In vitro characterization of TMPRSS2 inhibition in IPEC-J2 cells

Abstract

The transmembrane serine protease, TMPRSS2 is an important target in the treatment of seasonal influenza infections and contributes to prostate carcinogenesis and metastasis. In this study, the effect of the synthetic TMPRSS2 inhibitor I-432 on jejunal IPEC-J2 cell monolayers cultured on membrane inserts was characterized. Using a fluorogenic substrate, it was found that the apical addition of I-432 could suppress trypsin-like activity in the supernatants of IPEC-J2 cells. The inhibition of TMPRSS2 did not affect physiologically produced hydrogen peroxide levels in the apical and in basolateral compartments. Loss of expression of the TMPRSS2 serine protease domain (28 kDa) was also observed when cells were pre-exposed to I-432. Partial decrease in immunofluorescent signal intensities derived from the altered distribution pattern of TMPRSS2 was detected after a 48 h long incubation of IPEC-J2 cells with the inhibitor indicating the efficacy of TMPRSS2 inhibition via I-432 administration in vitro.

Keywords:

• 3-Amidinophenylalanine
• extracellular H₂O₂
• fluorogenic AMC substrate
• IPEC-J2 cells
• TMPRSS2
• trypsin-like serine protease

Acknowledgements

We are indebted to Dr. Jody Gookin and Dr. Stephen Stauffer, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA for providing IPEC-J2 cells and for the valuable advice on handling them and to Dr. Nóra Meggyesházi, Semmelweis University, 1st Department of Pathology and Experimental Cancer Research for contributing to taking fluorescent images of the IPEC-J2 samples. This project was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences and by the Hungarian Scientific Research Fund [grant number 115685].

Declaration of interest

The authors report no declaration of interest. The authors alone are responsible for the content and writing of this article. This research was supported by the 9877–3/2015/FEKUT grant of the Hungarian Ministry of Human Resources.