Screening the human druggable genome identifies ABHD17B as an anti-fibrotic target in hepatic stellate cells

Abstract

Hepatic stellate cells (HSCs) are activated with chronic liver injury and transdifferentiate into myofibroblasts, which produce excessive extracellular matrices that form the fibrotic scar. While the progression of fibrosis is understood to be the cause of end-stage liver disease, there are no approved therapies directed at interfering with the activity of HSC myofibroblasts. Here, we perform a high-throughput small interfering RNA (siRNA) screen in primary human HSC myofibroblasts to identify gene products necessary for the fibrotic phenotype of HSCs. We find that depletion of ABHD17B promotes the inactivation of HSCs, characterized by reduced COL1A1 and ACTA2 expression and accumulation of lipid droplets. Mice deficient in Abhd17b are also protected from fibrosis in the setting of in vivo liver injury. While ABHD17B is a depalmitoylase, our data suggest that ABHD17B promotes fibrosis through pathways independent of depalmitoylation that include interaction with MYO1B to modulate gene expression and HSC migration. Together, our results provide an analysis of the phenotypic consequences for siRNAs targeting RNAs from >9500 genes in primary human HSCs and identify ABHD17B as a potential therapeutic target to inhibit liver fibrosis.

Fig. 1. High-throughput screening identified siRNAs that induce HSC myofibroblast inactivation.

a Overview of the siRNA screen. b Results of the primary siRNA screen. Each dot represents the integrated score and toxicity value of three replicates of an experimental or control well. Data from all wells are shown. c The score and toxicity values for all experimental wells from the primary screen. d Summary of the validation screen results. e Summary of the siRNA knockdown efficiency in the cherrypick screen. f Grouping the final 71 hits by priority. g Expression of the 11 priority 1 genes in HSCs from the livers of CCl₄-treated and control mice based on single-cell RNA sequencing results. Circle size represents the fraction of cells expressing a gene, and color indicates mean expression level. Source data are provided as Supplementary Data 1–6 and a Source Data file.

Fig. 2. Depletion of ABHD17B inactivates HSCs.

a Fraction of Bodipy positive (top) and total cells (bottom) after two days in the indicated conditions. DMSO and nortriptyline (Nor) are negative and positive controls for Bodipy, respectively. siRNAs targeting UBB are a control for transfection efficiency, and siRNAs targeting GAPDH are a negative control for Bodipy. Error bars represent mean ± SEM (n = 10 biological replicates). One-way ANOVA test. b mRNA expression quantified by qRT-PCR two days after HSCs were transfected with the indicated siRNAs. Data represent three independent experiments. Error bars represent mean ± SEM (n = 3 biological replicates). One-way ANOVA test. c ABHD17B expression quantified by qRT-PCR three days after HSC transfection. Data represent two independent experiments from two donors (5 and 6), which are further analyzed in (d–g). Each dot represents the mean of biological replicates for pooled non-targeing control (NTC siP; n = 4 biological replicates), single non-targeting control (NTC si5; n = 4), and other indicated groups (n = 2). ABHD17B siP are compared to NTC siP; ABHD17B si1 and ABHD17B si4 are compared to NTC si5. HSCs from donor 5 (d) and donor 6 (e) were transfected with the indicated siRNAs prior to serum starvation and stimulation with scar-in-a-jar conditions for 72 h. α−smooth muscle actin (αSMA, red) and collagen (orange) were visualized by immunofluorescence in the same field of view. Nuclei were stained with Hoechst (blue). Representative images from two independent experiments are shown. White bars indicate 50 µm. Quantification of αSMA fibers (f) and collagen area (g) from scar-in-a-jar assay. Data represent two independent experiments for each of the two donors (5 and 6). Each dot represents averaged result from n = 4 biological replicates. Data from donors 5 and 6 are combined for four independent experiments. Error bars represent mean ± SEM. One-way ANOVA test. h Members of the ABHD17 family were depleted using pooled siRNAs. COL1A1 levels were quantified by qRT-PCR three days after transfection. Error bars represent mean ± SEM (n = 8 biological replicates for control groups of donors 1 and 4; n = 7 for the control group of donor 3; n = 4 for all other groups). One-way ANOVA test. Source data are provided as a Source Data file.

Fig. 3. Depalmitoylation activity of ABHD17B is not required for HSC activation or collagen expression.

a HEK-293 cells expressing wild-type (WT) ABHD17B-FLAG and ABHD17B-S170A-FLAG were lysed and treated with a desthiobiotin-labeled serine activity based probe (SABP). Serine activity was quantified by streptavidin (SA) HRP. Total ABHD17B protein was quantified by anti-FLAG antibody. Top: representative images. Bottom: each band was quantified three times using Fiji. Error bars represent mean ± SEM. Results are representative of two independent experiments. b COL1A1 and ABHD17B were quantified by qRT-PCR in HSCs transduced with lentivirus expressing GFP, ABHD17B-WT, or ABHD17B-S170A. Expression was normalized to PSMB2. Error bars represent mean ± SEM (n = 8 biological replicates). 2-tailed unpaired t-test between WT and S170A. Results are representative of two independent experiments. c HSCs were treated with nortriptyline (black) or palmostatin B (red) at indicated concentrations for 48 h. Cells were fixed and stained with Bodipy and Hoechst. Each dot represents the averaged value of ten biological replicates. Error bars represent mean ± SD (n = 10 biological replicates). d, e HEK-293 cells expressing wild-type ABHD17B-FLAG were treated with increasing concentrations of ABD957. Serine activity was measured following similar procedures as in (a). Band intensities were quantified three times using Fiji and normalized to FLAG signal (e). Error bars represent mean ± SEM. Results are representative of two independent experiments. f, g HSCs from donor 1 (f) and donor 3 (g) were treated with ABD957 for 48 h before Bodipy and Hoechst staining. Nanchangmycin (NCMC) served as a positive control. Error bars represent mean ± SEM. f: n = 10 biological replicates for DMSO, n = 5 for the other groups; g: n = 3 for DMSO, n = 4 for NCMC, and n = 5 for the other groups. One-way ANOVA test. h, i HSCs from donor 1 were treated with ABD957 or NCMC for 48 h. ACTA2 (h) and COL1A1 (i) levels were quantified. Error bars represent mean ± SEM (n = 8 biological replicates for DMSO, n = 4 for the other groups). One-way ANOVA test. Source data are provided as a Source Data file.

Fig. 4. ABHD17B interacts with pathways involving contractility, adhesion, ECM, and the cytoskeleton.

a RNA-seq and differential expression analysis were performed using nontargeting control (NTC) si5, and two siRNAs targeting ABHD17B (Supplementary Data 7). Dot plot displays the Gene Ontology (GO) terms most enriched in the genes repressed with depletion of ABHD17B. The color of each dot represents the adjusted p-value (hypergeometric test with Benjamini-Hochberg multiple testing correction), and the size of the dot represents gene count. b Heatmaps show expression of the repressed gene set for indicated GO categories for HSCs transfected with NTC (C), siRNA4 targeting ABHD17B (s4) and siRNA1 targeting ABHD17B (s1). Expression is centered and scaled by row (gene). c Proteins that interact with ABHD17B were identified by precipitation of ABHD17B-FLAG and GFP-FLAG followed by mass spectrometry (MS) (Supplementary Data 8). Enrichment of GO categories (Molecular Functions) for the 15 proteins showing the strongest interaction with ABHD17B (String-db.org) is shown. False discovery rate (FDR) was calculated by Benjamini-Hochberg procedure (String-db.org). d The interactions between the 15 proteins showing the strongest interaction with ABHD17B are displayed in addition to ABHD17B. Dark red lines indicate experimentally-determined interactions (String-db.org). e HSCs were transfected with lentivirus to express GFP-FLAG or ABHD17B-FLAG for 48 h before anti-FLAG precipitation followed by probing for MYO1B (top) and FLAG expression (bottom). Two precipitations were performed for each condition (#1 and #2) and compared to total lysates (L). Data are representative of two independent experiments. f Relative mRNA expression quantified by qRT-PCR in primary human HSCs (Donor 4) treated witih non-targeting siRNAs (NTC si5) and pooled siRNAs targeting MYO1B. Error bars represent mean ± SEM (n = 8 biological replicates). 2-tailed unpaired t-test. Data are representative of three independent experiments. g Wound healing assay was performed in HSCs (donor 4) transfected with siRNAs targeting MYO1B (green) and ABHD17B (blue). Normalized wound width was calculated at the indicated time points from three individual scratches. Error bars represent mean ± SEM (n = 3 biological replicates). One-way ANOVA test. Data are representative of two independent experiments. Source data are provided as Supplementary Data and a Source Data file.

Fig. 5. Loss of Abhd17b protects against development of liver fibrosis.

a Representative Sirius red staining results of livers from Abhd17b^+/+ and Abhd17b^−/− mice (n = 3 mice) receiving control olive oil via oral gavage 3 times a week for 4 weeks. Scale bar indicates 50 μm. b Hydroxyproline analysis was performed using mouse liver samples. Error bars represent mean ± SEM (n = 3 mice for olive oil groups, n = 14, 13 mice for CCl₄ treatment); 2-tailed unpaired t-test. c Collagen proportionate area (CPA) was calculated based on Sirius red staining. Error bars represent mean ± SEM (n = 3 mice for olive oil groups, n = 14, 12 mice for CCl₄ treatment); 2-tailed unpaired t-test. d Representative Sirius red staining results of livers from Abhd17b^+/+ (n = 14 mice) and Abhd17b^−/− (n = 13 mice) mice recieving CCl₄ via oral gavage 3 times a week for 4 weeks. Scale bar indicates 50 μm. e–i Relative mRNA expresion was analyzed for the indicated gene products by qRT-PCR from liver samples. Error bars represent mean ± SEM (n = 3 mice for olive oil groups, n = 14, 13 mice for CCl₄ treatment); 2-tailed unpaired t-test. j Representative IHC staining for Pdgfrb, Acta2 and Ck19 in livers from Abhd17b^+/+ (n = 2 median mice based on hydroxyproline level) and Abhd17b^−/− (n = 2 median mice based on hydroxyproline level) mice recieving either control olive oil or CCl₄ via oral gavage 3 times a week for 4 weeks. Scale bar indicates 50 μm. Red arrows indicate examples of Ck19 positive cells. Source data are provided as a Source Data file.