Role of Cathepsin-B in the Osteoclastogenic Effect of Porphyromonas Gingivalis- Phosphoglycerol Dihydroceramide

Nova Southeastern University, Davie, Florida, USA

Objective: This study aims to evaluate the impact of the PGDHC/CtsB axis on RANKL-mediated osteoclastogenesis. Background: Cathepsin-B (CtsB) is a lysosomal-cysteine-protease associated with pathological processes. CtsB regulates RANKL-mediated osteoclastogenesis through degradation of non-muscle myosin IIA (Myh9). We recently demonstrated that phosphoglycerol dihydroceramide (PGDHC) from Porphyromonas gingivalis upregulated osteoclastogenesis in a Myh9-dependent manner. However, activation of CtsB by PGDHC in periodontal bone loss is unclear. Methods: RANKL-stimulated RAW264.7 cells were exposed to PGDHC in the presence/absence of membrane impermeable CtsB inhibitor CA-074, and membrane permeable CtsB inhibitor CA-074 Me. TRAP staining was performed at day 4 to identify mature osteoclasts and pit formation assay was performed at day 7 to assess osteoclast activity. Intracellular CtsB activity and extracellular inflammatory cytokine expression were assessed after 48 and 96 hours. Results: PGDHC upregulated RANKL-induced osteoclastogenesis of RAW 264.7 cells. CA-074-Me, but not CA-074, inhibited osteoclastogenesis and mature osteoclast activity. PGDHC increased CtsB activity after 48h and induced extracellular expression of Tnf-α and IL-1β after 96h. However, only the overexpression of IL-1β was CtsB mediated. Conclusion: PGDHC promotes osteoclastogenesis via upregulation of intracellular CtsB activity in pre-osteoclasts. PGDHC does not induce an inflammatory effect on pre-osteoclasts but increases the production of inflammatory cytokines by mature osteoclasts through CtsB dependent and independent pathways. These results may lead to a better understanding of bacterial osteolysis, as in periodontitis, and development of novel anti-bone loss therapy. Grants: This study was funded by the NIH grant R03-7-16-2018 and NIDCR administrative research supplement to promote diversity in health-related research