Mechanical stimuli are important signals in articular cartilage, but what mediates them is unknown. We have shown that extracellular-signal-regulated kinase was activated on cutting and loading articular cartilage, and deduced that this was due to the release of bFGF (basic fibroblast growth factor) from the tissue. bFGF was shown to be extracellular, and by immunohistochemistry, was present in the pericellular matrix of articular chondrocytes attached to the heparan sulphate proteoglycan perlecan. We propose a novel mechanotransduction model, whereby pericellular bFGF, a short distance from the cell surface, becomes available to the cell surface tyrosine kinase receptors when articular cartilage is loaded.
- articular cartilage
- basic fibroblast growth factor (bFGF)
- mechanical loading
- pericellular matrix
In articular cartilage, mechanical loading leads to important anabolic tissue responses, but precisely how such mechanical signals modulate matrix synthesis is unknown. Integrin ligation is a favoured mechanism in tissue mechanotransduction, but integrins do not have intrinsic signalling capability, and published studies have been performed on isolated cells in the absence of a true matrix, through which these signals must surely travel [1–3].
We have shown that ERK (extracellular-signal-regulated kinase), one of the three MAPK (mitogen-activated protein kinase) pathways, is activated upon explanting or cutting cartilage. The response was shown to be due to the release of a soluble factor from the tissue, which was purified and identified by MS as bFGF [basic FGF (fibroblast growth factor)]. The activity in the medium from cut cartilage was inhibited by neutralizing antibodies to bFGF, and the tissue response to cutting was abrogated by pre-incubation with 50 nM SB402451, an FGF receptor tyrosine kinase inhibitor  (Figure 1). Cyclical loading of either ‘intact’ cartilage on bone or cartilage explants also caused activation of ERK, which was inhibited by SB402451  (Figure 2), suggesting that bFGF is a mechanotransducer for both injurious and non-injurious stimuli.
Articular cartilage is rich in extracellular matrix proteoglycans which might bind bFGF via heparan sulphate glycosaminoglycan chains. Treatment of the tissue with heparitinase, but not chondroitinase, caused release of bFGF, confirming a heparan sulphate-bound pool of bFGF in articular cartilage. Candidate heparan sulphate proteoglycans in cartilage include the cell surface molecules, the syndecans and glypicans, and the extracellular matrix proteoglycan perlecan. Perlecan, first described as the proteoglycan of basement membranes, is abundant in articular cartilage, and is predominantly heparan sulphated in mature tissue. It is an attractive candidate for bFGF binding because it has been implicated in facilitating bFGF signalling [6–9], and mutations in perlecan result in severe chondrodysplasias which are similar to those that result from bFGF receptor 3 gain-of-function mutations [10,11]. To determine whether bFGF was bound to perlecan in articular cartilage, we purified perlecan and the chondroitin sulphate proteoglycan aggrecan from porcine articular cartilage, and used surface plasmon resonance to show that perlecan, but not aggrecan, bound to recombinant bFGF. Binding was dependent on heparan sulphate, as pre-treatment of perlecan with heparitinase abrogated binding. Immunolocalization, by routine immunohistochemistry and confocal microscopy, has confirmed co-distribution of bFGF and perlecan in the pericellular matrix of articular chondrocytes.
The pericellular matrix of cartilage is a highly specialized matrix, devoid of type II collagen and aggrecan, and rich in the non-fibrillar collagen type VI. This results in markedly altered mechanical properties, which protect the cell from injury, and have been suggested to facilitate the transmission of mechanical signals from the further removed, territorial matrix. Type VI collagen is regarded as making up the ‘capsule’ of the pericellular matrix where it probably interacts indirectly with type II collagen and aggrecan. Perlecan is thought to be attached to the cell bound via its domain V to cell surface integrins.
Interestingly bFGF has been implicated as a mechanotransducer in other tissues: Kaye et al.  showed that bFGF was released by adult rat ventricular myocytes in response to increased mechanical pressure and in differentiated human skeletal-muscle cells bFGF was released in response to mechanical strain and sarcolemma injury . When smooth-muscle cells embedded in a three-dimensional collagen gel were subjected to a single compressive load, a 3-fold increase in thymidine incorporation occurred, which was inhibited by neutralizing antibodies to bFGF but not to PDGF (platelet-derived growth factor) . However, all of these studies were performed on isolated cells, not tissue, and most of the studies concluded that bFGF was liberated from the cell rather than from an extracellular store.
The mechanisms by which pericellular bFGF becomes available to the receptor on cutting, or mechanical loading of cartilage, are presently unknown. It is possible that on loading bFGF remains attached to perlecan and upon deformation of the matrix, it is able to bind the cell surface receptor directly. Alternatively, enzymatic cleavage of either the heparan sulphate or perlecan may take place, allowing soluble bFGF to diffuse to the cell surface. Finally, it is possible that ligand passing occurs, where bFGF is exchanged from the heparan sulphate of perlecan to cell surface heparan sulphates, or directly to the tyrosine kinase receptor.
So, does bFGF mediate the anabolic responses of articular cartilage to physiological loading? We have no data to support a direct anabolic role for bFGF, i.e. it did not increase sulphate incorporation, an indicator of proteoglycan synthesis, nor did it induce type II collagen production using metabolic labelling studies on cartilage in vitro. Our results suggest that bFGF is anticatabolic in articular cartilage. It induces members of the TIMP (tissue inhibitor of metalloproteinase) family, and it also strongly up-regulates activin, a member of the TGF-β (transforming growth factor-β) family. These responses support a key regulatory role for bFGF in articular cartilage turnover, and highlight its importance as a tissue mechanotransducer.
Cytokine–Proteoglycan Interactions: Biology and Structure: Biochemical Society Focused Meeting held at Royal Holloway University of London, Egham Hill, U.K., 9–10 January 2006. Organized and edited by B. Mulloy (NIBSC, U.K.) and C. Rider (Royal Holloway University of London, U.K.).
Abbreviations: FGF, fibroblast growth factor; bFGF, basic FGF; ERK, extracellular-signal-regulated kinase
- © 2006 The Biochemical Society