Mutant human cells with constitutive activation of NF-kappaB

Abstract

We have used a genetic approach to generate eight different mutant human cell lines in which NF-kappaB is constitutively activated. These independent clones have different phenotypes and belong to several different genetic complementation groups. In one clone inhibitor of kappaB(IkappaB) kinase is constitutively active, but in the seven others it is not, despite the fact that IkappaB is degraded in all eight clones. Thus, IkappaB kinase-independent mechanisms of IkappaB degradation and NF-kappaB activation are predominant in these mutants. Biochemical analyses of the mutants revealed that they fall into at least five different categories, differing in the sets of upstream kinases that are activated, confirming multiple mechanisms of NF-kappaB activation. By introducing a retroviral cDNA library into the Ras C6 cell line, with constitutively active NF-kappaB, followed by selection for functional complementation, we isolated a cDNA encoding a C-terminal fragment of enolase 1 and identified it as negative regulator of NF-kappaB.

Fig. 1.

Scheme for generating constitutive mutants in human embryonic kidney (HEK) cells and EMSA of parental and eight mutant clones. Cell extracts were made from parental C6 and mutant cells. Extracts were analyzed by EMSA for the ability of NF-κB to bind to the NF-κB-consensus sequence of the IP-10 gene. The faster moving band is the p50/p65 heterodimer, and the slower moving band is the p65 homodimer.

Fig. 2.

The E-selectin-luciferase reporter gene and the endogenous IL-8 gene are constitutively activated in the mutant cells. (A) Reporter assay. Cells were transfected transiently with the NF-κB-dependent reporter plasmid pE-selectin-luciferase (10 μg) and pSV2-βgal (1 μg) to normalize for transfection efficiencies. The cells were lysed 48 h after transfection and assayed for luciferase activity. Luciferase activity was normalized to β-galactosidase. The fold increase relative to C6 parental cells is shown. (B) IL-8 mRNA expression is constitutively activated in some of the mutant cells. Cells were either left untreated (-) or treated with TNF (T) at 20 ng/ml for 4 h. The cells were washed and lysed, and an equal amount of total RNA was assayed for the expression of IL-8 mRNA by the Northern method. A different analysis of the C6 cell line (Bottom) confirms that there is no basal expression of IL-8 in these cells.

Fig. 3.

Dominance and complementation. Each of the constitutive mutants tested was cotransfected independently with pBABE-Hygro and pBABE-Puro. The mutants P2Z1, Z2, and Z5 were fused either to parental C6 or C8 cells (dominance test) or to each other by using polyethylene glycol. The heterokaryons were selected in the presence of both hygromycin and puromycin. Pools of the clones resulting from each fusion were analyzed by EMSA by using an IP-10 probe.

Fig. 4.

IκBα and IκBβ are constitutively degraded in most mutant cell lines whereas IKK is activated above basal levels only in mutant Z12. (A) Parental C6 and mutant cells were either untreated (-) or treated with cycloheximide (CHX) for 3 h. Cells were washed and lysed, and whole cell extracts were prepared. The amounts of protein in the extracts were quantitated, and equal amounts of the protein were analyzed by SDS/PAGE by using antibodies against IκBα. The same blot was probed with anti-IκBβ. The proteins were visualized by enhanced chemiluminescence. (B) IKK is activated above basal levels only in mutant Z12. Parental C6 and mutant cells were either untreated or treated with IL-1 for 15 min. The cells were washed and lysed, and the extracts were incubated with anti-IKKα antibody overnight at 4°C. The IKK complex was pulled down by using Protein A beads. The beads were washed, and a kinase assay was performed for 1 h in the presence of γ-labeled ATP and GST-tagged IκBα (residues 1–54) as a substrate. The beads were boiled in loading buffer, the proteins were separated by SDS/PAGE, and labeled IκBα was visualized by autoradiography.

Fig. 5.

Akt and p90^rsk1 are activated in some constitutive mutants. (A) Akt is constitutively phosphorylated in mutants Z2, Z12, and Z3. Extracts of untreated and IL-1-treated (15 min) C6 cells and untreated mutant cells were analyzed by SDS/PAGE by using anti-phospho-Akt antibody. The faster migrating band corresponds to phosphorylated Akt (pAkt). The slower moving band in Z2, Z3, and Z12 lanes may correspond to a hyperphosphorylated or acetylated form of Akt. (B) The same extracts were analyzed by SDS/PAGE by using anti-phospho-p44/42 (Top), anti-p44/42 (Middle), and anti-phosphop90^rsk1 (Bottom). The proteins were visualized by enhanced chemiluminescence. p90^rsk1 and p44/42 are constitutively activated in mutants Z12, Z3, and Z13.

Fig. 6.

Phenotype of complemented clones of RasC6 cells. (A) EMSA for NF-κB in RasC6 cells and enolase 1-complemented clones (P2C8, P3C1, and P4C6; no cDNA was recovered from P1C4), untreated or treated with IL-1 for 20 min. (B) Enolase 1 inhibits IL-1-stimulated NF-κB promoter activity in the RasC6 cell line. Cells were cotransfected transiently with increasing amounts of pCR 3.1 enolase 1 and with 1 μg of the NF-κB-dependent reporter E-selectinluciferase and pSV2-β-gal plasmid. Forty-eight hours posttransfection, cells were incubated with IL-1 for 4 h. Luciferase activity was normalized to β-galactosidase to correct for transfection efficiency.