Wear particles enhance autophagy through up-regulation of CD147 to promote osteoclastogenesis

Abstract

Objectives: The study aimed to uncover the underlying mechanism linking wear particles to osteoclast differentiation, and we explored the effect of titanium particles of different sizes on CD147 expression and autophagy in macrophages.

Materials and methods: Effects of titanium particles on CD147 and RANKL mRNA were detected by QPCR; protein level of CD147 and Beclin-1 were detected by Western blot; soluble RANKL were detected by ELISA. To determine the effect of CD147 and autophagy, KG-1a cells were transfected with siRNA-CD147 or treated with autophagy inhibitor CQ (chloroquine), and then co-cultured with different sizes of titanium particles.

Results: Our results showed that 0.2-1.2 µm and 1.2-10 µm titanium particles up-regulate CD147 to activate autophagy, which increase the level of soluble RANKL to promote osteoclastogenesis. Suppression of CD147 with siRNA could diminish particle-induced autophagy and soluble RANKL expression. In addition, CQ could dramatically reduce particle-induced soluble RANKL expression.

Conclusion: Our findings suggested a possible mechanism underlying wear debris-induced osteolysis and identified CD147 as a potential therapeutic target in aseptic loosening.

Keywords: Autophagy; CD147; Osteoclastogenesis; Peri-implant osteolysis; RANKL.

Figure 1

Effects of titanium particles on mRNA and protein level of CD147. QPCR showed the expression of CD147 mRNA in KG-1a cells co-cultured with 0.2–1.2 µm (A), 1.2-10 µm(B) and >10 µm (C) titanium particles; the protein level of CD147 in KG-1a cells co-cultured with 0.2–1.2, 1.2-10 and >10µm titanium particles was detected by western blot (D). *P<0.05; **P<0.01; ***P<0.001

Figure 2

Effects of titanium particles on mRNA and soluble protein expression of RANKL. The mRNA of RANKL and soluble RANKL in KG-1a cells co-cultured with 0.2–1.2 µm (A, B), 1.2-10 µm (C, D) and >10 µm (E, F) titanium particles were detected by qPCR and ELISA respectively. *P<0.05; **P<0.01; ***P<0.001

Figure 3

Effects of CD147 siRNA on mRNA and soluble protein expression of RANKL. The mRNA and soluble protein expression levels of RANKL in KG-1a cells co-cultured with CD147 siRNA and 0.2–1.2 µm (A), 1.2-10 µm (B) were detected by qPCR and ELISA respectively. NC: Normal control. *P<0.05; **P<0.01; ***P<0.001. +P<0.05; ++P<0.01; +++P<0.001

Figure 4

Effects of titanium particles on Beclin-1 protein expression. Protein levels of Beclin-1 in KG-1a cells co-cultured with 0.2–1.2 µm, 1.2-10 µm and >10 µm titanium particles were detected by western blot

Figure 5

Effects of autophagy inhibitor CQ on mRNA and soluble protein expression of RANKL. The mRNA and soluble protein expression levels of RANKL in KG-1a cells cotreated with autophagy inhibitor CQ and 0.2–1.2 µm (A), 1.2-10 µm (B) were detected by qPCR and ELISA respectively. NC: Normal control. *P<0.05; **P<0.01; ***P<0.001. +P<0.05; ++P<0.01; +++P<0.001

Figure 6

Effects of CD147 siRNA on Beclin-1 protein expression. Protein levels of Beclin-1 in KG-1a cells co-cultured with CD147 siRNA and 0.2–1.2 µm, 1.2-10 µm titanium particles were detected by Western blot