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.