Psoriasis mutations disrupt CARD14 autoinhibition promoting BCL10-MALT1-dependent NF-κB activation

Abstract

Inherited and de novo mutations in the CARD14 gene promote the development of psoriasis, an inflammatory disease of the skin. Caspase recruitment domain-containing protein 14 (CARD14) is a member of the CARMA protein family that includes the structurally related CARD11 adaptor that mediates NF-κB activation by antigen receptors. We investigated the mechanism by which CARD14 mutation in psoriasis activates NF-κB. In contrast with wild-type CARD14, CARD14(E138A) and CARD14(G117S) psoriasis mutants interacted constitutively with BCL10 and MALT1, and triggered BCL10- and MALT1-dependent activation of NF-κB in keratinocytes. These alterations disrupted the inhibitory effect of the CARD14 linker region (LR) on NF-κB activation by facilitating BCL10 binding. Therefore, psoriasis mutations activated CARD14 by a mechanism analogous to oncogenic CARD11 mutations in non-Hodgkin B cell lymphomas. CARD14(E138A) also stimulated MALT1 paracaspase activity and activated both ERK1/2 and p38α MAP kinases. Inhibition of MALT1 with mepazine reduced CARD14(E138A)-induced expression of specific psoriasis-associated transcripts in keratinocytes. Our results establish the mechanism whereby gain-of-function CARD14 variants, which induce psoriatic disease in affected individuals, activate pro-inflammatory signalling.

Keywords: NF-κB; caspase recruitment domain-containing protein 14 (CARD14); keratinocytes; mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1); psoriasis.

Figure 1. Psoriasis-associated CARD14 mutants require BCL10 and MALT1 to activate NF-κB

(A) Schematic of CARD14 domains and psoriasis-associated mutations. (B, C) HEK293 cells were co-transfected with vectors encoding the indicated V5-CARD14 variants (0.1–1.2 μg), FLAG-MALT1 (1 μg) and HA-BCL10 (0.6–1.2 μg). Anti-FLAG immunoprecipitates were immunoblotted with the indicated antibodies. Representative of at least two independent experiments. (D, E) HaCaT keratinocytes were co-transfected with vectors encoding the indicated FLAG-CARD14 variants (0.125–0.2 μg) or EV, pNFκB-Luc and pRL-TK. After 24 h, NF-κB activity was determined by luciferase assay (mean ± S.E.M. of three independent experiments). FLAG-CARD14 expression was determined by immunoblotting of lysates. Significance determined by t test. (F, G) HaCaT keratinocytes were transfected with Bcl10 or Malt1 siRNA pools or control non-targeting pool. After 48 h, cells were transfected with FLAG-CARD14 variants (0.05–0.1 μg), pNFκB-Luc and pRL-TK vectors and cultured for a further 24 h. NF-κB activity was determined as in (D) (mean ± S.E.M. of three independent experiments). Representative expression of FLAG-CARD14, endogenous BCL10 and endogenous MALT1 was determined by immunoblotting of corresponding lysates. Significance determined by two-way ANOVA.

Figure 2. Abrogation of CARD14 inhibitory LR activity by deletion or psoriasis mutation induces BCL10 association

(A, B) HaCaT keratinocytes were co-transfected with FLAG-CARD14 (0.125–0.2 μg), pNFκB-Luc and pRL-TK plasmids. NF-κB activity was determined as in Figure 1(D) (mean ± S.E.M. of three independent experiments). Representative expression of FLAG-CARD14 and tubulin in corresponding lysates was determined by immunoblotting. Significance determined by−two-way ANOVA. FC; fold change. (C) FC NF-κB activation of mutant compared with WT CARD14, in the presence or absence of the LR (calculated from data in A and B). Mean ± S.E.M. of three independent experiments. Significance determined by t test. (D) HEK293 cells were co-transfected with vectors encoding the indicated V5-CARD14 variants (0.1–1 μg) and FLAG-BCL10 (0.6–0.8 μg). Anti-FLAG immunoprecipitates were immunoblotted with the indicated antibodies. Representative of three independent experiments.

Figure 3. CARD14^E138A mutation activates MALT1 paracaspase activity

(A) HEK293 cells were co-transfected with vectors encoding V5-CARD14 variants (0.1–0.4 μg), WT or C464 FLAG-MALT1 (0.8–1.2 μg) and HA-BCL10 (0.8–1.4 μg). Active MALT1 was assayed by ABP pull down and immunoblotting. Representative of three independent experiments. (B) HaCaT keratinocytes were co-transfected with FLAG-CARD14 variant (0.125–0.2 μg) or EV, pNFκB-Luc and pRL-TK vectors. 5 h after transfection, cells were treated with mepazine or DMSO vehicle and lysed after further 24 h culture. NF-κB activity was determined as in Figure 1(D) (mean ± S.E.M. of three independent experiments). Representative expression of FLAG-CARD14 was determined by immunoblotting of corresponding lysates. Significance determined by two-way ANOVA.

Figure 4. CARD14^E138A-induced gene expression is dependent on IKK2, MALT1, ERK1/2 and p38α activity

(A) HaCaT-TR or HaCaT-CARD14^E138A cells were treated with tetracycline for the indicated times. CARD14^E138A expression and phosphorylation of indicated proteins was determined by immunoblotting. Representative of three independent experiments using two independently generated stable cultures. (B–D) HaCaT-CARD14^E138A cells were pre-treated with BI605906 (10 μM, IKK2 inhibitor), mepazine (MALT1 inhibitor), PD0325901 (0.1 μM, MEK1/2 inhibitor), VX-745 (1 μM, p38α inhibitor) or DMSO vehicle for 1 h, and cultured for a further 6 h after subsequent tetracycline addition. Gene expression was assayed by qRT-PCR (mean ± S.D., n = 3). Significance determined by two-way ANOVA. Representative of at least two independent experiments using independently generated stable cultures.