S100 proteins are acidic low molecular weight calcium binding proteins that are only found in vertebrates and are expressed in many tissues in humans (1). The S100 protein family consists of 21 known members and is considered to be one of the largest subgroups of the EF-hand calcium binding protein family (1). S100 proteins regulate numerous intracellular functions, including protein phosphorylation, enzyme activation, cell motility, cell growth and differentiation, and calcium homeostasis (2). Interestingly, S100 proteins are also known to have extracellular functions. Studies have shown that S100B is released into the extracellular environment by neuronal cells, stimulates neurite extension, and promotes cell survival (3). The extracellular functions of S100 proteins are attributed to their ability to be released from cells and to interact with cell surface receptors, including the receptor for advanced glycation end products (RAGE) (4). Recent studies of chondrocytes have shown that when added extracellularly, S100 proteins stimulate the expression of matrix metalloproteinase 13 (MMP-13) (5) and promote chondrocyte hypertrophy (6) through stimulation of RAGE signaling.
S100A4 is a member of the S100 family that was originally isolated as a gene that was differentially expressed in mouse adenocarcinoma cells (7) and was subsequently found in other tissues (8). Recent studies have identified S100A4 in cartilage and have shown it to be up-regulated in tissues from patients with osteoarthritis (OA) or rheumatoid arthritis (RA) (5, 9). Like other members of the S100 family, S100A4 exerts intracellular and extracellular effects. With respect to its intracellular targets, S100A4 binds the p53 tumor suppressor and regulates its functions (10). S100A4 also interacts with the heavy chain of non-muscle myosin II and plays an active role in cell motility and adhesion in metastatic tumor cells (11). When applied extracellularly, S100A4 acts as a potent cytokine that stimulates neurite outgrowth in astrocytes (12) and angiogenesis in endothelial cells (13).
In addition, S100A4 has also been suggested to play an important role in matrix remodeling (14). We have previously shown that extracellular S100A4 binds to RAGE in articular chondrocytes and activates the RAGE signaling cascade, leading to increased production of MMP-13 (5). Recent studies have shown that extracellular S100A4 can induce the up-regulation of several MMPs, such as MMP-1, MMP-3, MMP-9, and MMP-13 in RA synovial fibroblasts (15). Taken together, these findings suggest that S100A4 may play an important role in the degradation of cartilage and the progression of arthritis.
Interleukin-7 (IL-7) was initially identified as a factor required for the growth of murine B cell precursors (16). However, subsequent studies have shown that IL-7 plays an important role in T cell, dendritic cell, and bone biology in humans (17). IL-7 has been studied in RA because of its elevated levels in RA patient serum (18) and its increased expression in RA synovium and synovial fibroblasts (19). Recently, we found that IL-7 is expressed in chondrocytes and that its expression is up-regulated in OA chondrocytes and in normal chondrocytes with aging (20). In addition, we also found that IL-7 expression was increased in chondrocytes in response to fibronectin fragment and IL-1 stimulation and that chondrocytes responded to IL-7 treatment with increased production of MMP-13 (20). These data suggest that IL-7 may play an important role in the cartilage degradation seen in OA as well as RA.
As discussed above, studies have shown that S100A4 is secreted into the extracellular environment (21); however, the mechanism of secretion is not known. The current study was therefore designed to determine if cytokines that are known to be active in cartilage can stimulate S100A4 secretion and to study the pathway involved in this process. Experiments were performed using human articular chondrocytes treated with different chemical inhibitors to define the involvement of cell signaling pathways, protein expression, and protein secretion. The data presented here demonstrate that chondrocytes secrete S100A4 in response to IL-7 activation of the JAK/STAT signaling pathway.
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Members of the S100 family of calcium binding proteins are unique in having both intracellular and extracellular functions. S100 proteins lack the classic signaling sequence for secretion, yet are released into the extracellular environment. The molecular mechanism of secretion of these proteins is poorly understood. In the present study, we found that S100A4 is secreted by human articular chondrocytes in response to IL-7 stimulation and that activation of the JAK/STAT pathway is required. IL-7–mediated S100A4 secretion can then act in an autocrine manner to stimulate MMP-13 production via RAGE.
We have recently identified IL-7 in chondrocytes and showed that IL-7 is produced by chondrocytes in response to fibronectin fragment and IL-1β stimulation (20). In the current study, we show that treatment of human articular chondrocytes with IL-7 resulted in an increase in both the mRNA expression and the secretion of S100A4. Pretreatment of chondrocytes with cycloheximide abolished the IL-7–mediated secretion of S100A4, suggesting that S100A4 mRNA expression and de novo protein synthesis play an important role in the IL-7–mediated secretion of S100A4 by chondrocytes. However, pretreatment of chondrocytes with brefeldin A did not have any effect on the IL-7–mediated secretion of S100A4, suggesting that the secretion of S100A4 in chondrocytes does not follow the ER–Golgi secretory pathway. Previous studies of other members of the S100 family of proteins have likewise demonstrated an alternative secretory pathway that is independent of the classic ER–Golgi route (27, 28) and the requirement for de novo protein synthesis (29).
Previous studies have shown that members of the S100 family of proteins can be secreted in response to external stimuli that activate signaling proteins, such as protein kinase C (27, 28) or ERK-1/2 (30). In the present study, IL-7 signaling required phosphorylation of JAK-3 and the downstream transcription factor STAT-3, which resulted in an increased DNA binding activity of the STAT. In addition, our studies also showed a delayed phosphorylation of JAK-1. Studies have shown that JAK-3 is constitutively associated with the carboxy-terminal region of the γ-chain component of the IL-7 receptor and that activation of JAK-3 is considered to be the first step in the signal transduction cascade induced by IL-7 binding (27). Activation of JAK-3 is followed by phosphorylation of JAK-1, which is associated with the α-chain of the receptor, and then leads to the recruitment of STAT protein (31, 32). Activated STATs translocate into the nucleus and activate their target genes (33, 34).
The JAK/STAT pathway has been shown to be operative in chondrocytes. Stimulation of chondrocytes with oncostatin M resulted in increased phosphorylation of JAK-3 and increased DNA binding activity of STAT-1. Inhibition of this signaling pathway by a JAK-3 inhibitor blocked the oncostatin M–induced expression of MMP-1, MMP-3, and MMP-13 genes in chondrocytes (35). Additionally, in support of our observation, a recent study has shown that IL-7 can stimulate increased phosphorylation of Pyk-2, a non–receptor tyrosine kinase and member of the focal adhesion kinase family, in chondrocytes (20). Studies have previously shown that Pyk-2 is activated by IL-7 via a JAK signaling pathway (36), and Pyk-2 was found in association with JAK-3, thus implicating Pyk-2 as an important component of the JAK/STAT signaling pathway (37). Taken together, these studies suggest an important role for IL-7–mediated JAK/STAT signaling in the secretion of S100A4 by chondrocytes.
Treatment of chondrocytes with IL-7 also resulted in the induction of MMP-13 production in conditioned media, which is consistent with our previous report (20). Pretreatment of chondrocytes with a JAK-3 inhibitor blocked both the IL-7–mediated secretion of S100A4 and the production of MMP-13, suggesting a relationship between the secretion of S100A4 and the production of MMP-13 in the presence of IL-7. Previously, we showed that stimulation of MMP-13 production by extracellular S100A4 required activation of RAGE signaling (5). In the present study, transient overexpression of dominant-negative RAGE in chondrocytes blocked the IL-7–mediated production of MMP-13, which is consistent with the hypothesis that S100A4 released by chondrocytes in response to IL-7 stimulation functions as an autocrine or paracrine factor to induce MMP-13 production via RAGE activation.
In summary, our data showed that IL-7 stimulates chondrocyte secretion of S100A4 via activation of the JAK/STAT signaling pathway. Extracellular S100A4 then functions as an autocrine factor and stimulates MMP-13 production via RAGE. Previous studies have shown that S100A4, RAGE, and IL-7 are increased in OA cartilage. Thus, IL-7 and S100A4 may contribute to cartilage destruction and the development of OA.