Activation of alternative NF-kappa B pathway by human herpes virus 8-encoded Fas-associated death domain-like IL-1 beta-converting enzyme inhibitory protein (vFLIP)

Abstract

The Kaposi's sarcoma-associated herpesvirus (KSHV, also called human herpesvirus 8) has been linked to KS and primary effusion lymphoma (PEL) in immunocompromised individuals. We report that PEL cell lines have constitutive active alternative NF-kappa B pathway and demonstrate high-level expression of NF-kappa B2/p100 precursor and its processed subunit p52. To elucidate the mechanism of activation of the alternative NF-kappa B pathway in PEL cells, we have investigated the role of KSHV-encoded viral Fas-associated death domain-like IL- beta 1-converting enzyme inhibitory protein (vFLIP) K13. We demonstrate that stable expression of K13, but not other FLIPs, in a variety of cell lines constitutively up-regulates p100/NF-kappa B2 expression and leads to its processing into the p52 subunit. K13-induced up-regulation and processing of p100 critically depends on the I kappa B kinase (IKK)alpha/IKK1 subunit of the IKK complex, whereas IKK beta/IKK2, receptor-interacting protein, and NF-kappa B-inducing kinase are dispensable for this process. Silencing of endogenous K13 expression by siRNA inhibits p100 processing and cellular proliferation. Our results demonstrate for the first time, to our knowledge, that KSHV vFLIP K13 is required for the growth and proliferation of PEL cells and alternative NF-kappa B pathway plays a key role in this process. Therapeutic agents targeting the alternative NF-kappa B pathway may have a role in the treatment of KSHV-associated lymphomas.

Fig. 1.

Status of alternating NF-κB pathway in vFLIP K13-expressing cells. (A) Alternative NF-κB pathway is constitutively active in PEL cell lines. Western blots demonstrating constitutive p100 up-regulation and processing in three PEL cell lines (BC-1, BC-3, and BCBL-1) as compared with CEM cells. The actin blot shows equal protein loading. The numbers below the blots represent the intensity of the p52 band relative to the actin band. (B) Expression of KSHV vFLIP K13, but not of other FLIPs, leads to p100 up-regulation and its processing into the p52 subunit. Cell lysates from H460 cells expressing an empty vector or the indicated Flag epitope-tagged FLIPs were analyzed by immunoblotting with an antibody against p52. The blot was reprobed with Flag and actin antibodies to demonstrate the expression of various FLIPs and equal loading of the lanes, respectively. (C and D) Retroviral-mediated expression of vFLIP K13 leads to p100 up-regulation and its processing into p52 subunit in HeLa and CEM cells, as measured by Western blotting by using a p52 antibody.

Fig. 2.

RIP and NIK are not required for K13-induced p100 up-regulation or processing. (A) Wild-type (WT) and RIP-deficient Jurkat cells were transduced with an empty retroviral vector or a vector expressing Flag epitope-tagged K13. Expression of p100/p52 (Top), Flag-K13 (Middle), and actin (Bottom) was determined by Western blotting with the indicated antibodies. (B) Wild-type (WT) and NIK knockout (NIK-/-) MEF cells were transduced with an empty retroviral vector or a vector-expressing Flag epitope-tagged K13. Expression of p100/p52, Flag-K13, and actin was determined by Western blotting with the indicated antibodies.

Fig. 3.

vFLIP K13-mediated p100 up-regulation and processing depends on IKK1/IKKα but is independent of IKK2/IKKβ. (A) Cellular extracts from MEFs (WT, IKK1-/-, and IKK2-/-) stably expressing empty vector or Flag-K13 were subjected to Western blot analysis with the indicated antibodies. K13-mediated p100 up-regulation and processing is markedly reduced in IKK1-/- cells, whereas loss of IKK2 has no significant effect. (B) Role of IKK1 and IKK2 in K13-induced canonical NF-κB activation as measured by phosphorylation of IκBα. Cellular extracts from MEF cells expressing an empty vector or K13 were immunoblotted with the indicated antibodies. (C) Kinase activity of IKK1 is required for p100 processing. The indicated constructs were transfected into 293T cells. The amount of dominant-negative IKK1 (IKK1-KM) construct (1.5 μg) was three times the amount of K13-Flag (0.5 μg) and p100-HA (0.5 μg) constructs, respectively, and the total amount of transfected DNA was kept constant by adding an empty vector. Approximately 36 h after transfection, cell lysates were prepared and immunoblotted with the indicated antibodies. The IKK1-KM mutant effectively blocks K13-mediated p100 processing.

Fig. 4.

vFLIP K13 directly interacts with p100 and induces p100 ubiquitination. (A) p100 phosphorylation is required for p100 processing into its p52 subunit. Wild-type p100 (p100-WT) and its mutant construct (p100-SS/AA; where Ser residues at sites 866 and 870 are mutated to Ala; SS866/870AA) were used to transfect 293 cells along with an empty vector or Flag-tagged K13. A GFP-encoding plasmid was cotransfected to monitor transfection efficiency. After 48 h, cells lysates were prepared and immunoblotted with the indicated antibodies. vFLIP K13 induces the processing of p100-WT but not of the p100SS/AA mutant. (B) vFLIP K13-induced p100 processing is associated with its ubiquitination. A quantity of 293 cells were cotransfected, as indicated with 500 ng of vector encoding Flag-p100 together with an empty vector or 500 ng of plasmid encoding HA-ubiquitin (Ubi-HA) and 500 ng of Flag-tagged vFLIP-K13 or Myc tagged-Tax. After 48 h, cells lysates were prepared and immunoblotted with the indicated antibodies. (C) KSHV vFLIP K13 directly interacts with p100. Whole-cell extracts from 293 cells transfected with HA-tagged p100 along with either an empty vector or Flag-K13 were immunoprecipitated by using mouse IgG control beads (lane C) or Flag beads (lane F). The interaction between K13 and p100 in the immunoprecipitates was detected by using rabbit polyclonal HA antibody. Western blot with a GFP antibody confirms equal transfection efficiency and antibody against the Flag-tag confirms the expression of Flag-tagged vFLIP K13. (D) vFLIP K13 enhances the interaction between p100 and IKK1 and stimulates p100 processing. Whole-cell extracts from 293 cells transfected with Flag-tagged IKK1 and HA-tagged p100 along with either an empty vector or K13 were immunoprecipitated by using mouse IgG control beads (lane C) or Flag beads (lane F). The interaction between IKK1 and p100 in the immunoprecipitates was detected by using rabbit polyclonal HA antibody. Western blot with a GFP antibody confirms equal transfection efficiency and with a rabbit polyclonal antibody against the K13 confirms the expression of vFLIP K13. (E) vFLIP K13 specifically interacts with endogenous p100 and IKK1. BC-3 cells were transduced with an empty retroviral vector or vectors expressing Flag epitope-tagged vFLIP K13 or Flag-tagged cFLIP_L/MRIT-α1. Lysates from stably transduced cells were immunoprecipitated by using mouse IgG control beads (lane C) or Flag beads (lane F), and the presence of coimmunoprecipitated p100 and IKK1 was analyzed by Western blotting with the indicated antibodies.

Fig. 5.

Effects of siRNA-mediated K13 down-regulation in a BC-3 PEL cell line. (A) Flow cytometry analysis demonstrating high-efficiency infection of BC-3 PEL cells with siRNA-encoding lentiviral vectors as determined by GFP expression. Analysis was performed 72 h after infection with the indicated lentiviral constructs. (B and C) BC-3 cells were left uninfected (UI) or infected with lentiviral vectors encoding a control siRNA or siRNAs directed against K13 and p100. Approximately 72 h after infection, cell lysates were prepared and used in Western blot with the indicated antibodies. Equal loading of protein was demonstrated by Western blotting with antibodies against actin or tubulin. The numbers below the blots represent the intensity of the K13 and p52 bands relative to tubulin and actin bands, respectively. (D and E) Effect of vFLIP-K13 and p100 gene silencing on BC-3 cell number. BC-3 cells were either left uninfected or infected with the indicated siRNA-encoding lentiviral constructs. At 72 h after infection, viable cell numbers were measured by using (3–4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (D), and Cell titer-Glo luminescence reagent (E). The data represents mean ± SD of a representative of three independent experiments with similar results. (F) Effect of vFLIP-K13 and p100 gene silencing on the proliferation of BC-3 cells. Cells were infected with the indicated siRNA-encoding lentiviral vectors and were grown for 24 h in the presence of BrdUrd reagent. ELISA was performed to detect the amount of BrdUrd-labeled DNA as suggested by the manufacturer (Exalpha Biologicals). The data represent mean ± SD of a representative of three independent experiments with similar results.