast, TNF strongly induced all of these, except for GM-CSF. PMN bound to E-selectin on venom-activated endothelial cells, apparently via counterreceptors different from those that bind E-selectin on TNFa-activated monolayers. Notably, PMN bound venom-activated monolayers only at intercellular junctions, did not polarize, and completely failed to migrate beneath the monolayer. Despite this, bound PMN demonstrated increased intracellular Ca2" levels and secreted primary and secondary granule markers. The latter event was suppressed by sulfones used to treat envenomation. We have defined a new endothelial cell agonist, Loxosceles venom, that differentially stimulates the inflammatory response of endothelial cells. This, in turn, leads to a dysregulated PMN response where adhesion and degranulation are completely dissociated from shape change and transmigration. (J. Clin. Invest. 1994.94:631-642.)

Cellular phenotype is determined not only by genetic transcription but also by subsequent translation of mRNA into protein. Extracellular signals trigger intracellular pathways that distinctly activate translation. The 70/85-kDa S6 kinase (pp70(S6k)) is a central enzyme in the signal-dependent control of translation, but its regulation in endothelial cells is largely unknown. Here we show that fluid flow (in the absence of an exogenous mitogen) as well as humoral agonists activate endothelial pp70(S6k). Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), and wortmannin, a phosphatidylinositol 3-kinase inhibitor, blocked flow-induced pp70(S6k) activation; FK-506, a rapamycin analog with minimal mTOR inhibitory activity, and PD-98059, an inhibitor of the flow-sensitive mitogen-activated protein kinase pathway, had no effect. Synthesis of Bcl-3, a protein whose translation is controlled by an mTOR-dependent pathway, was induced by flow and inhibited by rapamycin and wortmannin. Transcriptional blockade did not abolish the flow-induced upregulation of Bcl-3. Fluid forces may therefore modify endothelial phenotype by specifically regulating translation of certain mRNA transcripts into protein.