IkappaB kinase-independent IkappaBalpha degradation pathway: functional NF-kappaB activity and implications for cancer therapy

Antiapoptotic activity of NF-kappaB in tumors contributes to acquisition of resistance to chemotherapy. Degradation of IkappaB is a seminal step in activation of NF-kappaB. The IkappaB kinases, IKK1 and IKK2, have been implicated in both IkappaB degradation and subsequent modifications of NFkappaB. Using mouse embryo fibroblasts (MEFs) devoid of both IKK1 and IKK2 genes (IKK1/2(-/-)), we document a novel IkappaB degradation mechanism. We show that this degradation induced by a chemotherapeutic agent, doxorubicin (DoxR), does not require the classical serine 32 and 36 phosphorylation or the PEST domain of IkappaBalpha. Degradation of IkappaBalpha is partially blocked by phosphatidylinositol 3-kinase inhibitor LY294002 and is mediated by the proteasome. Free NF-kappaB generated by DoxR-induced IkappaB degradation in IKK1/2(-/-) cells is able to activate chromatin based NF-kappaB reporter gene and expression of the endogenous target gene, IkappaBalpha. These results also imply that modification of NF-kappaB by IKK1 or IKK2 either prior or subsequent to its release from IkappaB is not essential for NF-kappaB-mediated gene expression at least in response to DNA damage. In addition, DoxR-induced cell death in IKK1/2(-/-) MEFs is enhanced by simultaneous inhibition of NF-kappaB activation by blocking the proteasome activity. These results reveal an additional pathway of activating NF-kappaB during the course of anticancer therapy and provide a mechanistic basis for the observation that proteasome inhibitors could be used as adjuvants in chemotherapy.     

Acylpeptide hydrolase inhibition as targeted strategy to induce proteasomal down-regulation

Acylpeptide hydrolase (APEH), one of the four members of the prolyl oligopeptidase class, catalyses the removal of N-acylated amino acids from acetylated peptides and it has been postulated to play a key role in protein degradation machinery. Disruption of protein turnover has been established as an effective strategy to down-regulate the ubiquitin-proteasome system (UPS) and as a promising approach in anticancer therapy.Here, we illustrate a new pathway modulating UPS and proteasome activity through inhibition of APEH. To find novel molecules able to down-regulate APEH activity, we screened a set of synthetic peptides, reproducing the reactive-site loop of a known archaeal inhibitor of APEH (SsCEI), and the conjugated linoleic acid (CLA) isomers. A 12-mer SsCEI peptide and the trans10-cis12 isomer of CLA, were identified as specific APEH inhibitors and their effects on cell-based assays were paralleled by a dose-dependent reduction of proteasome activity and the activation of the pro-apoptotic caspase cascade. Moreover, cell treatment with the individual compounds increased the cytoplasm levels of several classic hallmarks of proteasome inhibition, such as NFkappaB, p21, and misfolded or polyubiquitinylated proteins, and additive effects were observed in cells exposed to a combination of both inhibitors without any cytotoxicity. Remarkably, transfection of human bronchial epithelial cells with APEH siRNA, promoted a marked accumulation of a mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), herein used as a model of misfolded protein typically degraded by UPS. Finally, molecular modeling studies, to gain insights into the APEH inhibition by the trans10-cis12 CLA isomer, were performed.Our study supports a previously unrecognized role of APEH as a negative effector of proteasome activity by an unknown mechanism and opens new perspectives for the development of strategies aimed at modulation of cancer progression.     

Treatment of COS-7 cells with proteasome inhibitors or gamma-interferon reduces the increase in caspase 3 activity associated with staurosporine-induced apoptosis.

Proteasomes play a major role in intracellular protein degradation and have been implicated in apoptosis. In this study we have investigated proteasome activity and the effects of inhibition of proteasomes or modulation of proteasome complexes on staurosporine-induced apoptosis in COS-7 cells. Staurosporine treatment of COS-7 cells had little direct effect on proteasome activity and did not cause dissociation of 26S proteasomes. There was also no major redistribution of proteasomes accompanying apoptosis in COS-7 cells. However, when the cells were pretreated with proteasome inhibitors, both the caspase 3 activity of the cells and the percentage of apoptotic cells measured by the TUNEL assay were reduced compared to staurosporine-treated cells, which had no inhibitor added. Proteasome inhibitors were also found to reduce the activation of caspase 3 in living cells which was assayed using a FRET-based method. However, proteasome inhibitors did not prevent some of the morphological changes associated with staurosporine-induced apoptosis. Pretreatment of cells with gamma-interferon, which increases immunoproteasomes and PA28 complexes and reduces 26S proteasome levels, had an antiapoptotic effect. These results are consistent with a role for 26S proteasomes in regulating the activation of caspase 3 through the degradation of key regulatory proteins.     

Distinct differences between TNF receptor 1- and TNF receptor 2-mediated activation of NFkappaB

Tumor necrosis factor (TNF) signaling is mediated via two distinct receptors, TNFR2 and TNFR1, which shows partially overlapping signaling mechanisms and biological roles. In the present study, TNFR2 and TNFR1 signal transduction mechanisms involved in activation of NFkappaB and CMV promoter-enhancer were compared with respect to their susceptibility towards inhibitors of intracellular signaling. For this, we used SW480 cells, where we have shown that TNF-signaling can occur independently through each of the two receptors. The TNFR1 response was inhibited by D609, bromophenacyl bromide (BPB), nordihydroguararetic acid (NDGA), and by sodium salicylate, while TNFR2-mediated activation of NFkappaB and CMV promoter-enhancer was resistant to these compounds. The signaling mechanisms known to be affected by these inhibitors include phospholipases as well as redox- and pH-sensitive intracellular components. Our results imply that TNFR2 signaling involved in NFkappaB activation proceeds independently of these inhibitor-sensitive signaling components, indicating distinct signaling pathways not shared with TNFR1.     

Okadaic acid induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha in human neutrophils

Human neutrophils differ from other cells by containing high amount of IkappaBalpha in the nucleus, and this increased nuclear IkappaBalpha accumulation is associated with the inhibition of NFkappaB activity and increased apoptosis. However, the mechanisms regulating NFkappaB activation and IkappaBalpha degradation in human neutrophils are little understood. The objective of this study was to provide a further insight into the mechanisms regulating NFkappaB activity and IkappaBalpha degradation in human neutrophils. We show that okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha, and increases interleukin-8 expression in the neutrophils. Furthermore, inhibitors of protein kinase C-delta (PKCdelta) and IkappaB kinase (IKK) inhibit the OA-induced activation of NFkappaB. Collectively, our results indicate that in human neutrophils, the sustained activation of NFkappaB is regulated by a continuous phosphorylation and degradation of the nuclear IkappaBalpha.     

The kinase activity of IL-1 receptor-associated kinase 4 is required for interleukin-1 receptor/toll-like receptor-induced TAK1-dependent NFkappaB activation

Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFkappaB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classic NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through dissociation of phosphorylated IkappaBalpha from NFkappaB without IkappaBalpha degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFkappaB activation pathway, but mediated IL-1-induced TAK1-independent NFkappaB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFkappaB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFkappaB activation.     

Delayed and sustained activation of p42/p44 mitogen-activated protein kinase induced by proteasome inhibitors through p21(ras) in PC12 cells

Proteolysis by the ubiquitin/proteasome pathway regulates the intracellular level of several proteins, some of which control cell proliferation and cell cycle progression. To determine what kinds of signaling cascades are activated or inhibited by proteasome inhibition, we treated PC12 cells with specific proteasome inhibitors and subsequently performed in-gel kinase assays. N-Acetyl-Leu-Leu-norleucinal and lactacystin, which inhibit the activity of the proteasome, induced the activation of p42/p44 mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinases (ERKs) 1 and 2]. In contrast, N-acetyl-Leu-Leu-methional, which inhibits the activity of calpains, but not of the proteasome, failed to induce ERK activation. Uniquely, the kinetics of MAP kinase activation induced by proteasome inhibitors are very slow compared with those resulting from activation by nerve growth factor; ERK activation is detectable only after a 5-h treatment with the inhibitors, and its activity remained unchanged for at least until 27 h. Proteasome inhibitor-initiated ERK activation is inhibited by pretreatment with the ERK kinase inhibitor PD 98059, as well as by overexpression of a dominant-negative form of Ras. Thus, proteasome inhibitors induce sustained ERK activation in a Ras-dependent manner. Proteasome inhibitor-induced neurite outgrowth, however, is not inhibited by PD 98059, indicating that sustained activation of ERKs is not the factor responsible for proteasome inhibitor-induced morphological differentiation. Our data suggest the presence of a novel mechanism for activation of the MAP kinase cascade that involves proteasome activity.