Surface calreticulin mediates muramyl dipeptide-induced apoptosis in RK13 cells

Calreticulin (CRT) is a binding protein for apoptotic N-acetylmuramyl-L-alanyl-D-isoglutamine (L,D-MDP) or peptidoglycan in RK13 cells. CRT on RK13 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 RK13 srCRT dose-dependently inhibited L,D-MDP- induced apoptosis. In RK13 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 RK13 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 RK13 cell CRT and promoted free L,D-MDP dissociation from RK13 cell CRT.MDP complex. Different concentrations of EGTA (0-40mM) 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 theL,D-MDP-induced apoptotic DNA ladders and caspase-3 activity in RK13 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 RK13 cell apoptosis through activating the TNFR1.TRADD-Fas-associated death domain protein apoptotic pathway.