Downregulation of a tumor suppressor RECK by hypoxia through recruitment of HDAC1 and HIF-1alpha to reverse HRE site in the promoter.
Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a tumor suppressor and the suppression of RECK is induced by Ras or Her-2/neu oncogenes. However, regulation of RECK under hypoxic microenvironment is largely unknown. Here, we identified that hypoxia significantly downregulates RECK mRNA and protein expression using semiquantitative RT-PCR, real-time RT-PCR and western blot analysis. This repression was reversed by the HDAC inhibitor, trichostatin A (TSA) and HIF-1 inhibitor, YC-1. Hypoxia-induced downregulation of RECK was abolished by knockdown of HDAC1 and HIF-1alpha with respective small interfering RNAs (siRNAs), whereas overexpression of HDAC1 and HIF-1alpha suppressed RECK expression similar to the level under hypoxic conditions. Transfection of a deletion mutant of the second reverse HRE (rHRE2, -2345 to -2333) site of RECK promoter completely removed RECK suppression under hypoxia, indicating that the rHRE2 site is responsible for the inhibition of RECK. Chromatin immunoprecipitation and DNA affinity precipitation assays demonstrated that HDAC1 and HIF-1alpha were recruited to the rHRE2 region of RECK promoter under hypoxic conditions, but the treatment of TSA or YC-1 inhibited their binding to the rHRE2 site. Moreover, TSA and YC-1 inhibited hypoxia-induced cancer cell migration, invasion and MMPs secretion. Taken together, we can conclude that hypoxia induces RECK downregulation through the recruitment of HDAC1 and HIF-1alpha to the rHRE2 site in the promoter and the inhibition of hypoxic RECK silencing would be a therapeutic and preventive target for early tumorigenesis.
Lee
Kyung Ju
KJ
School of Life Sciences and Biotechnology, Kyungpook National University, Daegu, 702-701, Republic of Korea.
Lee
Kwang Youl
KY
Lee
You Mie
YM
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D058851:GPI-Linked Proteins; D005786:Gene Expression Regulation; D016147:Genes, Tumor Suppressor; D056284:Histone Deacetylase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D058851:GPI-Linked Proteins; C497442:HIF1A protein, human; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008562:Membrane Glycoproteins; C115460:RECK protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C012589:trichostatin A; D008156:Luciferases; C110550:HDAC1 protein, human; D056284:Histone Deacetylase 1