KIAA0317 regulates pulmonary inflammation through SOCS2 degradation

Abstract

Dysregulated proinflammatory cytokine release has been implicated in the pathogenesis of several life-threatening acute lung illnesses such as pneumonia, sepsis, and acute respiratory distress syndrome. Suppressors of cytokine signaling proteins, particularly SOCS2, have recently been described as antiinflammatory mediators. However, the regulation of SOCS2 protein has not been described. Here we describe a mechanism of SOCS2 regulation by the action of the ubiquitin E3 ligase KIAA0317. KIAA0317-mediated degradation of SOCS2 exacerbated inflammation in vitro, and depletion of KIAA0317 in vivo ameliorated pulmonary inflammation. KIAA0317-knockout mice exhibited resistance to LPS-induced pulmonary inflammation, while KIAA03017 reexpression mitigated this effect. We uncovered a small molecule inhibitor of KIAA0317 protein (BC-1365) that prevented SOCS2 degradation and attenuated LPS- and P. aeruginosa-induced lung inflammation in vivo. These studies show KIAA0317 to be a critical mediator of pulmonary inflammation through its degradation of SOCS2 and a potential candidate target for therapeutic inhibition.

Keywords: Inflammation; Innate immunity; Pulmonology; Ubiquitin-proteosome system.

Figure 1. SOCS2 protein is ubiquitinated and degraded during pulmonary inflammation.

(A) Immunoblot analysis of leukocyte pellets from ARDS patients or control serum samples. Data represent mean ± SEM, (n = 7–10; ***P < 0.001 compared with control, Mann-Whitney U test). (B) Cell type clustering results of control lung tissue cells from single-cell RNA sequencing of SOCS2 transcript. (C) Immunoblotting following CHX (50 μg/mL), MG132 (20 μM), or leupeptin (50 μg/mL) treatment for the indicated times with ectopic expression of V5-tagged SOCS2 in MLE cells. (D) Immunoblotting following overexpression of ubiquitin (Ubi) in MLE cells. (E) UbiCRest analysis of SOCS2 ubiquitination following immunoprecipitation from MLE cells; digestion supernatant is shown below, with deubiquitinase enzymes indicated by asterisks. Ctrl, control; CD, catalytic domain. (F) Immunoblot analysis following expression of SOCS2-V5 and ubiquitin lysine-to-arginine mutants. (G) Immunoblotting analysis of MLE cells following SOCS2-HIS and HA-ubiquitin expression. Following HIS PD, eluate was immunoblotted for HA ubiquitin signal. (H) qPCR analysis of MLE cells treated with LPS for the indicated times. Data represent mean values ± SEM (n = 3; NS, P > 0.05 compared with 0 hours; Student’s 2-tailed unpaired t test). (C–G) Data are representative of n = 2–3 independent experiments.

Figure 2. KIAA0317 ubiquitinates and degrades SOCS2 in response to bacterial insult.

(A) Protein staining of eluate from control GST and SOCS2-GST bait and capture of BEAS-2B lysate prior to mass spectrometry analysis. (B) Immunoblotting following CHX treatment (50 μg/mL) for the indicated times with ectopic expression of empty plasmid or V5-tagged KIAA0317 in MLE cells. (C) SOCS2 protein densitometry (normalized to actin) for B (n = 2). (D) Immunoblotting following CHX treatment (50 μg/mL) for the indicated times with ectopic expression of Ctrl shRNA or Kiaa0317 shRNA in MLE cells. (E) SOCS2 protein densitometry (normalized to actin) for D (n = 2). (F and G) SOCS2 immunoblotting following (F) KIAA0317 and (G) UBE3B expression in MLE cells. (H) In vivo ubiquitination assay and SOCS2 immunoprecipitation following coexpression with KIAA0317 and ubiquitin. (I) In vitro ubiquitination assay of SOCS2 protein. (J) Immunoblot analysis of SOCS2 PD following KIAA0317 expression and LPS exposure. (K and L) NF-κB promoter activity assays in 293T cells transfected with control shRNA (CON) or KIAA0317 shRNA and exposed to (K) LPS (10 μg/mL) or (L) TNF (10 ng/mL) treatment for the indicated times. Data represent mean values ± SEM (n = 4; *P < 0.05; ****P < 0.0001; NS, P > 0.05 compared with the indicated treatment, with control shRNA 6-hour treatment (†), or with control shRNA 18-hour treatment (#). Two-way ANOVA with Bonferroni’s multiple-comparisons test. (F–J) Data are representative of n = 2–3 independent experiments.

Figure 3. KIAA0317 targets SOCS2 phosphodegron for binding and ubiquitination.

(A) Schematic of SOCS2 deletional or point mutants used in mechanistic studies. (B) Immunoblot analysis of MLE cells expressing SOCS2 lysine mutants followed by CHX (50 μg/mL) treatment for the indicated times. (C) Deletion mapping of SOCS2 site for binding KIAA0317. (D) Immunoblot analysis of MLE cells expressing SOCS2 S52N followed by CHX (50 μg/mL) treatment for the indicated times. (E) Immunoblot analysis of SOCS2 S52N binding KIAA0317. (F) Immunoblotting of MLE cells cotransfected with WT SOCS2 or the S52N or K173R mutant without or with KIAA0317. The lanes were run on the same gel but were noncontiguous. (G) Schematic of KIAA0317 deletional or point mutants used in mechanistic studies. (H and I) Deletion and point mapping of KIAA0317 site for binding SOCS2. (J) KIAA0317 P779L binding assay with SOCS2. (K) KIAA0317 Peptide binding assay. (L) Immunoblot analysis of MLE cells expressing KIAA0317 WT and P779L at increasing doses. In B–F and H–L, data are representative of n = 2 independent experiments.

Figure 4. Kiaa0317 knockdown ameliorates Pseudomonas-induced lung injury in vivo.

(A) Treatment strategy for lentiviral infection (1 × 10⁷ PFU/mouse) and PA103 exposure (1 × 10⁴ CFU/mouse). Mice were euthanized, and lungs were lavaged with saline and harvested. Con, control. (B–D) Protein concentration, cell count, and bacterial count measurements in BALF from treated mice (n = 6–8 mice per group). In D, box represents interquartile range; line, median; and whiskers, highest and lowest values. (E and F) ELISA analysis of BALF cytokines IL-6 and TNF. (G) Survival studies of mice exposed to PA103 (i.t. 1 × 10⁵ CFU/mouse, n = 8 mice per group). Mice were monitored over time; moribund, preterminal animals were immediately euthanized and recorded as deceased. Kaplan-Meier survival curves were generated and compared; data represent mean values (n = 8 mice per group). (H) Immunoblot analysis from representative murine lung homogenate; n = 4 per group. (I) Histology of murine lungs following H&E staining; scale bar: 100 μm. **P < 0.01, ***P < 0.001, ****P < 0.0001, NS: P > 0.05 compared with control shRNA, 2-tailed unpaired Student’s t test (B–F) or compared with control shRNA, log-rank (Mantel-Cox) test (G).

Figure 5. Kiaa0317 knockout confers protection from LPS-induced lung inflammation.

(A) sgRNA target site amplification from Kiaa0317^+/+, Kiaa0317^+/–, and Kiaa0317^–/– mice. (B) Kiaa0317 transcript levels among Kiaa0317^+/+, Kiaa0317^+/–, and Kiaa0317^–/– mice. Data represent mean values ± SEM (n = 3 mice). (C–G) Kiaa0317^+/+, Kiaa0317^+/–, and Kiaa0317^–/– mice were i.t. inoculated with LPS (3 mg/kg) for 18 hours. Data represent mean values ± SEM (n = 3 mice per group). (C and D) Protein concentration and cell count from BALF. (E) BALF IL-6 concentration. (F) BALF TNF concentration. (G) BALF IL-1β concentration. (H) Histology of murine lungs following H&E staining; scale bar: 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the indicated group or to sex-specific +/+ group; 1-way ANOVA with Tukey’s multiple-comparisons (B) or 1-way ANOVA with Dunnett’s multiple-comparisons test (C–G).

Figure 6. Reexpression of KIAA0317 in Kiaa0317^–/– mice ablates resistance to inflammation.

Mice were infected i.t. with empty lentivirus or lentivirus encoding KIAA0317 WT or KIAA0317 P779L (1 × 10⁷ PFU/mouse) prior to LPS exposure (3 mg/kg). Following exposure, mice were euthanized, and lungs were lavaged with saline and harvested. (A and B) Protein concentration and cell count measurements from BALF. (C–E) BALF cytokine concentrations. (F–H) BALF leukocyte differential. (A–H) Data represent mean values ± SEM (n = 4–7 mice; *P < 0.05; **P < 0.01; ***P < 0.001 compared with Kiaa0317^+/+ mice, 1-way ANOVA with Dunnett’s multiple comparisons). (I) Histology of murine lungs following H&E staining; scale bar: 200 μm. *P < 0.05, **P < 0.01, ***P < 0.001 compared with Kiaa0317^+/+ mice; 1-way ANOVA with Dunnett’s multiple-comparisons test (A–H).

Figure 7. Protected phenotype of Kiaa0317^–/– mice requires SOCS2.

Mice were infected i.t. with lentivirus encoding control shRNA or Socs2 shRNA (1 × 10⁷ PFU/mouse) prior to LPS exposure (3 mg/kg). Following exposure, mice were euthanized, and lungs were lavaged with saline and harvested. (A and B) Protein concentration and cell count measurements from BALF. (C–E) BALF cytokine concentrations. (F–H) BALF leukocyte differential. In A–H, data represent mean values ± SEM (n = 5–6 mice). (I) Histology of murine lungs following H&E staining; scale bar: 200 μm. *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001 compared with control shRNA-Kiaa0317^+/+ mice or as indicated; 1-way ANOVA with Tukey’s multiple-comparisons test.

Figure 8. Chemical inhibition of KIAA0317 prevents SOCS2 degradation and inflammation in vitro.

(A) Structural analysis of the KIAA0317 HECT domain revealed a major cavity within the C-terminus of the HECT domain. (B and C) Docking study of the candidate inhibitor BC-1365 with the KIAA0317-HECT domain. (D) BC-1365 competition assay. SOCS2 protein was incubated with KIAA0317-bound resin and a titration of BC-1365 prior to immunoblot analysis. The relative amounts of SOCS2 detected in the PDs was normalized to vehicle and quantified. Data represent mean values ± SEM (n = 3). (E) Immunoblot analysis of MLE cells following exposure to a titration of BC-1365. Data represent mean values ± SEM (n = 4). (F) MLE cells were exposed to a titration of BC-1365 for 18 hours before qPCR analysis. Data represent mean values ± SEM (n = 4). NS: P > 0.05, *P < 0.05, **P < 0.01 compared with vehicle treatment; 1-way ANOVA with Dunnett’s multiple-comparisons test (E and F).

Figure 9. KIAA0317 small molecule inhibitor is antiinflammatory in vivo.

(A–E) C57BL/6J mice were exposed to PA103 and treated with BC-1365 for 18 hours. Following exposure, mice were sacrificed, and lungs were lavaged with saline and harvested. Box represents the interquartile range; line, the median; and whiskers, highest and lowest values. (A–C) Bacterial count, protein concentration, and cell count measurements from BALF. (D–F) BALF cytokine concentrations. Veh, vehicle. In A–F, data represent mean values ± SEM; n = 4–8 mice. NS: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with control mice or as indicated; 1-way ANOVA with Tukey’s multiple-comparisons test. (G) Immunoblotting of mouse lung homogenate for Kiaa0317 and Socs2 protein signal; n = 3 mice per treatment. (H) Representative histology of murine lungs following H&E staining; scale bar: 100 μm.