Abstract
The importance of S100A4, a Ca2+-binding protein, in mediating tumour cell migration, both intracellularly and extracellularly, is well documented. Tissue transglutaminase (TG2) a Ca2+-dependent protein crosslinking enzyme, has also been shown to enhance cell migration. Here by using the well characterised non-metastatic rat mammary R37 cells (transfected with empty vector) and highly metastatic KP1 cells (R37 cells transfected with S100A4), we demonstrate that inhibition of TG2 either by TG2 inhibitors or transfection of cells with TG2 shRNA block S100A4-accelerated cell migration in the KP1cells and in R37 cells treated with exogenous S100A4. Cell migration was also blocked by the treatment with the non-cell permeabilizing TG2 inhibitor R294, in the human breast cancer cell line MDA-MB-231 (Clone 16, which has a high level of TG2 expression). Inhibition was paralleled by a decrease in S100A4 polymer formation. In vitro co-immunoprecipitation and Far Western blotting assays and cross-linking assays showed not only the direct interaction between TG2 and S100A4, but also confirmed S100A4 as a substrate for TG2. Using specific functional blocking antibodies, a targeting peptide and a recombinant protein as a competitive treatment, we revealed the involvement of syndecan-4 and α5β1 integrin co-signalling pathways linked by activation of PKCα in this TG2 and S100A4-mediated cell migration. We propose a mechanism for TG2-regulated S100A4-related mediated cell migration, which is dependent on TG2 crosslinking.
Highlights
The onset of tumour metastasis is a complicated process involving complex intracellular cell signalling network(s) elicited via cell contact with the extracellular matrix (ECM), and by crosstalk between tumour cells, stromal cells and immune cells
Given the cell surface co-localization of TG2 activity and S100A4, the ability of the non-cell permeable TG2 inhibitors R281 and R294 to reduce this and in parallel inhibit KP1 cell migration, our results suggest that the major action of TG2 on S100A4 is likely to be through an extracellular mediated signalling pathway involving the cell surface receptor syndecan-4
S100A4, acting as either an intracellular or extracellular protein, can promote cell migration using a number of different pathways, such as the activation of MMPs [30] through integrin receptors [6] or through the RAGE signalling pathway [5], but the exact mechanism is still not fully understood
Summary
The onset of tumour metastasis is a complicated process involving complex intracellular cell signalling network(s) elicited via cell contact with the extracellular matrix (ECM), and by crosstalk between tumour cells, stromal cells and immune cells. Intracellular S100A4 can bind directly to the myosin light chain to mediate cytoskeletal organization and in turn promote cell migration [3]. Via its direct interaction with NF-kB, S100A4 is reputed to be involved in cancer cell proliferation and differentiation [4]. S100A4 is found in the extracellular environment, where it can be externalised from cancer cells and surrounding stromal and immune cells via an unknown non-coventional secretion pathway. Extracellular S100A4, like the intracellular protein, can promote cell migration, but its mode of action is still not fully undertsood. It has been suggested that RAGE [5] or a6b4 integrin [6] could be the cell surface receptors involved in transducing the S100A4-mediated signalling, while other research suggests the involvement of cell surface heparan sulphates in the signal transduction process [7]
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