[Transcriptional activation of TMSG-1 by complex of KLF6 and Sp1].

Objective To investigate the regulatory mechanism of the transcription of tumor metastasis suppressor gene TMSG-1. Methods 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(ColP) was performed to analyze the interaction between KLF6 and Spl. 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. Results 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/Spl 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 Spl interacted with this region. CoIP also indicated a possible interaction between KLF6 and Spl 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. Conclusions Transcription factor complex of KLF6 and Spl 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. Key words: Neoplasm metastasis;  Transcription, genetic;  Genes, tumor suppressor;  Cell line, tumor