Hypothesis

Duration‑dependent JNK signaling switches AP‑1 from a transient regenerative factor to a stable SASP‑driving chromatin remodeler.

Mechanistic Basis

Recent work shows that mitochondrial ROS activates JNK in senescent cells (1) and that young wounds contain transient p16+/p21+ fibroblasts with regenerative programs, whereas aged tissues display persistent, pro‑inflammatory senescence (2). We propose that the length of JNK activity determines whether AP‑1 complexes recruit co‑activators such as p300/CBP to SASP enhancers. Brief JNK pulses allow AP‑1 to bind DNA and drive expression of regenerative genes (e.g., FGF2, VEGF) without stable enhancer remodeling. Prolonged JNK activity leads to sustained AP‑1 occupancy, which recruits p300, deposits H3K27ac, and remodels nucleosomes at IL6, IL8, and MMP promoters, locking in a chronic SASP (3, 4). This model integrates the known JNK‑NF‑κB synergy and adds a temporal chromatin‑state switch that explains why constitutive JNK inhibition fails to distinguish acute from chronic phases.

Testable Predictions

1. Kinetic correlation: Live‑cell reporters of JNK activity will show that fibroblasts exhibiting SASP markers (>24 h post‑stress) have JNK phosphorylation lasting >12 h, whereas those resolving senescence show JNK activity <6 h.
2. Chromatin transition: ChIP‑seq for phospho‑c‑Jun and p300 will reveal that early (≤6 h) AP‑1 binding is transient and lacks H3K27ac, while late (>12 h) binding coincides with stable H3K27ac peaks at SASP loci.
3. Phosphatase timer: The dual‑specificity phosphatase DUSP16 will be induced early after stress and decline with age; its overexpression will truncate JNK activity and prevent SASP despite persistent stress, whereas DUSP16 knockdown will prolong JNK and accelerate SASP.
4. Therapeutic window: Pharmacologic JNK inhibition applied only after the first 8 h of stress will reduce SASP without affecting early regenerative gene expression, whereas inhibition within the first 2 h will impair wound‑healing‑associated programs.

Experimental Approach

• Use primary human dermal fibroblasts subjected to sublethal H₂O₂ to induce senescence.
• Employ a FRET‑based JNK activity biosensor (e.g., JNK‑KTR) for real‑time imaging; categorize cells by integrated JNK signal over time.
• At defined intervals (0, 4, 8, 12, 24 h) perform ATAC‑seq, ChIP‑seq for phospho‑c‑Jun, p300, and H3K27ac, and RNA‑seq to link JNK duration to enhancer activation and SASP transcription.
• Manipulate DUSP16 via CRISPRa/i or siRNA and assess JNK kinetics and SASP output.
• Apply the JNK inhibitor SP600125 (5) in timed pulses (0‑2 h, 2‑8 h, 8‑24 h) and measure regenerative markers (FGF2, VEGF) versus SASP (IL6, IL8, MMP1, MMP3).
• Validate findings in murine wound models (young vs aged) using intravital JNK reporters and SASP staining.

Falsification: If SASP induction occurs equally after short (<4 h) and long (>12 h) JNK pulses, or if DUSP16 manipulation does not alter JNK duration or SASP, the hypothesis would be refuted.

This framework converts the phenomenological acute‑to‑chronic senescence transition into a testable, mechanism‑driven model that defines a precise therapeutic window for JNK‑targeted interventions in inflammaging.