IADR Abstract Archives
Role of Cathepsin-B in Osteoclastogenesis induced by Porphyromonas gingivalis-Phosphoglycerol Dihydroceramide
Objectives: Cathepsin-B (CtsB) is a lysosomal cysteine protease associated with Alzheimer’s disease, cancer, inflammation and viral infection susceptibility. It was reported that CtsB is associated with RANKL-mediated osteoclastogenesis via degradation of non-muscle myosin IIA (Myh9). Our group recently demonstrated that phosphoglycerol dihydroceramide (PGDHC) produced by Porphyromonas gingivalis (Pg) also upregulates osteoclastogenesis in a Myh9-dependent manner. Importantly, host-derived ceramides can directly activate CtsB catalytic function leading to the release of pro-inflammatory cytokines and apoptosis. However, it is unclear wether Pg-derived PGDHC can also activate CtsB in periodontal bone loss. This study aims to evaluate the possible effect of PGDHC on CtsB activity in RANKL-mediated osteoclastogenesis.
Methods: For the in vitro assessment, RANKL-stimulated RAW264.7 cells were treated with PGDHC in the presence or absence of various concentrations of: 1) membrane impermeable CtsB inhibitor CA-074 or 2) membrane permeable CtsB inhibitor CA-074 Me followed by TRAP staining at Day 5. For the in vivo studies, 1) PGDHC, 2) RANKL, or 3) an equal mixture of RANKL and PGDHC were injected every other day onto the calvaria periosteum of C56BL/6 mice (six-week-old, male). Ten days after injection, CtsB activity was evaluated by cutting-edge in situ imaging systems (Brucker) along with conventional immuno-histochemical staining for TRAP.
Results: As expected, PGDHC significantly upregulated RANKL-induced osteoclastogenesis in vitro and in vivo. CtsB inhibitor CA-074-Me, but not CA-074, significantly inhibited PGDHC-mediated osteoclastogenesis in a dose dependent manner in vitro. In the in vivo calvarial osteolytic model, local injection of the PGDHC/RANKL mixture significantly up-regulated CtsB activity and enhanced the differentiation of TRAP+ osteoclasts compared to PGDHC or RANKL injections alone.
Conclusions: PGDHC promotes osteoclastogenesis via upregulation of intracellular CtsB activity which can be suppressed by CtsB cell permeable inhibitor CA-074-Me. These results suggest the therapeutic potential of CtsB inhibitors to control bone loss in periodontitis.
Methods: For the in vitro assessment, RANKL-stimulated RAW264.7 cells were treated with PGDHC in the presence or absence of various concentrations of: 1) membrane impermeable CtsB inhibitor CA-074 or 2) membrane permeable CtsB inhibitor CA-074 Me followed by TRAP staining at Day 5. For the in vivo studies, 1) PGDHC, 2) RANKL, or 3) an equal mixture of RANKL and PGDHC were injected every other day onto the calvaria periosteum of C56BL/6 mice (six-week-old, male). Ten days after injection, CtsB activity was evaluated by cutting-edge in situ imaging systems (Brucker) along with conventional immuno-histochemical staining for TRAP.
Results: As expected, PGDHC significantly upregulated RANKL-induced osteoclastogenesis in vitro and in vivo. CtsB inhibitor CA-074-Me, but not CA-074, significantly inhibited PGDHC-mediated osteoclastogenesis in a dose dependent manner in vitro. In the in vivo calvarial osteolytic model, local injection of the PGDHC/RANKL mixture significantly up-regulated CtsB activity and enhanced the differentiation of TRAP+ osteoclasts compared to PGDHC or RANKL injections alone.
Conclusions: PGDHC promotes osteoclastogenesis via upregulation of intracellular CtsB activity which can be suppressed by CtsB cell permeable inhibitor CA-074-Me. These results suggest the therapeutic potential of CtsB inhibitors to control bone loss in periodontitis.
