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. 2011 Oct 9;12(11):1063-70.
doi: 10.1038/ni.2113.

The kinase LRRK2 is a regulator of the transcription factor NFAT that modulates the severity of inflammatory bowel disease

Zhihua Liu  1 , Jinwoo Lee, Scott Krummey, Wei Lu, Huaibin Cai, Michael J Lenardo
Affiliations

The kinase LRRK2 is a regulator of the transcription factor NFAT that modulates the severity of inflammatory bowel disease

Zhihua Liu et al. Nat Immunol. .

Abstract

Leucine-rich repeat kinase 2 (LRRK2) has been identified by genome-wide association studies as being encoded by a major susceptibility gene for Crohn's disease. Here we found that LRRK2 deficiency conferred enhanced susceptibility to experimental colitis in mice. Mechanistic studies showed that LRRK2 was a potent negative regulator of the transcription factor NFAT and was a component of a complex that included the large noncoding RNA NRON (an NFAT repressor). Furthermore, the risk-associated allele encoding LRRK2 Met2397 identified by a genome-wide association study for Crohn's disease resulted in less LRRK2 protein post-translationally. Severe colitis in LRRK2-deficient mice was associated with enhanced nuclear localization of NFAT1. Thus, our study defines a new step in the control of NFAT activation that involves an immunoregulatory function of LRRK2 and has important implications for inflammatory bowel disease.

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Figures

Fig. 1
Fig. 1
LRRK2 deficiency exacerbates experimental colitis in mice. (A) Relative levels of LRRK2 mRNA measured by qRT-PCR in bone-marrow derived macrophages (BMDM)(set as 100%), bone-marrow derived dendritic cells (BMDC) and CD4+ T cells. β-actin was used for total mRNA normalization. (B) Mean body weight as a percent of starting weight (left panel) and mean clinical scores (right panel) of WT (blue, n=5) and LRRK2−/− (red, n=5) mice treated with 3% DSS. *P < 0.05. **P < 0.02. (C) Hematoxylin and eosin (H&E) staining of colonic sections. Loss of epithelial crypts (blue arrowhead), transmural inflammation and thickened intestinal wall (green open double arrow) are noted. Scale bar, 200 µm. (D) Mean of histological scores of colon sections. **P < 0.02. (E) Serum cytokines at Day 8. *P < 0.05, **P < 0.02. (F) Mean body weight (left panel) and mean clinical scores (right panel) of WT mice that received either WT or LRRK2−/− bone marrow (BM) (n=6). Data are representative of three independent experiments (A–E) and two independent experiments (F). Error bars represent standard errors of the mean (SEM).
Fig. 2
Fig. 2
LRRK2 inhibits NFAT1 nuclear function. (A) Schematic of LRRK2 structure. (B) NFAT- and NF-κB-induced luciferase activity in 293T cells transfected with vector or LRRK2 stimulated with 0.5 µM ionomycin and phorbol myristate acetate (PMA) or TNF-α at indicated doses. Data are means of triplicate wells with SEM. (C) Immunoblotting of V5-LRRK2 and actin from 293T cells with a tet-regulated LRRK2 expression construct uninduced (−) or induced with 5 µg ml−1 doxycycline (DOX)(+), top panels. Confocal images of NFAT1-GFP transfected into 293T cells with DOX-inducible V5-LRRK2 before and after 1 µM ionomycin (Iono), Lower panels. Hoechst nuclear staining (blue). Scale bar, 5 µm. (D) Percentage of cells with nuclear NFAT1-GFP among ~100 GFP-positive cells for each condition in (C). Error bars indicate SEM for 10 fields in three experiments. **P < 0.01. (E) Representative immunoblotting of NFAT1 in nuclear vs. cytosolic fractions from BMDM treated with Iono at the indicated doses (µM) for 30’. Numbers below the NFAT1 blot are the quantified relative ratio of NFAT1 in nuclear fractions. (F) Representative images of NFAT1 immunohistochemical staining in colon sections. Cells with cytosolic NFAT1 staining (blue arrows) and nuclear NFAT1 staining (red arrows) are noted. Inset panels, enlarged cells. Scale bar, 25 µm. B–F are representative of three experiments.
Fig. 3
Fig. 3
LRRK2 affects NFAT1 cytoplasmic sequestration not phosphorylation. (A) NFAT-induced luciferase activity (mean ± SEM) in 293T cells transfected with vector or LRRK2 WT or kinase dead (KD) variants stimulated with 0.5 µM Iono and indicated doses of PMA. (B) Immunoblotting of NFAT1-GFP from 293T cells transfected with vector control or myc-LRRK2 at the indicated time points after washing out Iono treatment. NT, untreated. Open arrowheads indicate unphosphorylated NFAT1-GFP, and closed arrowhead marks highly phosphophorylated NFAT1-GFP. (C) Confocal images of caNFAT1-GFP transfected into 293T cells with DOX-inducible V5-LRRK2. Hoechst nuclear staining (blue). Scale bar, 5 µm. (D) Percent of cells with predominantly cytoplasmic (cyto), cytoplasmic and nuclear (cyto + nucl), or nuclear (nucl) distribution of caNFAT1-GFP among 100 GFP-positive cells for each condition. (E) Immunoblotting of caNFAT1-GFP in cytosolic vs nuclear fractions from 293T cells transfected with vector control or myc-LRRK2. (F) Immunoprecipitation and immunoblotting of proteins as indicated, using 293T overexpressing myc-LRRK2 as bait. Iso, isotype control Ig for immunoprecipitation. (G) Immunoprecipitation/RT-PCR of NRON using 293T cells with DOX-inducible V5-LRRK2. (H) Immunoprecipitation/immunoblotting for proteins as indicated in 293T cells. *small change; **large change. (I) Immunoprecipitation/immunoblotting, as indicated, using THP1 cells. (J) Immunoblotting of LRRK2 from BMDM treated with lipopolysaccharide (LPS) at the indicated doses for 1 hour (top panels) or at the indicated times (min) (bottom panels) with 1000 pg ml−1 LPS; (K) Immunoblotting of NFAT1 in nuclear vs cytosolic fractions from LPS-primed (1000 pg ml−1 for 1 hour) or untreated (NC) BMDM cells treated with Iono at the indicated doses (µM) for 30 min. A–K represent three experiments.
Fig. 4
Fig. 4
LRRK2 deficiency increases NFAT1 activation in BMDMs and underlies a CD susceptibility polymorphism. (A) IL-12/p40, IL-6 and TNF-α production (mean ± SEM) by WT or LRRK2−/− BMDMs with stimulation of 100 µg ml−1 zymosan or 10 µg ml−1 Pam3CSK4. IL-1β production by BMDMs with 25 ng ml−1 LPS and 5 mM ATP. **P < 0.02. (B) IL-12/p40 and IL-6 production by zymosan (µg ml−1) with or without 2 mM FK506 treatment. (C) NFAT1 immunoblotting in nuclear and cytosol fractions in BMDMs treated with 100 µg ml−1 zymosan for 30 minutes. Numbers below are the quantified relative ratio of NFAT1 in nuclear fractions. (D) Mean body weight (mean ± SEM) of WT and LRRK2−/− receiving DSS with or without 1 mg/mouse/day CsA treatment (n=5). **P < 0.02. P values were calculated between vehicle treated and CsA treated groups. (E) Immunohistochemical NFAT1 staining in colon sections from LRRK2−/− mice receiving vehicle (Mock) or CsA treatment, left panels. Scale bar, 25 µm. Percentage of cells with nuclear NFAT1 among ~100 NFAT1-positive cells for each group, right panel. (F) Immunoblotting of overexpressed M2397 and T2397 forms of mycLRRK2 in 293T cells (top panels). Hsp90 was used as loading control. Ratio of mycLRRK2 and Hsp90 from top panels (bottom panel). *P < 0.05 (G) Cycloheximide (CHX) chase assay of 293T transfected with M2397 or T2397 of mycLRRK2 plasmids treated with 100 µg ml−1 CHX, analyzed by immunoblot with anti-myc antibody to detect mycLRRK2 and anti-Hsp90 to detect Hsp90 as loading control (top panel). Quantification of relative amount of mycLRRK2 (bottom panel). The ratio of mycLRRK2, either M2397 or T2397, to Hsp90 was normalized to their respective starting points at 0 h (set as 1.0). (H) Immunoblotting of LRRK2 in purified peripheral B cells from humans homozygous at 2397, either M/M or T/T (top panels). Scatter plot of quantification of LRRK2 and Hsp90 from top panels (bottom panel). Error bars indicate SEM for each group. **P < 0.01. A–C and H represent three experiments. F and G represent five experiments. D and E represent two experiments.
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