TIMP-3 INDUCES CELL DEATH BY STABILIZING TNF-α RECEPTORS ON THE SURFACE OF HUMAN COLON CARCINOMA CELLS
摘要
Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) regulate the structural integrity of the extracellular matrix (ECM). Constitutive expression of human TIMP-3 in human DLD colon carcinoma cells renewed serum-responses and inhibited tumour formation in nude mice. To elucidate the mechanism of TIMP-3-mediated tumour suppression, we compared parental DLD and TIMP-3 expressing DLD cells (TIMP-3/DLD), finding them to be significantly different. TIMP-3/DLD cultures have fewer mitotic cells, are delayed in G1, and die after serum starvation. TIMP-3/DLD conditioned media activates cell death on fibroblast cells. The cell death induced by serum starvation and conditioned media was inhibited by 70%, in the presence of neutralizing tumour necrosis factor α (TNF-α) antibody. TIMP-3/DLD whole cell lysate contained p55 TNF-α receptor, while vector/DLD lysate had p55 TNF-α receptor and p46 soluble TNF-α inhibitor. Vector/DLD conditioned media had p46, while no soluble TNF-α receptor was detected in TIMP-3/DLD conditioned media. In addition, FACS analysis revealed that TIMP-3/DLD cells have more TNF-α surface binding sites, suggesting a direct correlation between TIMP-3 expression and surface receptors. The mechanism of tumorigenic reversion induced by TIMP-3 in DLD cells may involve protection of receptors from the proteolytic activity of MMPs. Putative TIMP-3-mediated inhibition of MMPs restores the TNF-α p55 signalling pathway and the carcinoma cell is killed by autocrine TNF-α. Thus, DLD cells have specific ECM MMPs that cleave cytokines and cytokine receptors. TIMP-3 specifically inhibits MMPs involved in receptor shedding.