Suman Govindaraju1, Beth S Lee. 1. Department of Physiology and Cell Biology, The Ohio State University College of Medicine and Ohio State Biochemistry Program, Columbus, Ohio, USA.
Abstract
BACKGROUND/AIMS: HuR is an RNA-binding protein that regulates the post-transcriptional life of thousands of cellular mRNAs and promotes cell survival. HuR is expressed as two mRNA transcripts that are differentially regulated by cell stress. The goal of this study is to define factors that promote transcription of the longer alternate form. METHODS: Effects of transcription factors on HuR expression were determined by inhibition or overexpression of these factors followed by competitive RT-PCR, gel mobility shift, and chromatin immunoprecipitation. Transcription factor expression patterns were identified through competitive RT-PCR and Western analysis. Stress responses were assayed in thapsigargin-treated proximal tubule cells and in ischemic rat kidney. RESULTS: A previously described NF-κB site and a newly identified Sp/KLF factor binding site were shown to be important for transcription of the long HuR mRNA. KLF8, but not Sp1, was shown to bind this site and increase HuR mRNA levels. Cellular stress in cultured or native proximal tubule cells resulted in a rapid decrease of KLF8 levels that paralleled those of the long HuR mRNA variant. CONCLUSIONS: These results demonstrate that KLF8 can participate in regulating expression of alternate forms of HuR mRNA along with NF-κB and other factors, depending on cellular contexts.
BACKGROUND/AIMS: HuR is an RNA-binding protein that regulates the post-transcriptional life of thousands of cellular mRNAs and promotes cell survival. HuR is expressed as two mRNA transcripts that are differentially regulated by cell stress. The goal of this study is to define factors that promote transcription of the longer alternate form. METHODS: Effects of transcription factors on HuR expression were determined by inhibition or overexpression of these factors followed by competitive RT-PCR, gel mobility shift, and chromatin immunoprecipitation. Transcription factor expression patterns were identified through competitive RT-PCR and Western analysis. Stress responses were assayed in thapsigargin-treated proximal tubule cells and in ischemicrat kidney. RESULTS: A previously described NF-κB site and a newly identified Sp/KLF factor binding site were shown to be important for transcription of the long HuR mRNA. KLF8, but not Sp1, was shown to bind this site and increase HuR mRNA levels. Cellular stress in cultured or native proximal tubule cells resulted in a rapid decrease of KLF8 levels that paralleled those of the long HuR mRNA variant. CONCLUSIONS: These results demonstrate that KLF8 can participate in regulating expression of alternate forms of HuR mRNA along with NF-κB and other factors, depending on cellular contexts.
Authors: Isabel López de Silanes; Jinshui Fan; Xiaoling Yang; Alan B Zonderman; Olga Potapova; Ellen S Pizer; Myriam Gorospe Journal: Oncogene Date: 2003-10-16 Impact factor: 9.867