Reactive-site mutants of N-TIMP-3 that selectively inhibit ADAMTS-4 and ADAMTS-5: biological and structural implications.

Published versio

Aberrant DNA hypermethylation of the ITIH5 tumor suppressor gene in acute myeloid leukemia

Epigenetic mechanisms such as DNA hypermethylation and modifications of histone amino acids are known to play an important role in the control of gene expression both in normal human development and tumorigenesis. Hypermethylation of CpG islands within promoter regions of tumor suppressor genes is associated with transcriptional inactivation and represents, in addition to genetic aberrations, an important mechanism of gene silencing in the pathogenesis of human cancer. Inter-α-trypsine inhibitors (ITIs) are a family of serine protease inhibitors consisting of one light chain (bikunin) and two heavy chains (ITI heavy chains, ITIHs). ITIHs stabilize the extracellular matrix (ECM) by interacting with hyaluronic acid, which is a major ECM component. Hypermethylation in the upstream region of the promoter-associated CpG island of ITIH5, the most recently described member of the ITIH family, has been previously detected in breast cancer and was associated with an adverse outcome. In this study, we determined the DNA methylation status of the promoter region near the transcription start site of the ITIH5 tumor suppressor gene in leukemia cell lines and primary samples from patients with acute myeloid leukemia (AML) as well as the potential use of demethylating agents to restore a demethylated state of the promoter. Aberrant ITIH5 promoter hypermethylation occurred in 15 of 104 (14.4%) diagnostic AML samples. There were no statistically significant correlations between the ITIH5 methylation status and clinical prognostic parameters. Our results indicate that aberrant ITIH5 promoter hypermethylation is a novel epigenetic event in AML

ADAM10 controls collagen signaling and cell migration on collagen by shedding the ectodomain of discoidin domain receptor 1 (DDR1)

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and transmits signals from various collagens in epithelial cells. However, how DDR1-dependent signaling is regulated has not been understood. Here we report that collagen binding induces ADAM10-dependent ectodomain shedding of DDR1. DDR1 shedding is not a result of an activation of its signaling pathway since DDR1 mutants defective in signaling were shed in an efficient manner. DDR1 and ADAM10 were found to be in a complex on the cell surface, but shedding did not occur unless collagen bound to DDR1. Using a shedding resistant DDR1 mutant, we found that ADAM10-dependent DDR1 shedding regulates the half-life of collagen-induced phosphorylation of the receptor. Our data also revealed that ADAM10 plays an important role in regulating DDR1 mediated cell adhesion to achieve efficient cell migration on collagen matrices

ADAM10 controls collagen signaling and cell migration on collagen by shedding the ectodomain of discoidin domain receptor 1 (DDR1)

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and transmits signals from various collagens in epithelial cells. However, how DDR1-dependent signaling is regulated has not been understood. Here we report that collagen binding induces ADAM10-dependent ectodomain shedding of DDR1. DDR1 shedding is not a result of an activation of its signaling pathway since DDR1 mutants defective in signaling were shed in an efficient manner. DDR1 and ADAM10 were found to be in a complex on the cell surface, but shedding did not occur unless collagen bound to DDR1. Using a shedding resistant DDR1 mutant, we found that ADAM10-dependent DDR1 shedding regulates the half-life of collagen-induced phosphorylation of the receptor. Our data also revealed that ADAM10 plays an important role in regulating DDR1 mediated cell adhesion to achieve efficient cell migration on collagen matrices

Regulation of insulin-like growth factor (IGF)-I action by matrix metalloproteinase-3 involves selective disruption of IGF-I/IGF-binding protein-3 complexes.

IGF-I and IGF-II play important roles in growth and development via interactions with cell-surface receptors; however, in nature, IGFs are sequestered by at least six soluble, high-affinity IGF-binding proteins (IGFBPs), namely IGFBPs 1-6. Herein, we demonstrate that the stromal cell-derived extracellular matrix-degrading metalloproteinase stromelysin 1 (matrix metalloproteinase 3) disrupts IGF/IGFBP-3 complexes and liberates free, intact IGFs, leading to phosphorylation of cell surface type 1 IGF receptors and cellular proliferation. Tissue inhibitor of metalloproteinases (TIMP-1) or an antibody to the type 1 IGF receptor mitigates IGF-mediated cellular proliferation. Thus, these studies suggest that matrix metalloproteinases, beyond their effects on extracellular matrix turnover, regulate cellular proliferation by modulating the bioavailability of IGFs, an event critical for such diverse phenomena as embryo development, morphogenesis, angiogenesis, and tumorigenesis

Catalytic properties of ADAM12 and its domain deletion mutants.

Human ADAM12 (a disintegrin and metalloproteinase) is a multidomain zinc metalloproteinase expressed at high levels during development and in human tumors. ADAM12 exists as two splice variants: a classical type 1 membrane-anchored form (ADAM12-L) and a secreted splice variant (ADAM12-S) consisting of pro, catalytic, disintegrin, cysteine-rich, and EGF domains. Here we present a novel activity of recombinant ADAM12-S and its domain deletion mutants on S-carboxymethylated transferrin (Cm-Tf). Cleavage of Cm-Tf occurred at multiple sites, and N-terminal sequencing showed that the enzyme exhibits restricted specificity but a consensus sequence could not be defined as its subsite requirements are promiscuous. Kinetic analysis revealed that the noncatalytic C-terminal domains are important regulators of Cm-Tf activity and that ADAM12-PC consisting of the pro domain and catalytic domain is the most active on this substrate. It was also observed that NaCl inhibits ADAM12. Among the tissue inhibitors of metalloproteinases (TIMP) examined, the N-terminal domain of TIMP-3 (N-TIMP-3) inhibits ADAM12-S and ADAM12-PC with low nanomolar Ki(app) values while TIMP-2 inhibits them with a slightly lower affinity (9-44 nM). However, TIMP-1 is a much weaker inhibitor. N-TIMP-3 variants that lack MMP inhibitory activity but retained the ability to inhibit ADAM17/TACE failed to inhibit ADAM12. These results indicate unique enzymatic properties of ADAM12 among the members of the ADAM family of metalloproteinases

Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing.

ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs) is a multidomain metalloproteinase belonging to the reprolysin family. The enzyme cleaves aggrecan core protein at several sites. Here we report that the non-catalytic ancillary domains of the enzyme play a major role in regulating aggrecanase activity, with the C-terminal spacer domain masking the general proteolytic activity. Expressing a series of domain deletion mutants in mammalian cells and examining their aggrecan-degrading and general proteolytic activities, we found that full-length ADAMTS-4 of 70 kDa was the most effective aggrecanase, but it exhibited little activity against the Glu(373)-Ala(374) bond, the site originally characterized as a signature of aggrecanase activity. Little activity was detected against reduced and carboxymethylated transferrin (Cm-Tf), a general proteinase substrate. However, it readily cleaved the Glu(1480)-Gly(1481) bond in the chondroitin sulfate-rich region of aggrecan. Of the constructed mutants, the C-terminal spacer domain deletion mutant more effectively hydrolyzed both the Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds. It also revealed new activities against Cm-Tf, fibromodulin, and decorin. Further deletion of the cysteine-rich domain reduced the aggrecanase activity by 80% but did not alter the activity against Cm-Tf or fibromodulin. Further removal of the thrombospondin type I domain drastically reduced all tested proteolytic activities, and very limited enzymatic activity was detected with the catalytic domain. Full-length ADAMTS-4 binds to pericellular and extracellular matrix, but deletion of the spacer domain releases the enzyme. ADAMTS-4 lacking the spacer domain has promiscuous substrate specificity considerably different from that previously reported for aggrecan core protein. Finding of ADAMTS-4 in the interleukin-1alpha-treated porcine articular cartilage primarily as a 46-kDa form suggests that it exhibits a broader substrate spectrum in the tissue than originally considered