Members of the cysteine-rich 61/connective tissue growth factor/nephroblastoma overexpressed (CCN1-3) family of genes (CCN-family) have been reported to be involved in central cell-biological processes such as cell adhesion, migration, proliferation, differentiation and survival [1–3]. Additional CCN proteins are the wnt induced secreted proteins WISP1/CCN4, WISP2/CCN5 and WISP3/CCN6. The CCN proteins share a common modular structure comprising an insulin-like growth factor binding motif, a von Willebrand type C domain and a thrombospondin type I domain . Based on the identical position of conserved cysteine residues within these proteins they are considered a structural family, but vary in function. CYR61 is considered as an angiogenic inducer, which binds to integrin receptors and proteoglycans [2, 3].
Various CCN-proteins have been implicated in chondrogenesis and bone formation as well as angiogenesis during development and adult life . These biological processes are also essential for skeletal tissue regeneration involving both endothelial and mesenchymal progenitor cells. Local recruitment of cells with the capacity to differentiate along the osteoblastic lineage for example is necessary for bone growth, remodeling and repair. The underlying cell-biological processes involve multipotent mesenchymal progenitor cells, which have been identified in bone marrow and various skeletal tissues including bone and cartilage [4–6]. Therefore, mechanisms regulating directed migration of undifferentiated cells might play an important role in skeletal physiology and pathology. Different chemokine receptors including CXCR4, CCR7, CCR1 and CX3CR1 have been identified on MSCs and stromal-derived factor (SDF)-1 has been shown to possess chemotactic activitiy for CXCR4-positive MSCs [7–9]. Further candidates for mediating chemotactic signals to bone marrow derived MSCs are growth factors, which are released during tissue remodeling or fracture healing. Thus it is not surprising that a chemotactic response of human bone marrow derived mesenchymal progenitor cells to factors like bone morphogenetic protein-2 (BMP-2), platelet-derived growth factor-isoforms (PDGF-AA, AB, BB), basic fibroblast growth factor (bFGF) or IGF-I and -II, has been demonstrated [10–14]. Some of these factors exhibited preferential or exclusive activity in osteoblastic cells while others showed enhanced activity in MSCs [13, 15]. Vascular endothelial growth factor-A (VEGF-A), which has been shown to act as a coupling factor for angiogenesis and bone formation, stimulated directed cell migration of endothelial cells mainly via VEGFR2 and that of MSCs via VEGFR1 [15, 16]. A dual function on bone formation and vascularisation has also been attributed to CYR61/CCN1 . Recently, overexpression of CYR61/CCN1 has been identified as a chemotactic stimulus for the mesenchymal stem cell line C3H10T1/2 .
The observation that mutations in WISP3/CCN6 lead to progressive pseudorheumatoid dysplasia (PPRD) and polyarticular juvenile idiopathic arthritis, diseases that are associated with cartilage growth plate defects and/or loss of adult articular cartilage indicates that this CCN-protein might be related to the preservation of cartilage integrity [19, 20]. It has been shown that WISP3/CCN6 stimulates collagen type II, aggrecan as well as superoxide dismutase expression and activity in chondrocytes [21, 22]. Potential effects on mesenchymal stem cells, however, have not been described so far, although a differential expression of CCN-family members in human MSCs during osteogenic, chondrogenic or adipogenic differentiation has been reported. Both CYR61 and WISP3 were found to be expressed in undifferentiated MSCs. CYR61 expression generally seized with any differentiation along different lineages while WISP3 expression seized in chondrogenic differentiation, indicating that both polypeptides play important roles early in commitment or even in uncommitted MSCs .
The aim of this study was to establish recombinant expression of WISP3/CCN6 and to quantify possible chemotactic effects on primary human MSCs in comparison to CYR61/CCN1. Our results indicate that both CCN-proteins are chemoattractive ligands for human mesenchymal stroma cells before and after osteogenic commitment and that the effect of WISP3/CCN6 but not CYR61/CCN1 on cell migration of MSCs is mediated by the integrin receptor ανß 5.