Inhibition of HTRA3 stimulates trophoblast invasion during human placental development

Controlled invasion of extravillous trophoblast (EVT) is necessary for implantation and placentation. The serine protease HTRA3 is highly expressed in decidual cells in the late secretory phase of the menstrual cycle and throughout pregnancy. During the first trimester it is expressed in most trophoblast cell types, but not in the invading interstitial trophoblast. HTRA3 and its family members are down-regulated in a number of cancers and are proposed as tumour-suppressors. The current study investigated whether inhibiting HTRA3 in a first trimester trophoblast cell line expressing high levels of HTRA3 would alter invasion. HTR-8/SVneo (HTR-8, derived from first trimester placenta) and a number of choriocarcinoma cells (JEG-3, AC-1M88 and AC-1M32) were screened for HTRA3 expression. Only HTR-8 cells expressed high levels of HTRA3 mRNA, consistent with HTRA3 being down-regulated in cancer. Western blotting and immunofluorescence confirmed HTRA3 protein expression and localisation in HTR-8 cells. HTRA3 was detected in conditioned medium of HTR-8 cells, confirming its secretory nature. For functional studies, both long and short forms of recombinant human HTRA3, wild type and protease-inactive mutant (S 305 A) were produced using wheat-germ cell-free technology. Both have a similar molecular size, but the mutants have negligible protease activity. In addition, the mutants significantly inhibited the wild type protease activity, supporting their dominant-negative inhibition and utility as specific inhibitors of the wild type protein. Inhibition of HTRA3 by exogenous addition of HTRA3 mutant resulted in a significant increase in HTR-8 cell invasion. These results strongly support the hypothesis that HTRA3 is an inhibitor of trophoblast invasion during placental development.