Mechanical Compression of Cartilage Explants Induces Multiple Time-dependent Gene Expression Patterns and Involves Intracellular Calcium and Cyclic AMP

Abstract

Chondrocytes are influenced by mechanical forces to remodel cartilage extracellular matrix. Previous studies have demonstrated the effects of mechanical forces on changes in biosynthesis and mRNA levels of particular extracellular matrix molecules, and have identified certain signaling pathways that may be involved. However, the broad extent and kinetics of mechano-regulation of gene transcription has not been studied in depth. We applied static compressive strains to bovine cartilage explants for periods between 1 and 24 h and measured the response of 28 genes using real time PCR. Compression time courses were also performed in the presence of an intracellular calcium chelator or an inhibitor of cyclic AMP-activated protein kinase A. Cluster analysis of the data revealed four main expression patterns: two groups containing either transiently up-regulated or duration-enhanced expression profiles could each be subdivided into genes that did or did not require intracellular calcium release and cyclic AMP-activated protein kinase A for their mechano-regulation. Transcription levels for aggrecan, type II collagen, and link protein were up-regulated ϳ2-3-fold during the first 8 h of 50% compression and subsequently down-regulated to levels below that of free-swelling controls by 24 h. Transcription levels of matrix metalloproteinases-3, -9, and -13, aggrecanase-1, and the matrix protease regulator cyclooxygenase-2 increased with the duration of 50% compression 2-16-fold by 24 h. Thus, transcription of proteins involved in matrix remodeling and catabolism dominated over anabolic matrix proteins as the duration of static compression increased. Immediate early genes c-fos and c-jun were dramatically up-regulated 6 -30-fold, respectively, during the first 8 h of 50% compression and remained up-regulated after 24 h. Articular cartilage is responsible for the smooth articulation of synovial joints during locomotion. Chondrocytes within cartilage constantly remodel the extracellular matrix (ECM) 1 of the tissue throughout life. The major load-bearing constituents of the ECM are type II collagen and aggregates of the proteoglycan, aggrecan, which provide the tensile and compressive stiffness of the tissue, respectively. Also present in the ECM are families of matrix proteinases, tissue inhibitors of matrix metalloproteinases (TIMPs), growth factors, and cytokines that together regulate ECM remodeling and turnover in health and disease (1). It is known that mechanical exercise of the knee joint in vivo increases the density of aggrecan in cartilage (2), whereas knee joint inactivity results in decreased aggrecan deposition (3, 4). Traumatic injury to cartilage diminishes mechanical strength and leads to excessive catabolism of the ECM, increasing the risk of osteoarthritis later in life (5).A number of model systems have been developed to simulate various aspects of the mechanical loading forces experienced by articular cartilage in vivo. Compressive and shear forces have been applied to cartilage explants and chondrocyte cul...

Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures

“…The role of elevated cAMP levels (and the possible involvement of PKA) in mechanotransduction pathways of bovine articular chondrocytes is also discussed in another study [2]. Rp-cAMP, the Rp-diastereomer of adenosine-3',5'-cyclic monophosphothioate, which is a membrane-permeable inhibitor that blocks cAMP-mediated effects, was able to prevent upregulation of certain clusters of genes in response to static compression [1], suggesting a determining role of signalling pathways that involve PKA. In our study, the loading regime resulted in fivefold elevation of cAMP levels compared to untreated control HD cultures.…”

“…Chondrocytes in articular cartilage are constantly exposed to compressive forces during normal activity, which is essential for maintenance of metabolic processes and healthy composition of cartilage extracellular matrix (ECM). Type II collagen and large proteoglycan (PG) aggregates with glycosaminoglycan (GAG) side chains are the main load-bearing constituents of the ECM, which account for the tensile and compressive stiffness of the tissue, respectively; furthermore, large amounts of cations and interstitial water bound to GAGs are also important factors in cartilage matrix biomechanics [1].…”

Association between a variation in LRCH1 and knee osteoarthritis: A genome‐wide single‐nucleotide polymorphism association study using DNA pooling

“…Disruption of this mechanism may contribute to the phenotype of primary OA. Compressive stress plays a role in chondrocyte metabolism and development (34,35). LRCH1 may play a role in this signaling pathway, such that chondrocytes do not respond normally to compressive stress, which leads to the changes in cartilage composition and structure that are characteristic of OA.…”

Mechanical injury of cartilage explants causes specific time‐dependent changes in chondrocyte gene expression

“…To distinguish the main expression trends, a k-means clustering algorithm was applied to the injury time-course data (28)(29)(30)(31). Each gene was grouped on the basis of correlations between the time-course expression profile and a set of randomly chosen starting genes.…”

“…The optimal solution was chosen as the grouping that had the highest overall correlation of genes with group profiles, by averaging over all of the genes (for details, see ref. 31). The number of groups was varied from 3 to 6.…”