Collagen Cross-Linking Influences Osteoblastic Differentiation

• Published in: Calcified tissue international 2008-05, Vol.82 (5), p.392-400
• Main Authors: Turecek, C., Fratzl-Zelman, N., Rumpler, M., Buchinger, B., Spitzer, S., Zoehrer, R., Durchschlag, E., Klaushofer, K., Paschalis, E. P., Varga, F.
• Format: Article
• Language: English
• Subjects: Amino Acids - chemistry; Amino Acids - metabolism; Aminopropionitrile - pharmacology; Animals; Biochemistry; Biomedical and Life Sciences; Bones; Cell Biology; Cell Differentiation - drug effects; Cell Differentiation - physiology; Cell Line; Cellular biology; Collagen - biosynthesis; Collagen - chemistry; Collagen Type I - chemistry; Collagen Type I - genetics; Collagen Type I - metabolism; Cross-Linking Reagents; Dipeptides - chemistry; Dipeptides - metabolism; Endocrinology; Extracellular Matrix - chemistry; Extracellular Matrix - drug effects; Extracellular Matrix - metabolism; Gene expression; Gene Expression Regulation - drug effects; Life Sciences; Mice; Orthopedics; Osteoblasts - cytology; Osteoblasts - drug effects; Osteoblasts - metabolism; Osteocalcin - genetics; Osteocalcin - metabolism; Protein Processing, Post-Translational; Protein-Lysine 6-Oxidase - chemistry; Protein-Lysine 6-Oxidase - genetics; Protein-Lysine 6-Oxidase - metabolism; RNA, Messenger - metabolism; Spectroscopy, Fourier Transform Infrared
• ISSN: 0171-967X; 1432-0827
• DOI: 10.1007/s00223-008-9136-3

Osteoblasts synthesize collagen matrix, which itself regulates the differentiation of precursor cells into mature osteoblasts. They express lysyl oxidase (LOX), which is involved in the collagen cross-linking process. Lathyrogens, like ß-aminopropionitrile (ßAPN), inhibit the formation of a stable matrix. The aim of the present study was to investigate the influence of cross-linking on osteoblastic differentiation. MC3T3-E1 cells were seeded and treated with or without 400 μM ßAPN for 1 week. Thereafter, living cells were removed and, on this extracellular matrix, new MC3T3-E1 cells were seeded and cultured for 1 week without ßAPN. RNA was isolated, and expression of specific marker genes was determined by quantitative reverse transcription-polymerase chain reaction. Changes in specific cross-links after ßAPN treatment were measured with Fourier-transform infrared spectroscopy. The collagen matrix that formed showed a significant reduction of two major cross-links of bone collagen, deH-DHLNL and pyr, compared to control cultures. Gene expression studies showed an increase of collagen α1 (I) (COL1A1) to 150%. Expression of LOX and osteocalcin (OCN) mRNA was significantly downregulated to about 75%. When fresh MC3T3-E1 cells were seeded on this altered matrix without ßAPN, COL1A1 mRNA expression was upregulated (140%), OCN was downregulated (60%), and LOX mRNA expression remained unaffected. These results indicate that ßAPN treatment not only disrupts collagen cross-link formation but also affects osteoblastic activity and expression. In conclusion, the disrupted matrix produced in the presence of lathyrogen influences, even in its absence, the expression of osteoblastic genes.