PP2 protects from keratin mutation–associated liver injury and filament disruption via SRC kinase inhibition in male but not female mice

Abstract

Background and Aims: Hepatocyte keratin polypeptides 8/18 (K8/K18) are unique among intermediate filaments proteins (IFs) in that their mutation predisposes to, rather than causes, human disease. Mice that overexpress human K18 R90C manifest disrupted hepatocyte keratin filaments with hyperphosphorylated keratins and predisposition to Fas-induced liver injury.We hypothesized that high-throughput screening will identify compounds that protect the liver from mutation-triggered predisposition to injury. Approach and Results: Using A549 cells transduced with a lentivirus K18 construct and high-throughput screening, we identified the SRC-family tyrosine kinases inhibitor, PP2, as a compound that reverses keratin filament disruption and protects from apoptotic cell death caused by K18 R90C mutation at this highly conserved arginine. PP2 also ameliorated Fas-induced apoptosis and liver injury in male but not female K18 R90C mice. The PP2 male selectivity is due to its lower turnover in male versus female livers. Knockdown of SRC but not another kinase target of PP2, protein tyrosine kinase 6, in A549 cells abrogated the hepatoprotective effect of PP2. Phosphoproteomic analysis and validation showed that the protective effect of PP2 associates with Ser/Thr but not Tyr keratin hypophosphorylation, and differs from the sex-independent effect of the Ser/Thr kinase inhibitor PKC412. Inhibition of RAF kinase, a downstream target of SRC, by vemurafenib had a similar protective effect to PP2 in A549 cells and male K18 R90C mice. Conclusions: PP2 protects, in a male-selective manner, keratin mutation-induced mouse liver injury by inhibiting SRC-triggered downstream Abbreviations: ALT, alanine aminotransferase; FasL, Fas ligand; GFP, green fluorescent protein; IF, intermediate filament protein; NMHC-IIA, nonmuscle myosin heavy chain-IIA; PTK6, protein tyrosine kinase 6; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

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

“…One potential benefit of identifying uniquely functioning drugs that may have clinical utility in keratin-and other IF-related disorders is that they may offer the opportunity to be used, after testing experimentally, as combination drugs. This applies to the demonstrated protective effect in keratin mutants by 2 separate Ser/Thr or Tyr kinase inhibitors (17)(18)(19) and the observation herein by targeting protein acetylation. Such an approach is likely to be particularly beneficial for drugs that are already in clinical use and, therefore, can be repurposed for keratin-and other IF-related disorders.…”

“…A total of 24 compounds were selected from the initial screening (performed in 384-well plates) for validation using chamber slides, with transduced A549 cells treated with the 24 compounds. From these compounds, PP2 (19) and PN had the most prominent effect in terms of decreasing the extent of K18-R90C filament collapse and dot formation. Further validation was then carried out by testing the dose response for PN (data not shown).…”

“…This approach has successfully identified the pan-kinase inhibitor PKC412 as a compound that reverts disrupted K18-R90C aggregates into a WT-like extended filament network by enhancing keratin association with nonmuscle myosin heavy chain IIA (NM-IIA) in a myosin-dephosphorylation-regulated manner (17), or in patient-derived keratinocytes expressing K14-R125C (18). Also, by using this high-throughput drug screening system, another compound termed PP2, a SRC-family tyrosine kinase inhibitor, was identified and demonstrated to protect cells from keratin-mutation-associated liver injury and filament disruption via SRC kinase inhibition (19). Our hypothesis is that compounds that convert the mutant keratin phenotype from dots/aggregates to filaments will protect cells and tissues from injury.…”

Single-cell morphological tracking of liver cell states to identify small-molecule modulators of liver differentiation

“…SFK activity and downstream signalling has previously been implicated in the progression of a number of liver disorders including hepatocellular carcinoma 44 and liver fibrosis 45 . In addition, a role for c-SRC in cell fate determination during endodermal commitment of human iPSCs has recently been described 46 , the kinase inhibitor PP2 has previously been shown to protect against Keratin mutation-induced mouse liver injury through Src inhibition (Li et al, 2023), and previous studies have demonstrated that genetic deletion of Src's binding partner focal adhesion kinase (FAK) accelerates liver regeneration 48 . It is our understanding that ours is the first study to demonstrate a role for small molecule SFK inhibitors in the promotion of hepatocyte progenitor differentiation and as a potential therapeutic opportunity for liver regeneration.…”