Activin A stimulates IkappaB-alpha/NFkappaB and RANK expression for osteoclast differentiation,but not AKT survival pathway in osteoclast precursors

Recent studies have reported that activin A enhances osteoclastogenesis in cultures of mouse bone marrow cells stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, the exact mechanisms by which activin A functions during osteoclastogenesis are not clear. RANKL stimulation of RANK/TRAF6 signaling increases nuclear factor-kappaB (NFkappaB) nuclear translocation and activates the Akt/PKB cell survival pathway. Here we report that activin A alone activates IkappaB-alpha, and stimulates nuclear translocation of NFkappaB and receptor activator of nuclear factor-kappaB (RANK) expression for osteoclastogenesis, but not Akt/PKB survival signal transduction including BAD and mammalian target of rapamycin (mTOR) for survival in osteoclast precursors in vitro. Activin A alone failed to activate Akt, BAD, and mTOR by immunoblotting, and it also failed to prevent apoptosis in osteoclast precursors. While activin A activated IkappaB-alpha and induced nuclear translocation of phosphorylated-NFkappaB, and it also enhanced RANK expression in osteoclast precursors. Moreover, activin A enhanced RANKL- and M-CSF-stimulated nuclear translocation of NFkappaB. Our data suggest that activin A enhances osteoclastogenesis treated with RANKL and M-CSF via stimulation of RANK, thereby increasing the RANKL stimulation. Activin A alone activated the NFkappaB pathway, but not survival in osteoclast precursors in vitro, but it is, thus, insufficient as a sole stimulus to osteoclastogenesis.     

Receptor activator of NF-kappaB (RANK) cytoplasmic motif, 369PFQEP373, plays a predominant role in osteoclast survival in part by activating Akt/PKB and its downstream effector AFX/FOXO4

Receptor activator of NF-kappaB ligand (RANKL) plays a crucial role in osteoclast differentiation, function, and survival. RANKL exerts its effect by activating its receptor RANK (receptor activator of NF-kappaB), which recruits various intracellular signaling molecules via specific motifs in its cytoplasmic tail. Previously, we identified three RANK cytoplasmic motifs (Motif 1, 369PFQEP373; Motif 2, 559PVQEET564; and Motif 3, 604PVQEQG609) mediating osteoclast formation and function. Here, we investigated RANK cytoplasmic motifs involved in osteoclast survival. Motif 1, in contrast to its minimal role in osteoclast formation and function, plays a predominant role in promoting osteoclast survival. Moreover, whereas Motif 2 and Motif 3 are highly potent in osteoclast formation and function, they exert a moderate effect on osteoclast survival. We also investigated the role of these motifs in activating Akt/protein kinase B (PKB), which has been implicated in RANKL-induced osteoclast survival. Motif 1, but not Motif 2 or Motif 3, is able to stimulate Akt/PKB activation. Because Akt/PKB has been shown to utilize distinct downstream effectors (glycogen synthase kinase-3beta, FKHR/FOXO1a, BAD, and AFX/FOXO4) to regulate cell survival, we next determined which downstream effector(s) is activated by Akt/PKB to promote osteoclast survival. Our data revealed that RANKL only stimulates AFX/FOXO4 phosphorylation, indicating that AFX/FOXO4 is a key downstream target activated by Akt/PKB to modulate osteoclast survival. Taken together, we conclude that Motif 1 plays a predominant role in mediating osteoclast survival in part by activating Akt/PKB and its downstream effector AFX/FOXO4.     

Novel pro-survival functions of the Kruppel-like transcription factor Egr2 in promotion of macrophage colony-stimulating factor-mediated osteoclast survival downstream of the MEK/ERK pathway

Determining the underlying mechanisms of macrophage colony-stimulating factor (M-CSF)-mediated osteoclast survival may be important in identifying novel approaches for treating excessive bone loss. This study investigates M-CSF-mediated MEK/ERK activation and identifies a downstream effector of this pathway. M-CSF activates MEK/ERK and induces MEK-dependent expression of the immediate early gene Egr2. Inhibition of either MEK1/2 or inhibition of Egr2 increases osteoclast apoptosis. In contrast, wild-type Egr2 or an Egr2 point mutant unable to bind the endogenous repressors Nab1/2 (caEgr2) suppresses basal osteoclast apoptosis and rescues osteoclasts from apoptosis induced by MEK1/2 or Egr2 inhibition. Mechanistically, Egr2 induces pro-survival Blc2 family member Mcl1 while stimulating proteasome-mediated degradation of pro-apoptotic Bim. In addition, Egr2 increased the expression of c-Cbl, the E3 ubiquitin ligase that catalyzes Bim ubiquitination. M-CSF, therefore, promotes osteoclast survival through MEK/ERK-dependent induction of Egr2 to control the Mcl1/Bim ratio, documenting a novel function of Egr2 in promoting survival.     

IL-1alpha stimulation of osteoclast survival through the PI 3-kinase/Akt and ERK pathways.

Osteoclasts, cells that resorb bone, die once fully differentiated. Several factors including interleukin-1 (IL-1) have been shown to regulate the survival of mature osteoclasts. However, information on the mechanism underlying the regulation of osteoclast survival has been limited. In this study, we investigated the mechanism for the IL-1-stimulated survival of osteoclasts. Treatment of purified osteoclasts with IL-1alpha led to activation of the serine-threonine kinases Akt and ERK. Blocking the activation of Akt with LY294002, a specific inhibitor of the Akt up-stream molecule PI 3-kinase, or an with adenoviral vector for a dominant-negative form of Akt prevented the stimulation of osteoclast survival by IL-1alpha. PD98059, a specific inhibitor of the ERK-activating kinase MEK1, also abolished the effects of IL-1alpha on ERK activation and osteoclast survival. IL-1alpha reduced the apoptosis of osteoclasts by reducing caspase 3 activity. The IL-1alpha-mediated suppression of apoptosis was abolished by the PI 3-kinase/Akt or MEK1/ERK pathway inhibitor. These findings implicate the PI 3-kinase/Akt and ERK signaling pathways in the promotion of osteoclast survival by IL-1alpha.     

Overexpression of 4EBP1, p70S6K,Akt1 or Akt2 differentially promotes Coxsackievirus B3-induced apoptosis in HeLa cells

Our previous studies have shown that the inhibition of phosphatidylinositol 3-kinase (PI3K) or mTOR complex 1 can obviously promote the Coxsackievirus B3 (CVB3)-induced apoptosis of HeLa cells by regulating the expression of proapoptotic factors. To further illustrate it, Homo sapiens eIF4E-binding protein 1 (4EBP1), p70S6 kinase (p70S6K), Akt1 and Akt2 were transfected to HeLa cells, respectively. And then, we established the stable transfected cell lines. Next, after CVB3 infection, apoptosis in different groups was determined by flow cytometry; the expressions of Bim, Bax, caspase-9 and caspase-3 were examined by real-time fluorescence quantitative PCR and western blot analysis; the expression of CVB3 mRNA and viral capsid protein VP1 were also analyzed by real-time fluorescence quantitative PCR, western blot analysis and immunofluorescence, respectively. At the meantime, CVB3 replication was observed by transmission electron microscope. We found that CVB3-induced cytopathic effect and apoptosis in transfected groups were more obvious than that in controls. Unexpectedly, apoptosis rate in Akt1 group was higher than others at the early stage after viral infection and decreased with the viral-infected time increasing, which was opposite to other groups. Compared with controls, the expression of CVB3 mRNA was increased at 3, 6, 12 and 24 h postinfection (p. i.) in all groups. At the meantime, VP1 expression in 4EBP1 group was higher than control during the process of infection, while the expressions in the other groups were change dynamically. Moreover, overexpression of 4EBP1 did not affect the mRNA expressions of Bim, Bax, caspase-9 and caspase-3; while protein expressions of Bim and Bax were decreased, the self-cleavages of caspase-9 and caspase-3 were stimulated. Meanwhile, overexpression of p70S6K blocked the CVB3-induced Bim, Bax and caspase-9 expressions but promoted the self-cleavage of caspase-9. In the Akt1 group, it is noteworthy that the expressions of Bim protein were higher than controls at 3 and 6 h p. i. but lower at 24 h p. i., and the expression of Bax protein were higher at 6 and 24 h p. i., while their mRNA expressions were all decreased. Furthermore, overexpression of Akt1 stimulated the procaspase-9 and procaspase-3 expression but blocked their self-cleavages. Overexpression of Akt2, however, had little effect on Bim, Bax and caspase-3, while prevented caspase-9 from self-cleavage at the late stage of CVB3 infection. As stated above, our results demonstrated that overexpression of 4EBP1, p70S6K, Akt1 or Akt2 could promote the CVB3-induced apoptosis in diverse degree via different mediating ways in viral replication and proapoptotic factors in BcL-2 and caspase families. As 4EBP1, p70S6K and Akt are the important substrates of PI3K and mammalian target of rapamycin (mTOR), we further illustrated the role of PI3K/Akt/mTOR signaling pathway in the process of CVB3-induced apoptosis.     

AG490, a Jak2-specific inhibitor, induces osteoclast survival by activating the Akt and ERK signaling pathways

Osteoclasts are multinucleated cells with the unique ability to resorb bone. Elevated activity of these cells under pathologic conditions leads to the progression of bone erosion that occurs in osteoporosis, periodontal disease, and rheumatoid arthritis. Thus, the regulation of osteoclast apoptosis is important for bone homeostasis. In this study, we examined the effects of the Janus tyrosine kinase 2 specific inhibitor AG490 on osteoclast apoptosis. We found that AG490 greatly inhibited osteoclast apoptosis. AG490 stimulated the phosphorylation of Akt and ERK. Adenovirus-mediated expression of dominant negative (DN)-Akt and DN-Ras in osteoclasts inhibited the survival of osteoclasts despite the presence of AG490. Cytochrome c release during osteoclast apoptosis was inhibited by AG490 treatment, but this effect was inhibited in the presence of LY294002 or U0126. AG490 suppressed the proapoptotic proteins Bad and Bim, which was inhibited in osteoclasts infected with DN-Akt and DN-Ras adenovirus. In addition, constitutively active MEK and myristoylated-Akt adenovirus suppressed the cleavage of pro-caspase-9 and -3 and inhibited osteoclast apoptosis induced by etoposide. Taken together, our results suggest that AG490 inhibited cytochrome c release into the cytosol at least partly by inhibiting the pro-apoptotic proteins Bad and Bim, which in turn suppressed caspase-9 and -3 activation, thereby inhibiting osteoclast apoptosis.     

PTEN regulates RANKL- and osteopontin-stimulated signal transduction during osteoclast differentiation and cell motility

PTEN (also known as MMAC-1 or TEP-1) is a frequently mutated tumor suppressor gene in human cancer. PTEN functions have been identified in the regulation of cell survival, growth, adhesion, migration, and invasiveness. Here, we characterize the diverse signaling networks modulated by PTEN in osteoclast precursors stimulated by RANKL and osteopontin (OPN). RANKL dose-dependently stimulated transient activation of Akt before activation of PTEN, consistent with a role for PTEN in decreasing Akt activity. PTEN overexpression blocked RANKL-activated Akt stimulated survival and osteopontin-stimulated cell migration while a dominant-negative PTEN increased the actions of RANKL and OPN. PTEN overexpression suppressed RANKL-mediated osteoclast differentiation and OPN-stimulated cell migration. The PTEN dominant-negative constitutively induced osteoclast differentiation and cell migration. Our data demonstrate multiple roles for PTEN in RANKL-induced osteoclast differentiation and OPN-stimulated cell migration in RAW 264.7 osteoclast precursors.     

MCP-1 is induced by receptor activator of nuclear factor-{kappa}B ligand, promotes human osteoclast fusion, and rescues granulocyte macrophage colony-stimulating factor suppression of osteoclast formation

Human osteoclast formation from monocyte precursors under the action of receptor activator of nuclear factor-kappaB ligand (RANKL) was suppressed by granulocyte macrophage colony-stimulating factor (GM-CSF), with down-regulation of critical osteoclast-related nuclear factors. GM-CSF in the presence of RANKL and macrophage colony-stimulating factor resulted in mononuclear cells that were negative for tartrate-resistant acid phosphatase (TRAP) and negative for bone resorption. CD1a, a dendritic cell marker, was expressed in GM-CSF, RANKL, and macrophage colony-stimulating factor-treated cells and absent in osteoclasts. Microarray showed that the CC chemokine, monocyte chemotactic protein 1 (MCP-1), was profoundly repressed by GM-CSF. Addition of MCP-1 reversed GM-CSF suppression of osteoclast formation, recovering the bone resorption phenotype. MCP-1 and chemokine RANTES (regulated on activation normal T cell expressed and secreted) permitted formation of TRAP-positive multinuclear cells in the absence of RANKL. However, these cells were negative for bone resorption. In the presence of RANKL, MCP-1 significantly increased the number of TRAP-positive multinuclear bone-resorbing osteoclasts (p = 0.008). When RANKL signaling through NFATc1 was blocked with cyclosporin A, both MCP-1 and RANTES expression was down-regulated. Furthermore, addition of MCP-1 and RANTES reversed the effects of cyclosporin A and recovered the TRAP-positive multinuclear cell phenotype. Our model suggests that RANKL-induced chemokines are involved in osteoclast differentiation at the stage of multinucleation of osteoclast precursors and provides a rationale for increased osteoclast activity in inflammatory conditions where chemokines are abundant.     

Phosphatidylinositol 3-kinase coordinately activates the MEK/ERK and AKT/NFkappaB pathways to maintain osteoclast survival

We have examined highly purified osteoclasts that were generated in vitro from murine co-culture of marrow precursors with stromal support cells and have found evidence of activation of the MEK/ERK and AKT/NFkappaB survival pathways. Many mature marrow-derived osteoclasts survived for at least 48 h in culture whether or not they are maintained with stromal cells. Moreover, supplementing purified osteoclasts with RANKL and/or M-CSF had no impact on their survival pattern. In addition, spleen-derived osteoclasts generated with RANKL and M-CSF treatment exhibited a similar survival pattern. Blocking MEK, AKT, or NFkappaB activity resulted in apoptosis of many, but not all, of the osteoclasts in purified marrow-derived osteoclasts, marrow-derived osteoclasts co-cultured with stromal cells, and spleen-derived osteoclasts maintained with RANKL and M-CSF. These data support that both the MEK/ERK and AKT/NFkappaB pathways contribute to osteoclast survival. Since PI3K has been shown to activate either of these pathways, we have examined its role in osteoclast survival. PI3K inhibition caused apoptosis of nearly all osteoclasts in purified and co-cultured marrow-derived osteoclasts and spleen-derived osteoclasts maintained with RANKL and M-CSF. Interestingly, in marrow-derived co-cultures, the apoptotic response was restricted to osteoclasts as there was no evidence of stromal support cell apoptosis. PI3K inhibition also blocked MEK1/2, ERK1/2, and AKT phosphorylation and NFkappaB activation in purified osteoclasts. Simultaneous blockage of both AKT and MEK1/2 caused rapid apoptosis of nearly all osteoclasts, mimicking the response to PI3K inhibition. These data reveal that PI3K coordinately activates two distinct survival pathways that are both important in osteoclast survival.     

Downregulation of Bim, a proapoptotic relative of Bcl-2, is a pivotal step in cytokine-initiated survival signaling in murine hematopoietic progenitors

Two distinct signaling pathways regulate the survival of interleukin-3 (IL-3)-dependent hematopoietic progenitors. One originates from the membrane-proximal portion of the cytoplasmic domain of the IL-3 receptor (betac chain), which is shared by IL-3 and granulocyte-macrophage colony-stimulating factor and is involved in the regulation of Bcl-x(L) through activation of STAT5. The other pathway emanates from the distal region of the betac chain and overlaps with downstream signals from constitutively active Ras proteins. Although the latter pathway is indispensable for cell survival, its downstream targets remain largely undefined. Here we show that the expression of Bim, a member of the BH3-only subfamily of cell death activators, is downregulated by IL-3 signaling through either of two major Ras pathways: Raf/mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/mammalian target of rapamycin. Akt/phosphokinase B does not appear to play a significant role in this regulatory cascade. Bim downregulation has important implications for cell survival, since enforced expression of this death activator at levels equivalent to those induced by cytokine withdrawal led to apoptosis even in the presence of IL-3. We conclude that Bim is a pivotal molecule in cytokine regulation of hematopoietic cell survival.     

Vascular endothelial growth factor receptors in osteoclast differentiation and function

Osteoclasts are derived from haematopoietic stem cell precursors of the monocyte/macrophage cell lineage, through interaction with factors that are believed to include M-CSF and RANKL. VEGF is a proangiogenic cytokine that has been shown to promote osteoclast differentiation and survival. In this study, we assessed the role of VEGF and its receptors in osteoclastogenesis, in vitro, by culturing osteoclast precursors in the presence of VEGF, VEGF receptor-specific ligands, and blocking antibodies to VEGF receptors. Activation of VEGFR1 in the presence of RANKL induces osteoclast differentiation. Stimulating the receptors individually induced increased resorption by osteoclasts compared to controls but not to the level observed when stimulating both receptors simultaneously. We have shown that VEGF induces osteoclast differentiation through its action on VEGFR1. The way in which VEGF mediates its effect on mature osteoclast activity, however, may be through its interaction with both receptor subtypes.     

Ubiquitous calpains promote both apoptosis and survival signals in response to different cell death stimuli

The mu- and m-calpain proteases have been implicated in both pro- or anti-apoptotic functions. Here we compared cell death responses and apoptotic or survival signaling pathways in primary mouse embryonic fibroblasts (MEFs) derived from wild type or capn4 knock-out mice which lack both mu- and m-calpain activities. Capn4(-/-) MEFs displayed resistance to puromycin, camptothecin, etoposide, hydrogen peroxide, ultraviolet light, and serum starvation, which was consistent with pro-apoptotic roles for calpain. In contrast, capn4(-/-) MEFs were more susceptible to staurosporine (STS) and tumor necrosis factor alpha-induced cell death, which provided evidence for anti-apoptotic signaling roles for calpain. Bax activation, release of cytochrome c, and activation of caspase-9 and caspase-3 all correlated with the observed cell death responses of wild type or capn4(-/-) MEFs to the various challenges, suggesting that calpain might play distinct roles in transducing different death signals to the mitochondria. There was no evidence that calpain cleaved Bcl-2 family member proteins that regulate mitochondrial membrane permeability including Bcl-2, Bcl-xl, Bad, Bak, Bid, or Bim. However, activation of the phosphatidylinositol 3 (PI3)-kinase/Akt survival signaling pathway was compromised in capn4(-/-) MEFs under all challenges regardless of the cell death outcome, and blocking Akt activation using the PI3-kinase inhibitor LY294002 abolished the protective effect of calpain to STS challenge. We conclude that the anti-apoptotic function of calpain in tumor necrosis factor alpha- and STS-challenged cells relates to a novel role in activating the PI3-kinase/Akt survival pathway.     

PI3K/Akt/mTOR信号通路及临床相关肿瘤抑制剂

哺乳动物雷帕霉素靶（mTOR）和蛋白激酶B（Akt/PKB）与肿瘤发生的密切关系已被广泛地认可.mTOR是一种丝/苏氨酸激酶,可以通过影响mRNA转录、代谢、自噬等方式调控细胞的生长.它既是PI3K的效应分子,也可以是PI3K的反馈调控因子.mTORC1 和mTORC2是mTOR的两种不同复合物. 对雷帕霉素敏感的mTORC1受到营养、生长因子、能量和应激4种因素的影响.生长因子通过PI3K/Akt信号通路调控mTORC1是最具特征性调节路径.而mTORC2最为人熟知的是作为Akt473磷酸化位点的上游激酶. 同样,Akt/PKB在细胞增殖分化、迁移生长过程中发挥着重要作用. 随着Thr308和Ser473两个位点激活,Akt/PKB也得以全面活化.因此,mTORC2-Akt-mTORC1的信号通路在肿瘤形成和生长中是可以存在的.目前临床肿瘤治疗中,PI3K/Akt/mTOR是重要的靶向治疗信号通路.然而,仅抑制mTORC1活性,不是所有的肿瘤都能得到预期控制.雷帕霉素虽然能抑制mTORC1,但也能反馈性地增加PI3K信号活跃度,从而影响治疗预后.近来发现的第二代抑制剂可以同时抑制mTORC1/2和PI3K活性,这种抑制剂被认为在肿瘤治疗上颇具前景.本综述着重阐述了PI3K/Akt/mTOR信号通路的传导、各因子之间的相互调控以及相关抑制剂的发展.     

Co-culture with endothelial progenitor cells promotes survival,migration, and differentiation of osteoclast precursors

In this study, we report the effect of endothelial progenitor cells (EPCs) on the biological behavior of osteoclast precursors in vitro by establishing an indirect co-culture system of mice EPCs and RAW 264.7 monocyte cells. Results show that the survival, migration, and differentiation of osteoclast precursors were greatly enhanced when co-cultured with EPCs. These phenotypic changes coincide with the upregulation of multiple genes affected cell behavior, including phospho-VEGFR-2, CXCR4, phospho-Smad2/3, phospho-Akt, phospho-ERK1, and phospho-p38 MAPK. The results collectively suggest that EPCs could modulate the survival, migration, and differentiation potential of osteoclast precursors, thus providing new insights in understanding of correlation between angiogenesis and bone homeostasis.     

Hyaluronan oligosaccharides inhibit anchorage-independent growth of tumor cells by suppressing the phosphoinositide 3-kinase/Akt cell survival pathway

Hyaluronan oligosaccharides (molecular weight: approximately 2.5 x 10(3)) inhibit growth of several types of tumors in vivo. In vitro, the oligomers inhibit anchorage-independent growth of several tumor cell types. In accordance with this finding, the oligomers also induce apoptosis and stimulate caspase-3 activity under anchorage-independent conditions. Since inhibitors of phosphoinositide 3-kinase (PI 3-kinase) mimic the action of hyaluronan oligomers and since the PI 3-kinase/Akt (protein kinase B) cell survival pathway has previously been implicated in anchorage-independent growth of tumor cells, we examined the effect of oligomers on PI 3-kinase and its downstream activities in TA3/St murine mammary carcinoma and HCT 116 human colon carcinoma cells. We observed that 50-150 microg/ml hyaluronan oligomers inhibit PI 3-kinase activity and phosphorylation of Akt to approximately the same extent as optimal doses of wortmannin and LY294002, known inhibitors of PI 3-kinase. Similar inhibition of downstream events, i.e. phosphorylation of BAD and FKHR, was also observed. These effects were not observed on treatment with similar concentrations of chitin oligomers, chondroitin sulfate, or hyaluronan polymer. High molecular weight (approximately 2 x 10(6)) and low molecular weight (approximately 8 x 10(4)) preparations of hyaluronan polymer were equally ineffective. The effects of hyaluronan oligomers on these parameters were similar in magnitude to the effect of treatment with activity-blocking antibody against CD44. We interpret these results to indicate that the oligomers competitively block binding of endogenous hyaluronan polymer to CD44, consequently giving rise to attenuated signaling. Finally, we observed that hyaluronan oligomers, but not chitin oligomers, chondroitin sulfate, or hyaluronan polymer, stimulate expression of PTEN, a phosphatase that degrades the major signaling product of PI 3-kinase action, phosphoinositide 3,4,5-trisphosphate. We conclude that perturbation of hyaluronan-CD44 binding leads to suppression of the PI 3-kinase/Akt cell survival pathway and consequently to inhibition of anchorage-independent growth in culture and tumor growth in vivo.     

Regulation of TRAIL expression by the phosphatidylinositol 3-kinase/Akt/GSK-3 pathway in human colon cancer cells

The intestinal mucosa is a rapidly-renewing tissue characterized by cell proliferation, differentiation, and eventual apoptosis with progression up the vertical gut axis. The inhibition of phosphatidylinositol (PI) 3-kinase by specific chemical inhibitors or overexpression of the lipid phosphatase PTEN enhances enterocyte-like differentiation in human colon cancer cell models of intestinal differentiation. In this report, we examined the role of PI 3-kinase inhibition in the regulation of apoptotic gene expression in human colon cancer cell lines HT29, HCT-116, and Caco-2. Inhibition of PI 3-kinase with the chemical inhibitor wortmannin increased TNF-related apoptosis-inducing ligand (TRAIL; Apo2) mRNA and protein expression. Similarly, overexpression of the tumor suppressor protein PTEN, an antagonist of PI 3-kinase signaling, resulted in the increased expression of TRAIL. Activation of PI 3-kinase by pretreatment with IGF-1, a gut trophic factor, markedly attenuated the induction of TRAIL by wortmannin. Moreover, overexpression of active Akt, a downstream target of PI 3-kinase, or inhibition of GSK-3, a downstream target of active Akt, completely blocked the induction of TRAIL by wortmannin. Consistent with findings that TRAIL is induced by agents that enhance intestinal cell differentiation, TRAIL expression was specifically localized to the differentiated cells of the colon and small bowel. Adenovirus-mediated overexpression of TRAIL increased DNA fragmentation of HCT-116 cells, demonstrating the functional activity of TRAIL induction. Taken together, our findings demonstrate induction of the TRAIL by inhibition of PI 3-kinase in colon cancer cell lines. These results identify TRAIL, a novel TNF family member, as a downstream target of the PI 3-kinase/Akt/GSK-3 pathway and may have important implications for better understanding the role of the PI 3-kinase pathway in intestinal cell homeostasis.