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 (0-40 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 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.