# Calpain/talin/MLCP axis in pulmonary endothelial barrier regulation

> **NIH NIH R01** · AUGUSTA UNIVERSITY · 2022 · $721,969

## Abstract

PROJECT SUMMARY
Acute lung injury (ALI) is characterized by lung vascular endothelial cell (EC) barrier compromise resulting in
pulmonary edema. While bacterial infections induced by E.coli or Streptococcus pneumoniae (Spn), and their
respective bacteria toxin lipopolysaccharides (LPS) and pneumolysin (PLY) are the major causes for ALI, the
molecular mechanisms involved in LPS- or PLY-induced ALI are ill-defined. The Scientific Premise comes from
our novel preliminary data, which show that both LPS and PLY activate endopeptidase calpain in human lung
microvascular ECs (HLMVECs) in ERK-dependent manner and that specific calpain inhibition prevents LPS-
and PLY-induced disruption of EC barrier in HLMVECs and LPS-induced pulmonary edema in ALI. Further, we
found that ERK-mediated calpain phosphorylation at Ser-50 and calpain activity were much higher in murine
lung microvascular ECs isolated from lungs exposed to LPS, indicating that ERK-calpain pathway is highly
related to ALI. We show that LPS/PLY induces talin cleavage into head and rod domain and talin phosphorylation
in HLMVECs and that overexpression of calpain causes talin cleavage, and Rho-mediated inhibition of myosin
light chain phosphatase (MLCP). Talin is activated through either talin cleavage or phosphorylation. Talin
cleavage separates head from rod domain thus removing auto-inhibition and stimulating talin head binding to
integrin and thus induces activation of focal adhesions (FAs), leading to RhoA-mediated MLCP inhibition and
increased lung EC permeability. We found that MLCP, which opposes EC barrier compromise in ALI, is down-
regulated in lungs of ARDS patients, highlighting the importance of MLCP inhibition in human ALI. MLCP binds
talin and induced talin dephosphorylation, which may lead to talin degradation followed by FAs disassembly.
Interestingly, MLCP dephosphorylates another calpain substrate, ezrin, leading to its deactivation and withdrawal
from membrane cytoskeleton. Activation of talin/ezrin axis is involved in FA formation and our data suggest that
talin is upstream of ezrin in LPS-induced EC cytoskeletal remodeling. It was shown that ezrin depletion
decreases calpain activity suggesting positive feedback effect of MLCP inhibition on LPS/PLY-induced calpain
activation and EC barrier compromise via ezrin phosphorylation/membrane attachment. These data led to our
novel hypothesis that calpain/MLCP crosstalk coordinates talin activation leading to lung EC barrier disruption
in ALI. To test this hypothesis we will employ unique molecular tools such as murine lung-targeting gene delivery
in vivo, EC-specific inducible calpain and MLCP KO mice and ERK insensitive knockin mouse (Capn2S50A). We
will: (1) evaluate whether ERK-mediated calpain activation is involved in lung microvascular EC barrier disruption
and cytoskeletal reorganization in ALI induced by bacterial toxins (LPS, PLY), live bacteria (E.coli, Spn) and in
sepsic conditions; (2) determine whether Gram- and Gram...

## Key facts

- **NIH application ID:** 10522290
- **Project number:** 1R01HL158909-01A1
- **Recipient organization:** AUGUSTA UNIVERSITY
- **Principal Investigator:** YUNCHAO SU
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $721,969
- **Award type:** 1
- **Project period:** 2022-09-01 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10522290

## Citation

> US National Institutes of Health, RePORTER application 10522290, Calpain/talin/MLCP axis in pulmonary endothelial barrier regulation (1R01HL158909-01A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10522290. Licensed CC0.

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