Platelet-derived growth factor (PDGF) is a major mitogen for fibroblasts and other cells of mesenchymal origin. Its biologic function is mediated by PDGF receptor tyrosine kinases, which are formed by homo- or heterodimers of α and β chains. The classic PDGF family consists of 2 members, PDGF-A and PDGF-B, which form homo- or heterodimers with distinct receptor binding abilities (1). Recently, 2 new family members have been discovered through database mining tools (2–4). Besides the core PDGF/vascular endothelial growth factor (VEGF) domain, PDGF-C and PDGF-D contain an additional CUB (complement subcomponents C1r/C1s, urchin epidermal growth factor–like protein, and bone morphogenetic protein 1) N-terminal domain, which is cleaved upon activation (2–4).
PDGF-C messenger RNA (mRNA) is expressed in many tissues, particularly the heart, liver, pancreas, and kidney. Although its detailed functional role remains to be investigated, PDGF-C seems to be a potent transforming factor, because it is expressed in many tumor cell lines (5), induces tumor formation, and efficiently transforms a murine fibroblast cell line (6). The ability to stimulate the growth of smooth muscle cells (5) and of microvessels in the murine cornea (7) also suggests an important role in angiogenesis. In addition, PDGF-C may contribute to fibrotic processes, since the hearts of PDGF-C–transgenic mice exhibit a progressive hypertrophy with strong proliferation of cardiac fibroblasts (2).
Transfection with PDGF-D results in transformation of murine NIH3T3 cells, which are then characterized by anchorage-independent growth, actin reorganization, an increased proliferation rate, and the capacity to induce tumor formation in mice (8, 9). In addition, PDGF-D is known to be involved in angiogenic processes (10).
Rheumatoid arthritis (RA) is characterized by progressive joint inflammation accompanied by synovial hyperplasia with some similarities to malignancy. Synovial cells located in pannus show an invasive behavior, are of variable cell size (11), and express high levels of protooncogenes (12) and tyrosine phosphorylated proteins (13). PDGF receptor (PDGFR) α and β chains are detected in fibroblast-like cells in the inflamed synovial membrane (SM) (14). PDGFRs, as well as PDGF-A and PDGF-B, are expressed to a significantly higher degree in the SM of RA patients compared with that of osteoarthritis (OA) patients (15). Furthermore, fibroblast-like cells from RA synovium respond to PDGF-BB stimulation with proliferation (16) and anchorage-independent growth in vitro (17), showing the importance of PDGFs in RA.
We therefore investigated the expression of the newly discovered PDGF-C and PDGF-D at the mRNA and protein levels in SM samples from patients with RA and OA, the cellular distribution of these factors within the SM, and the functional effects of PDGF-D on synovial fibroblasts.
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PDGFs are thought to play an important role in the synovial hyperplasia that is characteristic of RA. These effects are triggered by binding to PDGFR α and β chains, which results in activation of protooncogenes and subsequent proliferation of synoviocytes. This study is the first to demonstrate that, besides the classic PDGF-A and PDGF-B, the recently discovered isoforms PDGF-C and PDGF-D are expressed in the SM of RA and OA patients and that PDGF-D protein is detected at significantly higher levels in RA than in OA.
Interestingly, there was a discrepancy between the expression of mRNA and protein for PDGF-D in the SM of RA patients. However, similar discrepancies have been reported for the proteinase inhibitor maspin (23) and the jun/fos protooncogenes (24). These discrepancies were observed exclusively in SM from RA patients but not OA patients, suggesting disease-specific alterations of posttranscriptional processes.
PDGF-C and PDGF-D were detected in the synovial lining layer, diffuse infiltrates, and stroma of the SM, according to the expression previously reported for PDGF-A and PDGF-B (13, 15); this leads to the assumption that cells of these compartments are able to express all isoforms of PDGF. Double-labeling experiments with specific markers showed expression of both PDGF-C and PDGF-D by synovial fibroblasts and macrophages, which is consistent with recent findings of PDGF-C in dermal and lung fibroblasts (25) and in infiltrating inflammatory CD11b+ cells in coxsackievirus B3–induced chronic myocarditis (26).
PDGF-C and PDGF-D, although functional analogues of PDGF-A (2) and PDGF-B (3, 4), represent a new subfamily of growth factors that require proteolytic activation for receptor binding. This cleavage of the CUB domain results in the appearance of a 23-kd (PDGF-C) and a 32-kd (PDGF-D) protein under reducing conditions, representing the “core” growth factor domain monomers (2–4, 25). Although the respective bands were usually absent in the case of PDGF-C, active PDGF-D could be detected in all SM samples investigated (at the predicted molecular mass of 32 kd) and showed a significantly higher expression in RA SM. This is consistent with the increased proteolytic activity in the RA SM (for review, see ref. 27), in particular, with respect to the activity of plasmin, thrombin, and tissue plasminogen activator or urokinase plasminogen activator, which are reported to activate PDGF-C (2, 28) and PDGF-D (29), respectively.
Since little is known about the detailed functional roles of PDGF-C and PDGF-D, their importance in the pathogenesis of arthritic diseases remains speculative. Similar to PDGF-B, PDGF-D is important for cellular transformation and support of tumor development, probably due to exclusive or preferential signaling through PDGFRβ (8, 30). A similar function has been ascribed to the PDGFRα agonist PDGF-C (5, 6). Although signaling through PDGFRα alone is not able to efficiently transform fibroblastic cells (1), the transforming action of PDGF-C may be explained by the activation of PDGFRα/β heterodimers (25). Because both PDGFRs are expressed in the rheumatoid synovium (14), it is conceivable that binding of PDGF-D, and possibly also PDGF-C, to their receptors contributes to the proliferation and semitransformation of cells that is observed during pannus formation in RA (11). This hypothesis is further supported by the finding that the PDGF-D core protein stimulated the proliferation of synovial fibroblasts, as was previously shown for vascular fibroblasts transfected with the PDGF-D gene (31).
Recent reports indicate the involvement of both factors in fibrosis (2) or tissue remodeling (32), processes characterized by the synthesis of matrix molecules and the activity of MMPs. Indeed, in the present study, stimulation with PDGF-D increased the expression of MMP-1 mRNA in RA synovial fibroblasts. Similar results for PDGF-D have been reported in the case of MMP-2 in vascular smooth muscle cells (31), for MMP-9 in renal carcinoma cells (33), as well as for PDGF-C with respect to MMP-1 expression in dermal fibroblasts (32). Taken together, these findings indicate the relevance of the protease-activated PDGF isoforms in tissue remodeling, a process of major importance during the progression of arthritic diseases.
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Drs. A. Roth, R. Fuhrmann, and R. Winter (all from the Orthopedic Clinic, Waldkrankenhaus “Rudolf Elle,” Eisenberg, Germany) are gratefully acknowledged for providing synovial tissue and Dr. E. Palombo-Kinne for critical revision of the manuscript. We thank Prof. F.-D. Böhmer (Institute of Molecular Cell Biology, Jena, Germany) for support and Prof. U. Eriksson (Ludwig Institute for Cancer Research, Stockholm, Sweden) for providing the PDGF-D core protein.