Upregulation of MMP-13 and TIMP-1 expression in response to mechanical strain in MC3T3-E1 osteoblastic cells

Abstract

Background: Mechanical strain plays a significant role in the regulation of bone matrix turnover, which is mediated in part by matrix metalloproteinase (MMP)-13 and tissue inhibitors of matrix metalloproteinase (TIMP)-1. However, little is known about the correlation between mechanical strain and osteoblastic cell activities, including extracellular matrix (ECM) metabolism. Herein, we determined the effect of different magnitudes of cyclic tensile strain (0%, 6%, 12%, and 18%) on MMP-13 and TIMP-1 mRNA and protein expression in MC3T3-E1 osteoblasts. Furthermore, we employed specific inhibitors to examine the role of distinct signal transduction pathways known to mediate cellular responses to mechanical strain.

Results: We identified a magnitude-dependent increase in MMP-13 and TIMP-1 mRNA and protein levels in response to mechanical strains corresponding to 6%, 12%, and 18% elongation. The strain-induced increases in MMP-13 and TIMP-1 mRNA expression were inhibited by PD098059 and cycloheximide, respectively.

Conclusions: Our results suggest a mechanism for the regulation of bone matrix metabolism mediated by the differential expression of MMP-13 and TIMP-1 in response to increasing magnitudes of mechanical strain.

Figure 1

Effect of increasing tensile strain on MMP-13 mRNA and protein levels in mouse osteoblastic MC3T3-E1 cells. Cells were seeded at a density of 2 × 10⁵ cells/well on Flex I culture plates and cultured in α-MEM medium supplemented with 10% FBS for 24 h. The cells were then loaded with (6%, 12%, 18%) or without (0%) cyclic tensile strain at the indicated percent elongation at 6 cycles/min using a Flexercell strain unit for 24 h. The levels of MMP-13 mRNA and protein were determined by RT-PCR and immunoblot analysis, respectively, as described in the Materials and methods. (A) Agarose gel electrophoresis of the RT-PCR products using specific primers for MMP-13 or GAPDH. (B) MMP-13 mRNA expression levels normalized to GAPDH. (C) Immunoblot analysis of MMP-13 and GAPDH. (D) MMP-13 protein expression levels normalized to GAPDH. The results shown are means ± SD of five independent experiments. Significant differences from the 0% culture are shown by ANOVA and Student's t test (*P < 0.05, ** P < 0.01).

Figure 2

Effect of increasing tensile strain on TIMP-1 mRNA and protein levels in mouse osteoblastic MC3T3-E1 cells. Cells were seeded at a density of 2 × 10⁵ cells/well, cultured for 24 h, and were then loaded with (6%, 12%, 18%) or without (0%) tensile strain at the indicated percent elongation at 6 cycles/min using a Flexercell strain unit for 24 h. TIMP-1 mRNA and protein levels were determined by RT-PCR and Western blot analysis, respectively, as described in the Materials and methods. (A) Agarose gel electrophoresis of the RT-PCR products using primers specific for TIMP-1 or GAPDH. (B) TIMP-1 mRNA expression levels normalized to GAPDH mRNA levels. (C) Immunoblot analysis of TIMP-1 and GAPDH. (D) TIMP-1 protein expression levels normalized to GAPDH. The results shown are means ± SD of five independent experiments. Significant differences from the 0% culture are shown by ANOVA and Student's t test (*P < 0.05, ** P < 0.01).

Figure 3

Active-MMP-13 and TIMP-1 concentrations in conditioned medium induced by mechanical strain in MC3T3-E1 cells. Conditioned media harvested from the cultured cells were used as samples. The concentrations of active form of MMP-13 and TIMP-1 in these supernatants were assayed using a comercially available enzyme-linked immunosorbent assay (ELISA) kits, as described in the Materials and methods. (A) The concentrations of active form of MMP-13. (B) The concentrations of TIMP-1. The results shown are means ± SD of five independent experiments. Significant differences from the 0% culture are shown by ANOVA and Student's t test (*P < 0.05, ** P < 0.01).

Figure 4

Effects of inhibitors on tensile strain-induced expression of MMP-13 mRNA in MC3T3-E1 cells. Cells were seeded at a density of 2 × 10⁵ cells/well on Flex I culture plates and grown in α-MEM medium supplemented with 10% FBS for 24 h. Cells were then cultured in medium containing cycloheximide (10 μM), indomethacin (10 μM), genistein (20 μM), PD098059 (10 μM), or vehicle (control) for 30 min. The cells were cultured with (+) or without (-) loading with tensile strain at 18% elongation at 6 cycles/min for 24 h. Total RNA was extracted from the cells, and expression levels of MMP-13 mRNA were determined by RT-PCR as described in the Materials and methods. (A) Agarose gel electrophoresis of MMP-13 and GAPDH RT-PCR products. (B) MMP-13 mRNA expression levels normalized to GAPDH. The results shown are means ± SD of five independent experiments. **Indicates a significant difference from the strain (+) culture (P < 0.01)as determined by Student's t test.

Figure 5

The effects of inhibitors on tensile strain-induced expression of TIMP-1 mRNA in MC3T3-E1 cells. Cells were seeded at a density of 2 × 10⁵ cells/well on Flex I culture plates and were cultured in α-MEM medium supplemented with 10% FBS for 24 h. They were then cultured in medium containing cycloheximide (10 μM), indomethacin (10 μM), genistein (20 μM), PD098059 (10 μM), or vehicle (control) for 30 min. The cells were cultured with (+) or without (-) loading with tensile strain at 18% elongation at 6 cycles/min for 24 h. Total RNA was isolated, and TIMP-1 and GAPDH mRNA levels were determined by RT-PCR. (A) Agarose gel electrophoresis of the TIMP-1 and GAPDH RT-PCR products. (B) TIMP-1 mRNA levels normalized to GAPDH. The results shown are means ± SD of five independent experiments. **Indicates a significant difference from stress (+) culture as determined by Student's t test (P < 0.01).