The role of Hepsin in diaphragm development

Hepsin, a type II transmembrane serine protease, is mainly expressed in the liver. It was investigated that in vitro Hepsin can activate the human factor VIIa and hepatocyte growth factor precursor (pro-HGF). It been proposed that Hepsin may be involved in blood coagulation and hepatocyte growth. Whereas Hepsin knockout mice have normal blood clotting function, liver weight and liver regeneration ability. The physiological function of hepsin remains unclear. In our previous studies showed that Hepsin knockout mice diaphragm area, diameter and the distance between liver edge to the last ribs are significantly less than the wild-type (WT) mice. Moreover, Diaphragm central tendon part of knockout mice was thinner than those in the wild-type mice. Under Methacholine stimulation, the airway resistance and lung compliance of Hepsin knockout mice was inferior to wild-type mice. It was proposed that the deficiency of Hepsin may induce the eventration of the diaphragm. In this studies, the human Hepsin transgene mice (Tg-hHPN) were used to investigate whether Hepsin participate in diaphragm development. Firstly, Tg-hHPN mice were established to express wild-type human Hepsin or the mutant form human Hepsin, HepsinRS. After obtaining the founders (G0), those mice crossed with Hepsin knockout mice. To obtain the highest and stable human Hepsin expressing transgene mice, the Hepsin expression level was detected in the liver of each founder offspring by Western blots. Wherein establishing the Tg-hHPNWT mice of line-68 and the line-5, strain and established the Tg-hHPNRS mice of line-39 and line-54.The G2 generation of the transgenic mice was used for the analisus in this study. The results showed that the diaphragm area, diameter and the distance between liver edge to the last ribs human Hepsin expressing with mice Hepsin-deficiency transgenic mice (Tg-hHPN; HPN - / -) of the significantly greater than Hepsin knockout mice. It showed that expression of human Hepsin can rescue developmental defects in the diaphragm in knockout mice. To understand the role of Hepsin in diaphragm development, we also measured diaphragm precursor structure the pleuroperitoneal folds (PPF) area and the number of muscle progenitors cell and Fibroblast in the transgene mive embryo (E13.5). The results showed that Hepsin knockout mouse had no difference in the PPF area and the number of fibroblasts, while the number of muscle precursor cells less than the wild-type (but no significant difference) continue to accrue n value). Based on the results, Hepsin may affect the muscle precursor cells, which resulted in the development of the central tendon in the diaphragm and caused diaphragm eventration.