ABSTRACT
<p><b>OBJECTIVE</b>To investigate the regulatory mechanism of the transcription of tumor metastasis suppressor gene TMSG-1.</p><p><b>METHODS</b>Luciferase reporter assay and site-directed mutagenesis were used to analyze the regulatory region of TMSG-1. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were carried out to verify the interaction of KLF6 and Sp1 with the regulatory region of TMSG-1. Co-immunoprecipitation (CoIP) was performed to analyze the interaction between KLF6 and Sp1. TMSG-1 and wt-KLF6 mRNA expressions in cells with different metastatic capacities were quantitated by real-time PCR. Cell invasive capability was determined by Matrigel invasion assay.</p><p><b>RESULTS</b>A 63 bp inducible regulatory region (+59 bp - +123 bp) in exon 1 was identified by luciferase assay using reporter plasmids with a series of TMSG-1 regulatory region deletions. Mutations in KLF6/Sp1 binding sites of this region resulted in a decrease of luciferase activity, while cotransfection with KLF6 or Sp1 expressing plasmids led to a remarkable increase of luciferase activity. EMSA and ChIP demonstrated that KLF6 as well as Sp1 interacted with this region. CoIP also indicated a possible interaction between KLF6 and Sp1 proteins. In the highly metastatic cell sublines, a low level of wild type KLF6 was associated synchronously with a low TMSG-1 level. Prostate carcinoma cells overexpressing KLF6 exhibited a higher TMSG-1 level and a lower invasive capability.</p><p><b>CONCLUSIONS</b>Transcription factor complex of KLF6 and Sp1 may participate in the inducible transcriptional regulation of TMSG-1, and a decreased wild type KLF6 expression is likely associated with a low TMSG-1 level in the highly metastatic cell sublines.</p>
Subject(s)
Humans , Male , Binding Sites , Genetics , Cell Line, Tumor , Electrophoretic Mobility Shift Assay , Immunoprecipitation , Kruppel-Like Factor 6 , Kruppel-Like Transcription Factors , Genetics , Metabolism , Lung Neoplasms , Metabolism , Pathology , Membrane Proteins , Genetics , Metabolism , Mutagenesis, Site-Directed , Mutation , Neoplasm Invasiveness , Prostatic Neoplasms , Metabolism , Pathology , Proto-Oncogene Proteins , Genetics , Metabolism , RNA, Messenger , Metabolism , Sp1 Transcription Factor , Genetics , Metabolism , Sphingosine N-Acyltransferase , Genetics , Metabolism , Transcriptional Activation , Transfection , Tumor Suppressor Proteins , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To identify the putative specific localization signal sequence of tumor metastasis suppressor gene-1 (TMSG-1) and to explore the mechanism of subcellular localization of TMSG-1 protein.</p><p><b>METHODS</b>Vectors expressing green fluorescence protein (GFP) tagged different TMSG-1 fragments were generated and transfected into human embryo kidney 293 (HEK293) cells. The expression of those fusion proteins was detected by Western blotting and their subcellular localizations were observed by laser confocal microscope.</p><p><b>RESULTS</b>GFP was fused with the native TMSG-1(aa1-380) or different fragments including T1 (aa1-70), T2 (aa1-128), T3 (aa129-380), T4 (aa71-128), T5 (aa71-179) and T6 (aa71-380). Anti-GFP Western blotting showed that these fusion proteins were successfully expressed. Under laser confocal microscope, GFP fused with fragment T4 (aa71-128) localized mainly in the nucleolus; GFP fused with fragment T6 (aa71-380) localized diffusely in the nucleus; while other fusion proteins with TMSG-1 (aa1-380) or fragment T1 (aa1-70), T2 (aa1-128), T3 (aa129-380) and T5 (aa71-179) localized in the cytoplasm. Fragment T4(Δ119-128) was generated from T4 with deletion of 10 amino acid of the C terminal. GFP fused with fragment T4(Δ119-128) remained in the nucleus, but no longer in the nucleolus.</p><p><b>CONCLUSIONS</b>There is a nucleolar localization signal (aa119-128 RRRRNQDRPS) within TMSG-1. This finding may have laid the foundation for further investigations into subcellular localization and function of TMSG-1.</p>