John P. Hobson

Sphingosine 1-Phosphate Stimulates Cell Migration through a Gi-coupled Cell Surface Receptor POTENTIAL INVOLVEMENT IN ANGIOGENESIS

Sphingosine 1-phosphate (SPP) has been shown to inhibit chemotaxis of a variety of cells, in some cases through intracellular actions, while in others through receptor-mediated effects. Surprisingly, we found that low concentrations of SPP (10–100 nm) increased chemotaxis of HEK293 cells overexpressing the G protein-coupled SPP receptor EDG-1. In agreement with previous findings in human breast cancer cells (Wang, F., Nohara, K., Olivera, O., Thompson, E. W., and Spiegel, S. (1999) Exp. Cell Res. 247, 17–28), SPP, at micromolar concentrations, inhibited chemotaxis of both vector- and EDG-1-overexpressing HEK293 cells. Nanomolar concentrations of SPP also induced a marked increase in chemotaxis of human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC), which express the SPP receptors EDG-1 and EDG-3, while higher concentrations of SPP were less effective. Treatment with pertussis toxin, which ADP-ribosylates and inactivates Gi-coupled receptors, blocked SPP-induced chemotaxis. Checkerboard analysis indicated that SPP stimulates both chemotaxis and chemokinesis. Taken together, these data suggest that SPP stimulates cell migration by binding to EDG-1. Similar to SPP, sphinganine 1-phosphate (dihydro-SPP), which also binds to this family of SPP receptors, enhanced chemotaxis; whereas, another structurally related lysophospholipid, lysophosphatidic acid, did not compete with SPP for binding nor did it have significant effects on chemotaxis of endothelial cells. Furthermore, SPP increased proliferation of HUVEC and BAEC in a pertussis toxin-sensitive manner. SPP and dihydro-SPP also stimulated tube formation of BAEC grown on collagen gels (in vitro angiogenesis), and potentiated tube formation induced by basic fibroblast growth factor. Pertussis toxin treatment blocked SPP-, but not bFGF-stimulated in vitro angiogenesis. Our results suggest that SPP may play a role in angiogenesis through binding to endothelial cell Gi-coupled SPP receptors.

Neuropeptide Y induces migration, proliferation, and tube formation of endothelial cells bimodally via Y1, Y2, and Y5 receptors

Previously we discovered that NPY induces ischemic angiogenesis by activating Y2 and Y5 receptors. The receptors that mediate specific steps of the complex process of angiogenesis are unknown. Here, we studied in vitro NPY receptors subtypes involved in migration, proliferation, and differentiation of human endothelial cells. In cells that expressed Y1, Y2, and Y5 receptors, NPY bimodally stimulated migration and proliferation with a 2‐fold increase at 10−12 M and 10−8 M (high‐ and low‐affinity peaks, respectively). Preincubation of cells with NPY up‐regulated the Y5 receptor and markedly enhanced endothelial cell migration and proliferation. NPY‐induced endothelial cell migration was mimicked by agonists and fully blocked by antagonists for any specific NPY receptors (Y1, Y2, or Y5), while proliferation was blocked by any two antagonists (Y1+Y2, Y1+Y5, or Y2+Y5), and capillary tube formation on Matrigel was blocked by all three (Y1+Y2+Y5). Thus, NPY‐induced angiogenesis requires participation of Y1, Y2, and Y5 receptor subtypes, with the Y5 receptor acting as an enhancer. We propose that these receptors form heteromeric complexes, and the Y1/Y2/Y5 receptor oligomer may be the uncloned Y3 receptor.—Movafagh, S., Hobson, J. P., Spiegel, S., Kleinman, H. K., Zukowska, Z. Neuropeptide Y (NPY) induces migration, proliferation, and tube formation of endothelial cells bimodally via Y1, Y2, and Y5 receptors. FASEB J. 20, 1327–1337 (2006)

Regulation of cell surface protease matriptase by HAI2 is essential for placental development, neural tube closure and embryonic survival in mice

Hypomorphic mutations in the human SPINT2 gene cause a broad spectrum of abnormalities in organogenesis, including organ and digit duplications, atresia, fistulas, hypertelorism, cleft palate and hamartoma. SPINT2 encodes the transmembrane serine protease inhibitor HAI2 (placental bikunin), and the severe developmental effects of decreased HAI2 activity can be hypothesized to be a consequence of excess pericellular proteolytic activity. Indeed, we show here that HAI2 is a potent regulator of protease-guided cellular responses, including motogenic activity and transepithelial resistance of epithelial monolayers. Furthermore, we show that inhibition of the transmembrane serine protease matriptase (encoded by St14) is an essential function of HAI2 during tissue morphogenesis. Genetic inactivation of the mouse Spint2 gene led to defects in neural tube closure, abnormal placental labyrinth development associated with loss of epithelial cell polarity, and embryonic demise. Developmental defects observed in HAI2-deficient mice were caused by unregulated matriptase activity, as both placental development and embryonic survival in HAI2-deficient embryos were completely restored by the simultaneous genetic inactivation of matriptase. However, neural tube defects were detected in HAI2-deficient mice even in the absence of matriptase, although at lower frequency, indicating that the inhibition of additional serine protease(s) by HAI2 is required to complete neural development. Finally, by genetic complementation analysis, we uncovered a unique and complex functional interaction between HAI2 and the related HAI1 in the regulation of matriptase activity during development. This study indicates that unregulated matriptase-dependent cell surface proteolysis can cause a diverse array of abnormalities in mammalian development.

Co‐localization of the channel activating protease prostasin/(CAP1/PRSS8) with its candidate activator, matriptase

Prostasin (CAP1/PRSS8) is a glycosylphosphatidylinositol‐anchored membrane serine protease believed to be critical for the regulation of epithelial sodium channel (ENaC) activity. Prostasin is synthesized as an inactive zymogen that requires a site‐specific endoproteolytic cleavage to be converted to an active protease. We have recently reported that the tumor‐associated type II transmembrane serine protease, matriptase is necessary and sufficient for prostasin activation in the epidermis. In this study, the interrelationship between the two membrane serine proteases was investigated further by using enzymatic gene trapping combined with immunohistochemistry to delineate the spatial expression of matriptase and prostasin in mouse tissues. We utilized a knock‐in mouse with a promoterless β‐galactosidase marker gene inserted into the matriptase locus, as a unique tool for precise assessment of endogenous matriptase expression. The spatial expression of matriptase and prostasin in mouse tissues was delineated by combining in situ β‐galactosidase matriptase staining with immunohistochemical detection of prostasin. We report that prostasin displays a near‐ubiquitous co‐localization with its candidate activator matriptase in a variety of normal epithelial tissues. These include simple, stratified, and pseudo‐stratified epithelium of the integumentary system, digestive tract, respiratory tract, and urogenital tract. However, matriptase and prostasin expression segregates during epithelial multi‐stage carcinogenesis to eventually become localized in separate compartments of the tumor. These data suggest that a matriptase‐prostasin zymogen activation cascade may be functionally operative in multiple epithelial tissues, but matriptase promotes epithelial carcinogenesis independent of prostasin. J. Cell. Physiol. 213: 237–245, 2007.

Potent Inhibition and Global Co-localization Implicate the Transmembrane Kunitz-type Serine Protease Inhibitor Hepatocyte Growth Factor Activator Inhibitor-2 in the Regulation of Epithelial Matriptase Activity

Hepatocyte growth factor activator inhibitors (HAI)-1 and -2 are recently identified and closely related Kunitz-type transmembrane serine protease inhibitors. Whereas HAI-1 is well established as an inhibitor of the serine proteases matriptase and hepatocyte growth factor activator, the physiological targets of HAI-2 are unknown. Here we show that HAI-2 displays potent inhibitory activity toward matriptase, forms SDS-stable complexes with the serine protease, and blocks matriptase-dependent activation of its candidate physiological substrates proprostasin and cell surface-bound pro-urokinase plasminogen activator. To further explore the potential functional relationship between HAI-2 and matriptase, we generated a transgenic mouse strain with a promoterless β-galactosidase marker gene inserted into the endogenous locus encoding HAI-2 protein and performed a global high resolution mapping of the expression of HAI-2, matriptase, and HAI-1 proteins in all adult tissues. This analysis showed striking co-localization of HAI-2 with matriptase and HAI-1 in epithelial cells of all major organ systems, thus strongly supporting a role of HAI-2 as a physiological regulator of matriptase activity, possibly acting in a redundant or partially redundant manner with HAI-1. Unlike HAI-1 and matriptase, however, HAI-2 expression was also detected in non-epithelial cells of brain and lymph nodes, suggesting that HAI-2 may also be involved in inhibition of serine proteases other than matriptase.

Sphingosine 1-Phosphate, Present in Serum-derived Lipoproteins, Activates Matriptase

We describe here a novel biological function of sphingosine 1-phosphate (S1P): the activation of a serine protease, matriptase. Matriptase is a type II integral membrane serine protease, expressed on the surface of a variety of epithelial cells; it may play an important role in tissue remodeling. We have previously reported that the activation of matriptase is regulated by serum. We have now identified the bioactive component from serum. First, the activity was observed to co-purify with lipoproteins by conventional liquid chromatography and immunoaffinity chromatography. The ability of lipoproteins to induce the activation of matriptase was further confirmed with commercial preparations of low density lipoprotein (LDL) and very low density lipoprotein (VLDL). Next, we observed that the bioactive component of LDL is associated with the phospholipid components of LDL. Fractionation of lipid components of LDL by thin layer chromatography (TLC) revealed that the bioactive component of LDL comigrates with S1P. Nanomolar concentrations of commercially obtained S1P were then observed to induce the rapid activation of matriptase on the surfaces of nontransformed human mammary epithelial cells. Other structurally related sphingolipids, including dihydro-S1P, ceramide 1-phosphates, and sphingosine phosphocholine as well as lysophosphatidic acid, can also induce the activation of matriptase, but at significantly higher concentrations than S1P. Furthermore, S1P-dependent matriptase activation is dependent on Ca2+ but not via Gi protein-coupled receptors. Our results demonstrate that bioactive phospholipids can function as nonprotein activators of a cell surface protease, suggesting a possible mechanistic link between S1P and normal and possibly pathologic tissue remodeling.

Matriptase-3 is a novel phylogenetically preserved membrane-anchored serine protease with broad serpin reactivity.

We report in the present study the bioinformatic identification, molecular cloning and biological characterization of matriptase-3, a novel membrane-anchored serine protease that is phylogenetically preserved in fish, birds, rodents, canines and primates. The gene encoding matriptase-3 is located on syntenic regions of human chromosome 3q13.2, mouse chromosome 16B5, rat chromosome 11q21 and chicken chromosome 1. Bioinformatic analysis combined with cDNA cloning predicts a functional TTSP (type II transmembrane serine protease) with 31% amino acid identity with both matriptase/MT-SP1 and matriptase-2. This novel protease is composed of a short N-terminal cytoplasmic region followed by a transmembrane domain, a stem region with one SEA, two CUB and three LDLRa (low-density lipoprotein receptor domain class A) domains and a C-terminal catalytic serine protease domain. Transcript analysis revealed restricted, species-conserved expression of matriptase-3, with the highest mRNA levels in brain, skin, reproductive and oropharyngeal tissues. The full-length matriptase-3 cDNA directed the expression of a 90 kDa N-glycosylated protein that localized to the cell surface, as assessed by cell-surface biotin labelling. The purified activated matriptase-3 serine protease domain expressed in insect cells hydrolysed synthetic peptide substrates, with a strong preference for Arg at position P(1), and showed proteolytic activity towards several macromolecular substrates, including gelatin, casein and albumin. Interestingly, activated matriptase-3 formed stable inhibitor complexes with an array of serpins, including plasminogen activator inhibitor-1, protein C inhibitor, alpha1-proteinase inhibitor, alpha2-antiplasmin and antithrombin III. Our study identifies matriptase-3 as a novel biologically active TTSP of the matriptase subfamily having a unique expression pattern and post-translational regulation.

Imaging specific cell-surface proteolytic activity in single living cells.

We describe a simple, sensitive and noninvasive assay that uses nontoxic, reengineered anthrax toxin–β-lactamase fusion proteins with altered protease cleavage specificity to visualize specific cell-surface proteolytic activity in single living cells. The assay could be used to specifically image endogenous cell-surface furin, urokinase plasminogen activator and metalloprotease activity. We have adapted the assay for fluorescence microscopy, flow cytometry and fluorescent plate reader formats, and it is amenable for automation and high-throughput analysis.

The sphingosine-1-phosphate receptor EDG-1 is essential for platelet-derived growth factor-induced cell motility.

EDG-1, encoded by the endothelial differentiation gene-1, is a heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) for sphingosine-1-phosphate (SPP) that has been shown to stimulate angiogenesis and cell migration in cultured endothelial cells. Unexpectedly, EDG-1 knockout embryos had a normal blood vessel network, vasculogenesis and angiogenesis, but died in utero owing to massive haemorrhaging as a result of failure of smooth muscle cells and pericytes to migrate around the circumference and reinforce endothelial tubes [Liu, Wada, Yamashita, Mi, Deng, Hobson, Rosenfeldt, Nava, Chae, Lee, et al. (2000) J. Clin. Invest. 106, 951-961]. This vascular maturation defect is similar to the phenotype of mice homozygous for disrupted alleles of platelet-derived growth factor B-subunit homodimer (PDGF-BB) or its receptor PDGFR-beta. We found that fibroblasts from EDG-1 null embryos did not migrate toward PDGF or SPP, and inhibition of motility correlated with defective activation of the small guanosine triphosphatase Rac, which is required for lamellipodia formation and directional locomotion [Hobson, Rosenfeldt, Barak, Olivera, Poulton, Caron, Milstien, and Spiegel (2001) Science 291, 1800-1803]. Moreover, we showed that PDGF-directed cell migration requires both sphingosine kinase activation and expression of EDG-1, suggesting a functional link between PDGF signalling and EDG-1. Indeed, treatment of wild-type cells with PDGF transactivated EDG-1 as determined by translocation of beta-arrestin and phosphorylation of EDG-1. These findings reveal a new paradigm for receptor cross-communication in which activation of a GPCR by a receptor tyrosine kinase is critical for cell motility. Our observations might also clarify the role of EDG-1 in vascular maturation and angiogenesis.

Quantitative high-throughput screening identifies inhibitors of anthrax-induced cell death

Abstract Here, we report the results of a quantitative high-throughput screen (qHTS) measuring the endocytosis and translocation of a β-lactamase-fused-lethal factor and the identification of small molecules capable of obstructing the process of anthrax toxin internalization. Several small molecules protect RAW264.7 macrophages and CHO cells from anthrax lethal toxin and protected cells from an LF-Pseudomonas exotoxin fusion protein and diphtheria toxin. Further efforts demonstrated that these compounds impaired the PA heptamer pre-pore to pore conversion in cells expressing the CMG2 receptor, but not the related TEM8 receptor, indicating that these compounds likely interfere with toxin internalization.