ABSTRACT
Increased expression of metalloprotease-disintegrin ADAM12 is a hallmark of several pathological conditions, including cancer, cardiovascular disease, and certain inflammatory diseases of the central nervous system or the muscoskeletal system. We show that transforming growth factor beta1 (TGFbeta1) is a potent inducer of ADAM12 mRNA and protein in mouse fibroblasts and in mouse and human mammary epithelial cells. Induction of ADAM12 is detected within 2 h of treatment with TGFbeta1, is Smad2/Smad3-dependent, and is a result of derepression of the Adam12 gene. SnoN, a negative regulator of the TGFbeta signaling pathway, is a master regulator of ADAM12 expression in response to TGFbeta1 stimulation. Overexpression of SnoN in NIH3T3 cells reduces the magnitude of ADAM12 induction by TGFbeta1 treatment. Down-regulation of SnoN expression by short hairpin RNA enhances TGFbeta1-induced expression of ADAM12. In a panel of TGFbeta1-responsive cancer cell lines with high expression of SnoN, induction of ADAM12 by TGFbeta1 is significantly impaired, suggesting that the endogenous SnoN plays a role in regulating ADAM12 expression in response to TGFbeta1. Identification of SnoN as a repressor of the ADAM12 gene should contribute to advances in the studies on the role of ADAM12 in tumor progression and in the development of other pathologies.
Subject(s)
ADAM Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/genetics , Proto-Oncogene Proteins/metabolism , Transforming Growth Factor beta1/pharmacology , ADAM Proteins/biosynthesis , ADAM12 Protein , Animals , Cell Line , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enzyme Induction/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Humans , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/biosynthesis , Mice , Proteasome Endopeptidase Complex/metabolism , Protein Processing, Post-Translational/drug effects , Proto-Oncogene Proteins/genetics , Smad2 Protein/deficiency , Smad2 Protein/metabolism , Smad3 Protein/deficiency , Smad3 Protein/metabolism , Transcription, Genetic/drug effectsABSTRACT
ADAM12 has recently emerged as a Candidate Cancer Gene in a comprehensive genetic analysis of human breast cancers. Three somatic mutations in ADAM12 were observed at significant frequencies in breast cancers: D301H, G479E and L792F. The first 2 of these mutations involve highly conserved residues in ADAM12, and our computational sequence analysis confirms that they may be cancer-related. We show that the corresponding mutations in mouse ADAM12 inhibit the proteolytic processing and activation of ADAM12 in NIH3T3, COS-7, CHO-K1 cells and in MCF-7 breast cancer cells. The D/H and G/E ADAM12 mutants exert a dominant-negative effect on the processing of the wild-type ADAM12. Immunofluorescence analysis and cell surface biotinylation experiments demonstrate that the D/H and G/E mutants are retained inside the cell and are not transported to the cell surface. Consequently, the D/H and G/E mutants, unlike the wild-type ADAM12, are not capable of shedding Delta-like l, a ligand for Notch receptor, at the cell surface, or of stimulating cell migration. Our results suggest that the breast cancer-associated mutations interfere with the intracellular trafficking of ADAM12 and result in loss of the functional ADAM12 at the cell surface.
Subject(s)
ADAM Proteins/genetics , ADAM Proteins/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutation , ADAM12 Protein , Animals , Aspartic Acid , Biotinylation , Blotting, Western , Cell Movement , Female , Fluorescent Antibody Technique , Glutamic Acid , Glycine , Histidine , Humans , Immunoprecipitation , Leucine , Mice , PhenylalanineABSTRACT
Thymocytes exposed to the pro-oxidant tert-butyl-hydroperoxide (ButOOH) display a number of dramatic changes in morphology similar to those observed in the case of dexamethasone-treated cells. Both reagents induce nuclear chromatin peripheral aggregation below the nuclear membrane. Some nuclei themselves break up producing two or more fragments. ButOOH-treated cells are morphologically characterised by cell shrinkage, extensive surface blebbing and, finally, fragmentation into membrane-bound apoptotic bodies composed of cytoplasm and tightly packed with or without nuclear fragments. An increased level of lipid hydroxyperoxides was detected after exposure of thymocytes to ButOOH. Both oxidative stress markers and morphological damage to cells were prevented by the antioxidant 4-OH-TEMPO.
