To determine if membrane type-1 matrix metalloproteinase (MT1-MMP) will respond to cyclic compression of chondrocytes grown in vitro and the regulatory mechanisms underlying this response.
Cyclic compression (30min, 1kPa, 1Hz) was applied to bovine chondrocytes (6-9-month-old animals) grown on top of a biodegradable substrate within 3 days of initiating culture. Luciferase assays using bovine articular chondrocytes were undertaken to demonstrate the mechanosensitivity of MT1-MMP. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis were used to establish the time course of gene and protein upregulation in response to cyclic compression. The regulation of MT1-MMP was assessed by electrophoretic mobility shift assays, RT-PCR and western blot analysis. As well, an MT1-MMP decoy oligonucleotide and an extracellular signal-regulated kinase 1/2 (ERK1/2) pharmacological inhibitor were utilized to further characterize MT1-MMP regulation.
After cyclic compression, MT1-MMP showed a rapid and transient increase in gene expression. Elevated protein levels were detected within 2h of stimulation which returned to baseline by 6h. During cyclic compression, phosphorylation of the mitogen activated protein kinase ERK1/2 increased significantly. This was followed by increased gene and protein expression of the transcription factor; early growth factor-1 (Egr-1) and Egr-1 binding to the MT1-MMP promoter. Blocking Egr-1 DNA binding with a decoy MT1-MMP oligonucleotide, downregulated MT1-MMP gene expression. The ERK1/2 inhibitor U0126 also reduced Egr-1 DNA binding activity to MT1-MMP promoter sequences and subsequent transcription of MT1-MMP.
These data suggest that cyclic compression of chondrocytes in vitro upregulates MT1-MMP via ERK1/2 dependent activation of Egr-1 binding. Delineation of the regulatory pathways activated by mechanical stimulation will further our understating of the mechanisms influencing tissue remodeling.