Abstract
Purpose :
Nitrogen mustard (NM), an analog of the chemical warfare agent sulfur mustard, is an alkylating agent that modifies the genome guardian p53. At the acute stage of corneal injury, NM injury also induces citrullination, a post translational modification. Here we hypothesize that perturbation of p53 by NM may lead to activation of peptidyl arginine deiminases (PADs), the enzymes responsible for corneal hypercitrullination.
Methods :
C57Bl6 line mouse corneas (male and female aged 2-5 months, N=8/group) were topically injured with 1% NM for 5 min under isoflurane anesthesia. Mice were euthanatized at specific timepoints, and enucleated eyes subjected to cryosectioning. Tissue sections were stained with antibodies to quantify global citrullination (F95), citrullinated histone H3 (cit-H3), PAD4 and p53 by immunohistochemistry (IHC). Immunoprecipitation (IP) of corneal proteins and western blotting (WB) were employed to examine p53-PAD interactions and cit-H3. In other experiments, injured eyes were enucleated, and the globe placed in DMEM culture medium at 37oC in a CO2 incubator with PAD inhibitor Cl-amidine. Statistical analysis was performed with Student’s t-test (α=0.05).
Results :
NM injury significantly increases p53 expression by 1h in the injured corneal epithelium (P<0.0001), which appears in the stroma by 1d by IHC analysis (P=0.0003). NM increases nuclear cit-H3 at 30 min which was sustained through 1d, as shown by IHC and WB (P=0.0286). IP-WBs revealed p53 binds PAD4 and pulls down also citrullinated proteins from injured corneas. Interestingly, there was no difference in PAD4 levels between the injured and uninjured groups by IP-WBs. In the ex vivo culture model, inhibition of PAD4 even 1h post injury caused a reduction in global citrullination but not of increased cit-H3 levels when measured at 1d.
Conclusions :
NM induces the acute nuclear expression of p53 and cit-H3 (a nuclear target of PAD4) in injured corneal epithelium. PAD4 binds p53 at this acute post injury stage, causing rapid activation of PAD4 leading to increased cit-H3 expression. Thus, acute citrullination of nuclear histones by PAD4, as well as global citrullination targets affected at later times, may be regulated by PAD4-substrate preferences. These findings suggest that PAD4 governs corneal citrullination in a time and cell type-specific manner where altered functions of distinct targets may drive epithelial cell degeneration in vesicant injury.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.