Deacetylation via SIRT2 prevents keratin-mutation-associated injury and keratin aggregation
Jingyuan Sun, … , Li Liu, M. Bishr Omary
Jingyuan Sun, … , Li Liu, M. Bishr Omary
Published July 24, 2023
Citation Information: JCI Insight. 2023;8(14):e166314. https://doi.org/10.1172/jci.insight.166314.
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Research Article Dermatology Hepatology

Deacetylation via SIRT2 prevents keratin-mutation-associated injury and keratin aggregation

Abstract

Keratin (K) and other intermediate filament (IF) protein mutations at conserved arginines disrupt keratin filaments into aggregates and cause human epidermolysis bullosa simplex (EBS; K14-R125C) or predispose to mouse liver injury (K18-R90C). The challenge for more than 70 IF-associated diseases is the lack of clinically utilized IF-targeted therapies. We used high-throughput drug screening to identify compounds that normalized mutation-triggered keratin filament disruption. Parthenolide, a plant sesquiterpene lactone, dramatically reversed keratin filament disruption and protected cells and mice expressing K18-R90C from apoptosis. K18-R90C became hyperacetylated compared with K18-WT and treatment with parthenolide normalized K18 acetylation. Parthenolide upregulated the NAD-dependent SIRT2, and increased SIRT2-keratin association. SIRT2 knockdown or pharmacologic inhibition blocked the parthenolide effect, while site-specific Lys-to-Arg mutation of keratin acetylation sites normalized K18-R90C filaments. Treatment of K18-R90C–expressing cells and mice with nicotinamide mononucleotide had a parthenolide-like protective effect. In 2 human K18 variants that associate with human fatal drug-induced liver injury, parthenolide protected K18-D89H– but not K8-K393R–induced filament disruption and cell death. Importantly, parthenolide normalized K14-R125C–mediated filament disruption in keratinocytes and inhibited dispase-triggered keratinocyte sheet fragmentation and Fas-mediated apoptosis. Therefore, keratin acetylation may provide a novel therapeutic target for some keratin-associated diseases.

Authors

Jingyuan Sun, Pei Li, Honglian Gui, Laure Rittié, David B. Lombard, Katrin Rietscher, Thomas M. Magin, Qing Xie, Li Liu, M. Bishr Omary

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Figure 1

Parthenolide (PN) normalizes K18-R90C filament disruption and protects against Fas-induced apoptosis in cultured cells and mice.

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Parthenolide (PN) normalizes K18-R90C filament disruption and protects a...
(A) A549 cells were transduced with lentivirus expressing GFP-tagged K18-R90C and K8-WT (cotransfection with type I and II keratins is needed for physiologic heteropolymer filament formation). After 2 days, cells were treated with DMSO or PN (5 μM, 48 hours) and then fixed and stained with DAPI. Average percentage of cells with dots/group for DMSO vs. PN is shown. P < 0.001, from 3 independent experiments). Scale bar: 50 μm. (B) GFP-K18-WT and GFP-K18-R90C lentiviruses were transduced into A549 cells followed by treatment with DMSO or PN (5 μM, 48 hours) and then apoptosis was induced using IFN-γ and Fas ligand (Fas-L). Cell lysates were analyzed by blotting using antibodies against the indicated antigens (Coomassie stain shows equal loading). The average relative intensity of the indicated bands from 3 individual experiments is included below each blot. *P < 0.05, ***P < 0.001 using unpaired, 2-tailed Student’s t test (comparing DMSO with PN). (C) Representative TUNEL staining of GFP-K18-R90C–transduced A549 cells treated with DMSO or PN and then challenged with IFN-γ plus Fas-L. The percentage of TUNEL+ cells in DMSO vs. PN was 82% vs. 33%, respectively (P < 0.001). Scale bar: 50 μm. For panels A–C, all experiments were repeated at least 3 times. *P < 0.05, **P < 0.005 using unpaired, 2-tailed Student’s t test. (D) Transgenic mice that express K18-R90C were treated daily with DMSO or PN (1 mg/kg mouse weight; i.p.) for 4 days. Liver sections were double stained with anti-K18 antibody and DAPI. The percentage of cells with dots/group is shown in Supplemental Figure 1F (P < 0.001). Scale bar: 50 μm. (E) Mice were treated with DMSO or PN as in panel D and then challenged with Fas-L. Livers were collected after 5 hours and analyzed by immunoblotting (each lane represents a different liver). (F) Alanine transaminase (ALT) was measured using sera collected from the indicated number of mice. **P = 0.002 using 1-way ANOVA followed by Tukey’s post hoc test. (G) Representative images of hematoxylin and eosin–stained liver sections. Arrows highlight areas of hemorrhage. Scale bar: 100 μm. (H) Quantification of the hemorrhage after Fas-L treatment of the mice is included. *P = 0.013 using unpaired, 2-tailed Student’s t test. For each group (DMSO or PN), 10 sections for each liver and 7 independent livers were used.