ABSTRACT
A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrPSc, in the central nervous system of prion-affected humans and animals. PrPSc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrPSc. Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrPSc is tightly associated with proteins found in the systemic extracellular matrix, mostly fibronectin (FN). The interaction of PrPSc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich extracellular matrix while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrPSc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues.
Subject(s)
Fibronectins , Prion Diseases , Prions , Scrapie , Animals , Humans , Cell Line , Fibronectins/therapeutic use , Prion Diseases/drug therapy , Prion Diseases/prevention & control , Prions/metabolism , Scrapie/metabolism , SheepABSTRACT
UNLABELLED: In lethal prion neurodegenerative diseases, misfolded prion proteins (PrP(Sc)) replicate by redirecting the folding of the cellular prion glycoprotein (PrP(C)). Infections of different durations can have a subclinical phase with constant levels of infectious particles, but the mechanisms underlying this plateau and a subsequent exit to overt clinical disease are unknown. Using tandem biophysical techniques, we show that attenuated accumulation of infectious particles in presymptomatic disease is preceded by a progressive fall in PrP(C) level, which constricts replication rate and thereby causes the plateau effect. Furthermore, disease symptoms occurred at the threshold associated with increasing levels of small, relatively less protease-resistant oligomeric prion particles (oPrP(Sc)). Although a hypothetical lethal isoform of PrP cannot be excluded, our data argue that diminishing residual PrP(C) levels and continuously increasing levels of oPrP(Sc) are crucial determinants in the transition from presymptomatic to symptomatic prion disease. IMPORTANCE: Prions are infectious agents that cause lethal brain diseases; they arise from misfolding of a cell surface protein, PrP(C) to a form called PrP(Sc). Prion infections can have long latencies even though there is no protective immune response. Accumulation of infectious prion particles has been suggested to always reach the same plateau in the brain during latent periods, with clinical disease only occurring when hypothetical toxic forms (called PrP(L) or TPrP) begin to accumulate. We show here that infectivity plateaus arise because PrP(C) precursor levels become downregulated and that the duration of latent periods can be accounted for by the level of residual PrP(C), which transduces a toxic effect, along with the amount of oligomeric forms of PrP(Sc).
Subject(s)
Brain/metabolism , PrPSc Proteins/metabolism , Prion Diseases/metabolism , Protein Folding , Protein Multimerization , Animals , Brain/pathology , Mice , Mice, Transgenic , PrPSc Proteins/genetics , Prion Diseases/genetics , Prion Diseases/pathologyABSTRACT
UNLABELLED: Prion diseases are fatal neurodegenerative disorders associated with the conversion of cellular prion protein (PrPC) into its aberrant infectious form (PrPSc). There is no treatment available for these diseases. The bile acids tauroursodeoxycholic acid(TUDCA) and ursodeoxycholic acid (UDCA) have been recently shown to be neuroprotective in other protein misfolding disease models, including Parkinson's, Huntington's and Alzheimer's diseases, and also in humans with amyotrophic lateral sclerosis.Here, we studied the therapeutic efficacy of these compounds in prion disease. We demonstrated that TUDCA and UDCA substantially reduced PrP conversion in cell-free aggregation assays, as well as in chronically and acutely infected cell cultures. This effect was mediated through reduction of PrPSc seeding ability, rather than an effect on PrPC. We also demonstrated the ability of TUDCA and UDCA to reduce neuronal loss in prion-infected cerebellar slice cultures. UDCA treatment reduced astrocytosis and prolonged survival in RML prion-infected mice. Interestingly, these effects were limited to the males, implying a gender-specific difference in drug metabolism. Beyond effects on PrPSc, we found that levels of phosphorylated eIF2 were increased at early time points, with correlated reductions in postsynaptic density protein 95. As demonstrated for other neurodegenerative diseases, we now show that TUDCA and UDCA may have a therapeutic role in prion diseases, with effects on both prion conversion and neuroprotection. Our findings, together with the fact that these natural compounds are orally bioavailable, permeable to the blood-brain barrier, and U.S. Food and Drug Administration-approved for use in humans, make these compounds promising alternatives for the treatment of prion diseases. IMPORTANCE: Prion diseases are fatal neurodegenerative diseases that are transmissible to humans and other mammals. There are no disease-modifying therapies available, despite decades of research. Treatment targets have included inhibition of protein accumulation,clearance of toxic aggregates, and prevention of downstream neurodegeneration. No one target may be sufficient; rather, compounds which have a multimodal mechanism, acting on different targets, would be ideal. TUDCA and UDCA are bile acids that may fulfill this dual role. Previous studies have demonstrated their neuroprotective effects in several neurodegenerative disease models, and we now demonstrate that this effect occurs in prion disease, with an added mechanistic target of upstream prion seeding. Importantly, these are natural compounds which are orally bioavailable, permeable to the blood-brain barrier, and U.S.Food and Drug Administration-approved for use in humans with primary biliary cirrhosis. They have recently been proven efficacious in human amyotrophic lateral sclerosis. Therefore, these compounds are promising options for the treatment of prion diseases.
Subject(s)
Neurons/metabolism , PrPC Proteins/metabolism , PrPSc Proteins/metabolism , Prion Diseases/metabolism , Prion Diseases/physiopathology , Taurochenodeoxycholic Acid/metabolism , Ursodeoxycholic Acid/metabolism , Animals , Bile Acids and Salts/metabolism , Cell Death , Cell Survival , Female , Humans , Male , Mice , Mice, Inbred C57BL , Neurons/cytology , Species SpecificityABSTRACT
Mycoplasma bovis continues to cause significant disease in feedlots and dairy farms. The ability of the micro-organism to evade the immune system of the host combined with the lack of effective vaccines makes this disease difficult to control. Bacterin-based vaccines have not been successful in field trials and in some cases enhance the disease. In an attempt to develop a sub-unit vaccine, we used the conserved M. bovis glyceraldehyde-3-phosphate (GAPDH) protein in combination with a protein extract prepared from three M. bovis isolates to immunize feedlot animals. After challenge with a combination of three M. bovis isolates, there were differences in the proportion of weight loss between the control and vaccinated groups but no differences in rectal temperature and survival rate in all the groups. In addition, there were no significant differences between the proportions of lungs lesions in all the groups despite the percentages of lesions being higher in the vaccinated groups. These findings indicate that the M. bovis GAPDH protein is not a suitable antigen for a vaccine against this pathogen.
Subject(s)
Bacterial Vaccines/immunology , Cattle Diseases/immunology , Glyceraldehyde-3-Phosphate Dehydrogenases/immunology , Immunity, Humoral , Mycoplasma Infections/veterinary , Mycoplasma bovis/immunology , Animals , Antibodies, Bacterial/immunology , Bacterial Vaccines/administration & dosage , Bacterial Vaccines/genetics , Cattle , Cattle Diseases/microbiology , Cattle Diseases/prevention & control , Female , Glyceraldehyde-3-Phosphate Dehydrogenases/administration & dosage , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Male , Mycoplasma Infections/immunology , Mycoplasma Infections/microbiology , Mycoplasma Infections/prevention & control , Mycoplasma bovis/genetics , VaccinationABSTRACT
Besides the well characterized role in glycolysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been implicated in virulence of pathogenic micro-organisms and because of its cell surface location, it has been shown to act as an adhesin for colonization of tissue surfaces both for pathogenic and non-pathogenic normal microflora. These novel properties of GAPDH make this protein a target for studies in pathogenesis and a candidate for vaccine development against several diseases. Previously, we have isolated the GAPDH protein of Mycoplasma bovis and we are currently using this protein as a test antigen to develop a vaccine to protect feedlot animals from M. bovis-related diseases. As part of our vaccine studies, we are testing several novel immune modulators, some of which are host-defence peptides (HDP). HDP are small protein molecules that are part of the innate immune system of the host possess antimicrobial activities and can act as adjuvants. These novel compounds have been used as part of chimeric proteins composed of viral antigens fused to HDP and these chimeras were found to promote immune responses. The first step in the use of the M. bovis GAPDH protein and HDP as components of a vaccine was to construct M. bovis GAPDH-HDP chimeric proteins. The three M. bovis GAPDH-HDP chimeric proteins constructed here: GAPDH-BMAP28 (sGap-M), GAPDH-indolicidin (sGap-I), and GAPDH-TAP (Gap-T) retained properties associated with the individual components, namely GAPDH enzymatic and HDP antimicrobial activities.
Subject(s)
Defensins/pharmacology , Glyceraldehyde-3-Phosphate Dehydrogenases/pharmacology , Mycoplasma bovis/metabolism , Recombinant Fusion Proteins/pharmacology , Amino Acid Sequence , Animals , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antigens, Viral/biosynthesis , Bacterial Vaccines/biosynthesis , Bacterial Vaccines/chemistry , Bacterial Vaccines/genetics , Bacterial Vaccines/pharmacology , Cattle , Defensins/biosynthesis , Defensins/chemistry , Defensins/genetics , Gene Expression Regulation, Bacterial , Glyceraldehyde-3-Phosphate Dehydrogenases/biosynthesis , Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Immunity, Innate , Microbial Sensitivity Tests , Molecular Sequence Data , Mycoplasma bovis/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/geneticsABSTRACT
To determine if previous exposure to bovine viral diarrhea virus (BVDV) and bovine herpes virus 1 (BHV-1) type 2 affects the onset of disease caused by Mycoplasma bovis, 6- to 8-month-old beef calves were exposed to BVDV or BHV-1 4 d prior to challenge with a suspension of 3 clinical isolates of M. bovis. Animals were observed for clinical signs of disease and at necropsy, percent abnormal lung tissue and presence of M. bovis were determined. Most animals pre-exposed to BHV-1 type 2 but not BVDV developed M. bovis-related respiratory illness. In a second trial, we determined that a 100-fold reduction in the number of M. bovis bacteria administered to BHV-1 exposed animals reduced the percentage of abnormal lung tissue but not the severity of clinical signs. We conclude that previous exposure to BHV-1 but not BVDV type 2 was a necessary cause of M. bovis-related respiratory diseases in our disease model.
Subject(s)
Cattle Diseases/microbiology , Cattle Diseases/virology , Coinfection/veterinary , Herpesvirus 1, Bovine/pathogenicity , Mycoplasma bovis/pathogenicity , Respiratory Tract Infections/veterinary , Animals , Cattle , Coinfection/microbiology , Coinfection/virology , Diarrhea Virus 2, Bovine Viral/pathogenicity , Respiratory Tract Infections/microbiology , Respiratory Tract Infections/virologyABSTRACT
Mycoplasma bovis is a small, cell wall-less bacterium that contributes to a number of chronic inflammatory diseases in both dairy and feedlot cattle, including mastitis and bronchopneumonia. Numerous reports have implicated M. bovis in the activation of the immune system, while at the same time inhibiting immune cell proliferation. However, it is unknown whether the specific immune-cell population M. bovis is capable of attaching to and potentially invading. Here, we demonstrate that incubation of M. bovis Mb1 with bovine peripheral blood mononuclear cells (PBMC) resulted in a significant reduction in their proliferative responses while still remaining viable and capable of gamma interferon secretion. Furthermore, we show that M. bovis Mb1 can be found intracellularly (suggesting a role for either phagocytosis or attachment/invasion) in a number of select bovine PBMC populations (T cells, B cells, monocytes, γδ T cells, dendritic cells, NK cells, cytotoxic T cells, and T-helper cells), as well as red blood cells, albeit it at a significantly lower proportion. M. bovis Mb1 appeared to display three main patterns of intracellular staining: diffuse staining, an association with the intracellular side of the cell membrane, and punctate/vacuole-like staining. The invasion of circulating immune cells and erythrocytes could play an important role in disease pathogenesis by aiding the transport of M. bovis from the lungs to other sites.
Subject(s)
Erythrocytes/microbiology , Leukocytes, Mononuclear/microbiology , Mycoplasma Infections/immunology , Mycoplasma Infections/pathology , Mycoplasma bovis/pathogenicity , Animals , Apoptosis/physiology , Cattle , Erythrocytes/cytology , Interferon-gamma/metabolism , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lymphocyte Activation , Mycoplasma Infections/microbiology , Mycoplasma bovis/isolation & purification , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/microbiologyABSTRACT
Proteinase-activated receptor (PAR)(2) is activated by trypsin-like serine proteinases and has been implicated in intestinal inflammation. However, its role in the regulation of intestinal mucosal function remains unclear. Using the intestinal epithelial cell line, SCBN, we have studied the stimulus-secretion coupling mechanisms of PAR(2)-induced epithelial chloride transport, focusing on cyclooxygenase (COX)-1 and COX-2 activities and prostaglandin (PG) E(2) secretion. SCBN monolayers were grown on Snapwell supports, mounted in modified Ussing chambers, and exposed to the activating peptide, SLIGRL-NH(2) (50 microM), to activate PAR(2). Pretreatment with inhibitors of cytosolic PLA(2) (cPLA(2)) (AACOCF3, arachidonyltrifluoromethyl ketone), COX-1 [SC560, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole], and COX-2 (celecoxib) resulted in a significant concentration-dependent attenuation of PAR(2)-induced changes in short-circuit current. Immunoblot analysis showed a PAR(2)-induced increase in cPLA(2) phosphorylation that was blocked by the mitogen-activated protein kinase kinase inhibitor, PD98059 [2-(2-amino-3methoxyphenyl)-4H-1benzopyran-4-one, C(16)H(13)NO(3)], and the pan-protein kinase C inhibitor, GFX (bisindolylmaleimide). PAR(2) stimulation also resulted in a large increase in the production of PGE(2) as determined by enzyme-linked immunosorbent assay and was also blocked by PD98059 and GFX. Immunofluorescence and immunoblot analysis determined that EP2 and EP4 are expressed at the basolateral membrane of SCBN cells. Through the use of selective inhibitors (EP2, AH6809 [6-isopropoxy-9-oxoxanthene-2-carboxylic acid]; EP4, GW627368X [N-[2[4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl] acetyl]benzene sulphonamide]), it was found that both EP2 and EP4 were involved in mediating the PAR(2)-induced chloride secretory response. We conclude that basolateral PAR(2) activation induces epithelial chloride secretion that is mediated by cPLA(2), COX-1, COX-2, and the subsequent release of PGE(2). The production of PGE(2) results in an autocrine secretory response that is dependent on basolateral EP2 and EP4 receptors.
Subject(s)
Cyclooxygenase 1/physiology , Cyclooxygenase 2/physiology , Dinoprostone/physiology , Intestinal Mucosa/drug effects , Ion Transport , Receptor, PAR-2/metabolism , Animals , Cell Line , Chlorine/metabolism , Cyclooxygenase 1/biosynthesis , Cyclooxygenase 2/biosynthesis , Cyclooxygenase Inhibitors/pharmacology , Dinoprostone/metabolism , Dogs , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/drug effects , Epithelial Cells/enzymology , Epithelial Cells/metabolism , Immunoblotting , Intestinal Mucosa/cytology , Intestinal Mucosa/enzymology , Intestinal Mucosa/metabolism , Ion Transport/drug effects , Receptors, Prostaglandin E/antagonists & inhibitors , Receptors, Prostaglandin E, EP2 Subtype , Receptors, Prostaglandin E, EP4 SubtypeABSTRACT
Prion diseases are a family of fatal neurodegenerative diseases that involve the misfolding of a host protein, PrPC. Measuring prion infectivity is necessary for determining efficacy of a treatment or infectivity of a prion purification procedure; animal bioassays are, however, very expensive and time consuming. The Standard Scrapie Cell Assay (SSCA) provides an alternative approach. The SSCA facilitates quantitative in vitro analysis of prion strains, titres and biological properties. Given its robust nature and potential for high throughput, the SSCA has substantial utility for in vitro characterization of prions and can be deployed in a number of settings. Here we provide an overview on establishing the SSCA, its use in studies of disease dissemination and pathogenesis, potential pitfalls and a number of remaining challenges.
Subject(s)
Cytological Techniques/methods , Prions/analysis , Prions/isolation & purification , Scrapie/diagnosis , Animals , Cell Line , Humans , Mice , Prions/metabolism , RabbitsABSTRACT
Prion diseases reflect the misfolding of a self-protein (PrP(C)) into an infectious, pathological isomer (PrP(Sc)). By targeting epitopes uniquely exposed by misfolding, our group developed PrP(Sc)-specific vaccines to 3 disease specific epitopes (DSEs). Here, antibodies induced by individual DSE vaccines are evaluated for their capacity to neutralize prions in vitro. For both purified antibodies and immunoreactive sera, the PrP(Sc)-specific antibodies were equally effective in neutralizing prions. Further, there was no significant increase in neutralizing activity when multiple DSEs were targeted within an assay. At a low antibody concentration, the PrP(Sc)-specific antibodies matched the neutralization achieved by an antibody that may act via both PrP(C) and PrP(Sc). At higher doses, however, this pan-specific antibody was more effective, potentially due to a combined deactivation of PrP(Sc) and depletion of PrP(C).
Subject(s)
Antibodies/immunology , PrPSc Proteins/immunology , Prions/antagonists & inhibitors , Prions/immunology , Animals , Antibodies, Neutralizing/immunology , Humans , Vaccines/immunologyABSTRACT
The cellular prion protein (PrP(C)) comprises a natively unstructured N-terminal domain, including a metal-binding octarepeat region (OR) and a linker, followed by a C-terminal domain that misfolds to form PrP(S) (c) in Creutzfeldt-Jakob disease. PrP(C) ß-endoproteolysis to the C2 fragment allows PrP(S) (c) formation, while α-endoproteolysis blocks production. To examine the OR, we used structure-directed design to make novel alleles, 'S1' and 'S3', locking this region in extended or compact conformations, respectively. S1 and S3 PrP resembled WT PrP in supporting peripheral nerve myelination. Prion-infected S1 and S3 transgenic mice both accumulated similar low levels of PrP(S) (c) and infectious prion particles, but differed in their clinical presentation. Unexpectedly, S3 PrP overproduced C2 fragment in the brain by a mechanism distinct from metal-catalysed hydrolysis reported previously. OR flexibility is concluded to impact diverse biological endpoints; it is a salient variable in infectious disease paradigms and modulates how the levels of PrP(S) (c) and infectivity can either uncouple or engage to drive the onset of clinical disease.
Subject(s)
PrPC Proteins/chemistry , PrPC Proteins/metabolism , Prion Diseases/pathology , Prion Diseases/physiopathology , Protein Processing, Post-Translational , Animals , Cell Line , DNA Mutational Analysis , Disease Models, Animal , Histocytochemistry , Humans , Mice, Transgenic , Microscopy , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Conformation , ProteolysisABSTRACT
The symptoms of prion infection can take years or decades to manifest following the initial exposure. Molecular markers of prion disease include accumulation of the misfolded prion protein (PrPSc), which is derived from its cellular precursor (PrPC), as well as downregulation of the PrP-like Shadoo (Sho) glycoprotein. Given the overlapping cellular environments for PrPC and Sho, we inferred that PrPC levels might also be altered as part of a host response during prion infection. Using rodent models, we found that, in addition to changes in PrPC glycosylation and proteolytic processing, net reductions in PrPC occur in a wide range of prion diseases, including sheep scrapie, human Creutzfeldt-Jakob disease, and cervid chronic wasting disease. The reduction in PrPC results in decreased prion replication, as measured by the protein misfolding cyclic amplification technique for generating PrPSc in vitro. While PrPC downregulation is not discernible in animals with unusually short incubation periods and high PrPC expression, slowly evolving prion infections exhibit downregulation of the PrPC substrate required for new PrPSc synthesis and as a receptor for pathogenic signaling. Our data reveal PrPC downregulation as a previously unappreciated element of disease pathogenesis that defines the extensive, presymptomatic period for many prion strains.
Subject(s)
Prion Diseases/physiopathology , Animals , Arvicolinae , Brain/metabolism , Cell Line , Creutzfeldt-Jakob Syndrome/metabolism , Creutzfeldt-Jakob Syndrome/physiopathology , Disease Progression , Down-Regulation , Glycosylation , Humans , Mesocricetus , Mice , Mice, Transgenic , PrPC Proteins/chemistry , PrPSc Proteins/chemistry , Prion Diseases/metabolism , Protein Isoforms/chemistry , Scrapie/metabolism , Scrapie/physiopathology , Signal Transduction , Time Factors , Wasting Disease, ChronicABSTRACT
Serine proteases play important physiological roles through their activity at G protein-coupled protease-activated receptors (PARs). We examined the roles that specific phospholipase (PL) C and protein kinase (PK) C (PKC) isoforms play in the regulation of PAR(2)-stimulated chloride secretion in intestinal epithelial cells. Confluent SCBN epithelial monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short-circuit current (I(sc)) responses to basolateral application of the selective PAR(2) activating peptide, SLIGRL-NH(2), were monitored as a measure of net electrogenic ion transport caused by PAR(2) activation. SLIGRL-NH(2) induced a transient I(sc) response that was significantly reduced by inhibitors of PLC (U73122), phosphoinositol-PLC (ET-18), phosphatidylcholine-PLC (D609), and phosphatidylinositol 3-kinase (PI3K; LY294002). Immunoblot analysis revealed the phosphorylation of both PLCbeta and PLCgamma following PAR(2) activation. Pretreatment of the cells with inhibitors of PKC (GF 109203X), PKCalpha/betaI (Gö6976), and PKCdelta (rottlerin), but not PKCzeta (selective pseudosubstrate inhibitor), also attenuated this response. Cellular fractionation and immunoblot analysis, as well as confocal immunocytochemistry, revealed increases of PKCbetaI, PKCdelta, and PKCepsilon, but not PKCalpha or PKCzeta, in membrane fractions following PAR(2) activation. Pretreatment of the cells with U73122, ET-18, or D609 inhibited PKC activation. Inhibition of PI3K activity only prevented PKCdelta translocation. Immunoblots revealed that PAR(2) activation induced phosphorylation of both cRaf and ERK1/2 via PKCdelta. Inhibition of PKCbetaI and PI3K had only a partial effect on this response. We conclude that basolateral PAR(2)-induced chloride secretion involves activation of PKCbetaI and PKCdelta via a PLC-dependent mechanism resulting in the stimulation of cRaf and ERK1/2 signaling.
Subject(s)
Biological Transport/physiology , Chlorides/metabolism , Intestinal Mucosa/metabolism , Protein Kinase C/metabolism , Receptor, PAR-2/physiology , Signal Transduction/physiology , Type C Phospholipases/metabolism , Animals , Cell Line , Cell Membrane/metabolism , Dogs , Enzyme Inhibitors/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , ErbB Receptors/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Intestinal Mucosa/cytology , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Models, Biological , Oligopeptides/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phospholipase C beta/metabolism , Phospholipase C gamma/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C beta , Protein Kinase C-delta/antagonists & inhibitors , Protein Kinase C-delta/metabolism , Protein Kinase C-epsilon/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Transport/drug effects , Protein Transport/physiology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-raf/antagonists & inhibitors , Proto-Oncogene Proteins c-raf/metabolism , Receptor, PAR-2/antagonists & inhibitors , Signal Transduction/drug effects , Type C Phospholipases/antagonists & inhibitorsABSTRACT
We examined the stimulus-secretion pathways whereby proteinase-activated receptor 2 (PAR-2) stimulates Cl(-) secretion in intestinal epithelial cells. SCBN and T84 epithelial monolayers grown on Snapwell supports and mounted in modified Ussing chambers were activated by the PAR-2-activating peptides SLIGRL-NH(2) and 2-furoyl-LIGRLO-NH(2). Short-circuit current (I(sc)) was used as a measure of net electrogenic ion transport. Basolateral, but not apical, application of SLIGRL-NH(2) or 2-furoyl-LIGRLO-NH(2) caused a concentration-dependent change in I(sc) that was significantly reduced in Cl(-)-free buffer and by the intracellular Ca(2+) blockers thapsigargin and BAPTA-AM, but not by the Ca(2+) channel blocker verapamil. Inhibitors of PKA (H-89) and CFTR (glibenclamide) also significantly reduced PAR-2-stimulated Cl(-) transport. PAR-2 activation was associated with increases in cAMP and intracellular Ca(2+). Immunoblot analysis revealed increases in phosphorylation of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase, Src, Pyk2, cRaf, and ERK1/2 in response to PAR-2 activation. Pretreatment with inhibitors of cyclooxygenases (indomethacin), tyrosine kinases (genistein), EGFR (PD-153035), MEK (PD-98059 or U-0126), and Src (PP1) inhibited SLIGRL-NH(2)-induced increases in I(sc). Inhibition of Src, but not matrix metalloproteinases, reduced EGFR phosphorylation. Reduced EGFR phosphorylation paralleled the reduction in PAR-2-stimulated I(sc). We conclude that activation of basolateral, but not apical, PAR-2 induces epithelial Cl(-) secretion via cAMP- and Ca(2+)-dependent mechanisms. The secretory effect involves EGFR transactivation by Src, leading to subsequent ERK1/2 activation and increased cyclooxygenase activity.