Fractionated radiation suppresses Kruppel-like factor 2 pathway to a greater extent than by single exposure to the same total dose

Ratan Sadhukhan,Sarthak Garg,Alena V Savenka,Kimberly J Krager,Justin W C Leung,Rupak Pathak,Alexei G Basnakian

doi:10.1038/s41598-020-64672-3

Abstract

Kruppel-like factor 2 (KLF2) is a positive transcriptional regulator of several endothelial protective molecules, including thrombomodulin (TM), a surface receptor, and endothelial nitric oxide synthase (eNOS), an enzyme that generates nitric oxide (NO). Loss of TM and eNOS causes endothelial dysfunction, which results in suppressed generation of activated protein C (APC) by TM-thrombin complex and in upregulation of intercellular adhesion molecule 1 (ICAM-1). Mechanistic studies revealed that activation of extracellular signal-regulated kinase 5 (ERK5) via upregulation of myocyte enhancer factor 2 (MEF2) induces KLF2 expression. Radiation causes endothelial dysfunction, but no study has investigated radiation’s effects on the KLF2 pathway. Because fractionated radiation is routinely used during cancer radiotherapy, we decided to delineate the effects of radiation dose fractionation on the KLF2 signaling cascade at early time points (up to 24 h). We exposed human primary endothelial cells to radiation as a series of fractionated or as a single exposure, with the same total dose delivered to each group. We measured the expression and activity of critical members of the KLF2 pathway at subsequent time points, and determined whether pharmacological upregulation of KLF2 can reverse the radiation effects. Compared to single exposure, fractionated radiation profoundly suppressed KLF2, TM, and eNOS levels, subdued APC generation, declined KLF2 binding ability to TM and eNOS promoters, enhanced ICAM-1 expression, and decreased expression of upstream regulators of KLF2 (ERK5 and MEF2). Pharmacological inhibitors of the mevalonate pathway prevented fractionated-radiation–induced suppression of KLF2, TM, and eNOS expression. Finally, fractionated irradiation to thoracic region more profoundly suppressed KLF2 and enhanced ICAM-1 expression than single exposure in the lung at 24 h. These data clearly indicate that radiation dose fractionation plays a critical role in modulating levels of KLF2, its upstream regulators, and its downstream target molecules in endothelial cells. Our findings will provide important insights for selecting fractionated regimens during radiotherapy and for developing strategies to alleviate radiotherapy-induced toxicity to healthy tissues.

Highlights

Endothelium—the inner lining of blood and lymphatic vessels―is composed of endothelial cells and forms a critical component of all the body’s organs
Kruppel like factor-2 (KLF2) is suppressed at a higher level by fractionated radiation than by a single exposure to the same total radiation dose
We used immunofluorescence to examine KLF2 expression and localization, and these results confirmed those of the Western blot analyses, indicating that KLF2 expression was more profoundly suppressed by fractionated radiation than by a single exposure (Fig. 1c)

Summary

Introduction

Endothelium—the inner lining of blood and lymphatic vessels―is composed of endothelial cells and forms a critical component of all the body’s organs. Dysfunctional endothelium results in loss of barrier integrity and impaired blood flow, which causes tissue edema, ischemia, and hypoxia that subsequently trigger other pathological cascades, including inflammation, oxidative stress, excessive thrombosis, and fibrotic changes[8,13,14]. All these adverse events increase the risk of developing cardiovascular, pulmonary, neurological, and gastrointestinal disease[15,16]. It is not known whether these mevalonate pathway inhibitors can modify the radiation effects on KLF2

Methods

Results

Conclusion