Mechanism of Methylprednisolone-Induced Primary Cilia Formation Disorder and Autophagy in Osteoblasts

Abstract

Objective: To study the role of primary cilia formation disorder and osteoblasts autophagy in the pathogenesis of steroid-induced avascular necrosis of the femoral head (SANFH).

Methods: Osteoblasts were isolated from rabbit bones and treated with 1 μM Methylprednisolone for 0, 12, 24, 48, and 72 h. The Beclin1, MAP1LC3, Atg-5, Atg-12, IFT20 and OFD1 mRNAs and proteins were detected by PCR and Western blotting, and their correlation was statistically analyzed. The lengths of osteoblast cilia were measured under a laser confocal microscope, and the autophagy flux was tracked by transfecting the osteoblasts with GFP-RFP-LC3 lentivirus.

Results: Methylprednisolone significantly upregulated Beclin1, MAP1LC3, Atg-5, Atg-12 and OFD1 mRNAs and proteins in a time-dependent manner, and decreased that of IFT20 (P < 0.05). In addition, the autophagy flux in the osteoblasts also increased and the ciliary length decreased in a time-dependent manner after Methylprednisolone treatment. The length of the cilia were 5.46 ± 0.11 um at 0 h, 4.08 ± 0.09 um at 12 h, 3.07 ± 0.07 um at 24 h, 2.31 ± 0.10 um at 48 h, and finally 1.15 ± 0.04 um at 72 h. Methylprednisolone treatment also affects primary cilium numbers in cultures, for 0 to 72 h. The autophagy regulatory genes, Beclin1, MAP1LC3, Atg-5 and Atg-12, were found to be negatively correlated with IFT20, with an average correlation coefficient of -0.81. A negative correlation was also found between OFD1 and IFT20, with an average correlation coefficient of -0.53.

Conclusion: Methylprednisolone inhibits primary cilia formation and promotes autophagy, which could be the pathological basis of SANFH. The exact regulatory mechanism needs to be further studied in vivo.

Keywords: Autophagy; Methylprednisolone; Osteoblasts; Osteonecrosis; Primary cilia.