Cell Type-specific Expression of the IκB Kinases Determines the Significance of Phosphatidylinositol 3-Kinase/Akt Signaling to NF-κB Activation*

Phosphatidylinositol (PI) 3-kinase/Akt signaling activates NF-κB through pleiotropic, cell type-specific mechanisms. This study investigated the significance of PI 3-kinase/Akt signaling to tumor necrosis factor (TNF)-induced NF-κB activation in transformed, immortalized, and primary cells. Pharmacological inhibition of PI 3-kinase blocked TNF-induced NF-κB DNA binding in the 293 line of embryonic kidney cells, partially affected binding in MCF-7 breast cancer cells, HeLa and ME-180 cervical carcinoma cells, and NIH 3T3 cells but was without significant effect in H1299 and human umbilical vein endothelial cells, cell types in which TNF activated Akt. NF-κB is retained in the cytoplasm by inhibitory proteins, IκBs, which are phosphorylated and targeted for degradation by IκB kinases (IKKα and IKKβ). Expression and the ratios of IKKα and IKKβ, which homo- and heterodimerize, varied among cell types. Cells with a high proportion of IKKα (the IKK kinase activated by Akt) to IKKβ were most sensitive to PI 3-kinase inhibitors. Consequently, transient expression of IKKβ diminished the capacity of the inhibitors to block NF-κB DNA binding in 293 cells. Also, inhibitors of PI 3-kinase blocked NF-κB DNA binding in Ikkβ–/– but not Ikkα–/– or wild-type cells in which the ratio of IKKα to IKKβ is low. Thus, noncoordinate expression of IκB kinases plays a role in determining the cell type-specific role of Akt in NF-κB activation.

extracellular stimuli such as cytokines or UV radiation, IB proteins are phosphorylated, polyubiquitinated, and then degraded by the 26 S proteasome (6 -13). Dissociation from IBs unmasks the nuclear localization sequence of NF-B, permitting it to move into the nucleus, bind the promoters of target genes, and alter gene expression and cell function (10,13,14). The demonstration that phosphorylation of IB proteins initiates events necessary for activation of NF-B led to the discovery of IB kinase (IKK) complexes composed of IKK␣, IKK␤, and IKK␥ (NEMO) (15)(16)(17)(18)(19). IKK␣ and IKK␤ are serine-threonine kinases, and IKK␥ is a scaffolding protein essential for the function of IKK␣ and IKK␤. IKK␣ and IKK␤ share a high degree of amino acid homology and domain organization. The kinases are composed of an N-terminal kinase domain, a leucine zipper that facilitates homo-and heterodimerization, and a helix-loop-helix domain (20).
Akt may affect NF-B through multiple mechanisms. We demonstrated previously that TNF activates Akt, which phosphorylates and activates IKK␣, thus promoting NF-B function (42). TNF and interleukin-1 can also increase the transactivation potential of the RelA/p65 subunit of NF-B through a mechanism in which Akt has been implicated (43)(44)(45). PI 3-kinase activated by phorbol esters or lipopolysaccharide and PI 3-kinase/Akt signaling induced by signaling through CD40, interleukin-1, or G protein-coupled receptors activates NF-B (43, 46 -48). However, PI 3-kinase/Akt signaling induced by TNF in human umbilical vein endothelial cells inhibits apoptosis without playing a significant role in activation of NF-B (49). Furthermore, Akt can activate a member of the mitogenactivated protein kinase kinase kinase (MAP3K) family, Cot, and indirectly affect IKK activity and NF-B (50). Thus, PI 3-kinase/Akt signaling is upstream of diverse pathways that activate NF-B. However, the mechanisms through which PI 3-kinase/Akt signaling activates NF-B are cell type-specific, and surrogate pathways also affect NF-B.
The goal of the present study was to determine the basis for the cell type specificity with which PI 3-kinase/Akt signaling activates NF-B. We demonstrate that the ability of PI 3-kinase inhibitors to impair TNF-induced DNA binding of NF-B is cell type-specific. Such specificity does not necessarily result from alterations in the ability of TNF to activate Akt, as this capacity was observed in seven different cell types, including primary cells. Rather, the proportion of IKK␣ to IKK␤ varies among cells, and those cells in which the ratio of IKK␣, the target for Akt phosphorylation, to IKK␤ is high are most susceptible to the ability of PI 3-kinase inhibitors to impair NF-B DNA binding.

EXPERIMENTAL PROCEDURES
Materials-Recombinant human tumor necrosis factor was a gift from Genentech Inc. (South San Francisco, CA). Antibodies to IKK␣ and IKK␤ were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-Akt and anti-Akt were from Cell Signaling, Inc. (Beverly, MA).

RESULTS
We previously showed that PI 3-kinase/Akt signaling induces NF-B DNA binding in 293 cells (42). However, this observation has not been observed in human umbilical vein endothelial cells (49). Experiments were therefore conducted to test whether the effect of PI 3-kinase/Akt signaling on NF-B DNA binding is cell type-specific. To accomplish this, we used EMSAs to test the effect of the inhibitors of PI 3-kinase, LY294002 and wortmannin, on TNF-induced NF-B DNA binding in seven different cell types. As illustrated in Fig. 1, NF-B DNA binding was induced by TNF in each cell type. The NF-B DNA binding complex was identified by supershifting with an antibody to p65 (RelA) (Fig. 1, far right lane in each EMSA). LY294002 or wortmannin variably affected TNF-induced NF-B DNA binding. The inhibitors completely blocked TNF-induced NF-B DNA binding in 293 cells, partially blocked binding in MCF-7, ME-180, HeLa, and NIH 3T3 cells, but had little effect on binding in H1299 cells and HUVEC.
The variable efficacy with which PI 3-kinase inhibitors blocked TNF-induced NF-B DNA binding led us to test whether Akt activation might be attenuated or defective in some cell types. Akt activity was assayed by probing Western blots prepared from lysates of TNF-treated cells with an antibody that exclusively recognizes the Akt, which is phosphorylated on serine 473 and therefore is active. TNF activated Akt in each cell type used to characterize the effect of PI 3-kinase inhibitors on NF-B DNA binding in Fig. 1, although the kinetics and extent of activation varied from one cell type to another (Fig. 2).
Activation of Akt by TNF could not account for the varied cellular sensitivity to LY294002. Because our previous work suggested that IKK␣ is the IKK kinase upon which Akt acts (42), we focused on the possibility that expression of the IKK kinases might be a determinant in the capacity of PI 3-kinase inhibitors to affect NF-B DNA binding. Expression of IKK␣ and IKK␤ in cell lines was therefore examined. Western blots showed that the amount of IKK␣ and IKK␤ varies among cell types (Fig. 3a). Additionally, we reproducibly observed faster and slower migrating forms of IKK␤ in cells. Although the basis for this heterogeneity is not yet defined, it might result from phosphorylation of IKK␤ by diverse kinases in cells. Densitometry quantified the proportion of IKK␣ to IKK␤ (Fig. 3b). In 293 cells the proportion of IKK␣ to IKK␤ was highest among the cell types tested. H1299 cells and HUVEC contain a very low proportion of IKK␣ to IKK␤. In ME180, HeLa, MCF-7, and NIH 3T3 cells, the proportion of IKK␣ to IKK␤ favors IKK␤ but not so greatly as in H1299 cells and HUVEC. These observations show that the proportion of IKK␣ to IKK␤ differs among cells. Also, a high proportion of IKK␣ to IKK␤ correlates with the ability of PI 3-kinase inhibitors to block TNF-induced NF-B DNA binding.
To demonstrate whether the proportion of IKK␣ to IKK␤ affects the role of PI 3-kinase in NF-B DNA binding, the proportion of the kinases was altered by transient transfection of 293 cells, in which the IKK␣/IKK␤ proportion is high. These, or vector-transfected control cells, were treated with wortmannin and stimulated with TNF or medium, and NF-B DNA binding was assayed. Wortmannin abrogated TNF-induced NF-B DNA binding in 293 cells transfected with empty vector but had lesser effect in cells transfected with IKK␤ (Fig. 4a). Quantitation of the observations just described showed that inhibition of PI 3-kinase in cells transfected with empty vector diminished NF-B DNA binding by 66%, whereas NF-B DNA binding was diminished by only 33% in cells transfected with IKK␤. Reproducibly, expression of IKK␤ in 293 cells increased DNA binding, an effect that may relate to its ability to phosphorylate IB␣ more effectively than IKK␣ (22,27,51,52). Western blot analysis confirmed that the cellular content of IKK␤ was greatly increased in cells transfected with IKK␤ (Fig. 4b). This experiment showed that by altering the proportion of IKK␣ to IKK␤ in favor of the latter, the sensitivity of NF-B DNA binding to PI 3-kinase inhibition is diminished. These observations support the conclusion that the proportion of IKK␣ to IKK␤ determines the sensitivity of NF-B DNA binding to blockade by PI 3-kinase inhibitors.
To support these observations, wild-type MEFs or MEFs from IKK␣-or IKK␤-deficient mice were used to assess the sensitivity of NF-B DNA binding to inhibitors of PI 3-kinase. Serum-starved wild-type, Ikk␣ Ϫ/Ϫ, and Ikk␤ Ϫ/Ϫ MEFs treated for 1 h with Me 2 SO or 20 M LY294002 were incubated with phosphate-buffered saline or 1 nM TNF for 30 min. TNF increased DNA binding in the parental or knockout MEFs, but LY294002 inhibited NF-B DNA binding only in Ikk␤ Ϫ/Ϫ cells  (Fig. 5a). Supershifting using anti-p65 showed that NF-B DNA binding complexes from parental, Ikk␤Ϫ/Ϫ, and Ikk␣Ϫ/Ϫ MEFs contained this subunit. These observations causally relate the effect of PI 3-kinase inhibitors on NF-B DNA binding and the proportion and expression of IKK␣ and IKK␤.
Our previous observations (Ref. 42 and figures herein) suggested that IKK␣ is the target for Akt in IB kinase complexes. The inability of PI 3-kinase inhibitors to affect NF-B DNA binding in wild-type MEFs therefore led us to predict that this cell type would have a low proportion of IKK␤ relative to IKK␣ when compared with other cell types. To evaluate this prediction, we first confirmed the phenotypes of wild-type, Ikk␣Ϫ/Ϫ, and Ikk␤Ϫ/Ϫ MEFs insofar as IB kinase expression is concerned. Western blot analysis of the expression of the IB kinases revealed that wild-type MEFs express IKK␣ and IKK␤, Ikk␣Ϫ/Ϫ MEFs express IKK␤ but not IKK␣, and Ikk␤Ϫ/Ϫ MEFs express IKK␣ but not IKK␤ (Fig. 5b). Having confirmed the phenotypes of the cells in this study, we compared the expression of IKK␣ to IKK␤ in H1299 cells and wild-type MEFs. It was observed (Fig. 5c) that the proportion of IKK␣ to IKK␤ was similarly low in H1299 cells and wild-type MEFs. Also, IKK␣ in human H1299 cells displayed a somewhat greater apparent molecular weight than IKK␣ in murine wildtype MEFs. The results obtained from these experiments are consistent with the conclusion that in comparisons among different cell types, a high proportion of IKK␤ relative to IKK␣ diminishes the ability of inhibitors of PI 3-kinase to block NF-B DNA binding. DISCUSSION A high molecular weight IB kinase complex called a signalsome (15, 21, 52, 53), which can be composed of IKK␣, IKK␤, and IKK␥, is activated by a group of serine-threonine kinases (21-28, 54 -57). Activated signalsomes phosphorylate IB pro-teins, promoting their dissociation from NF-B (20). Once released from the inhibitory proteins, NF-B translocates into the nucleus and activates target genes. The present study addressed the cell type specificity with which PI 3-kinase/Akt signaling affects NF-B. Investigation of transformed, immortalized, and primary cells shows that pharmacological blockade of PI 3-kinase can entirely inhibit, partially impair, or have minimal effect on NF-B DNA binding induced by TNF. In some cell types, the failure of pharmacological blockade can result from an inability of TNF to activate Akt. For example, in a comparison of PC-3 and DU-145 prostate cancer cells, we observed that TNF activated Akt in the former but not the latter cell type (58). This distinction was associated with the absence of PTEN expression in PC-3 cells and robust expression of PTEN in DU-145 cells. PTEN dephosphorylates the lipid mediators that mediate PI 3-kinse function and thereby blocks Akt activation (39,59,60).
In most cell types that we have tested, however, the different responsiveness of NF-B DNA binding to blockade by inhibitors of PI 3-kinse did not arise from inability of TNF to activate Akt. Rather, distinct cellular sensitivities arose from varied proportions of IKK␣ to IKK␤. In 293 cells the proportion of IKK␣ to IKK␤ favors the former, relative to most other cell types, in which this relationship is reversed. In HUVEC, H1299 cells, and mouse embryo fibroblasts the proportion greatly favors IKK␤. Cells containing a high proportion of IKK␣ to IKK␤ are most susceptible to inhibition of NF-B DNA binding by PI 3-kinase inhibitors. This is explained by our demonstration that Akt associates with, phosphorylates, and activates IKK␣ but not IKK␤ (42). Two additional observations causally demonstrate that IKK␣ is the target for PI 3-kinase/ Akt signaling in the IKK complex. Alteration of the proportion of IKK␣ to IKK␤ in 293 cells by transient expression of IKK␤ a, serum-starved MEFs were treated with Me 2 SO or 20 M LY294002 for 1 h prior to stimulation with vehicle or 1 nM TNF for 30 min at 37°C. NF-B DNA binding was assayed by EMSA using equal amounts of protein from lysates. WT, wild type; ab, antibody. b, equal amounts of protein from lysates of wild-type, Ikk␣Ϫ/Ϫ, or Ikk␤Ϫ/Ϫ mouse embryo fibroblasts were fractionated by SDS-PAGE, and a Western blot was probed with antibodies to IKK␣, IKK␤, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). c, a Western blot prepared from lysates of H1299 cells and wild-type mouse embryo fibroblasts was probed with antibodies to IKK␣, IKK␤, and glyceraldehyde-3-phosphate dehydrogenase.
all but eliminated the ability of wortmannin to inhibit NF-B DNA binding. In addition, studies with mouse embryo fibroblasts showed that inhibitors of PI 3-kinase had little or no effect on TNF-induced DNA binding in wild type or Ikk␣ Ϫ/Ϫ cells, the former having low expression and the latter no expression of IKK␣. However, LY294002 abrogated NF-B DNA binding induced by TNF in Ikk␤ Ϫ/Ϫ cells. The demonstration that PI 3-kinase/Akt signaling is directed toward IKK␣ explains a report (49), confirmed here, that Akt does not play a role in the activation of NF-B by TNF in HUVEC, as these cells express little IKK␣.
IKK␣ and IKK␤ are homologous but have different functions. Homozygous deletion of the IKK␤ gene decreases cytokineinduced NF-B activation and results in embryonic lethality in mice, because of severe apoptosis in the liver (61)(62)(63). Cytokine-induced NF-B activity is modestly diminished or unaffected in cells from IKK␣ knockout animals (64 -66), but they die shortly after birth because of severe skin and bone defects. IKK␣ but not IKK␤ induces processing of NF-B2 (p100) to p52 (67) and is a component of a pathway that controls mammary epithelial cell proliferation in response to receptor activator of NF-B (RANK) signaling (68).
Both kinases phosphorylate IB␣ in vitro, but IKK␤ is far more effective than IKK␣ (17,22,27,51). IKK␣ and IKK␤ homo-and heterodimerize, and heterogeneous signalsomes have been isolated from cells (17). IKK␣/IKK␤ heterodimers are components of a 900-kDa multicomponent complex, which additionally contains NEMO/IKK␥/IKKAP1, a protein that lacks kinase activity but appears to be necessary for proper organization of the complex (15, 17-19, 52, 69). In heterodimers, which form in preference to homodimers, IKK␣ inhibits basal IKK␤ activity. TNF, lipopolysaccharide, and ectopically expressed NIK (NF-B-inducing kinase), Cot, MEKK1, and Tax activate heterodimeric signalsomes (15, 16, 21, 23, 24, 54 -57, 70) by relieving repression of IKK␤ activity by IKK␣ (71). IKK␣ homodimers can be isolated from high molecular weight multimeric complexes, whereas IKK␤ homodimers are recovered from lower molecular weight complexes (17). Our demonstration that the expression of IKK␣ to IKK␤ in cells is variable and that the proportions of the kinases to one another varies among cell types is important in understanding the cell type-specific mechanisms through which NF-B can be activated. HeLa cells express a high proportion of IKK␤ to IKK␣, which is consistent with the isolation of IKK␣-IKK␤ heterodimers and IKK␤ homodimers from these cells (17). Thus, the proportion of IKK␣ to IKK␤, defined by Western blot analysis in this study, finds its counterpart in the composition of signalsomes in cells. That Akt acts on and through IKK␣ but not IKK␤ explains the cell type specificity with which PI 3-kinase/Akt signaling affects NF-B induction. The substrate specificity of Akt is consistent with its initiating activation of IKK␣-IKK␤ heterodimers and its having the capacity to activate IKK␣ but not IKK␤ homodimers. Variable expression of IKK␣ and IKK␤, as well as the presence of upstream kinases that preferentially activate one or the other IB kinase, identifies a mechanism that can account for cell type-specific regulation of NF-B activity.