Anti-SPINT1 monoclonal antibody, clone 200843

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.

Hepatocyte growth factor activator inhibitor-1 (HAI-1), encoded by the serine protease inhibitor Kunitz type 1 (SPLVT1) gene, is a membrane-associated proteinase inhibitor that potently inhibits a variety of serine proteinases, including those that are membrane bound. Although HAI-1/SPINT1 is widely expressed by epithelial cells and cancer cells, its functional role is still unclear, particularly in cancer. Here, we show that stable knockdown of HAI-1/SPINT1 in the human pancreatic cancer cell line SUIT-2 induces an elongated spindle-like morphology associated with accelerated invasion, thereby mimicking an epithelial to mesenchymal transition (EMT). We found that HAI-1/SPINT1 knockdown significantly reduced the expression of E-cadherin and was accompanied by up-regulation of Smad-interacting protein 1 (SUP1), an E-cadherin transcriptional repressor. In addition, matrix metalloproteinase-9 (MMP-9) was up-regulated. Similar results were obtained in the HLC-1 lung carcinoma cell line. Moreover, a metastatic variant of SUIT-2 (S2-CP8) that showed loss of E-eadherin expression also showed a significantly reduced level of HAI-1/SPINT1. Engineered overexpression of HAI-1/SPINT1 in S2-CP8 resulted in reversion of E-cadherin expression and SIPI down-regulation, which accompanied reestablishment of epithelial morphology in culture. The EMT caused by HAI-1/SPINT1 knockdown seemed to be mediated at least partly, by membrane-bound serine proteinases: matriptase/ST14 and TMPRSS4, as knockdown of matriptase/ST14 or TMPRSS4 in HAI-1/SPINT1 knockdown SLTIT-2 cells and HLC-1 cells resulted in reversion of SIP1 and/or MMP-9 expression levels. We suggest that interactions between HAI-1/SPINT1 and membrane-bound serine proteinases contribute to transcriptional and functional changes involved in EMT in certain carcinoma cells. [Cancer Res 2009;69(5):1828-35]