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1.
BMC Immunol ; 11: 53, 2010 Oct 28.
Article in English | MEDLINE | ID: mdl-21029417

ABSTRACT

BACKGROUND: Protease-Activated Receptors (PARs), members of G-protein-coupled receptors, are activated by proteolytic activity of various proteases. Activation of PAR1 and PAR2 triggers innate immune responses in human oral keratinocytes (HOKs), but the signaling pathways downstream of PAR activation in HOKs have not been clearly defined. In this study, we aimed to determine if PAR1- and PAR2-mediated signaling differs in the induction of innate immune markers CXCL3, CXCL5 and CCL20 via ERK, p38 and PI3K/Akt. RESULTS: Our data show the induction of innate immunity by PAR1 requires both p38 and ERK MAP kinases, while PAR2 prominently signals via p38. However, inhibition of PI3K enhances expression of innate immune markers predominantly via suppressing p38 phosphorylation signaled by PAR activation. CONCLUSION: Our data indicate that proteases mediating PAR1 and PAR2 activation differentially signal via MAP kinase cascades. In addition, the production of chemokines induced by PAR1 and PAR2 is suppressed by PI3K/Akt, thus keeping the innate immune responses of HOK in balance. The results of our study provide a novel insight into signaling pathways involved in PAR activation.


Subject(s)
Keratinocytes/metabolism , Periodontitis/immunology , Periodontitis/metabolism , Receptor, PAR-1/metabolism , Receptor, PAR-2/metabolism , Cells, Cultured , Chemokine CCL20/biosynthesis , Chemokine CCL20/genetics , Chemokine CCL20/immunology , Chemokine CXCL5/biosynthesis , Chemokine CXCL5/genetics , Chemokine CXCL5/immunology , Chemokines, CXC/biosynthesis , Chemokines, CXC/genetics , Chemokines, CXC/immunology , Down-Regulation , Humans , Immunity, Innate , Keratinocytes/immunology , Keratinocytes/pathology , Mitogen-Activated Protein Kinase 3/metabolism , Mouth/pathology , Periodontitis/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptor, PAR-1/immunology , Receptor, PAR-2/immunology , Signal Transduction/immunology , p38 Mitogen-Activated Protein Kinases/metabolism
2.
Immunol Lett ; 131(2): 113-9, 2010 Jul 08.
Article in English | MEDLINE | ID: mdl-20219537

ABSTRACT

Protease-activated receptors (PARs), nucleotide-binding oligomerization domain (NOD) receptors and Toll-like receptors (TLRs) play a role in innate immunity, but little is known about interaction between these receptors. The goal of this study was to investigate how silencing one receptor affects the expression of other receptors and downstream innate immune markers in response to bacteria. Human gingival epithelial cells (GECs) were transfected with siRNA specific for PAR1 or PAR2, then stimulated with periopathogen Porphyromonas gingivalis, bridging organism between pathogens and non-pathogens Fusobacterium nucleatum, or non-pathogen Streptococcus gordonii. PAR1 or PAR2 knock-down resulted in up-regulated NOD1 and NOD2 expression with P. gingivalis or F. nucleatum stimulation (p<0.01), as well as enhanced TLR2 and TLR4 expression when cells were stimulated by bacteria that utilize TLR2 or TLR4, respectively. Involvement of PARs for induction of CC chemokine ligand 20 (CCL20), a cytokine with antimicrobial properties, was observed following stimulation of the three bacterial species. Furthermore, results from multiple cytokine ELISA array showed receptors utilized in the induction of various innate immune markers are tailored to individual bacterium tested. Our data suggest complex interplay of several receptors is required for appropriate innate immune responses to the different types of bacteria present within the oral cavity and that receptor expression itself is altered depending on which organism the cell encounters.


Subject(s)
Bacteroidaceae Infections/immunology , Epithelial Cells/metabolism , Fusobacterium Infections/immunology , Fusobacterium nucleatum/immunology , Immunity, Innate , Porphyromonas gingivalis/immunology , Streptococcal Infections/immunology , Streptococcus gordonii/immunology , Bacteroidaceae Infections/genetics , Bacteroidaceae Infections/metabolism , Cells, Cultured , Chemokine CCL20/biosynthesis , Chemokine CCL20/genetics , Epithelial Cells/immunology , Epithelial Cells/pathology , Fusobacterium Infections/genetics , Fusobacterium Infections/metabolism , Gingiva/pathology , Humans , Immunity, Mucosal , Nod1 Signaling Adaptor Protein/biosynthesis , Nod1 Signaling Adaptor Protein/genetics , Nod2 Signaling Adaptor Protein/biosynthesis , Nod2 Signaling Adaptor Protein/genetics , Porphyromonas gingivalis/pathogenicity , RNA, Small Interfering/genetics , Receptor Cross-Talk , Receptor, PAR-1/genetics , Receptor, PAR-2/genetics , Streptococcal Infections/genetics , Streptococcal Infections/metabolism , Toll-Like Receptor 2/biosynthesis , Toll-Like Receptor 2/genetics , Toll-Like Receptor 4/biosynthesis , Toll-Like Receptor 4/genetics
3.
Am J Physiol Cell Physiol ; 298(5): C1151-8, 2010 May.
Article in English | MEDLINE | ID: mdl-20164376

ABSTRACT

A-kinase (PKA) anchoring proteins (AKAPs) are essential for targeting type II PKA to specific locales in the cell to control function. In the present study, AKAP5 (formerly AKAP150) and AKAP6 were identified in mouse parotid acini by type II PKA regulatory subunit (RII) overlay assay and Western blot analysis of mouse parotid cellular fractions, and the role of AKAP5 in mouse parotid acinar cell secretion was determined. Mice were euthanized with CO(2). Immunofluorescence staining of acinar cells localized AKAP5 to the basolateral membrane, whereas AKAP6 was associated with the perinuclear region. In functional studies, amylase secretion from acinar cells of AKAP5 mutant [knockout (KO)] mice treated with the beta-adrenergic agonist, isoproterenol, was reduced overall by 30-40% compared with wild-type (WT) mice. In contrast, amylase secretion in response to the adenylyl cyclase (AC) activator, forskolin, and the cAMP-dependent protein kinase (PKA) activator, N(6)-phenyl-cAMP, was not statistically different in acini from WT and AKAP5 KO mice. Treatment of acini with isoproterenol mimicked the effect of the Epac activator, 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT-2'-O-Me-cAMP), in stimulating Rap1. However, in contrast to isoproterenol, treatment of acini with 8-pMeOPT-2'-O-Me-cAMP resulted in stimulation of amylase secretion from both AKAP5 KO and WT acinar cells. As a scaffolding protein, AKAP5 was found to coimmunoprecipitate with AC6, but not AC8. Data suggest that isoproterenol-stimulated amylase secretion occurs via both an AKAP5/AC6/PKA complex and a PKA-independent, Epac pathway in mouse parotid acini.


Subject(s)
A Kinase Anchor Proteins/metabolism , Amylases/metabolism , Parotid Gland/enzymology , A Kinase Anchor Proteins/genetics , Adenylyl Cyclases/metabolism , Animals , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Glucose Oxidase , Guanine Nucleotide Exchange Factors/metabolism , Isoproterenol/pharmacology , Lactoperoxidase , Mice , Mice, Knockout , Parotid Gland/drug effects , Parotid Gland/metabolism , Protein Transport , Sympathomimetics/pharmacology
4.
Cell Signal ; 16(2): 157-65, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14636886

ABSTRACT

Several regulated Ca2+ entry pathways have been identified, with capacitative Ca2+ entry (CCE) being the most characterized. In the present study, we examined Ca2+ entry pathways regulated by arachidonic acid (AA) in mouse parotid acini. AA induced Ca2+ release from intracellular stores, and increased Ca2+ entry. AA inhibited thapsigargin (Tg)-induced CCE, whereas AA activated Ca2+ entry when CCE was blocked by gadolinium (Gd3+). AA-induced Ca2+ entry was associated with depletion of calcium from ryanodine-sensitive stores; both AA-induced Ca2+ release and Ca2+ entry were inhibited by tetracaine and the nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI). The nitric oxide (NO) donor, 1,2,3,4-ox-triazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162), but not 8-bromo-cGMP, mimicked the effects of AA in inhibiting CCE. Results suggest that AA acts via nitric acid to inhibit the CCE pathway that is selective for Ca2+, and to activate a second Ca2+ entry pathway that is dependent on depletion of Ca2+ from ryanodine-sensitive stores.


Subject(s)
Arachidonic Acid/pharmacology , Calcium Signaling/drug effects , Calcium/metabolism , Cyclic GMP/analogs & derivatives , Nitric Oxide/metabolism , Parotid Gland/metabolism , Animals , Calcium Signaling/physiology , Cells, Cultured , Cyclic GMP/pharmacology , Enzyme Inhibitors/pharmacology , Fura-2/chemistry , Gadolinium/pharmacology , Indazoles/pharmacology , Mice , Ryanodine/pharmacology , Tetracaine/pharmacology , Thapsigargin/pharmacology , Triazoles/pharmacology
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