Suppressing phosphoinositide-specific phospholipases Cγ1 promotes mineralization of osteoarthritic subchondral bone osteoblasts via increasing autophagy, thereby ameliorating articular cartilage degeneration

Introduction: Phosphoinositide-specific phospholipases C-γ1 (PLC-γ1) signaling has been shown to modulate osteoarthritis (OA) chondrocyte metabolism. However, the role of PLC-γ1 in OA osteoblasts remains unclear. Herein, whether and how PLC-γ1 was involved in mineralization in OA subchondral bone osteoblasts were investigated.

Methods: Primary non-OA and OA osteoblasts of human and rat isolated from the subchondral bone or the calvaria were cultured in vitro, as well as mouse pre-osteoblastic cell line MC3T3-E1 cells. Rat knee OA model was induced by anterior cruciate ligament transection (ACLT), in which bone canal was carried out from the surface of lateral epicondyle of femur using micro-electric drill. Morphological characteristics of subchondral bone structure and articular cartilage were assessed using CT, micro-CT, and Safranin O/Fast green staining, respectively. Mineralization was measured by alizarin red staining. The expression and production of genes involved in osteoblastic phenotype and mineralization were evaluated by qPCR, western blotting, and immunohistochemistry assays, respectively. The inhibitions were performed using inhibitors and ShRNAs.

Results: The decreased relative bone density and thickness in the early stage of OA and the increased one in the late stage of OA were observed in subchondral bone of ACLT-rat model. Decreased ALP and OCN levels and absorbance values of ARS content were observed in in vitro osteoblasts isolated from 2 w post-ACLT rat model, as well as IL-1β-treated (for maintaining and mimicking inflammatory status) human OA and rat osteoblasts. Decreased Atg7 level and LC3BII/I ratio in combination with an increase in the p62 level, was concomitant with decreased ALP and OCN mRNA levels and absorbance values of ARS content in OA or IL-1β-treated osteoblasts. Specific inhibition of PLC-γ1 by ShRNAs or inhibitor (U73122) elevated ALP and OCN mRNA levels and absorbance values of ARS content accompanied with increased Atg7 level and LC3BII/I ratio in combination with a decrease in the p62 level in OA osteoblasts. Furthermore, the promoting effect of PLC-γ1 inhibition on ALP and OCN mRNA levels and absorbance values of ARS content was reversed by endoplasmic reticulum (ER) stress activator HA15, as well as autophagic inhibitors CQ and 3MA. Injection with PLC-γ1 inhibitor U73122 from the surface of lateral epicondyle of femur reduced aberrant subchondral bone formation and attenuated articular cartilage degeneration in ACLT-rat.

Conclusion: Aberrant changes of OA subchondral bone structure were concomitant with altered osteoblastic phenotype and mineralization. Impaired Autophagy contributed to decreased osteoblastic mineralization in the early stage of OA. PLC-γ1 inhibition promoted osteoblastic mineralization through increasing Autophagy in OA osteoblasts, which was partially attributed to suppression of ER stress. Targeting PLC-γ1 in subchondral bone osteoblasts could be more efficacious for OA therapy through treating the bone and cartilage at the same time. In summary, we hypothesize that suppressing PLCγ1 promotes mineralization of osteoarthritic subchondral bone osteoblasts via increasing Autophagy, thereby ameliorating articular cartilage degeneration.