Hypothesis

We propose that the transition from acute JNK‑AP‑1 protection to chronic inflammaging is governed by the kinetic accumulation of minority mitochondrial outer membrane permeabilization (miMOMP) events, which generate oscillatory bursts of mitochondrial ROS. These bursts modify redox‑sensitive cysteines on JNK‑activating scaffolds (e.g., MKK4/7) and on AP‑1 subunits themselves, shifting the dimer composition from c‑Jun homodimers that repress proliferation to c‑Jun/c‑Fos heterodimers that drive SASP transcription. In tissues where ASK1 preferentially signals through p38 MAPK, the phosphatase MKP‑1 is induced by p38 activity and accelerates JNK dephosphorylation, thereby raising the miMOMP threshold required for chronic JNK‑AP‑1 activation. Consequently, skin fibroblasts exhibit a lower miMOMP threshold and rapid SASP onset, whereas hepatocytes require sustained miMOMP stress before JNK‑AP‑1 dominates over ASK1‑p38 signaling.

Predictions

1. Single‑cell live imaging of mitochondrial ROS (using MitoSOX) combined with a FRET‑based JNK activity reporter will show that discrete ROS spikes precede sustained JNK phosphorylation only after a critical number of miMOMP events (≈3‑5 per cell) have occurred.
2. Mutating the redox‑sensitive Cys‑252 of c‑Jun to serine will lock AP‑1 in the homodimeric state, preventing SASP induction even when miMOMP is experimentally increased (e.g., by low‑dose BAX activator).
3. Pharmacological inhibition of MKP‑1 in hepatocytes will sensitize ASK1‑p38‑dominant cells to miMOMP‑driven JNK‑AP‑1 activation, converting their SASP profile to resemble that of skin fibroblasts.
4. In vivo, transient miMOMP induced by a brief pulse of hypoxia‑reoxygenation will produce a biphasic SASP: early IL‑6 suppression followed by late IL‑6 elevation, with the switch timing correlating to the measured miMOMP frequency in tissue‑specific biopsies.

Experimental Approach

• Induce miMOMP titratably using a CRISPR‑activatable BAX construct or low concentrations of the BAX agonist compound‑X, while monitoring mitochondrial outer membrane integrity with cytochrome c‑GFP release assays.
• Measure JNK‑AP‑1 dynamics with a dual‑reporter system: a JNK‑KTR translocation sensor and an AP‑1‑dependent luciferase construct containing either a c‑Jun homodimer‑binding site or a c‑Jun/c‑Fos heterodimer‑binding site.
• Quantify SASP components (IL‑6, IL‑8, MMPs) by multiplex ELISA at 0‑6‑12‑24‑48‑72 h post‑induction.
• Perturb redox switches via CRISPR‑mediated Cys‑252‑Ser knock‑in of c‑Jun or pharmacological MKP‑1 inhibition (using compound Y).
• Compare tissues by isolating primary human skin fibroblasts and hepatocytes, performing the same timeline, and calculating the miMOMP‑to‑JNK activation ratio.
• Statistical test: compare the area under the JNK activity curve before and after the predicted miMOMP threshold using a two‑tailed t‑test; falsify the hypothesis if no significant change in SASP output is observed after crossing the threshold.

If the data show that SASP induction strictly depends on surpassing a defined miMOMP‑ROS oscillation threshold, and that manipulating redox‑sensitive AP‑1 cysteines or MKP‑1 levels shifts this threshold as predicted, the model gains mechanistic support. Conversely, a lack of correlation between miMOMP frequency and JNK‑AP‑1 state would falsify the proposed gatekeeping role of miMOMP.