PLAP-1/Asporin Negatively Regulates Inflammatory Responses Through the Direct Interaction With TLR2

Objectives: PLAP-1/asporin is an extracellular matrix protein which is predominantly expressed in periodontal ligament (PDL). PLAP-1 is classified in class I of small leucine rich repeat proteoglycan family including decorin and biglycan. Recently, it was reported that decorin and biglycan function as an endogenous ligand for TLR2. We have hypothesized that PLAP-1 may also regulate inflammatory responses by regulating TLR2 signaling.
Methods: Mouse PDL cells were infected by adenovirus vectors expressing PLAP-1 and subsequently stimulated with Porphyromonas gingivalis (P.g.) LPS. Then, we assessed the expressions of Il-6 and Cxcl10 mRNA and the CXCL10 concentration in the culture supernatants by real-time PCR and ELISA, respectively. To evaluate the molecular basis of regulatory mechanisms of PLAP-1 to TLR2, we performed a solid-phase binding assay in which microplate wells coated with recombinant PLAP-1 were incubated with recombinant TLR2. Then, binding of TLR2 to PLAP-1 was quantified by ELISA. We also carried out co-immunoprecipitation assay using cell lysates derived from FLAG-tagged PLAP-1 transfected HEK–Blue hTLR2 cells, which were engineered HEK293 cells that stably expressed TLR2. Binding of TLR2 to PLAP-1 was assessed by SDS-PAGE and western blotting analysis using anti-FLAG and anti-TLR2 antibodies.
Results: PLAP-1 inhibits pro-inflammatory cytokine expressions induced by P.g. LPS in mouse PDL cells. Solid-phase binding assay revealed that recombinant TLR2 bound to immobilized PLAP-1 in a dose-dependent manner. Co-immunoprecipitation assay, followed by SDS-PAGE and western blotting analysis demonstrated that TLR2 co-precipitated with PLAP-1.
Conclusions: We demonstrated that PLAP-1 inhibited P.g. LPS-induced inflammatory responses in mouse PDL cells and showed the binding capacity to TLR2. These data suggest that PLAP-1 negatively regulates inflammatory responses through the direct interaction with TLR2 and may influence the pathological condition of periodontal disease.