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

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.


Background
Bone is continuously remodeled throughout life in order to meet the functional demands of its physiological and mechanical environment [1][2][3]. Furthermore, active remodeling of alveolar bone must occur in order to cope with orthodontic force and mechanical loading generated during orthodontic tooth movement. This remodeling process requires a complex turnover of the bone extracellular matrix, which is mediated in part by matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) [4][5][6][7]. MMP-13, a member of the collagenase subgroup of MMP proteins, plays a key role in bone matrix degradation and is expressed highly in osteoblasts [3,[8][9][10][11][12]. MMP-13 is likely to contribute to bone healing [13,14], bone development [15,16], and bone loss [17]. Previous studies also indicated that mRNA and protein levels of MMP-13 increase significantly following the application of orthodontic forces [18,19]. In contrast, TIMP-1 is an endogenous inhibitor of bone matrix degradation that binds tightly to active MMP-13, thereby downregulating MMP-13 activity [20][21][22].
The cellular response to mechanical strain is regulated by the type, frequency, magnitude, and duration of the mechanical strain imposed. In response to mechanical loading of bone tissue, osteoblasts exhibit changes in enzymatic activity and in protein production. The effect of mechanical force on the expression of MMPs has been demonstrated using various cell types [23][24][25][26]. However, few reports have examined the correlation between varying magnitudes of mechanical strain and osteoblastic cell activities, including extracellular matrix (ECM) metabolism. The relationship between mechanical strain and the expression of MMP-13 and TIMP-1 in osteoblasts is not known, particularly with respect to increasing magnitudes of mechanical strain.
In this study, we investigated the effect of different magnitudes of mechanical strain on MMP-13 and TIMP-1 expression in osteoblasts. MC3T3-E1 osteoblastic cells were subjected to 0%, 6%, 12%, or 18% elongation using the Flexercell Strain Unit, followed semi-quantitative reverse transcriptase-PCR (RT-PCR) and immunoblot analysis to determine MMP-13 and TIMP-1 mRNA and protein expression levels, respectively. Finally, we used specific inhibitors to determine the signal transduction pathways that regulate MMP-13 and TIPM-1 upregulation in response to mechanical strain.

Application of strain force
Cells were plated onto six-well, flexible-bottomed plates at a density of 2 × 10 5 cells/well. After overnight incubation, the cells were nearly confluent and were subjected to mechanical strains of 6%, 12%, or 18% elongation at 6 cycles/min for 24 h using a Flexercell Strain Unit (FX 3000, Flexcell International), as described previously [27]. Control cells (0% elongation) were cultured on similar plates and were maintained in the same incubator without mechanical strain.

Immunoblot Analysis
Immunoblot analysis was performed using monoclonal antibodies against MMP-13 or TIMP-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Total protein lysates were collected from cells, and the protein concentration was determined using the Bradford assay (Bio-Rad); BSA was used to generate the standard curve. Approximately 50 μg of total protein was loaded in each well of a 10% SDS PAGE gel; GAPDH served as the control for protein loading. Proteins were transferred electrophoretically onto nitrocellulose membranes (Hybond C, Amersham), followed by incubation in PBS-T buffer [PBS (pH 7.5) buffer containing 5% (w/v) blocking reagent (Amersham) and 1.0% (w/v) Tween 20 (Sigma, St. Louis, MO, USA)]. Individual membranes were incubated overnight in PBS-T buffer containing antibodies against MMP-13 or TIMP-1, followed by a 1 h incubation with goat anti-rabbit IgG conjugated to horseradish peroxidase (ECL Western blotting analysis system, Amersham). Protein bands corresponding to MMP-13, TIMP-1, and GAPDH were visualized, and the density of the protein bands was analyzed.
Enzyme-linked immunosorbent assay for the quantitative determination of active-MMP-13 and TIMP-1 concentrations 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 (R&D Systems, Minneapolis, MN). Each sample was assayed in triplicate. For active-MMP-13 quantification, the samples and standards were quantified fluorometrically with a fluorescence plate reader (MFX Microtiter1 Plate Fluorometer, Dynex technologies, Chantilly, VA, USA) with excitation wavelength set to 320 nm and emission wavelength set to 405 nm. For TIMP-1 quantification, the samples and standards were quantified with a microplate reader (MRX Microplate reader, Dynex Technologies, Chantilly, VA, USA) capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm. The concentrations of active form of MMP-13 and TIMP-1 were determined using a standard curve and normalized to the total cell number in each sample.

Inhibitors
We used specific inhibitors to identify signal transduction pathways that mediate the cellular response to tensile stress. We employed cycloheximide (10 μM) to inhibit de novo protein synthesis, indomethacin (10 μM) to inhibit cyclooxygenase (COX), genistein (20 μM) to inhibit tyrosine kinase activity, and PD098059 (10 μM) to specifically inhibit extracellular signal-related kinase (ERK). All inhibitors were purchased from Sigma (St. Louis, MO, USA), and the concentrations used correspond to effective doses reported previously [5,28]. MC3T3-E1 cells were pre-incubated in the presence of each inhibitor for 30 min, followed by the application of cyclic tensile strain at 18% elongation and 6 cycles/min in culture for 24 h. Total RNA was extracted from the cells, and MMP-13, TIMP-1, and GAPDH mRNA levels were determined by RT-PCR.

Statistical analysis
We performed these experiments using samples from at least five different cell preparations, and reproducibility was confirmed by using the same cell sample in triplicate. Values were calculated as means ± standard deviation (SD). Some data were subjected to multiple measurement analyses of variance (ANOVA), and a Student's t test was used to determine differences between the groups tested. P values of less than 0.05 were considered significant. Figure 1 shows how different magnitudes of strain affect MMP-13 mRNA and protein expression levels in the MC3T3-E1 cells. The expression of MMP-13 mRNA and protein were increased significantly and in a magnitude-dependent manner by mechanical strain with 6%, 12% or 18% elongation in comparison with the control (0% elongation) cultures. Figure 2 shows how different magnitudes of strain affect the TIMP-1 mRNA and protein expression levels in the MC3T3-E1 cells. Mechanical strain at 6%, 12% or 18% elongation caused a significant magnitudedependent increase in TIMP-1 mRNA and protein expression compared to that in control (0% elongation) cultures.

Results
The elevated expressions of MMP-13 and TIMP-1 in the stretched cells were further confirmed by ELISA for quantifying active-MMP-13 and TIMP-1 concentrations in conditioned medium from these cells. Active-MMP-13 and TIMP-1 concentrations in conditioned medium induced by mechanical strain in MC3T3-E1 cells also increased significantly in a magnitude-dependent manner compared to that in the control (See figure  3).
To determine whether the stretch-induced increases in MMP-13 and TIMP-1 mRNA expression were dependent on de novo protein synthesis, cyclooxygenase activity, tyrosine kinase activity, or extracellular signal-related kinase activity, MC3T3-E1 cells were treated with specific inhibitors, including cycloheximide, indomethacin, genistein, or PD098059, respectively. We found that only the MEK inhibitor PD098059 was capable of blocking the strain-induced upregulation of MMP-13 mRNA (See figure 4). In contrast, the signaling pathway linked to the induction of TIMP-1 mRNA expression was blocked only by cycloheximide (See figure 5).

Discussion
It has been demonstrated that matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate matrix degradation. The balance between the activities of MMPs and TIMPs is believed to determine the rate of this process. Previous studies indicate that MMP-13 plays a key role in bone remodeling and mechanical strain can affect the expression of MMP-13 mRNA in osteoblasts [3]. As we know the effects of mechanical strain on cells are dependent on the magnitude, duration, and frequency of mechanical strain, however, to date, very few reports have shown the effects of different magnitudes of mechanical strain on the mRNA and protein expression levels of MMP-13 and TIMP-1 in osteoblasts. The molecular biology mechanism of the regulation of extracellular matrix metabolism of osteoblasts under mechanical strain is not clear that progressive study should be carried out to  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).
clarify how different magnitudes of mechanical strain affect the expression of MMP-13 and TIMP-1 in osteoblasts. Yang et al. [3] reported previously that MMP-13 mRNA levels exhibited a time-dependent increase in MC3T3-E1 cells following the application of mechanical strain corresponding to 8% elongation. But, the correlation between varying magnitudes of mechanical strain and the expression of MMP-13 in osteoblasts is not known. The present study demonstrates that MC3T3-E1 cells subjected to 6%, 12%, or 18% elongation exhibit a magnitude-dependent increase in MMP-13 mRNA and protein levels (See figure 1). Active-MMP-13 concentration in conditioned medium induced by mechanical strain in MC3T3-E1 cells also increased significantly in a magnitude-dependent manner compared to that in the control (See figure 3). Several lines of evidence suggest that low levels of mechanical strain (1.8% -6% elongation) generate potent anti-inflammatory signals, whereas high levels of mechanical strain (12% -18% elongation) generate an inflammatory signal and induce PGE 2 and IL-1β, both implicated in matrix degradation [29][30][31][32]. In the present study, we found that the increase in MMP-13 expression was magnitude-dependent. Furthermore, mechanical strain corresponding to 6%, 12%, or 18% elongation led to a magnitude-dependent increase in TIMP-1 mRNA and protein expression (See figure 2). TIMP-1 concentration in conditioned medium induced by 12%, or 18% elongation mechanical strain increased significantly compared to that in the control (See figure 3). Collectively, these results suggest that the increase in MMP-13 levels observed in osteoblastic cells  under mechanical strain levels higher than those found under normal physiological conditions is accompanied by a corresponding increase in TIMP-1. TIMP-1 may in turn bind to activated MMP-13, thereby inhibiting MMP-13 activity and suppressing the degradation of bone matrix as part of the body's defense system. Our present findings may help to explain the lack of pathological inflammatory changes during orthodontic tooth movement when mechanical strain loads the alveolar bone. It appears that the essential functions of osteoblasts in bone remodeling are affected by the magnitude of mechanical strain and that cellular responses to mechanical strain are crucial to maintaining homeostasis and adapting to the bone environment.
The molecular mechanisms translating mechanical strain into a signal that activates increased gene expression are complicated. Mechanotransduction pathways may initiate signal transduction through many possible mechanisms including integrins, receptor tyrosine kinases, ion channel, gap junction, membrane fluidity, etc. To determine whether the stretch-induced increases in MMP-13 and TIMP-1 mRNA expression were dependent on de novo protein synthesis, cyclooxygenase activity, tyrosine kinase activity, or extracellular signal-related kinase activity, MC3T3-E1 cells were treated with specific inhibitors, including cycloheximide, indomethacin, genistein, or PD098059, respectively. We found that only the MEK inhibitor PD098059 was capable of blocking the strain-induced upregulation of MMP-13 mRNA (See figure 4). This result indicates that MMP-13 induction is mediated by signaling molecules related to extracellular signal-regulated kinase (ERK), which is supported by a previous study [3]. Therefore, the ERK-MAPK pathway is likely to contribute to the straininduced increase in MMP-13 mRNA expression. In contrast, the signaling pathway linked to the induction of TIMP-1 mRNA expression was blocked by cycloheximide (See figure 5), indicating that strain-induced upregulation of TIMP-1 mRNA required de novo protein synthesis, and suggesting that the strain-induced upregulation of TIMP-1 mRNA is not the primary response; rather, TIMP-1 upregulation required the synthesis of an as yet unidentified protein(s).

Conclusions
In conclusion, the application of different magnitudes of cyclic tensile strain (0%, 6%, 12%, or 18%) induced a magnitude-dependent increase in MMP-13 and TIMP-1 expression in cultured osteoblasts. Furthermore, we provide evidence that PD098059 and cyclohexamide treatment suppressed the strain-induced upregulation of MMP-13 and TIMP-1 expression, respectively. These results indicate that MMP-13 and TIMP-1 expression are differentially regulated in response to increasing magnitudes of mechanical strain in osteoblasts. This effect may regulate bone matrix metabolism, and suggests novel mechanisms through which osteoblasts may respond and adapt to mechanical strain, including occlusal and orthodontic forces, in order to maintain homeostasis and adapt to the bone environment.