IADR Abstract Archives
PGE2 stimulation increases RANKL in primary bone cells and enhance THP1 differentiation
Objectives: To investigate whether PGE2 simulation can increase RANKL derived from human mandibular-derived primary bone cells (HmBCs) and drive THP1 human monocytes (THP1) differentiation into osteoclasts
Methods: HmBCs were stimulated with 0.1 µM PGE2 for 24h and soluble RANKL (sRANKL) released in culture supernatant was measure by ELISA at 24, 48, and 72h post PGE2 treatment. At 48h following PGE2 treatment, hmBCs were transferred to the co-culture. THP1 were incubated with 40ng/mL vitamin D for 24h before cells were seeded in a lower chamber of the transwell system to co-culture with hmBCs in a upper chamber, or 30 ng/mL human sRANKL, for 5 or 7 days. THP1 were collected for RNA extraction and RT-PCR was performed. The relative expression of NFAT (nuclear factor of activated Tcells) c1, or cathepsin K mRNA, expressed by treated THP1 was determined by PCR band density visualized on 1.5% agarose gel. TRAP (tartrate-resistant acid phosphatase) mRNA level was also determined by SYBR qPCR. The hmBCs without PGE2 stimulation was added for a control.
Results: HmBCs increased sRANKL in culture supernatant after PGE2 stimulation as shown by ELISA. NFATc1, cathepsin K, and TRAP mRNA level was upregulated in THP1 on day5 and day7 with sRANKL treatment, or by co-culture with PGE2-stimulated hmBCs. Unstimulated HmBCs release low level of sRANKL and upregulated NFATc1, cathepsin K, and TRAP mRNA in THP1, but lower than stimulated HmBCs.
Conclusions: An increase of NFATc1, cathepsin K, and TRAP mRNA in THP1 suggested a process of osteoclast differentiation that is driven by treatment of vitamin D and sRANKL. Thus, hmBCs may be a source of sRANKL protein causing osteoclast differentiation.
Methods: HmBCs were stimulated with 0.1 µM PGE2 for 24h and soluble RANKL (sRANKL) released in culture supernatant was measure by ELISA at 24, 48, and 72h post PGE2 treatment. At 48h following PGE2 treatment, hmBCs were transferred to the co-culture. THP1 were incubated with 40ng/mL vitamin D for 24h before cells were seeded in a lower chamber of the transwell system to co-culture with hmBCs in a upper chamber, or 30 ng/mL human sRANKL, for 5 or 7 days. THP1 were collected for RNA extraction and RT-PCR was performed. The relative expression of NFAT (nuclear factor of activated Tcells) c1, or cathepsin K mRNA, expressed by treated THP1 was determined by PCR band density visualized on 1.5% agarose gel. TRAP (tartrate-resistant acid phosphatase) mRNA level was also determined by SYBR qPCR. The hmBCs without PGE2 stimulation was added for a control.
Results: HmBCs increased sRANKL in culture supernatant after PGE2 stimulation as shown by ELISA. NFATc1, cathepsin K, and TRAP mRNA level was upregulated in THP1 on day5 and day7 with sRANKL treatment, or by co-culture with PGE2-stimulated hmBCs. Unstimulated HmBCs release low level of sRANKL and upregulated NFATc1, cathepsin K, and TRAP mRNA in THP1, but lower than stimulated HmBCs.
Conclusions: An increase of NFATc1, cathepsin K, and TRAP mRNA in THP1 suggested a process of osteoclast differentiation that is driven by treatment of vitamin D and sRANKL. Thus, hmBCs may be a source of sRANKL protein causing osteoclast differentiation.