Summary We report an unexpected role for protease signaling in neural tube closure and formation of the central nervous system. Mouse embryos lacking protease-activated receptor 1 and 2 showed defective hindbrain and posterior neuropore closure and developed exencephaly and spina bifida, important human congenital anomalies. Par1 and Par2 were expressed in surface ectoderm, Par2 selectively along the line of closure. Ablation of Gi/z and Rac1 function in these Par2-expressing cells disrupted neural tube closure, further implicating G protein-coupled receptors and identifying a likely effector pathway. Cluster analysis of protease and Par2 expression patterns revealed a group of membrane-tethered proteases often co-expressed with Par2. Among these, matriptase activated Par2 with picomolar potency, and hepsin and prostasin activated matriptase. Together, our results suggest a role for protease-activated receptor signaling in neural tube closure and identify a local protease network that may trigger Par2 signaling and monitor and regulate epithelial integrity in this context.

The homotrimeric human serine protease HtrA1 is homologous to bacterial HtrA proteases regarding the trypsin-like catalytic and PDZ domains but differs by the presence of an N-terminal domain with IGFBP and Kazal homology. The crystal structures and SAXS analysis presented herein reveal the rare tandem of IGFBP- and Kazal-like modules, a protease active site that adopts a competent conformation in the absence of substrate or inhibitor and a model for the intact protein in solution. Highly sensitive enzymatic assays and binding studies demonstrate that the N-terminal tandem has no apparent effect on protease activity, and in accordance with the structure-based predictions, neither the IGFBP- nor Kazal-like module retains the function of their prototype proteins. Our structures of the unliganded HtrA1 active site suggest two-state equilibrium and a "conformational selection" model, in which substrate binds to the active conformer.

Financial support: All authors are paid employees of the Janssen Pharmaceuticals, Johnsons & Johnson Group of companies and receiving salary and other compensation.

Hepsin, a cell surface protease, is widely reported to be overexpressed in more than 90% of human prostate tumors. Hepsin expression correlates with tumor progression, making it a significant marker and target for prostate cancer. Recently, it was reported that in a prostate cancer mouse model, hepsin up-regulation in tumor tissue promotes progression and metastasis. The underlying mechanisms, however, remain largely uncharacterized. Hepsin transgenic mice displayed reduced laminin-332 (Ln-332) expression in prostate tumors. This is an intriguing cue, since proteolytic processing of extracellular matrix macromolecules, such as Ln-332, is believed to be involved in cancer progression, and Ln-332 expression is lost during human prostate cancer progression. In this study, we provide the first direct evidence that hepsin cleaves Ln-332. Cleavage is specific, since it is both inhibited in a dose-dependent manner by a hepsin inhibitor (Kunitz domain-1) and does not occur when catalytically inactive hepsin is used. By Western blotting and mass spectrometry, we determined that hepsin cleaves the ␤3 chain of Ln-332. N-terminal sequencing identified the cleavage site at ␤3 Arg 245 , in a sequence context (SQLR 245 2LQGSCFC) conserved among species and in remarkable agreement with reported consensus target sequences for hepsin activity. In vitro cell migration assays showed that hepsin-cleaved Ln-332 enhanced motility of DU145 prostate cancer cells, which was inhibited by Kunitz domain-1. Further, hepsin-overexpressing LNCaP prostate cancer cells also exhibited increased migration on Ln-332. Direct cleavage of Ln-332 may be one mechanism by which hepsin promotes prostate tumor progression and metastasis, possibly by up-regulating prostate cancer cell motility.Prostate cancer is the second leading cause of cancer death in men in the United States; according to the American Cancer Society, 186,320 new prostate cancer cases and 28,660 deaths from prostate cancer are projected to occur in 2008 (1). This high rate of mortality is largely due to metastasis of the primary tumor (2). For metastasis to occur, primary tumor cells must breach the basement membrane (BM) 3 by degrading extracellular matrix (ECM) molecules to initiate the invasion process (3). Escaped tumor cells interact with neighboring ECM molecules to promote this activity. This interaction sometimes promotes remodeling of the ECM to create a more conducive environment for tumor cell migration and invasion and thereby aids in cancer progression (4). The remodeling of certain ECM molecules has been reported to occur as a direct result of protease processing, which results in increased tumor cell migration and invasion (5-7).Laminin-332 (Ln-332; previously known as laminin-5), an ECM molecule, is an important component of BM (8). It is a trimeric glycoprotein consisting of disulfide-bonded subunits: ␣3, ␤3, and ␥2 polypeptide chains (9). The importance of Ln-332 in BM assembly was established by the discovery of the occurrence of a lethal skin blistering disorder, jun...