Functional Analyses of the Crohn's Disease Risk Gene LACC1 - PubMed (original) (raw)

. 2016 Dec 13;11(12):e0168276.

doi: 10.1371/journal.pone.0168276. eCollection 2016.

Liselotte Vesterlund 1, Ferdinando Bonfiglio 1, Luca Mazzurana 2, Lina Cordeddu 1, Danika Schepis 3, Jenny Mjösberg 2, Sabrina Ruhrmann 4, Alessia Fabbri 5, Vladana Vukojevic 4, Piergiorgio Percipalle 6 7, Florian A Salomons 8, Jurga Laurencikiene 9, Leif Törkvist 10, Jonas Halfvarson 11, Mauro D'Amato 1 12

Affiliations

Functional Analyses of the Crohn's Disease Risk Gene LACC1

Ghazaleh Assadi et al. PLoS One. 2016.

Abstract

Background: Genetic variation in the Laccase (multicopper oxidoreductase) domain-containing 1 (LACC1) gene has been shown to affect the risk of Crohn's disease, leprosy and, more recently, ulcerative colitis and juvenile idiopathic arthritis. LACC1 function appears to promote fatty-acid oxidation, with concomitant inflammasome activation, reactive oxygen species production, and anti-bacterial responses in macrophages. We sought to contribute to elucidating LACC1 biological function by extensive characterization of its expression in human tissues and cells, and through preliminary analyses of the regulatory mechanisms driving such expression.

Methods: We implemented Western blot, quantitative real-time PCR, immunofluorescence microscopy, and flow cytometry analyses to investigate fatty acid metabolism-immune nexus (FAMIN; the LACC1 encoded protein) expression in subcellular compartments, cell lines and relevant human tissues. Gene-set enrichment analyses were performed to initially investigate modulatory mechanisms of LACC1 expression. A small-interference RNA knockdown in vitro model system was used to study the effect of FAMIN depletion on peroxisome function.

Results: FAMIN expression was detected in macrophage-differentiated THP-1 cells and several human tissues, being highest in neutrophils, monocytes/macrophages, myeloid and plasmacytoid dendritic cells among peripheral blood cells. Subcellular co-localization was exclusively confined to peroxisomes, with some additional positivity for organelle endomembrane structures. LACC1 co-expression signatures were enriched for genes involved in peroxisome proliferator-activated receptors (PPAR) signaling pathways, and PPAR ligands downregulated FAMIN expression in in vitro model systems.

Conclusion: FAMIN is a peroxisome-associated protein with primary role(s) in macrophages and other immune cells, where its metabolic functions may be modulated by PPAR signaling events. However, the precise molecular mechanisms through which FAMIN exerts its biological effects in immune cells remain to be elucidated.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. LACC1 mRNA expression in immune-related and gastrointestinal human tissues.

(A) qRT-PCR analysis of LACC1 relative expression in a panel of human tissues identifies spleen and lymph nodes as tissues with higher LACC1 expression. Reactions were done in triplicates. (B) LACC1 mRNA expression data extracted from Genevisible (

https://genevestigator.com/gv/

).

Fig 2

Fig 2. Characterization of anti-FAMIN antibodies and FAMIN expression in HeLa and THP-1 cell lines.

(A) Anti-FAMIN mouse monoclonal antibodies (H-6, E-7 and E-12) were used in WB analyses of cell extracts from HeLa cells untransfected (mock) or transfected with FAMIN, GFP-FAMIN or GFP (control vector). (B) FAMIN expression is induced upon PMA differentiation of THP-1 cells (lanes 1–2 cells transfected with scrambled-control siRNA). Complete knockdown of FAMIN expression was obtained upon transfection of THP-1 cells with siRNA targeting LACC1 transcript (lanes 3–4). β-actin was used for equal loading control. Data are representative of three independent experiments.

Fig 3

Fig 3. LPS stimulation of THP-1 cells results in FAMIN upregulation.

(A) qRT-PCR analysis of LACC1 mRNA expression in THP-1 cells stimulated with PMA and LPS. (B) WB analysis of FAMIN protein expression in THP-1 cells stimulated with PMA and LPS. Relative intensity (RI) from densitometric analysis is reported. Data are representative of three independent experiments. Abbreviation; UN: unstimulated control.

Fig 4

Fig 4. FACS analyses of FAMIN expression in PBMCs and granulocytes.

PBMCs and granulocytes from human healthy donors were co-stained for different cell markers and FAMIN. Neutrophils, monocytes and DCs were shown to be FAMIN+. The events in the displayed graphs were first identified and gated by forward and side scatter parameters. (A) Gating strategy for FAMIN+ monocytes (CD14+, black) and CD14- neutrophils (CD15+, purple and CD16+, orange). (B) Gating strategy for FAMIN+ myeloid DCs (CD11c+, brown) and plasmacytoid DCs (BDCA2+, green) was obtained from gating the CD3-CD19- population followed by gating of CD14-HLADR+ cells. (C) Gating strategy for FAMIN+ T-cells (CD3+, purple) and B-cells (CD19+, orange). Numbers in the outlined areas indicate percent cells in each cell subtype. Data are representative of three independent experiments.

Fig 5

Fig 5. Characterization of FAMIN subcellular localization.

PMA differentiated THP-1 cells were co-stained with a panel of antibodies directed towards different organelle markers and anti-FAMIN antibodies. Magnifications are shown for each staining on the right side of the pictures, together with indication of the target protein and corresponding compartment or cell organelle. Co-localization of FAMIN with PMP70 and catalase were previously shown and serve as positive control, Some additional co-localization could also be detected for endomembrane structure proteins detected at the level of endoplasmic reticulum (ER; calnexin, calreticulin and PDI), lysosomes (LAMP-1) and mitochondrion (COX-IV). Data are representative of six independent experiments.

Fig 6

Fig 6. Analysis of human recombinant FAMIN laccase activity.

Four phenolic substrates (DMP, Guaiacol, L-DOPA and SGZ) were tested in order to evaluate laccase activity of the C-terminal MYC/DDK-tagged recombinant FAMIN protein, as indicated. Data are representative of three independent experiments.

Fig 7

Fig 7. Effect of PPAR agonists FAMIN expression in THP-1 cells.

WB analysis of FAMIN expression in THP-1 macrophages after 24hrs treatment with PPARα (WY14643) or PPARγ (rosiglitazone) as indicated. β-actin were used as a loading control. Relative intensity (RI) from densitometric analysis is reported. Data are representative of three independent experiments. Abbreviation; UN: unstimulated control.

Fig 8

Fig 8. Effect of siRNA-mediated FAMIN knockdown on peroxisome number and peroxisome protein expression.

(A) WB analysis of PMA-induced THP-1 macrophages show diminished FAMIN expression upon siRNA knockdown. Subsequent treatment with WY14643 does not affect peroxisome protein expression. (B) However, a possible trend towards increasing numbers of peroxisomes per cell in THP-1 macrophages could be observed after treatment with PPARα agonist WY14643, independent of FAMIN presence or absence. Data are representative of three independent experiments.

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This study was supported by funds from the Swedish Research Council, Vetenskapsrådet (VR; http://www.vr.se/) to MDA. GA is the recipient of a Karolinska Institutet KID PhD fellowship.

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