Articular cartilage has little or no capacity for repair following injury (1). Damaged cartilage is prone to degeneration. For example, scoring of the articular surface of rabbit knees leads, within weeks, to cartilage degeneration similar to that observed in osteoarthritis (OA) (2). In humans, joint injury, either directly to the articular surface or via destabilizing ligamentous or meniscal tears, is a risk factor for the development of OA (3). It is likely that joint tissue microinjury, secondary to congenital or acquired biomechanical factors, accounts for further and perhaps a larger proportion of cases of progressive OA. In an attempt to understand the response of chondrocytes to cartilage injury, several groups of investigators have studied the effects of high or repeated impact loading, strain, or cutting of cultured cartilage or chondrocyte monolayers (4–6).
Our group has studied the effects of either dissecting cartilage from the intact articular surface or scoring the surface, because these are simple, reproducible modes of experimental injury that can be delivered rapidly to amounts of tissue sufficient for biochemical investigation. Within minutes, this type of sharp injury to the intact articular surface activated the 3 MAPKs, namely, JNK, p38 MAPK, and ERK (7, 8). Some degradation of IκBα was also apparent, implying possible activation of NF-κB. The injured cartilage rapidly released a factor that strongly activated ERK in cultured chondrocytes and was identified as basic fibroblast growth factor 2 (FGF-2), which was stored in the pericellular matrix (7, 9, 10). Although FGF-2, which signals via a receptor tyrosine kinase (predominantly FGF receptor 1 [FGFR-1]) accounted in part for ERK activation seen upon injury, this did not explain the strong JNK activation or activation of NF-κB. This pattern of signaling pathway activation is typically associated with inflammatory stimuli such as interleukin-1 (IL-1), tumor necrosis factor α (TNFα), or microbial products acting via Toll-like receptors.
The mechanism by which sterile tissue injury activates inflammatory signaling is obscure. Soluble ligands such as heat-shock proteins and high mobility group proteins released by injured cells can directly activate the pathways (11). However, no soluble JNK- or NF-κB–activating factor released from cartilage upon injury has been reported.
We previously observed that one response of freshly dissected cartilage was to produce activin A, a cytokine related to transforming growth factor β. We used an inhibitor of the FGFR tyrosine kinase (PD173074, also known as SB402451) to show that this action was FGF dependent (7, 12, 13). The effect of the Src family protein tyrosine kinase inhibitor PP2 on induction of activin A was also tested, because Src family kinases are commonly activated by growth factor receptor tyrosine kinases such as FGFR (12, 14). PP2 also inhibited activin A production. We therefore examined whether or not cartilage injury activated protein tyrosine kinase activity distinct from that activated by FGFR, and, if there was Src activation, whether it was downstream of FGF-2. Here, we show that cartilage injury rapidly stimulates Src-like protein kinase activity independently of FGF-2, and that this activity contributes to the overall gene response to injury. We also show that the injury activates IKK within seconds, and that FGF serves to amplify the MAPK and IKK signals.
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Inflammation is usually considered as a process in vascularized tissue that increases blood flow and causes diapedesis of leukocytes. However, as shown in Figure 4D, we demonstrated that even in avascular tissue such as cartilage, trauma causes intracellular signaling and gene expression characteristic of an inflammatory response. The signaling included activation of Src or Src-like kinases as well as MAPKs and IKK.
A striking feature of the response to injury was that activation of Src, IKK, and JNK occurred only upon damaging an intact articular surface; cutting cartilage adapted to culture (while still releasing FGF-2) did not cause these effects. Furthermore, no detectable soluble factor was released from the injured cartilage that reproduced the effects on cultured chondrocytes. It is possible that a soluble factor is unstable and thus undetectable. Alternatively, it is possible that the chondrocytes directly sense injury to the matrix, and that there is propagation of a signal by direct cell interactions. It is intriguing that whatever the sensing mechanism, it is an alarm that is not reset when the tissue is cultured.
This is the first report of Src activation and its effect on the gene response following injury of intact cartilage. Phosphorylation of FAK has been observed upon mechanical stimulation of cultured chondrocytes and was suggested to be integrin dependent (29). Src-like signaling appears to contribute to injury-induced gene expression independently of classic inflammatory signaling pathways such as NF-κB and the MAPKs. Src family kinases have been implicated in NF-κB activation by bacterial lipopolysaccharide and TNF family members (30, 31) but have also been reported to activate inflammatory gene transcription independently (32).
This injury response is not restricted to porcine articular cartilage. Preliminary experiments suggest that a similar response also occurs following injury to other connective tissue. For example, Src, IKK, and MAPK activation follows dissection of porcine synovium (Watt FE and Saklatvala J: unpublished observations). Similar signaling or gene expression is also seen in the connective tissue of mice, for example, following dissection of mesentery or in response to avulsion injury of the hip osteochondral junction (Ismail HM, et al: unpublished observations).
FGF-2 appears to be an important contributor to the response in cartilage, although its role is complex: FGF-2 suppresses IL-1–induced aggrecanolysis in vitro but at the same time does not inhibit the induction of several IL-1–induced genes (33). FGF-2 was shown to be chondroprotective in a murine model of OA (34), and yet here we observed that it amplifies inflammatory signaling and gene expression immediately upon cartilage injury.
The inflammatory gene response that follows cartilage injury is not simply the same as that caused by IL-1α in culture. For example, IL-8, HAS-1, and iNOS were all induced by IL-1 but not by cartilage injury. Interestingly, IL-1α, inhibin βA (the subunit of activin A), and MMP-1 mRNA were all regulated more strongly by injury than by IL-1 alone; this may illustrate that amplification by FGF-2 or Src-like signaling is important in gene expression following injury. Src and FGF-2 appear to contribute to this gene response independently; some Src-dependent genes (IL-1α, MCP-1, and SAA2) were not regulated by FGF-2. Given that pharmacologic inhibitors of signaling pathways may not be completely specific, these observations further support the signaling scheme presented in Figure 4D.
Is an injury-induced gene response important in vivo, and, specifically, in posttraumatic OA? In a recently published study by our group, 21 genes were significantly up-regulated in murine whole joint and articular cartilage immediately following surgical destabilization of the medial meniscus, which causes OA (35). All of the genes induced by dissection of porcine cartilage were among these and included IL-1, IL-6, COX-2, inhibin βA, MMP-3, ADAMTS-4, MCP-1 (CCL2), and SAA3 (the murine equivalent of SAA2). The exception was MMP-1 (which is not functional in the mouse). Interestingly, joint immobilization abrogates much of the murine gene response and protects against OA, implicating this immediate tissue injury response in the development of posttraumatic OA, at least in the mouse.
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Watt had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Watt, Didangelos, Peirce, Vincent, Wait.
Acquisition of data. Watt, Ismail, Didangelos, Peirce, Wait.
Analysis and interpretation of data. Watt, Ismail, Wait, Saklatvala.