Role of low-density lipoprotein receptor related protein 1 (LRP1) in CCN2/connective tissue growth factor (CTGF) protein transport in chondrocytes

LRP1 is known to be a receptor for signal transmission and endocytosis. We formerly reported that LRP1 regulates WNT/β-catenin and protein kinase C signaling in chondrocytes and represses the hypertrophy of chondrocytes during endochondral ossification, and that LRP1 is co-localized with a ligand, CCN2, which conducts endochondral ossification, on chondrocytes. However, the role of LRP1 in endocytotic transport of CCN2 in chondrocytes is not yet understood. In the present study, we investigated the interaction between LRP1 and CCN2 during endocytotic trafficking. RNAi-mediated knockdown of LRP1 in chondrocytic HCS-2/8 cells showed that the amount of exogenous CCN2 binding/incorporation was decreased in the LRP1 down-regulated cells. Importantly, we observed that CCN2 internalization in chondrocytes was dependent on clathrin and internalizated CCN2 was co-localized with an early or recycling endosome marker. Transcytosis of CCN2 through HCS-2/8 cells was confirmed by performing experiments with a trans-well apparatus, and the amount of transcytosed CCN2 was decreased by an LRP1 antagonist. These findings rule out possible leakage and confirm the critical involvement of LRP1 during experimental transcytosis. Moreover, under the hypoxic condition mimicking the cartilaginous microenvironment, the production level of LRP1 and the amount of transcytosed CCN2 were increased, which increases were neutralized by the LRP1 antagonist. The distribution of LRP1 and its antagonist in the growth plate in vivo was consistent with that of CCN2 therein, which was produced by and transported from the chondrocytes in the prehypertrophic layer. These findings suggest that LRP1 mediates the transcytosis of CCN2, which may be a critical event that determines the distribution of CCN2 in cartilage.