Biomedical subjects

John P Hobson

Publications and source records attributed to John P Hobson.

6 recordsLinked to original sources

PubMed2006

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.

Capillaries↗

PubMed2006

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

We describe a simple, sensitive and noninvasive assay that uses nontoxic, reengineered anthrax toxin-beta-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.

Antigens, Bacterial↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2004

Mouse DESC1 is located within a cluster of seven DESC1-like genes and encodes a type II transmembrane serine protease that forms serpin inhibitory complexes.

We report the identification and functional analysis of a type II transmembrane serine protease encoded by the mouse differentially expressed in squamous cell carcinoma (DESC) 1 gene, and the definition of a cluster of seven homologous DESC1-like genes within a 0.5-Mb region of mouse chromosome 5E1. This locus is syntenic to a region of human chromosome 4q13.3 containing the human orthologues of four of the mouse DESC1-like genes. Bioinformatic analysis indicated that all seven DESC1-like genes encode functional proteases. Direct cDNA cloning showed that mouse DESC1 encodes a multidomain serine protease with an N-terminal signal anchor, a SEA (sea urchin sperm protein, enterokinase, and agrin) domain, and a C-terminal serine protease domain. The mouse DESC1 mRNA was present in epidermal, oral, and male reproductive tissues and directed the translation of a membrane-associated 60-kDa N-glycosylated protein with type II topology. Mouse DESC1 was synthesized in insect cells as a zymogen that could be activated by exposure to trypsin. The purified activated DESC1 hydrolyzed synthetic peptide substrates, showing a preference for Arg in the P1 position. DESC1 proteolytic activity was abolished by generic inhibitors of serine proteases but not by other classes of protease inhibitors. Most interestingly, DESC1 formed stable inhibitory complexes with both plasminogen activator inhibitor-1 and protein C inhibitor that are expressed in the same tissues with DESC1, suggesting that type II transmembrane serine proteases may be novel targets for serpin inhibition. Together, these data show that mouse DESC1 encodes a functional cell surface serine protease that may have important functions in the epidermis, oral, and reproductive epithelium.

Amino Acid Sequence↗

PubMed2004

Role of sphingosine-1-phosphate phosphatase 1 in epidermal growth factor-induced chemotaxis.

Sphingosine-1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors that regulate a wide variety of cellular functions, including cytoskeletal rearrangements and cell motility. Because of the pivotal role of S1P, its levels are low and tightly regulated in a spatial-temporal manner through its synthesis catalyzed by sphingosine kinases and degradation by an S1P lyase and specific S1P phosphatases (SPP). Surprisingly, down-regulation of SPP-1 enhanced migration toward epidermal growth factor (EGF); conversely, overexpression of SPP-1, which is localized in the endoplasmic reticulum, attenuated migration toward EGF. To determine whether the inhibitory effect on EGF-induced migration was because of decreased S1P or increased ceramide as a consequence of acylation of increased sphingosine by ceramide synthase, we used fumonisin B1, a specific inhibitor of ceramide synthase. Although fumonisin B1 blocked ceramide production and increased sphingosine, it did not reverse the negative effect of SPP-1 expression on EGF- or S1P-induced chemotaxis. EGF activated the epidermal growth factor receptor to the same extent in SPP-1-expressing cells, yet ERK1/2 activation was impaired. In agreement, PD98059, an inhibitor of the ERK-activating enzyme MEK, decreased EGF-stimulated migration. We next examined the possibility that intracellularly generated S1P might be involved in activating a G protein-coupled S1P receptor important for EGF-directed migration. Treatment with pertussis toxin to inactivate Galpha(i) suppressed EGF-induced migration. Moreover, expression of regulator of G protein signaling 3, which inhibits S1P receptor signaling and completely prevented ERK1/2 activation mediated by S1P receptors, not only reduced migration toward S1P but also markedly reduced migration toward EGF. Collectively, these results suggest that metabolism of S1P by SPP-1 is important for EGF-directed cell migration.

Blotting, Western↗

PubMed2002

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 Ca(2+) but not via G(i) 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.

Blotting, Western↗