Reconstitution of TIMP-2 expression in SH-SY5Y neuroblastoma cells by 5-azacytidine is mediated transcriptionally by NF-Y through an inverted CCAAT site.
Advanced stage neuroblastomas (NB) exhibit a tissue inhibitor of metalloproteinase (TIMP)-2/matrix metalloproteinase (MMP) imbalance, considered a prerequisite for MMP involvement in tumor progression in vivo. Human SH-SY5Y NB cells exhibit a similar TIMP-2/MMP imbalance that promotes in vitro invasive behavior that is inhibited by exogenous TIMP-2. The DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) redresses this TIMP-2/MMP imbalance, reconstituting TIMP-2 expression, without effecting that of MMP-2, by stimulating TIMP-2 transcription and inhibiting in vitro invasivity of SH-SY5Y cells. 5-AzaC stimulated transcription from a nonmethylated TIMP-2 promoter reporter gene construct consistent with regulation of a TIMP-2 transactivator. Promoter deletion and point-mutation analysis localized this effect to an inverted CCAAT element at position -73. This element bound specific complexes containing NF-YA and NF-YB proteins in SH-SY5Y nuclear extracts, the binding of which was augmented by 5-AzaC in association with enhanced levels of NF-YB protein and the function of which was confirmed by inhibition using dominant-negative NF-YA. The data highlight a novel indirect methylation-mediated mechanism for regulating the TIMP/MMP equilibrium in NB cells, involving repression of TIMP-2 relative to MMP-2 expression, dependent upon suboptimal NF-Y transcription factor function, which can be reversed by methyltransferase inhibition.
Section of Molecular Pathology, Department of Experimental Medicine, University of L'Aquila, 67100 L'Aquila, Italy.
Lucia
L
Cappabianca
Antonietta Rosella
AR
Farina
Antonella
A
Tacconelli
Roberto
R
Mantovani
Alberto
A
Gulino
Andrew Reay
AR
Mackay
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000494:pharmacology; D023081:CCAAT-Binding Factor / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002648:Child; D019175:DNA Methylation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics*; D005809:Genes, Regulator / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009154:Mutation / Q000187:drug effects / Q000235:genetics; D009447:Neuroblastoma / Q000201:enzymology* / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D012097:Repressor Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D023081:CCAAT-Binding Factor; D012097:Repressor Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D008780:Methyltransferases; D001374:Azacitidine