Regulation of Integrin Activity by MIA

Abstract

MIA (melanoma inhibitory activity) has been identified as a small protein secreted from malignant melanoma cells, which interacts with extracellular matrix proteins including fibronectin. Here, we show that MIA negatively regulates the activity of the mitogen-activated protein kinase pathway in malignant melanoma. Using far Western blotting and co-immunoprecipitation we searched for MIA-binding cell surface proteins. We found that MIA interacts with integrin ␣4␤1 and ␣5␤1, leading to down-regulation of integrin activity and reduction of mitogen-activated protein kinase signaling. These findings also suggest that MIA may play a role in tumor progression and the spread of malignant melanomas via mediating detachment of cells from extracellular matrix molecules by modulating integrin activity. Inhibiting MIA functions in vivo may therefore provide a novel therapeutic strategy for metastatic melanoma disease.We have previously identified MIA (melanoma inhibitory activity), an 11-kDa protein secreted into the tissue culture supernatant from malignant melanoma cells (1). MIA expression in vivo correlates with progressive malignancy of melanocytic tumors (2). Additionally, in recent studies we detected enhanced MIA protein levels specifically in the serum of patients with metastatic melanomas (3). In vitro studies revealed a role for MIA in supporting the invasive and migratory potential of melanoma cells. In vivo studies in two animal model systems confirmed the importance of MIA in melanoma metastasis. MIA expression levels parallel closely the capability of melanoma cells to form metastases in syngeneic animals (4, 5).Three-dimensional analyses of MIA by multidimensional NMR (6 -8) or x-ray crystallography (9) indicate that MIA defines a novel family of secreted proteins that adopt an SH3 domain-like fold in solution. Furthermore, NMR spectra revealed that MIA interacts with peptides matching to type III human fibronectin repeats that are closely related to ␣4␤1 integrin-binding sites (6). These data support a model in which MIA regulates attachment to specific components of the extracellular matrix. Based on these results and on the observation that MIA alters melanoma cell morphology, we determined that MIA treatment results in cell detachment by decreasing interactions between melanoma cells and extracellular matrix molecules (10). The study presented here was performed to find additional MIA-interacting proteins and to identify signaling pathways regulated by MIA. EXPERIMENTAL PROCEDURESCell Lines and Culture Conditions-The melanoma cell lines Mel Im and Mel Ei, have been described in detail previously (11). The cell line Mel Ei was derived from a primary cutaneous melanoma, and the cell line Mel Im was derived from a metastasis of malignant melanoma. To establish fibronectin-deficient fibroblast-like cells, primary embryonic fibroblasts were isolated from E13.5 fibronectin (flox/flox) embryos and immortalized by retroviral transduction of the SV-40 large T antigen and cloned (12). Subsequently, two clonal lines...

Migration-associated secretion of melanoma inhibitory activity at the cell rear is supported by KCa3.1 potassium channels

“…In line with this assumption, external addition of MIA protein does not support migration but results in unpolarized cell detachment and reduced invasiveness in Boyden chamber assays [2]. To account for the pro-migratory effect of endogenously expressed and secreted MIA protein, we proposed that MIA secretion is directed to the rear end of a migrating cell where it can bind to integrins and thereby promote cell retraction [7]. MIA-integrin complexes are subsequently internalized at the rear of the cell [8].…”

“…MIA protein was described to directly interact with cell adhesion receptors, integrin α 4 β 1 and integrin α 5 β 1 [7], as well as with extracellular matrix molecules including fibronectin, tenascin and laminin [2]. As a result, matrix structures surrounding the cell and integrin adhesion molecules are masked by MIA protein, which consequently impacts melanoma cell attachment.…”

“…Further, we show that secretion of MIA protein is significantly augmented by KCa3.1 potassium channel activity. In summary, we provide experimental evidence for our model, first hypothesized in 2006 [7], of facilitated retraction at the rear of migrating cells controlled in part by directed secretion of MIA protein.…”

“…MIA protein strongly contributes to the invasive and migratory potential of melanoma cells and promotes the formation of tumor metastases [2]; however, the mechanistic link between MIA protein and cell motility remains unclear. Recent data describe a direct interaction of MIA protein with cell adhesion receptors, integrin α 4 β 1 and integrin α 5 β 1 , followed by facilitation of cell detachment [7]. Cell migration requires adhesion to the extracellular matrix to provide traction points and to stabilize protrusions at the cell front.…”

Processing of MIA protein during melanoma cell migration

“…In previous studies using far Western blotting and co-immunoprecipitation MIA protein was identified to bind to the cell surface proteins integrin a 4 b 1 and integrin a 5 b 1 . 12 Thus, MIA protein modulates integrin activity and thereby mediates detachment of cells from extracellular matrix proteins, resulting in enhanced invasive and migratory potential of melanoma cells.…”