Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway.     

Pseudomonas aeruginosa delays Kupffer cell death via stabilization of the X-chromosome-linked inhibitor of apoptosis protein

Kupffer cells are important for bacterial clearance and cytokine production during infection. We have previously shown that severe infection with Pseudomonas aeruginosa ultimately results in loss of Kupffer cells and hepatic bacterial clearance. This was associated with prolonged hepatic inflammation. However, there is a period of time during which there is both preserved hepatic bacterial clearance and increased circulating TNF-alpha. We hypothesized that early during infection, Kupffer cells are protected against TNF-alpha-induced cell death via activation of survival pathways. KC13-2 cells (a clonal Kupffer cell line) were treated with P. aeruginosa (strain PA103), TNF-alpha, or both. At early time points, TNF-alpha induced caspase-mediated cell death, but PA103 did not. When we combined the two exposures, PA103 protected KC13-2 cells from TNF-alpha-induced cell death. PA103, in the setting of TNF exposure, stabilized the X-chromosome-linked inhibitor of apoptosis protein (XIAP). Stabilization of XIAP can occur via PI3K and Akt. We found that PA103 activated Akt and that pretreatment with the PI3K inhibitor, LY294002, prevented PA103-induced protection against TNF-alpha-induced cell death. The effects of LY294002 included decreased levels of XIAP and increased amounts of cleaved caspase-3. Overexpression of Akt mimicked the effects of PA103 by protecting cells from TNF-alpha-induced cell death and XIAP cleavage. Transfection with a stable, nondegradable XIAP mutant also protected cells against TNF-alpha-induced cell death. These studies demonstrate that P. aeruginosa delays TNF-alpha-induced Kupffer cell death via stabilization of XIAP.     

Epidermal growth factor receptor-dependent,NF-kappaB-independent activation of the phosphatidylinositol 3-kinase/Akt pathway inhibits ultraviolet irradiation-induced caspases-3, -8, and -9 in human keratinocytes

Both phosphatidylinositol 3-kinase (PI3K)/Akt and NF-kappaB pathways function to promote cellular survival following stress. Recent evidence indicates that the anti-apoptotic activity of these two pathways may be functionally dependent. Ultraviolet (UV) irradiation causes oxidative stress, which can lead to apoptotic cell death. Human skin cells (keratinocytes) are commonly exposed to UV irradiation from the sun. We have investigated activation of the PI3K/Akt and NF-kappaB pathways and their roles in protecting human keratinocytes (KCs) from UV irradiation-induced apoptosis. This activation of PI3K preceded increased levels (3-fold) of active/phosphorylated Akt. UV (50 mJ/cm2 from UVB source) irradiation caused rapid recruitment of PI3K to the epidermal growth factor receptor (EGFR). Pretreatment of KCs with EGFR inhibitor PD169540 abolished UV-induced Akt activation/phosphorylation, as did the PI3K inhibitors LY294002 or wortmannin. This inhibition of Akt activation was associated with a 3-4-fold increase of UV-induced apoptosis, as measured by flow cytometry and DNA fragmentation ELISA. In contrast to Akt, UV irradiation did not detectably increase nuclear localization of NF-kappaB, indicating that it was not strongly activated. Consistent with this observation, interference with NF-kappaB activation by adenovirus-mediated overexpression of dominant negative IKK-beta or IkappaB-alpha did not increase UV-induced apoptosis. However, adenovirus-mediated overexpression of constitutively active Akt completely blocked UV-induced apoptosis observed with PI3K inhibition by LY294002, whereas adenovirus mediated overexpression of dominant negative Akt increased UV-induced apoptosis by 2-fold. Inhibition of UV-induced activation of Akt increased release of mitochondrial cytochrome c 3.5-fold, and caused appearance of active forms of caspase-9, caspase-8, and caspase-3. Constitutively active Akt abolished UV-induced cytochrome c release and activation of caspases-9, -8, and -3. These data demonstrate that PI3K/Akt is essential for protecting human KCs against UV-induced apoptosis, whereas NF-kappaB pathway provides little, if any, protective role.     

Activation of cAMP-guanine exchange factor confers PKA-independent protection from hepatocyte apoptosis

cAMP has previously been shown to promote cell survival in a variety of cell types, but the downstream signaling pathway(s) of this antiapoptotic effect is unclear. Thus the role of cAMP signaling through PKA and cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs) in cAMP's antiapoptotic action was investigated in the present study. cAMP's protective effect against bile acid-, Fas ligand-, and TNF-alpha-induced apoptosis in rat hepatocytes was largely unaffected by the selective PKA inhibitor, Rp-8-(4-chlorophenylthio)-cAMP (Rp-cAMP). In contrast, a novel cAMP analog, 8-(4-chlorophenylthio)-2'-O-methyl (CPT-2-Me)-cAMP, which activated cAMP-GEFs in hepatocytes without activating PKA, protected hepatocytes against apoptosis induced by bile acids, Fas ligand, and TNF-alpha. The role of cAMP-GEF and PKA on activation of Akt, a kinase implicated in cAMP survival signaling, was investigated. Inhibition of PKA with RP-cAMP had no effect on cAMP-mediated Akt phosphorylation, whereas CPT-2-Me-cAMP, which did not activate PKA, induced phosphatidylinositol 3-kinase (PI3-kinase)-dependent activation of Akt. Pretreatment of hepatocytes with the PI3-kinase inhibitor, Ly-294002, prevented CPT-2-Me-cAMP's protective effect against bile acid and Fas ligand, but not TNF-alpha-mediated apoptosis. Glucagon, CPT-cAMP, and CPT-2-Me-cAMP all activated Rap 1, a downstream effector of cAMP-GEF. These results suggest that a PKA-independent cAMP/cAMP-GEF/Rap pathway exists in hepatocytes and that activation of cAMP-GEFs promotes Akt phosphorylation and hepatocyte survival. Thus a cAMP/cAMP-GEF/Rap/PI3-kinase/Akt signaling pathway may confer protection against bile acid- and Fas-induced apoptosis in hepatocytes.     

Phosphatidylinositol 3-Kinase/Akt Plays a Part in Tumor Necrosis Factor-alpha-induced Interleukin-6 Synthesis in Osteoblasts.

We previously showed that tumor necrosis factor-alpha (TNF-alpha) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (Akt) is involved in TNF-alpha-stimulated IL-6 synthesis in MC3T3-E1 cells. TNF-alpha induced the phosphorylation of Akt depending upon time. Akt inhibitor, 1L-6-hydroxymethyl- CHIRO-inositol 2-( R)-2- O-methyl-3- O-octadecylcarbonate, significantly suppressed the TNF-alpha-stimulated IL-6 synthesis, but the inhibitory effect was partial. The phosphorylation of Akt induced by TNF-alpha was markedly attenuated by LY294002 and wortmannin, inhibitors of PI3-kinase. Wortmannin and LY294002 significantly reduce the TNF-alpha-induced IL-6 synthesis. On the contrary, the suppressive effects of Akt inhibitor, wortmannin or LY294002 on TNF-alpha-induced phosphorylation of p44/p42 MAP kinase were minor. PD98059, a specific inhibitor of MEK, had little effect on the TNF-alpha-induced phosphorylation of Akt. A combination of Akt inhibitor and PD98059 suppressed the TNF-alpha-induced IL-6 synthesis in an additive manner. These results strongly suggest that PI3-kinase/Akt plays a role in the TNF-alpha-stimulated IL-6 synthesis mainly independent of p44/p42 MAP kinase in osteoblasts.     

Hepatocytes sensitized to tumor necrosis factor-alpha cytotoxicity undergo apoptosis through caspase-dependent and caspase-independent pathways

Hepatocytes can be sensitized to tumor necrosis factor (TNF)-alpha toxicity by repression of NF-kappaB activation or inhibition of RNA synthesis. To determine whether both forms of sensitization lead to TNF-alpha cytotoxicity by similar mechanisms, TNF-alpha-induced cell death in RALA255-10G hepatocytes was examined following infection with an adenovirus, Ad5IkappaB, that blocks NF-kappaB activation or following cotreatment with actinomycin D (ActD). TNF-alpha treatment of Ad5IkappaB-infected cells resulted in 44% cell death within 6 h. ActD/TNF-alpha induced no death within 6 h but did lead to 37% cell death by 24 h. In both instances, cell death occurred by apoptosis and was associated with caspase activation, although caspase activation in ActD-sensitized cells was delayed. CrmA and chemical caspase inhibitors blocked Ad5IkappaB/TNF-alpha-induced cell death but did not inhibit ActD/TNF-alpha-induced apoptosis. A Fas-associated protein with death domain (FADD) dominant negative decreased Ad5IkappaB/TNF-alpha- and ActD/TNF-alpha-induced cell death by 81 and 47%, respectively. However, downstream events differed, since Ad5IkappaB/TNF-alpha but not ActD/TNF-alpha treatment caused mitochondrial cytochrome c release. These results suggest that NF-kappaB inactivation and inhibition of RNA synthesis sensitize RALA255-10G hepatocytes to TNF-alpha toxicity through distinct cell death pathways that diverge below the level of FADD. ActD-induced hepatocyte sensitization to TNF-alpha cytotoxicity occurs through a FADD-dependent, caspase-independent pathway of apoptosis.     

The anti-apoptotic function of human ��A-crystallin is directly related to its chaperone activity

αA-crystallin is a molecular chaperone and an antiapoptotic protein. This study investigated the mechanism of inhibition of apoptosis by human αA-crystallin and determined if the chaperone activity of αA-crystallin is required for the antiapoptotic function. αA-crystallin inhibited chemical-induced apoptosis in Chinese hamster ovary (CHO) cells and HeLa cells by inhibiting activation of caspase-3 and -9. In CHO cells, it inhibited apoptosis induced by the overexpression of human proapoptotic proteins, Bim and Bax. αA-crystallin inhibited doxorubicin-mediated activation of human procaspase-3 in CHO cells and it activated the PI3K/Akt cell survival pathway by promoting the phosphorylation of PDK1, Akt and phosphatase tensin homologue in HeLa cells. The phosphoinositide 3 kinase (PI3K) activity was increased by αA-crystallin overexpression but the protein content was unaltered. Downregulation of PI3K by the expression of a dominant-negative mutant or inhibition by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 abrogated the ability of αA-crystallin to phosphorylate Akt. These antiapoptotic functions of αA-crystallin were enhanced in a mutant protein (R21A) that shows increased chaperone activity than the wild-type (Wt) protein. Interestingly, a mutant protein (R49A) that shows decreased chaperone activity was far weaker than the Wt protein in its antiapoptotic functions. Together, our study results show that αA-crystallin inhibits apoptosis by enhancing PI3K activity and inactivating phosphatase tensin homologue and that the antiapoptotic function is directly related to its chaperone activity.     

Tumor necrosis factor-alpha-stimulated cell proliferation is mediated through sphingosine kinase-dependent Akt activation and cyclin D expression

Tumor necrosis factor-alpha (TNF-alpha) has been shown to activate sphingosine kinase (SphK) in a variety of cell types. The extent to which SphK signaling mediates the pleiotropic effects of TNF-alpha is not entirely clear. The current study examined the role of SphK activity in TNF-alpha-stimulated cell proliferation in 1321N1 glioblastoma cells. We first demonstrated that pharmacological inhibitors of SphK markedly decrease TNF-alpha-stimulated DNA synthesis. Signaling mechanisms through which SphK mediated the effect of TNF-alpha on DNA synthesis were then examined. Inhibition of Rho proteins with C3 exoenzyme or of Rho kinase with Y27632 attenuated TNF-alpha-stimulated DNA synthesis. However, RhoA activation by TNF-alpha was not blocked by SphK inhibition. ERK activation was also required for TNF-alpha-stimulated DNA synthesis but likewise TNF-alpha-induced ERK activation was not blocked by inhibition of SphK. Thus, neither RhoA nor ERK activation are the SphK-dependent transducers of TNF-alpha-induced proliferation. In contrast, TNF-alpha-stimulated Akt phosphorylation, which was also required for DNA synthesis, was attenuated by SphK inhibition or SphK1 knockdown by small interfering RNA. Furthermore, cyclin D expression was increased by TNF-alpha in a SphK- and Akt-dependent manner. Additional studies demonstrated that TNF-alpha effects on DNA synthesis, ERK, and Akt phosphorylation are not mediated through cell surface Gi -coupled S1P receptors, because none of these responses were inhibited by pertussis toxin. We conclude that SphK-dependent Akt activation plays a significant role in TNF-alpha-induced cyclin D expression and cell proliferation.     

Bcl-xL mediates a survival mechanism independent of the phosphoinositide 3-kinase/Akt pathway in prostate cancer cells

Among various molecular strategies by which prostate cancer cells evade apoptosis, phosphoinositide 3-kinase (PI3K)/Akt signaling represents a dominant survival pathway. However, different prostate cancer cell lines such as LNCaP and PC-3 display differential sensitivity to the apoptotic effect of PI3K inhibition in serum-free media, reflecting the heterogeneous nature of prostate cancer in apoptosis regulation. Whereas both cell lines are equally susceptible to LY294002-mediated Akt dephosphorylation, only LNCaP cells default to apoptosis, as evidenced by DNA fragmentation and cytochrome c release. In PC-3 cells, Akt deactivation does not lead to cytochrome c release, suggesting that the intermediary signaling pathway is short-circuited by an antiapoptotic factor. This study presents evidence that Bcl-xL overexpression provides a distinct survival mechanism that protects PC-3 cells from apoptotic signals emanating from PI3K inhibition. First, the Bcl-xL/BAD ratio in PC-3 cells is at least an order of magnitude greater than that of LNCaP cells. Second, ectopic expression of Bcl-xL protects LNCaP cells against LY294002-induced apoptosis. Third, antisense down-regulation of Bcl-xL sensitizes PC-3 cells to the apoptotic effect of LY294002. The physiological relevance of this Bcl-xL-mediated survival mechanism is further underscored by the protective effect of serum on LY294002-induced cell death in LNCaP cells, which is correlated with a multifold increase in Bcl-xL expression. In contrast to Bcl-xL, Bcl-2 expression levels are similar in both cells lines, and do not respond to serum stimulation, suggesting that Bcl-2 may not play a physiological role in antagonizing apoptosis signals pertinent to BAD activation in prostate cancer cells.     

Contribution of caveolin-1 alpha and Akt to TNF-alpha-induced cell death

We used retrovirus insertion-mediated random mutagenesis to generate tumor necrosis factor-alpha (TNF-alpha)-resistant lines from L929 cells. Using this approach, we discovered that caveolin-1 alpha is required for TNF-alpha-induced cell death in L929 cells. The need for caveolin-1 alpha in TNF-alpha-induced cell death was confirmed by the restoration of sensitivity to TNF-alpha after ectopic reconstitution of caveolin-1 alpha/beta expression. This caveolin-1 alpha-mutated line was also resistant to H(2)O(2) and staurosporine, but not to lonidamine. HepG2 cells are known to lack endogenous caveolins. HepG2 cells stably transfected with caveolin-1 alpha/beta were found to be much more sensitive to TNF-alpha than either parental cells transfected with caveolin-1 beta or parental cells transfected with an empty vector. In contrast to its extensively documented antiapoptotic effect, the elevated activity of Akt appears to be important in sensitizing caveolin-1-expressing cells to TNF-alpha, since pretreatment of cells with the phosphatidylinositide 3-kinase (PI3K) inhibitor LY-294002 or wortmannin completely blocked PI3K activation and markedly improved the survival of TNF-alpha-treated L929 cells. The survival rates of caveolin-1 alpha-normal and caveolin-1 alpha-deficient L929 cells were comparable after treatment with PI3K inhibitor and TNF-alpha. Similar results were obtained with HepG2 cells that stably expressed caveolin-1 alpha/beta or -beta and parental cells transfected with an empty vector. In summary, our results indicate that caveolin-1 alpha preferentially sensitizes L929 cells to TNF-alpha through the activation of a PI3K/Akt signaling pathway.     

Carbon monoxide activates NF-kappaB via ROS generation and Akt pathways to protect against cell death of hepatocytes

Heme oxygenase overexpression or exogenous carbon monoxide (CO) protects against hepatocyte apoptosis and fulminant hepatitis. The prevention of hepatocyte apoptosis by CO has been shown to require activation of NF-kappaB. The purpose of these investigations was to determine the mechanism of CO-induced hepatocyte NF-kappaB activation and protection against apoptosis. Primary rat or mouse hepatocytes and Hep3B cells were utilized. CO exposure was performed at 250 parts per million. Main outcome measures included cell viability, reactive oxygen species (ROS) generation, and changes in the levels of the intracellular antioxidants glutathione and ascorbate. Western blotting was performed for phospho-Akt, total Akt, and IkappaBalpha. NF-kappaB activation was determined by electrophoretic mobility shift assay and luciferase reporter assays. We found that CO treatment of hepatocytes prevents spontaneous apoptosis and leads to an increase in ROS production in association with Akt phosphorylation and IkappaB degradation. CO did not increase ROS production in respiration-deficient (rho0) Hep3B cells. Both Akt phosphorylation and IkappaB degradation can be inhibited by the addition of antioxidants. Furthermore, CO-induced NF-kappaB activation is reversed by phosphatidylinositol 3-kinase (PI3-K) inhibitor (LY294002) or antioxidants. Additionally, prevention of spontaneous hepatocyte apoptosis by CO is reversed by PI3-K inhibition and antioxidants. In conclusion, these data implicate a survival pathway of CO-induced ROS, Akt phosphorylation, and NF-kappaB activation in cultured hepatocytes. This pathway may prove to be important in maintenance of hepatic function in both physiological and pathophysiological conditions.     

Rapid activation of protein kinase B/Akt has a key role in antiapoptotic signaling during liver regeneration

Liver regeneration is controlled by multiple signaling pathways induced by a variety of growth factors, hormones, and cytokines. Here we report that protein kinase B (PKB)/Akt, part of a key cell survival signaling pathway, is markedly activated after partial hepatectomy (PHX). The antiapoptotic protein Bad, a downstream target of PKB/Akt, is also phosphorylated. This cascade can be activated by various factors in primary hepatocytes, with the strongest activation by insulin and the alpha1-adrenergic agonist phenylephrine (PE), followed by IL-6, epidermal growth factor (EGF), and hepatocyte growth factor (HGF). Pretreatment of cells with the specific PI3 kinase inhibitor LY294002 abolished insulin- or PE-activation of PKB/Akt, suggesting that activation of PKB/Akt is mediated by a PI3 kinase-dependent mechanism. In vivo administration of PE, insulin, IL-6, HGF, or EGF to mice markedly stimulated PKB/Akt in the liver, with the strongest stimulation induced by insulin and PE. Moreover, HGF and insulin were able to attenuate transforming growth factor beta-induced apoptosis in hepatic cells, and these effects were antagonized by LY294002. Taken together, these findings suggest that rapid activation of PKB/Akt is a key antiapoptotic signaling pathway involved in liver regeneration.     

Inhibition of phosphatidylinositol-3 kinase/Akt or mitogen-activated protein kinase signaling sensitizes endothelial cells to TNF-alpha cytotoxicity.

Bovine carotid artery endothelial (BAE) cells are resistant to tumor necrosis factor-alpha (TNF), like most other cells. We examined if mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 (PI3) kinase/Akt pathways are involved in this effect. In BAE cells, TNF activates MAP kinase in a MAP kinase kinase 1 (MEK1) manner and Akt in PI3-kinase-dependent manner. Pretreatment with either the MEK1 inhibitor U0126 or PI3-kinase inhibitor LY294002 sensitized BAE cells to TNF-induced apoptosis. Neither U0126 nor LY294002 pretreatment affected TNF-induced activation of NF-kappaB, suggesting that the MAP kinase or PI3-kinase/Akt-mediated anti-apoptotic effect induced by TNF was not relevant to NF-kappaB activation. Both MAP kinase and PI3-kinase/Akt -mediated signaling could prevent cytochrome c release and mitochondrial transmembrane potential (Deltapsi) decrease. PI3-kinase/Akt signaling attenuated caspase-8 activity, whereas MAP kinase signaling impaired caspase-9 activity. These results suggest that TNF-induced MAP kinase and PI3-kinase/Akt signaling play important roles in protecting BAE cells from TNF cytotoxicity.