Coordinated change between complement C1s production and chondrocyte differentiation in vitro

Abstract

In vitro synthesis of the first component of complement C1s was examined by using hamster epiphyseal chondrocytes (HAC) and human chondrosarcoma cell line HCS-2/8. Hamster and human C1s produced by the cells were quantified by immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA), respectively. It was possible to measure active and inactive C1s by sandwich ELISA, when we used anti-human C1s monoclonal antibodies, M241 recognizing only active C1s, and M365 and M81 recognizing both active and inactive C1s. Approximately 40% of C1s secreted from HCS-2/8 was found to be activated in the culture medium, whereas C1s from HAC was not. C1s production increased in accordance with chondrocyte differentiation induced by ascorbic acid. In contrast, transforming growth factor-beta1 and basic fibroblast growth factor, which inhibited differentiation, suppressed C1s production. These results confirmed our previous observation showing that C1s synthesis increased with differentiation into hypertrophic chondrocytes in vivo.

Establishment of a Reliable Method for Direct Proteome Characterization of Human Articular Cartilage

“…We also found 16 proteins from the extracellular matrix, mostly in an unprocessed, intracellular form. Seven complement factors were identified, including both premature and processed forms, in agreement with previous demonstration of their expression by chondrocytes in vivo (27). In total, 105 identified proteins actually originated from inside the chondrocytes, confirming a selective extraction of intracellular proteins.…”

“…In this sense, our protocol constitutes much of a conceptual improvement compared with those that involve chondrocyte culture. One possibly direct consequence of this improvement is the finding of several complement factors originating from chondrocytes (27). These are not only obvious regulators of the immune system but also apparent modulators of cartilage matrix degradation.…”

Complement Cls, a classical enzyme with novel functions at the endochondral ossification center: immunohistochemical staining of activated Cls with a neoantigen-specific antibody

“…Degrading enzymes may play a major role in the cleaning mechanisms of cartilage. We have shown that Cls synthesis increases in accordance with chondrocyte differentiation both in vivo (Toyoguchi et al 1996) and in vitro (Nakagawa et al, 1997). Cls synthesized by chondrocytes as well as that delivered from the blood stream may participate in chondrocyte and matrix degradation.…”

“…Recently, however, we have demonstrated that Cls degrades type II collagen (Yamaguchi et al 1990), a major constituent of cartilage matrix and gelatin, and activates zymogen of matrix metalloproteinase 9 (MMP-9) (Sakiyama et al 1994). MMP-9 is known to be synthesized by chondrocytes in vivo (Toyoguchi et al 1996) and in vitro (Nakagawa et al, 1997), and colocalized with Cls at the primary ossification center of human femur (Sakiyama et al 1994). Therefore, Cls is thought to have a role in cartilage degradation.…”

Establishment and characterization of tartrate-resistant acid phosphatase and alkaline phosphatase double positive cell lines

“…Chondrocytes were obtained from 3-day-old hamster epiphyseal cartilage and cultured in MEM supplemented with 10% fetal calf serum (FCS) and 50 µg/ml ascorbic acid as described previously (Nakagawa et al 1997). When overconfluency was established they were irradiated with X-rays (20 Gy), and hamster bone marrow cells (approximately 10 7 cells) were inoculated.…”