Age‑Dependent Myosin Light Chain Kinase Hyperactivity Precedes Tight Junction Loss and Drives Early Endotoxin Translocation in Human Colon

Evidence shows aging colons lose ZO‑1, occludin and JAM‑A, yet it remains unclear whether this loss initiates inflammaging or merely reflects it 12. We hypothesize that an age‑related increase in basal myosin light chain kinase (MLCK) activity generates sustained perijunctional contractile tension that phosphorylates occludin and remodels ZO‑1 before any measurable decline in total protein levels, thereby increasing LPS permeability and triggering systemic inflammation.

Mechanistic rationale

• With age, colonic submucosal fibrosis raises tissue stiffness, enhancing integrin α5β1‑mediated mechanotransduction to epithelial cells 3. This chronic tensile bias primes MLCK for heightened activity independent of inflammatory cytokines.
• Persistent MLCK‑driven phosphorylation of occludin at Ser408 disrupts its binding to ZO‑1, causing rapid reorganization of the perijunctional F‑actin ring and opening of paracellular pathways 4.
• Because total occludin/ZO‑1 protein synthesis remains unchanged early in this process, standard immunostaining may miss the functional deficit until prolonged phosphorylation triggers proteolytic degradation.
• The resulting rise in paracellular LPS flux engages TLR4 on lamina propria macrophages, activating NF‑κB and MAPK pathways and elevating circulating IL‑6 and TNF‑α 5.

Testable predictions

1. In a longitudinal cohort of healthy adults aged 40‑80, baseline colonic biopsies will show higher phospho‑occludin (Ser408) and MLCK activity levels in individuals who later develop a decline in junctional ZO‑1/occludin signal.
2. Increases in phospho‑occludin/MLCK activity will precede detectable rises in serum LPS‑binding protein (LBP) and subsequent elevations in plasma IL‑6/TNF‑α by at least 12‑24 months.
3. Pharmacologic inhibition of MLCK (e.g., with ML‑7) in ex‑vivo aged colonic explants will reduce occludin phosphorylation, preserve ZO‑1 localization, and blunt LPS flux compared with vehicle controls.

Study design

• Recruit 200 participants without IBD, diabetes, or immunosuppression.
• Collect sigmoid colon biopsies at baseline and every 2 years for up to 8 years; assay total and junctional ZO‑1/occludin (immunofluorescence quantification), phospho‑occludin (Western blot), and MLCK activity (ELISA for phosphorylated MLC2).
• Simultaneously measure fecal LPS concentration, serum LBP, IL‑6, and TNF‑α.
• Use mixed‑effects modeling to test whether baseline phospho‑occludin/MLCK predicts future changes in junctional protein levels and inflammatory markers, adjusting for age, BMI, and medication use.

Falsifiability If longitudinal data reveal that declines in junctional ZO‑1/occludin occur without prior increases in phospho‑occludin or MLCK activity, or if LPS and cytokine elevations appear independently of measurable changes in MLCK signaling, the hypothesis would be refuted. Conversely, confirmation would establish MLCK‑mediated tension as an early, actionable driver of age‑related barrier failure and inflammaging, redirecting therapeutic focus toward mechanoprotective strategies.