Intracellular autoactivation and surface location of hepsin, TMPRSS2, and TMPRSS13

Hepsin (HPN), a Type II transmembrane serine protease (TTSP), is involved in hepatocyte metabolism and various diseases. It undergoes autoactivation on the surface of human hepatoma cells, a mechanism not observed in other cell types. This study aims to explore HPN activation and surface expression in endometrial epithelial cells.

We studied HPN zymogen activation and cell surface expression in human embryonic kidney 293 and endometrial epithelial AN3CA and Ishikawa cells using site-directed mutagenesis, Western blotting, flow cytometry, and immunostaining. Treatments with brefeldin A (BFA) and monensin, along with co-transfection assays, were employed to assess HPN activation and expression before reaching the cell surface. We also analyzed the activation and expression of TMPRSS2 and TMPRSS13 and examined the effect of the serine protease inhibitor HAI-1 on these proteases.

HPN zymogen autoactivates in the endoplasmic reticulum (ER) and Golgi apparatus. Its active form reduces cell surface expression through trans-autodegradation, a mechanism also applicable to in TMPRSS2 and TMPRSS13. Additionally, HAI-1 interacts with these TTSPs in different ways: it inhibits HPN activation and stabilizes its cell-surface expression; it inhibits TMPRSS2 activation without affecting its cell-surface expression; and it facilitates TMPRSS13 activation, protecting it from degradation and stabilizing its cell surface expression.

These results revealed an intracellular autoactivation and expression mechanism of HPN, TMPRSS2, and TMPRSS13, differing from the extracellular activated TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation, potentially aiding in treating TTSP-related endometrial diseases.