Altered mRNA level of matrix metalloproteinase-13 in MH7A synovial cells under mechanical loading and unloading

Abstract

In an effort to elucidate the interplay between mechanical load and proteolytic gene expression in arthritic tissue degradation, we investigated cellular morphology and mRNA levels of matrix metalloproteinase-13 (MMP-13) genes under mechanical stress in human MH7A synovial cells. The cells were isolated from the knee joint of a rheumatoid arthritis patient. Using a reverse transcription–polymerase chain reaction procedure, we found that loading by an oscillatory shaker transiently decreased the level of MMP-13 mRNA and unloading by a clinostat increased its mRNA level. The unloaded cells appeared to be rounded and displayed a poorly developed track of peripheral fibers, whereas the cells under loading tended to align to the shear flow and were elongated. We also found that altering the oscillatory direction of mechanical loads contributed to a further reduction in mRNA expression of MMP-13. Our results demonstrate the role of mechanical loading and unloading in the transcriptional regulation of MMP-13 in synovial cells, and suggest the potential value of physical therapy for arthritic joints.

Introduction

Rheumatoid arthritis is an autoimmune disease characterized by inflammation of the membrane lining of the joints. The joint can lose its shape and alignment, resulting in pain and loss of movement. Although the exact cause is yet to be understood, advances in molecular biology have provided numerous new data on the cascades of molecular interactions, including primary response genes, cytokines, prostaglandins, adhesion molecules, and matrix-degrading enzymes.7, 15, 16 Among families of matrix-degrading enzymes, matrix metalloproteinases (MMPs) have been considered the most influential group of proteinases in joint tissues. Despite the interplay of biochemical and biomechanical factors on the function and the pathology of joints, it is largely unknown whether mechanical loading or unloading plays any significant role in the process of tissue degradation of rheumatic joints and, more specifically, in the expression of MMP genes.

The human MMP family contains at least 24 identified genes, including groups such as collagenases, gelatinases, stromelysins, and membrane types.13, 20 Collagenases have the unique ability to cleave types I, II, and III collagens at a single site, producing one-quarter and three-quarter helical fragments. Gelatinases have substrate specificity for denatured collagens and gelatins, and stromelysins have specificity to collagen and gelatin. We focused specifically on the expression of MMP-13 in this study, because the elevated level of MMP-13 has previously been reported in synovial tissues of rheumatoid arthritis and osteoarthritis.3, 25 MMP-13 belongs to a group of collagenases. Type II collagen, which constitutes 80%–85% of the total collagen content in joint tissues, is a substrate of MMP-13.4 Although upregulation of collagenase gene expression was reported in rabbit synovial cells using agents such as 12-O-tetra-decanoylphorbol-13-acetate promoting cell rounding,23, 24 no study has been performed to test effects of mechanical loading and unloading in human synovial cells.

In this study, we investigated the effects of mechanical stress on MMP-13 mRNA levels. To simulate “mechanical loading” and “mechanical unloading,” we employed an oscillatory shaker and a clinostat, respectively. A clinostat is a rotating device around a horizontal axis, commonly used for simulating weightlessness. Using human MH7A synovial cells isolated from the knee joint of a rheumatoid arthritis patient MMP-13 mRNA levels were determined by quantitative reverse transcription–polymerase chain reaction (RT-PCR). Although MMP activity can be controlled in several steps from transcription to posttranslational modification,17 transcription is the first key regulatory step. In this study, we demonstrate that expression of the MMP-13 gene is downregulated by mechanical loading and upregulated by unloading. Our results indicate the potential of physical therapy at the molecular level for rheumatoid arthritic joints.