The plasminogen activator/plasmin system is thought to play an important role, in addition to clot lysis, in a number of pathophysiological processes such as inflammation, wound healing, vascular remodeling, tumor invasion and metastasis by regulating various cellular activities involved in these processes (1
). In tumor metastasis, the system appears to be involved not only in tumor invasion but also in tumor angiogenesis (32
). Although uPA, uPAR and PAI-1 were shown to regulate tumor cell migration and angiogenesis by plasmin-independent mechanisms (32
), plasmin appears to play a crucial role in facilitating these processes by breaking anatomical barriers by degrading proteins in basement membranes and ECM directly or indirectly via activation of other proteases (36
). Hydrolysis of matrix components by plasmin may lead to the release of biologically active fragments of the matrix and/or release of matrix-bound growth factors. This would facilitate the diffusion of biologically active peptide fragments and growth factors to more distant sites. Earlier studies showed that plasmin released or cleaved growth factors, such as bFGF (7
) and TGFß-1 (5
), from the extracellular matrix. In the present study, we show that plasmin also cleaves a pluripotent matricellular signaling protein, CCN1, and liberates a biologically active peptide fragment of CCN1 from tumor cells/matrix that could support endothelial cell migration.
CCN1 is a novel ECM-associated signaling protein, which was shown to influence not only tumor angiogenesis but also a myriad of cellular functions that contributes to many pathophysiological conditions (12
). CCN1 is composed of four discrete structural domains, insulin-like growth factor binding protein-like module (IGFBP), Von Willebrand factor type C-like module (VWC), thrombospondin 1-like module (TSP1) and cystein-knot containing family of growth regulator-like module (CT). A variable region connects domains I and II with III and IV. TSP1 repeat (WSxCSxxCG) is thought to be involved in binding to matrix macromolecules, particularly to sulfated glycoconjugates (13
). CT domain contains two heparin binding motifs (27
) that may participate in interaction with ECM (28
). Our present data show that plasmin cleaves CCN1 in the thrombospondin module region, probably at R250
) or R259
) since the ~28 kDa peptide fragment of the cleavage products was detected by the antibody that recognizes the variable region (163 to 240 aa) and not the C-terminus. Thus, the 28 kDa fragment represents the N-terminus portion of the protein. Multiple attempts of N-terminal sequencing of the 21 kDa C-terminus peptide fragment (using ~100 picomoles or more of the fragment) to identify the precise cleavage site were unsuccessful.
The observation that the 28kDa fragment of CCN1 was found in overlying media and not in cell lysates suggests that the binding motifs that participate in CCN1 binding to cell surfaces or ECM lie out side of this region, i.e., in the C-terminus portion of TSP1 domain and/or in the CT domain. At present, it is unclear whether the C-terminal fragment of CCN1 is still attached to the ECM/cell surfaces or released into the overlying media since we were unable to detect this band, either in the cell lysate or in the overlying media, on immunoblot analysis with the C-20 antibody.
CCN1 is a member of the newly established CCN family that plays an important regulatory role in development, wound healing, vascular diseases and cancer (12
) CCN1 is a growth factor-inducible immediate-early gene expressed at very low levels in various cells but rapidly induced by growth factors, proteases and other stimuli (11
). Upon synthesis, the CCN1 protein is secreted and associates with cell surfaces and ECM (14
). CCN1 has been shown to support cell adhesion, cell migration and augment growth factor-induced cell proliferation in various cell types through interactions with integrins and cell surface heparan sulfate proteoglycans (HSPGs) in cell type and context-specific manner (12
). In endothelial cells, CCN1 was shown to induce cell migration through interactions with integrin αv
). Recent studies identified a novel 20-residue sequence in the VWC domain of CCN1 (residues 116–135) as a functional binding site for integrin αv
). Our present observation that the N-terminal fragment of plasmin-cleaved CCN1 supports endothelial cell migration fits with the above finding.
Recent studies showed that CCN1 is highly expressed in breast cancers and elevated levels of this protein in primary breast cancer is associated with more advanced disease (23
). Tsai et al. (22
) examined the expression of CCN1 in many human breast cancer cell lines and found that the expression of CCN1 is strongly correlated with the ability of breast cancer cells to invade in vitro
and metastasize in vivo
. Furthermore, overexpression of CCN1 in MCF-12A normal breast cells was shown to induce tumor formation and vascularization in nude mice (46
). Similarly, expression of CCN1 cDNA under the regulation of constitutive promoter in RF-1 gastric adenocarcinoma cells significantly enhanced the tumor growth and vascularization (21
). Since CCN1 protein has been shown to influence many cellular activities, it is possible that CCN1 could contribute to tumor growth by multiple mechanisms (26
). However, the ability of CCN1 to promote angiogenesis is thought to be responsible primarily for tumor growth and vascularization (21
). Angiogenesis requires the coordinated execution of a series of cellular processes, starting from the degradation of basement membranes and ECM surrounding the parent vessel to proliferation and migration of endothelial cells from the parent vessel toward an angiogenic signal to form new capillary sprouts. A number of studies showed that tumor cells, including breast cancer cells, express plasminogen activator uPA and tPA, and specific receptors for uPA and plasminogen (see rev (33
)). In agreement with this, primary breast cancers were shown to express active enzymes capable of catalyzing plasmin formation (47
). Hyperpermeability of local microvasculature associated with solid tumors (48
) would allow the circulating plasminogen to enter into tumor tissues, which could be readily converted to plasmin on the tumor cell surface. Plasmin, by virtue of its ability to degrade ECM proteins and activate MMPs, can facilitate many steps involved in tumor growth and invasion. The ability of plasmin to liberate the biologically active CCN1 fragment adds a novel step to this complex process.
Plasmin cleavage of CCN1 may have wider biological implications because it permits the CCN1 partition into the soluble phase, rather than into the insoluble matrix, and therefore allows the CCN1 to diffuse freely within the tissue and interact with its plasma membrane receptors on various cell types. The 28 kDa fragment could act as either agonist or antagonist for full length CCN1 in a cell and context-specific fashion since CCN1 interacts with specific integrins in specific cell types, either requiring the CT domain or independent of the CT domain (12
). Since a number of pathological conditions are associated with upregulation of CCN1 (45
) and many cell types express the plasminogen activator/plasmin system, which could be upregulated further by pathological conditions (33
), it is likely that the truncated CCN1 fragments would be generated in vivo
. If so, the 28 kDa fragment of CCN1 may serve as a marker for pathogenesis of disease, particularly cancer and cardiovascular diseases. Although shorter isoforms for other proteins in the CCN family have been detected in biological fluids (26
), we are not aware of any published studies that examined the presence of truncated form of CCN1 in biological fluids either in normal or diseased conditions. Thus it will be interesting to test in the future whether the truncated form of CCN1 can be found in plasma or biological fluids of cancer patients and patients with cardiovascular diseases, and whether its levels correlate with disease parameters. It is also of interest to see whether plasmin cleaves other members of the CCN family. In this context, it may be relevant to note that there is 60 to 70% protein sequence homology among various member of CCN family in the region where plasmin cleaves the CCN1 (between aa 243 to 267 in CCN1) and the putative plasmin cleavage site R250↓Val251 (of CCN1) is preserved in 5 of 6 members of the CCN family (26