title: Surface calreticulin mediates muramyl dipeptide-induced apoptosis in RK13 cells.
abstract: Calreticulin (CRT) is a binding protein for apoptotic N-acetylmuramyl-L-alanyl-D-isoglutamine (L,D-MDP) or peptidoglycan in RK(13) cells. CRT on RK(13) cell surface (srCRT) forms complex(es) with tumor necrosis factor receptor 1 (TNFR1) and TNFR-associated death domain (TRADD) protein of the cell membrane. CRT polyclonal or monoclonal antibody binding to RK(13) srCRT dose-dependently inhibited L,D-MDP-induced apoptosis. In RK(13) cells, L,D-MDP up-regulated the TNFR1.TRADD complex of the plasma membrane and subsequently induced cytosolic TRADD-Fas-associated death domain protein complex. Biotinylated srCRT was capable of calcium-dependent binding of Sepharose-immobilized L,D-MDP or peptidoglycan. However, Toll-like receptors TLR-2 and TLR-4, Nod2, and CD14 of RK(13) cells did not specifically bind Sepharose-immobilized L,D-MDP. High concentrations (5-40 mm) of EGTA dose-dependently inhibited free L,D-MDP binding to purified RK(13) cell CRT and promoted free L,D-MDP dissociation from RK(13) cell CRT.MDP complex. Different concentrations of EGTA (0-40 mm) added to Dulbecco's modified essential medium with 1.8 mm calcium or phosphate-buffered saline with 0.18 mm calcium have different effects on medium free calcium concentrations but have identical inhibiting effects on L,D-MDP-induced apoptosis. More inhibition of the L,D-MDP-induced apoptotic DNA ladders and caspase-3 activity in RK(13) cells was obtained with EGTA pretreatment (83%) than just EGTA + L,D-MDP (47%). The knocking down of srCRT by antisense oligonucleotide CRTAS121 (250 nmol/ml) and stealth small interfering RNA CRT_siR479 (150 pm/ml) for 2 days (44 and 66%, respectively), resulted in the inhibition of L,D-MDP-induced caspase-3 activity (47 and 65%, respectively). The results suggest that (a) the binding of L,D-MDP to srCRT is calcium-dependent, i.e. on srCRT-bound calcium, and (b) it is srCRT, not TLR-2, TLR-4, Nod2 or CD14, that mediates L,D-MDP-induced RK(13) cell apoptosis through activating the TNFR1. TRADD-Fas-associated death domain protein apoptotic pathway.
authors.affiliation: Departments of Ophthalmology and Microbiology & Immunology, Louisiana State University Health Sciences Center, 1501 Kingshighway, Shreveport, LA 71130, USA. dchen@lsuhsc.edu
authors.forename: Dequan
authors.initials: D
authors.lastname: Chen
authors.forename: Donald E
authors.initials: DE
authors.lastname: Texada
authors.forename: Chris
authors.initials: C
authors.lastname: Duggan
authors.forename: Chanping
authors.initials: C
authors.lastname: Liang
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Reden
authors.forename: Lakshmana M
authors.initials: LM
authors.lastname: Kooragayala
authors.forename: Marlyn P
authors.initials: MP
authors.lastname: Langford
mesh_terms: D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000494:pharmacology*; D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D017209:Apoptosis; D001681:Biological Assay; D019921:Biotinylation; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D037282:Calreticulin / Q000378:metabolism / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004533:Egtazic Acid / Q000737:chemistry / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis; D008562:Membrane Glycoproteins / Q000737:chemistry; D051379:Mice; D053473:Nod2 Signaling Adaptor Protein; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D010457:Peptidoglycan / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D015854:Up-Regulation
chemical_list: D000276:Adjuvants, Immunologic; D000911:Antibodies, Monoclonal; D037282:Calreticulin; D007074:Immunoglobulin G; D047908:Intracellular Signaling Peptides and Proteins; D018950:Lipopolysaccharide Receptors; D008562:Membrane Glycoproteins; D053473:Nod2 Signaling Adaptor Protein; C495945:Nod2 protein, mouse; D016376:Oligonucleotides, Antisense; D010457:Peptidoglycan; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; C495342:TLR2 protein, human; C495345:TLR4 protein, human; C495343:Tlr2 protein, rat; C495347:Tlr4 protein, rat; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D004533:Egtazic Acid; D000119:Acetylmuramyl-Alanyl-Isoglutamine; D004247:DNA; D012685:Sepharose; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D002118:Calcium