Differential regulation of MMP-13 by chemical modified tetracyclines in osteoblasts

Tetracyclines have been shown to regulate matrix metalloproteinase (MMP) expression in numerous cell types with various periodontal disease models. MMP-13, or collagenase-3, has been shown to be induced by a number of osteotropic cytokines and hormones in osteoblastic cells. In this study, we studied MMP-13 gene expression and regulation in osteoblasts by chemically modified tetracyclines (CMTs). Preliminary cytotoxicity studies indicated that 1-10 microg/ml of CMT-8 did not result in statistically significant cell death. Additional fluorescent microscopy experiments indicated that CMT-8 but not CMT-5 had a nuclear distribution within one hour of addition CMTs. Using primary rat calvarial osteoblastic cells obtained from 21-day old neonatal rats, we determined MMP-13 gene expression when stimulated by parathyroid hormone (PTH), interleukin (IL)-1b, or tumour necrosis factor (TNF)-alpha in the presence or absence of CMT-8 or -5. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) using total RNA was used to determine relative expression of MMP-13 compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a constitutively expressed housekeeping gene. Results indicate that all agents were consistently able to induce MMP-13 expression in primary calvarial osteoblastic cells. However, CMT-8 inhibited MMP-13 expression from IL-1b and PTH stimulated cells while having little effect on TNF-alpha stimulated MMP-13 expression. In contrast, CMT-5 only inhibited PTH stimulated MMP-13 expression, with no effect on IL-1b or TNF-alpha stimulated MMP-13 expression. These results suggest that CMT-8 and CMT-5 may differentially affect cell signaling pathways in osteoblastic cells that mediate MMP-13 gene expression.