Cucurbitacin B suppresses the transactivation activity of RelA/p65

Cucurbitacin B, a natural triterpenoid is well-known for its strong anticancer activity, and recent studies showed that the compound inhibits JAK/STAT3 pathway. In this study, we demonstrate for the first time that cucurbitacin B is also a potent inhibitor of NF-κB activation. Our results showed that cucurbitacin B inhibited TNF-α-induced expression of NF-κB reporter gene and NF-κB target genes in a dose-dependent manner, however, it did not prevent either stimuli-induced degradation of IκBα or nuclear translocation and DNA-binding activity of NF-κB. On the other hand, cucurbitacin B dose-dependently suppressed not only NF-κB activation induced by overexpression of RelA/p65 but also transactivation activity of RelA/p65 subunit of NF-κB. Consistently, treatment of HeLa cells with the compound significantly suppressed TNF-α-induced activation of Akt and phosphorylation of Ser536 in RelA/p65, which is required for transactivation activity. Consequently, cucurbitacin B inhibited TNF-α-induced expression of NF-κB-dependent anti-apoptotic proteins such as c-IAP1, c-IAP2, XIAP, TRAF1, and TRAF2 and sensitized TNF-α-induced cell death. Taken together, our results demonstrated that cucurbitacin B could be served as a valuable candidate for the intervention of NF-κB-dependent pathological condition such as cancer.     

RelA/p65 regulation of IkappaBbeta

IkappaB inhibitor proteins are the primary regulators of NF-kappaB. In contrast to the defined regulatory interplay between NF-kappaB and IkappaBalpha, much less is known regarding the regulation of IkappaBbeta by NF-kappaB. Here, we describe in detail the regulation of IkappaBbeta by RelA/p65. Using p65(-/-) fibroblasts, we show that IkappaBbeta is profoundly reduced in these cells, but not in other NF-kappaB subunit knockouts. This regulation prevails during embryonic and postnatal development in a tissue-specific manner. Significantly, in both p65(-/-) cells and tissues, IkappaBalpha is also reduced, but not nearly to the same extent as IkappaBbeta, thus highlighting the degree to which IkappaBbeta is dependent on p65. This dependence is based on the ability of p65 to stabilize IkappaBbeta protein from the 26S proteasome, a process mediated in large part through the p65 carboxyl terminus. Furthermore, IkappaBbeta was found to exist in both a basally phosphorylated and a hyperphosphorylated form. While the hyperphosphorylated form is less abundant, it is also more stable and less dependent on p65 and its carboxyl domain. Finally, we show that in p65(-/-) fibroblasts, expression of a proteolysis-resistant form of IkappaBbeta, but not IkappaBalpha, causes a severe growth defect associated with apoptosis. Based on these findings, we propose that tight control of IkappaBbeta protein by p65 is necessary for the maintenance of cellular homeostasis.     

p65/RelA binds and activates the beclin 1 promoter

We unveiled novel p65/RelA consensus sites in the promoter of the beclin 1 gene and demonstrate that p65/RelA positively modulates canonical autophagy in various human cell lines both under basal conditions and upon induction by ceramide. Interestingly, we find that T cell receptor-dependent activation of Jurkat cells triggers an increase in the binding of p65/RelA to the beclin 1 promoter accompanied by enhanced autophagy, suggesting that p65/RelA could regulate T-cell activation and homeostasis through autophagy.     

Cell-type-specific roles of IGF-1R and EGFR in mediating Zn2+-induced ERK1/2 and PKB phosphorylation

Zn²⁺ exerts insulin-mimetic and antidiabetic effects in rodent models of insulin resistance, and activates extracellular-signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), key components of the insulin signaling pathway. Zn²⁺-induced signaling has been shown to be associated with an increase in the tyrosine phosphorylation of insulin receptor (IR), as well as of insulin-like growth factor 1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) in several cell types. However, the specific contribution of these receptor protein tyrosine kinases (R-PTKs) in mediating Zn²⁺-induced responses in a cell-specific fashion remains to be established. Therefore, using a series of pharmacological inhibitors and genetically engineered cells, we have investigated the roles of various R-PTKs in Zn²⁺-induced ERK1/2 and PKB phosphorylation. Pretreatment of Chinese hamster ovary (CHO) cells overexpressing a human IR (CHO-HIR cells) with AG1024, an inhibitor for IR protein tyrosine kinase (PTK) and IGF-1R-PTK, blocked Zn²⁺-induced ERK1/2 and PKB phosphorylation, but AG1478, an inhibitor for EGFR, was without effect in CHO cells. On the other hand, both of these inhibitors were able to attenuate Zn²⁺-induced phosphorylation of ERK1/2 and PKB in A10 vascular smooth muscle cells. In addition, in CHO cells overexpressing tyrosine kinase deficient IR, Zn²⁺ was still able to induce the phosphorylation of these two signaling molecules, whereas the insulin effect was significantly attenuated. Furthermore, both Zn²⁺ and insulin-like growth factor 1 failed to stimulate ERK1/2 and PKB phosphorylation in IGF-1R knockout cells. Also, Zn²⁺-induced responses in CHO-HIR cells were not associated with an increase in the tyrosine phosphorylation of the IR β-subunit and insulin receptor substrate 1 in CHO-HIR cells. Taken together, these data suggest that distinct R-PTKs mediate Zn²⁺-evoked ERK1/2 and PKB phosphorylation in a cell-specific manner.