Hsp90 Inhibitors Suppress P53 Phosphorylation in LPS‐Induced Endothelial Inflammation
Authors: Uddin Mohammad Afaz , Catravas John D , Barabutis Nektarios 
Published: June 2021

INTRODUCTION

Dysregulation of vascular barrier integrity is considered a cause and a consequence of inflammation. We have recently reported that P53 orchestrates meticulous responses to repair toxin ‐ induced injury to the human vasculature. Hsp90 inhibition increased P53 expression levels and deactivated the actin severing activity of cofilin, while LPS opposed those effects. To further elucidate the mechanisms that mediate the protective role of “the guardian of the genome” against endothelial barrier dysfunction and acute lung injury (ALI), we investigated the effects of Hsp90 inhibition on LPS – induced P53 phosphorylation in an in vivo and in vitro model of ALI.

METHODS

Bovine Pulmonary Arterial Endothelial Cells (BPAEC) were maintained at 37°C in a humidified atmosphere of 5% CO2 − 95% air, and were cultured in DMEM supplemented with 10% FBS. Protein levels were measured by Western Blot and analyzed by Image J. The antibodies were purchased from Cell Signaling (Danvers, MA). Mouse bronchoalveolar lavage fluid (BALF) was obtained by instilling and withdrawing 1ml 1x PBS in male C57BL/6 mice (7–8 weeks old) via a tracheal cannula. Cytokines were measured using a MAGPIX instrument (EMD Millipore).

RESULTS

BPAEC exposed to LPS (1 μg/ml or 10 μg/ml for 30, 60 and 120 minutes) exhibited increased phosphorylation of P53 at Ser6, Ser15, Ser33 and Ser392. Furthermore, LPS triggered the activation of myosin light chain 2 (MLC2). Pretreatment with the Hsp90 inhibitor 17DMAG (5 μM for 16 hrs.), prevented both the P53 and MLC2 phosphorylation. In our in vivo model of ALI, mice exposed to LPS (1.5 mg/kg, i.t) demonstrated elevated levels of the pro‐inflammatory cytokines IL‐2 and IL‐10 in the BALF. In bold contrast, animals that were post‐treated with the HSP90 inhibitor AUY922 (10 mg/kg each for 48 hrs.) were protected against the LPS‐induced elevation of IL‐2 and IL‐10 concentrations in their BALF.

CONCLUSIONS

Our study supports our previous findings on the mechanisms that regulate endothelial barrier function, and suggests that P53 is an appealing target in the investigation of new therapeutic avenues towards inflammation.

Support or Funding Information

This work was supported by 1) Start ‐ up funds (5RSPEC 300010 271008) to N.B from the College of Pharmacy, University of Louisiana Monroe, 2) The Faculty Research Support Program from the Dean's Office (5CALHN‐260615) to N.B from the College of Pharmacy, University of Louisiana Monroe, 3) The Malcolm Feist Partners Across Campuses Seed Program (Grant Index # BRS005) to N.B (co‐PI), Center for Cardiovascular Diseases and Sciences, Louisiana State University Health Shreveport, 4) The Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health (5 P20 GM103424‐15 and 3 P20 GM103424‐15S1) to N.B. and 5) NHLBI P01‐HL101902.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.