UBAC1/KPC2 Regulates TLR3 Signaling in Human Keratinocytes through Functional Interaction with the CARD14/CARMA2sh-TANK Complex

Abstract

CARD14/CARMA2 is a scaffold molecule whose genetic alterations are linked to human inherited inflammatory skin disorders. However, the mechanisms through which CARD14/CARMA2 controls innate immune response and chronic inflammation are not well understood. By means of a yeast two-hybrid screening, we identified the UBA Domain Containing 1 (UBAC1), the non-catalytic subunit of the E3 ubiquitin-protein ligase KPC complex, as an interactor of CARMA2sh, the CARD14/CARMA2 isoform mainly expressed in human keratinocytes. UBAC1 participates in the CARMA2sh/TANK complex and promotes K63-linked ubiquitination of TANK. In human keratinocytes, UBAC1 negatively regulates the NF-κF-activating capacity of CARMA2sh following exposure to poly (I:C), an agonist of Toll-like Receptor 3. Overall, our data indicate that UBAC1 participates in the inflammatory signal transduction pathways involving CARMA2sh.

Keywords: BCL10; CARD14; CARMA2sh; NF-κB; TANK; UBAC1; Ubiquitin; psoriasis.

Figure 1

UBAC1 associates to CARMA2sh. (A) HEK293T cells were co-transfected with a plasmid encoding for FLAG-tagged UBAC1 together with an HA-tagged expression vector empty or encoding for CARMA2sh. A total of 24 h later, lysates were immunoprecipitated with anti-HA antibodies and analyzed for co-precipitating FLAG-UBAC1 by western blot assay. Data shown are representative of three independent experiments. (B) Affinity purified GST-UBAC1 was incubated with HaCaT cell lysates and western blot analysis was carried out with anti-CARMA2 and re-probing the membrane with anti-GST antibody. Data shown are representative of three independent experiments. (C,D) HEK293T cells were co-transfected with a plasmid encoding for FLAG-tagged UBAC1 together with expression vectors encoding for the indicated HA-tagged polypeptides. A total of 24 h later, lysates were immunoprecipitated with anti-FLAG antibodies and analyzed for coprecipitating proteins by western blot assay. Data shown are representative of three independent experiments.

Figure 2

UBAC1 represses TLR3 signaling. (A) NHEK cells were infected with a lentiviral vector expressing UBAC1 or control GFP. A total of 48 h later, cells were left in PBS or exposed poly (I:C) (20 μg/mL) for 24 h, and the expression level of selected NF-κB and IRF3 target genes was monitored by real time PCR. Graphs show the fold-change with respect to the cells left in PBS. Data shown are representative of five independent experiments done in triplicate. Data were analyzed by Student’s t-test (* p-value ≤ 0.05; ** p-value ≤ 0.02). (B) NHEK cells were infected with retroviral vectors encoding for two different shRNAs targeting UBAC1 or a control shRNA (scramble). After selection, cells were left in PBS or exposed to poly (I:C) (20 μg/mL) for 24 h, and the expression level of selected NF-κB target genes were quantified by real-time PCR.

Figure 3

UBAC1 promotes K63-linked ubiquitination of TANK. (A) HEK293 cells were transfected with the indicated expression vectors. A total of 24 h later, cell lysates were immunoprecipitated with anti-HA antibody, separated by SDS-PAGE and transferred onto membranes subsequently probed with anti-ubiquitin. Statistical analysis for the blot shown in provided as supplementary Figure S1. (B) HEK293 cells were co-transfected with HA-tagged ubiquitin mutants and FLAG-tagged UBAC1 and TANK. The number indicates the only lysine residue remaining in the ubiquitin molecule. Immunoprecipitates with anti-TANK antibody were resolved by SDS-PAGE and blotted onto a membrane subsequently probed with anti-HA or anti- ubiquitin (P4D1). Statistical analysis for the blot shown in provided as supplementary Figure S1.

Figure 4

UBAC1 affects viability and proliferation in primary keratinocytes. (A) NHEK cells were infected with a lentiviral vector empty or expressing UBAC1. A total of 48 h later, cells were trypsinized, counted in a Neubauer chamber, and 10⁴ cells were seeded in each well of a 96-multi well plate. Cell viability was determined by MTT assay at seeding (right after cell attached to the bottom of the well) and 24, 48 and 72 h later. Representative phase contrast micrographs of transfected NHEK are also shown (magnification 4×). Graphs show the relative Optical Density at 570 nm at different time points. Data shown are representative of five independent experiments done in triplicate. Data were analyzed by Student’s t-test (* p-value ≤ 0.05). (B) NHEK cells were infected with a lentiviral vector expressing UBAC1-silencing shRNAs or scramble. A total of 48 h later, cells were trypsinized, counted with Neubauer chamber, and 10⁴ (lower confluence) or 3 × 10⁴ (higher confluence) were seeded in each well of a 96-multi well plate. MTT data and micrographs and statistical analysis were performed as in Figure 4A. When seeded at higher confluence, differences between UBAC1-silenced NHEK and control cells are evident only 144 h after seeding (micrographs not shown).