Rapamycin antagonizes NF-kappaB nuclear translocation activated by TNF-alpha in primary vascular smooth muscle cells and enhances apoptosis

Several lines of evidence support the view that rapamycin inhibits NF-kappaB. TNF-alpha, a potent inducer of NF-kappaB, is released after artery injury (e.g., balloon angioplasty) and plays an important role in inflammation and restenosis. We investigated the effect of rapamycin on NF-kappaB activation and apoptosis in vascular smooth muscle cells (VSMCs) stimulated with TNF-alpha. Using EMSA, we found that TNF-alpha caused NF-kappaB nuclear translocation in VSMCs after 1 h of incubation. Rapamycin inhibited IkappaBalpha degradation, thereby preventing nuclear translocation. Activation of NF-kappaB was accompanied by an increase of Bcl-xL and Bfl-1/A1 proteins, detected by Western blot assay, whereas rapamycin prevented the TNF-alpha-induced enhancement of these antiapoptotic proteins. The extent of apoptosis of VSMCs exposed to TNF-alpha was significantly enhanced by rapamycin. The effect of rapamycin appeared to be independent of the phosphatidylinositol 3-kinase/Akt-protein kinase B survival pathway, because the phosphatidylinositol 3-kinase inhibitor wortmannin neither prevented IkappaBalpha degradation nor increased apoptosis of cells incubated with TNF-alpha. Finally, we demonstrate that the large immunophilin FK-506 binding protein FKBP51 is essential for TNF-alpha-induced NF-kappaB activation in VSMCs. Our findings show that rapamycin inhibits NF-kappaB activation and acts in concert with TNF-alpha in induction of VSMC apoptosis.     

PG490 (triptolide) cooperates with tumor necrosis factor-alpha to induce apoptosis in tumor cells

Progress in the treatment of solid tumors has been slow and sporadic. The efficacy of conventional chemotherapy in solid tumors is limited because tumors frequently have mutations in the p53 gene. Also, chemotherapy only kills rapidly dividing cells. Members of the tumor necrosis factor (TNF) family, however, induce apoptosis regardless of the p53 phenotype. Unfortunately, the cytotoxicity of TNF-alpha is limited by its activation of NF-kappaB and activation of NF-kappaB is proinflammatory. We have identified a compound called PG490, that is composed of purified triptolide, which induces apoptosis in tumor cells and sensitizes tumor cells to TNF-alpha-induced apoptosis. PG490 potently inhibited TNF-alpha-induced activation of NF-kappaB. PG490 also blocked TNF-alpha-mediated induction of c-IAP2 (hiap-1) and c-IAP1 (hiap-2), members of the inhibitor of apoptosis (IAP) family. Interestingly, PG490 did not block DNA binding of NF-kappaB, but it blocked transactivation of NF-kappaB. Our identification of a compound that blocks TNF-alpha-induced activation of NF-kappaB may enhance the cytotoxicity of TNF-alpha on tumors in vivo and limit its proinflammatory effects.     

DNA synthesis precedes gliotoxin-induced apoptosis

The toxin gliotoxin induces apoptosis or programmed cell death in a variety of immune cells including thymocytes. Apoptosis induced by gliotoxin in thymocytes is unaffected by protein synthesis inhibitors nor is it associated with early changes in intracellular calcium levels (Beaver and Waring, 1994). This work shows that the cell lines P815 and WEHI7 and murine thymocytes when treated with gliotoxin show an early incorporation of tritiated thymidine over the concentration range which causes apoptosis. Proliferating cell nuclear antigen (PCNA), a marker for S phase, is elevated in cells following gliotoxin treatment and S phase DNA content is increased. Thymidine incorporation is inhibited by hydroxyurea, an inhibitor of replicative DNA synthesis not repair. Free radical scavangers have no effect on apoptosis induced by gliotoxin in thymocytes. Hydrogen peroxide-treated cells showed no enhanced thymidine incorporation and no apoptosis. Thus oxidative stress does not appear to be a factor in gliotoxin-induced apoptosis. Thymocytes treated with gliotoxin show increased phosphorylation of a 16.3 kDa protein, and apoptosis is inhibited by the tyrosine kinase inhibitor genistein, which also inhibited the increased thymidine incorporation in P815 cells. We conclude that one mechanism by which gliotoxin can cause apoptosis may be the induction of inappropriate entry of cells into the cell cycle followed by death.     

NF-kappaB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells

The maintenance of intestinal mucosal integrity depends on a balance between cell renewal and cell death, including apoptosis. The natural polyamines, putrescine, spermidine, and spermine, are essential for mucosal growth, and decreasing polyamine levels cause G(1) phase growth arrest in intestinal epithelial (IEC-6) cells. The present study was done to determine changes in susceptibility of IEC-6 cells to apoptosis after depletion of cellular polyamines and to further elucidate the role of nuclear factor-kappaB (NF-kappaB) in this process. Although depletion of polyamines by alpha-difluoromethylornithine (DFMO) did not directly induce apoptosis, the susceptibility of polyamine-deficient cells to staurosporine (STS)-induced apoptosis increased significantly as measured by changes in morphological features and internucleosomal DNA fragmentation. In contrast, polyamine depletion by DFMO promoted resistance to apoptotic cell death induced by the combination of tumor necrosis factor-alpha (TNF-alpha) and cycloheximide. Depletion of cellular polyamines also increased the basal level of NF-kappaB proteins, induced NF-kappaB nuclear translocation, and activated the sequence-specific DNA binding activity. Inhibition of NF-kappaB binding activity by sulfasalazine or MG-132 not only prevented the increased susceptibility to STS-induced apoptosis but also blocked the resistance to cell death induced by TNF-alpha in combination with cycloheximide in polyamine-deficient cells. These results indicate that 1) polyamine depletion sensitizes intestinal epithelial cells to STS-induced apoptosis but promotes the resistance to TNF-alpha-induced cell death, 2) polyamine depletion induces NF-kappaB activation, and 3) disruption of NF-kappaB function is associated with altered susceptibility to apoptosis induced by STS or TNF-alpha. These findings suggest that increased NF-kappaB activity after polyamine depletion has a proapoptotic or antiapoptotic effect on intestinal epithelial cells determined by the nature of the death stimulus.     

Inhibition of the NF-kappaB survival pathway via caspase-dependent cleavage of the IKK complex scaffold protein and NF-kappaB essential modulator NEMO

Apoptosis is mediated by cysteine-dependent, aspartate-directed proteases of the caspase family that proteolyse strategic intracellular substrates to induce cell suicide. We describe here that engagement of apoptotic processes by Fas triggering or by staurosporine stimulation leads to the caspase-dependent inactivation of the nuclear factor kappa B (NF-kappaB) pathway after cleavage of IKK1 (IkappaB kinase 1) and NEMO (NF-kappaB essential modulator), which are needed to transduce NF-kappaB activation signals. In this study, we have analyzed in more detail, the role of NEMO cleavage, as NEMO, but not IKK1, is important for the pro-survival actions of NF-kappaB. We demonstrate that NEMO is cleaved after Asp355 to remove the last 64 C-terminal amino acids. This short form was unable to rescue NF-kappaB activation by tumor necrosis factor-alpha (TNF-alpha) when transfected in NEMO-deficient cells. Consequently, inactivation of NEMO resulted in an inhibition of the expression of antiapoptotic NF-kappaB-target genes coding for caspase inhibitors (cIAP-1, cIAP-2) or adaptors of the TNF receptor family. NEMO-deficient Jurkat cells transiently expressing a non-cleavable mutant of NEMO were less sensitive to TNF-alpha-induced apoptosis. Therefore, downmodulation of NF-kappaB activation via the proteolytic cleavage of NEMO could represent an amplification loop for apoptosis.     

Synergistic induction of nuclear factor-kappaB by transforming growth factor-beta and tumour necrosis factor-alpha is mediated by protein kinase A-dependent RelA acetylation

The TGF-beta (transforming growth factor-beta) pathway represents an important signalling pathway involved in regulating diverse biological processes, including cell proliferation, differentiation and inflammation. Despite the critical role for TGF-beta in inflammatory responses, its role in regulating NF-kappaB (nuclear factor-kappaB)-dependent inflammatory responses still remains unknown. In the present study we show that TGF-beta1 synergizes with proinflammatory cytokine TNF-alpha (tumour necrosis factor-alpha) to induce NF-kappaB activation and the resultant inflammatory response in vitro and in vivo. TGF-beta1 synergistically enhances TNF-alpha-induced NF-kappaB DNA binding activity via induction of RelA acetylation. Moreover, synergistic enhancement of TNF-alpha-induced RelA acetylation and DNA-binding activity by TGF-beta1 is mediated by PKA (protein kinase A). Thus the present study reveals a novel role for TGF-beta in inflammatory responses and provides new insight into the regulation of NF-kappaB by TGF-beta signalling.     

Selective sensitization to tumor necrosis factor-alpha-induced apoptosis by blockade of NF-kappaB in primary glomerular mesangial cells.

Recent data have implicated nuclear factor kappaB (NF-kappaB) in the prevention of apoptosis in transformed cell lines exposed to tumor necrosis factor alpha (TNF-alpha). However, it is obscure whether NF-kappaB plays an anti-apoptotic role in nontransformed cells, and it is not clear whether NF-kappaB inhibits apoptosis triggered by other mediators. We investigated the effect of specific inhibition of NF-kappaB on cytokine-induced apoptosis of glomerular mesangial cells, which is important in determining the outcome of glomerulonephritis. Cultured rat mesangial cells were stably transfected with the dominant negative mutant inhibitor of NF-kappaB (IkappaBalphaM). IkappaBalphaM was resistant to stimulus-dependent degradation and suppressed NF-kappaB activation induced by TNF-alpha (10 ng/ml) or IL-1beta (10 ng/ml). IkappaBalphaM significantly sensitized mesangial cells to TNF-alpha-induced apoptosis in a dose- and time-dependent manner but had no significant effects on the level of apoptosis in the presence of proinflammatory or apoptosis-inducing stimuli including Fas ligand, IL-1alpha, IL-1beta, hydrogen peroxide, lipopolysaccharide, cycloheximide, or serum deprivation. Moreover, IkappaBalphaM-mediated sensitization to TNF-alpha overcame the protective effect of mesangial cell survival factors present in serum, which usually inhibit killing of mesangial cells by the proapoptotic stimuli used. These data show that inhibition of NF-kappaB selectively sensitizes primary adult glomerular mesangial cells to TNF-induced apoptosis but not to other mediators of cell death including the Fas ligand.