Malt1-dependent RelB cleavage promotes canonical NF-kappaB activation in lymphocytes and lymphoma cell lines

Abstract

The protease activity of the paracaspase Malt1 contributes to antigen receptor-mediated lymphocyte activation and lymphomagenesis. Malt1 activity is required for optimal NF-κB activation, but little is known about the responsible substrate(s). Here we report that Malt1 cleaved the NF-κB family member RelB after Arg-85. RelB cleavage induced its proteasomal degradation and specifically controlled DNA binding of RelA- or c-Rel-containing NF-κB complexes. Overexpression of RelB inhibited expression of canonical NF-κB target genes and led to impaired survival of diffuse large B-cell lymphoma cell lines characterized by constitutive Malt1 activity. These findings identify a central role for Malt1-dependent RelB cleavage in canonical NF-κB activation and thereby provide a rationale for the targeting of Malt1 in immunomodulation and cancer treatment.

Fig. 1.

Malt1 cleaves RelB. (A–E) Indicated lymphocyte cell lines or primary lymphocytes were stimulated using PMA and ionomycin (PMA+iono) or cross-linked anti-CD3 and anti-CD28 antibodies (CD3/CD28) for 30 min or the indicated times, and postnuclear lysates were analyzed by Western blotting (WB). Where indicated, cells were treated with the Malt1 inhibitor VRPR-fmk or solvent control before and during stimulation. In some experiments (B–E), cells were pretreated with the proteasome inhibitor MG132 or solvent alone (DMSO) before stimulation. (D) Splenocytes from Malt1 wild-type, heterozygous, or knockout mice were pretreated and stimulated as indicated. (E) Jurkat T cells were transduced with either control or Carma1 shRNA and pretreated with MG132 before stimulation. In all figure parts, black and open arrowheads indicate uncleaved and cleaved forms, respectively, of the Malt1 substrates RelB and Bcl-10. Data are representative of at least three (A–C) or two (D and E) independent experiments.

Fig. 2.

Malt1 cleaves human RelB after Arg-85. (A and B) Expression constructs for Malt1 (WT or catalytically inactive C464A mutant), Bcl-10, and RelB (WT or noncleavable R85G mutant) were cotransfected in the indicated combinations into 293T cells and postnuclear lysates were analyzed by Western blotting as indicated. (C) Alignment of Malt1-dependent cleavage sites in human and mouse Bcl-10 (hBcl-10 and mBcl-10), human A20 (hA20), and human RelB (hRelB). Cleavage occurs after the conserved Arg residue (R) shown in bold, the preceding Ser residue (S) is underlined. (D) The in vitro proteolytic activity of Strep–Malt1 bound to Streptactin beads was assessed by incubation with either LRSR-amc or LVSR-amc (corresponding to the Bcl-10 or RelB cleavage site, respectively) in cleavage assay buffer at 30 °C. Graph shows cleavage activity using LVSR-amc relative to LRSR-amc. Results are expressed as means ± SD (n = 3). Data are representative of at least three (A and B) or two (D) independent experiments. Black and open arrowheads indicate uncleaved and cleaved proteins, respectively.

Fig. 3.

RelB inhibits expression of NF-κB gene targets in T cells. (A and B) Jurkat T cells (A) or Jurkat T cells stably expressing an IL-2 reporter construct (B) were transfected with the indicated expression constructs, treated with or without PMA and ionomycin for 16 h, and assessed for NF-κB activation (A) or IL-2 gene activation (B) by luciferase reporter assay. The corresponding expression level of the constructs is shown by Western blot. Data are representative of at least three independent experiments. (C) Naïve human primary T cells were transfected with either scrambled (scr) or RelB siRNA. Forty-eight hours after transfection, cells were stimulated with agonistic anti-CD3 and anti-CD28 antibodies and IL-2 secretion was quantified 16 h later by ELISA. (D) Jurkat cells were lysed and assessed for the binding of endogenous RelB to the indicated proteins. Position of the Ig heavy chain (Ig) is indicated. (E) Jurkat T cells were pretreated for 30 min with the Malt1 inhibitor VRPR-fmk and stimulated as indicated. (F) Jurkat cells transduced with RelB or control vector were stimulated for the indicated times with PMA and ionomycin and DNA binding of indicated Rel subunits was assessed by TransAM DNA-binding ELISA. In all figure parts, results are expressed as means ± SD from three independent experiments (**P ≤ 0.005).

Fig. 4.

RelB is constitutively cleaved in ABC DLBCL cell lines. (A) DLBCL cell lines of the GCB (BJAB) or ABC type (all others) were treated with the proteasome inhibitor MG132 or solvent control for the indicated times, in the presence or absence of the Malt1 inhibitor VRPR-fmk. Postnuclear lysates were analyzed for expression and cleavage of RelB. Bcl-10 and IκBα blots serve as control for efficient Malt1 and proteasome inhibition, respectively. A nonspecific band (n.s.) of the anti–p-IκBα blot serves as a loading control (B) DLBCL cell lines of the GCB (BJAB), or ABC subtype (all others) were pretreated with 75 μM of the Malt1 inhibitor VRPR-fmk or solvent control for 48 h, and RelB expression was assessed by Western blotting of postnuclear cell extracts. Black and open arrowheads indicate uncleaved and cleaved proteins, respectively. Data in A and B are representative of at least three independent experiments. (C) OCI-Ly3, OCI-Ly10, and HBL-1 cells were treated with 50 μM of the Malt1 inhibitor VRPR-fmk or solvent control for 16 h, and DNA binding of individual NF-κB subunits was assessed by TransAM assay. Data are expressed as means ± SD (n = 3). (D) Gene expression profiling of the ABC DLBCL cell line OCI-Ly10 was performed following induction of RelB expression at the indicated time points (24, 48, 72, and 96 h). Gene expression changes are depicted according to the color scale shown. (Lower) Significance of the RelB-mediated down-regulation of the NF-κB signature. Gene expression measurements for the component genes in the NF-κB signature (Upper) were averaged for each of the indicated time points.

Fig. 5.

RelB overexpression impairs the survival of ABC DLBCL lines. Cell lines derived from the indicated DLBCL subtypes were retrovirally transduced with an expression vector for human RelB together with green fluorescent protein (GFP). Live GFP⁺ cells were enumerated by flow cytometry on the indicated days postretroviral transduction and normalized to the value at day 2 following retroviral transduction. Data are representative of three independent experiments.