Post-translational modifications of collagen upon BMP-induced osteoblast differentiation

Abstract

The pattern of collagen cross-linking is tissue specific primarily determined by the extent of hydroxylation and oxidation of specific lysine residues in the molecule. In this study, murine pre-myoblast cell line, C2C12 cells, were transdifferentiated into osteoblastic cells by bone morphogenetic protein (BMP)-2 treatment, and the gene expression of lysyl hydroxylases (LH1, 2a/b, and 3) and lysyl oxidase (LOX)/lysyl oxidase-like proteins (LOXL1-4), and the extent of hydroxylysine were analyzed. After 24h of treatment, the expression of most isoforms were upregulated up to 96h whereas LH2a and LOXL2 decreased with time. In the treated cells, both hydroxyproline and hydroxylysine were detected at day 7 and increased at day 14. The ratio of hydroxylysine to hydroxyproline was significantly increased at day 14. The results indicate that LHs and LOX/LOXLs are differentially responsive to BMP-induced osteoblast differentiation that may eventually lead to the specific collagen cross-linking pattern seen in bone.

Fig. 1

Transdifferentiation of C2C12 cells from myoblasts to osteoblasts induced by rhBMP-2. The cells were cultured for 4 (a, c) or 14 days (b, d), without (a, b) or with 100 ng/ml of rhBMP-2 (c, d) and observed (X 40). Arrowheads indicate the presence of myotubes. At 4 days of treatment, cell shape changed to osteoblast-like round shape (c). At day 14, typical myotubes were well formed without rhBMP-2 treatment (b) whereas cell/matrices layer was clearly formed with the treatment (d). At day 4, ALP activity was increased with rhBMP-2 treatment in a dose dependent manner (e). mRNA expression of myogenic and osteogenic genes with or without rhBMP-2 treatment at various time points analyzed by real-time PCR (f–j). Note that osteogenic genes were upregulated (h–j) whereas myogenic genes suppressed (f,g) with time.

Fig. 2

mRNA expression of type 1 collagen and collagen modifying enzymes with or without rhBMP-2 treatment at various time points analyzed by real-time PCR (ai). After 24 hours, only LOXL2 expression was decreased while the others were increased with time. Note that LH2 variants (LH2a and b) were not distinguished by this assay (see Fig 3). See text for the description of each abbreviation.

Fig. 3

mRNA expression of LH2a and LH2b splicing variants in response to rhBMP-2 analyzed by RT-PCR. LH2a, identified as a 158 bp PCR product was decreased with rhBMP-2 treatment while LH2b, identified as a 221 bp fragment, increased with time.