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Metastasis is a sequential process that allows cells to move from the primary tumor and grow elsewhere. Because of their ability to cleave a variety of extracellular signaling and adhesion molecules, metalloproteases have been long considered key components of the metastatic program. However, the function of certain metalloproteases, such as ADAMTS1, is not clear and seems to depend on the cellular environment and/or the stage of tumor progression. To characterize the function of ADAMTS1, we performed two alternative proteomic approaches, difference gel electrophoresis and stable isotope labeling by amino acids in cell culture, to identify novel substrates of the metalloprotease. Both techniques showed that overexpression of ADAMTS1 leads to the release of semaphorin 3C from the extracellular matrix. Although semaphorins are well known regulators of axon guidance, accumulating evidence shows that they may also participate in tumor progression. Here, we show that the cleavage of semaphorin 3C induced by ADAMTS1 promotes the migration of breast cancer cells, indicating that the co-expression of these molecules in tumors may contribute to the metastatic program.

ADAMTS1 is an extracellular metalloproteinase known to participate in a variety of biological processes that includes inflammation, angiogenesis, and development of the urogenital system. Many of its functions rely on its catalytic activity, which thus far has been limited to the cleavage of the matrix proteoglycans aggrecan and versican. However, it is likely that other substrates exist. Using a yeast two-hybrid screen, we identified the Kunitz-type inhibitor, tissue factor pathway inhibitor-2 (TFPI-2), as a binding partner of ADAMTS1. The interaction was confirmed by several biochemical and cell-based assays. In addition, our studies revealed alterations in the pattern of TFPI-2-secreted isoforms and in its extracellular location caused by the specific action of ADAMTS1. Interestingly, we found that TFPI-2 is a novel substrate of ADAMTS1. The cleavage removes a protease-sensitive C-terminal region in TFPI-2, altering its binding properties. The proposed role of TFPI-2 as a maintenance factor of extracellular remodeling suggests the indirect function of ADAMTS1 as an additional homeostatic player by its ability to alter the extracellular location of TFPI-2 and, therefore, to disrupt the remodeling machinery, a phenomenon directly associated to pathologies such as atherosclerosis and tumor progression.

The use of steroid hormones in postmenopausal replacement therapy has been associated with prevention of cardiovascular disease. Although the contribution of estradiol to endothelial cell function has been addressed, little information is available on the effect of progestins on this cell type. Here, we provide direct evidence for the presence of functional nuclear progesterone receptor in endothelial cells and demonstrate that physiological levels of progesterone inhibit proliferation through a nuclear receptor-mediated mechanism. The effects of progesterone were blocked by pretreatment with a progesterone receptor antagonist, and progesterone receptor-deficient endothelial cells failed to respond to the hormone. We evaluated the effect of progesterone by analysis of aorta re-endothelialization experiments in wild-type and progesterone receptor knockout mice. The rate of re-endothelialization was significantly decreased in wild-type mice when in the presence of progesterone, whereas there was no difference between control and progesterone-treated progesterone receptor knockout mice. FACS analysis showed that progestins arrest endothelial cell cycle in G1. The lag in cell cycle progression involved reduction in cyclin-dependent kinase activity, as shown by down-regulation in retinoblastoma protein phosphorylation. In addition, treatment of endothelial cells with progestins altered the expression of cyclin E and A in accordance with G1 arrest. These results have important implications to our current knowledge of the effect of steroids on endothelial cell function and to the overall contribution of progesterone to vascular repair.

GLUT1 and GLUT4 glucose transporter expression is highly regulated in muscle and adipose tissue during perinatal life. Here we have investigated the role of thyroid hormones in the regulation of GLUT4 induction and GLUT1 repression associated to neonatal development. Perinatal hypothyroidism markedly impaired GLUT4 protein induction in heart. This effect was heart specific, and a greater expression of GLUT4 was detected in brown adipose tissue from neonatal hypothyroid rats compared with controls. These changes in GLUT4 protein expression were not detected in brown adipose tissue or heart when hypothyroidism was induced in adult rats. These results indicate that GLUT4 induction during perinatal life is highly sensitive to thyroid hormones in both heart and adipose tissue. Perinatal hypothyroidism was characterized by decreased cardiac GLUT4 mRNA concentrations. T3 injection caused a marked increase in cardiac levels of GLUT4 mRNA in hypothyroid neonates. Thus, in 13-day-old hypothyroid rats, GLUT4 mRNA levels increased 3-fold 1 h after T3 injection. Under these conditions, retinoic acid also caused a rapid increase in cardiac GLUT4 mRNA levels from hypothyroid neonates. In addition, cardiac levels of GLUT4 protein markedly increased in fetuses and in neonates 24 h after T3 injection. These findings suggest that a direct effect of thyroid hormones is the promotion of cardiac GLUT4 gene expression. GLUT1 protein expression was markedly enhanced in brown adipose tissue and heart during neonatal hypothyroidism as well as in hypothyroidism induced in adult rats. This was concomitant to greater levels of GLUT1 mRNA in hearts from hypothyroid neonates. Immunofluorescence analysis indicated that cardiomyocytes from hypothyroid pups contained an enhanced level of GLUT1 protein. Furthermore, T3 injection caused a decrease in cardiac levels of GLUT1 mRNA in hypothyroid neonates. These results indicate that thyroid hormone manipulation leads to inverse regulation of GLUT1 and GLUT4 glucose transporter gene expression in the neonatal heart. We conclude that thyroid hormones play a pivotal role controlling the transition of glucose transporter carriers from fetal to neonatal levels in heart and brown adipose tissue.