3.4 NVP-BHG712 inhibited the RANKL-induced expression of osteoclast differentiation-related genes.
CTSK is a key enzyme in the process of bone resorption by osteoclasts. Under acidic conditions, activated CTSK participates in the degradation of collagen type I, osteopontin and other bone matrix proteins in the bone matrix, and it promotes the bone resorption function of osteoclasts. To observe whether the small molecule compound NVP-BHG712, which targets CTSK, can inhibit the protein expression of CTSK, Western blotting was used to measure the expression of CTSK in RANKL-treated BMMs-derived osteoclasts. The results showed that 0.4 μM NVP-BHG712 could inhibit CTSK expression in RANKL-treated BMMs-derived osteoclasts. (Fig. 4A, B). To investigate the effect of NVP-BHG712 on osteoclast differentiation-related genes, Western blotting was employed to measure the proteins levels of MMP9 and CTR. The results showed that the 0.4 μM concentration of NVP-BHG712 could significantly inhibit the RANKL-induced proteins expression of MMP9 and CTR in osteoclasts (Fig. 4A, C, D). Next, we measured the mRNA expression of MMP9 and CTR, the results showed that the mRNA expression of MMP9 and CTR was significantly inhibited by NVP-BHG712 (Fig. 4E, F).
Next, we measured the RANKL-induced protein expression of CTSK in BMMs-derived osteoclasts after adding 0, 0.1, 0.2, and 0.4 μM NVP-BHG712 to determine the effect of the different concentrations of NVP-BHG712 on the protein expression of CTSK in osteoclasts. The protein expression of CTSK in BMMs-derived osteoclasts treated with RANKL was measured after incubation with 0.4 μM VP-BHG712 for 1, 2, 3, and 4 days to determine the effect of different treatment times on the protein expression of CTSK in osteoclasts. The results revealed that the protein expression of CTSK was significantly decreased when the concentrations of 0.1, 0.2, and 0.4 μM NVP-BHG712 were added to the BMMs culture system (Fig. 4G, H). The expression of CTSK protein was not significantly changed after the addition of 0.4 μM VMP-BHG712 to the osteoclast differentiation and maturation culture system for 1 and 2 days; however, the protein expression of CTSK decreased significantly after 3 days and 4 days of culture (Fig. 4I, J). The mRNA expression levels of IP3R1, IP3R2, IP3R3, NFATc1, OC-STAMP, DC-STAMP, αV-integrin and Atp6v1c1 were measured by qPCR. We found that the mRNA expression of MMP9, CTR, IP3R1, IP3R3 and OC-STAMP was significantly inhibited by NVP-BHG712 (Fig. 5A-H). Therefore, NVP-BHG712 could inhibit the RANKL-induced expression of osteoclast differentiation-related genes in BMMs.