SPINT1 peptide

Hepatocyte growth factor activator inhibitor type 1/serine protease inhibitor Kunitz type 1 (HAI-1/SPINT1) is a membrane-bound Kunitz-type serine protease inhibitor that is abundantly expressed on the surface of cytotrophoblasts, and is critically required for the formation of the placenta labyrinth in mice. HAI-1/SPINT1 regulates several membrane-associated cell surface serine proteases, with matriptase being the most cognate target. Matriptase degrades extracellular matrix protein such as laminin and activates other cell surface proteases including prostasin. This study aimed to analyze the role of HAI-1/SPINT1 in pericellular proteolysis of trophoblasts. In HAI-1/SPINT1-deficient mouse placenta, laminin immunoreactivity around trophoblasts was irregular and occasionally showed an intense punctate pattern, which differed significantly from the linear distribution along the basement membrane observed in wild-type placenta. To explore the molecular mechanism underlying this observation, we analyzed the effect of HAI-1/SPINT1 knock down (KD) on pericellular proteolysis in the human trophoblast cell line, BeWo. HAI-1/SPINT1-KD BeWo cells had increased amounts of cellular laminin protein and decreased laminin degradation activity in the culture supernatant. Subsequent analysis indicated that cell-associated matriptase was significantly decreased in KD cells whereas its mRNA level was not altered, suggesting an enhanced release and/or dislocation of matriptase in the absence of HAI-1/SPINT1. Moreover, prostasin activation and pericellular total serine protease activities were significantly suppressed by HAI-1/SPINT1 KD. These observations suggest that HAI-1/SPINT1 is critically required for the cell surface localization of matriptase in trophoblasts, and, in the absence of HAI-1/SPINT1, physiological activation of prostasin and other protease(s) initiated by cell surface matriptase may be impaired.

Healthy placental development is essential for reproductive success; failure of the feto-maternal interface results in pre-eclampsia and intrauterine growth retardation. We found that grainyhead-like 2 (GRHL2), a CP2-type transcription factor, is highly expressed in chorionic trophoblast cells, including basal chorionic trophoblast (BCT) cells located at the chorioallantoic interface in murine placentas. Placentas from Grhl2-deficient mouse embryos displayed defects in BCT cell polarity and basement membrane integrity at the chorioallantoic interface, as well as a severe disruption of labyrinth branching morphogenesis. Selective Grhl2 inactivation only in ep cells rescued all placental defects but phenocopied intraembryonic defects observed in global Grhl2 deficiency, implying the importance of Grhl2 activity in trophectoderm-derived cells. ChIP-seq identified 5282 GRHL2 binding sites in placental tissue. By integrating these data with placental gene expression profiles, we identified direct and indirect Grhl2 targets and found a marked enrichment of GRHL2 binding adjacent to genes downregulated in Grhl2(-/-) placentas, which encoded known regulators of placental development and epithelial morphogenesis. These genes included that encoding the serine protease inhibitor Kunitz type 1 (Spint1), which regulates BCT cell integrity and labyrinth formation. In human placenta, we found that human orthologs of murine GRHL2 and its targets displayed co-regulation and were expressed in trophoblast cells in a similar domain as in mouse placenta. Our data indicate that a conserved Grhl2-coordinated gene network controls trophoblast branching morphogenesis, thereby facilitating development of the site of feto-maternal exchange. This might have implications for syndromes related to placental dysfunction.