Abstract

Endometriosis lesions require sustained inflammatory and tissue-remodeling signals after the initial neutrophil response subsides. Senescent endometrial stromal fibroblasts (eSFs) may support this persistence through cytoplasmic chromatin fragments (CCFs), which can activate cGAS–STING signaling and reinforce the senescence-associated secretory phenotype. Peptidylarginine deiminase 4 (PADI-4) is a calcium-dependent nuclear enzyme that citrullinates histones, reducing their positive charge and weakening histone–DNA interactions. This study investigated whether PADI-4-mediated chromatin remodeling facilitates CCF formation during fibroblast senescence. Primary IMR90 fibroblasts were treated with 250 nM doxorubicin to induce senescence and cultured under untreated conditions or with Cl-amidine, an irreversible pan-PAD inhibitor, or GSK484, a selective reversible PADI-4 inhibitor. Cells were fixed from 6 hours (immediately post-DNA damage) to 10 days (established senescence) and examined by confocal immunofluorescence for nuclear morphology, histone H3 citrullination, Lamin A/C, Lamin B receptor, DAPI, and γH2AX. All senescent conditions developed enlarged morphology, persistent DNA-damage signaling, nuclear blebbing, and progressive loss of nuclear organization, indicating that general senescence-associated nuclear deterioration was PADI-4-independent. However, discrete DAPI-positive, H3Cit17-positive, and Lamin A/C-negative CCF-like structures were observed in approximately 5–10% of established wild type senescent cells but were not detected under either PADI-inhibitor condition. H3Cit17 also redistributed from a diffuse nucleoplasmic pattern to the nuclear periphery before extrusion. These qualitative findings support a model in which nuclear-envelope deterioration creates an opportunity for chromatin escape, while PADI-4-mediated citrullination facilitates chromatin decompaction and extrusion. If validated quantitatively in hormone-responsive endometrial stromal cells, this pathway could explain how senescent fibroblasts provide a persistent nuclear-DNA stimulus for cGAS–STING signaling and endometriotic lesion maintenance.

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