ABSTRACT
Meta-analyses of microarray data indicate that GATA3 is co-expressed with estrogen receptor alpha (ER) in breast cancer cells. While the significance of this remains unclear, it is thought that GATA3 may serve as a prognostic indicator in breast tumors and may play a role in ER signaling. Recently, reciprocal regulation of GATA3 and ER transcription was demonstrated, suggesting that control of their expression is intertwined. We sought to determine whether GATA3 and ER expression was also coordinately regulated at other levels. Unlike ER, GATA3 was not under epigenetic control and was not re-expressed in the presence of DNMT or HDAC inhibitors in ER/GATA3-negative cells. However, like ER, these inhibitors decreased GATA3 expression in ER/GATA3-positive cell lines. We have previously reported that ER mRNA stability is increased through binding of the RNA-binding protein HuR/ELAV1 to the 3'untranslated region (UTR) and that DNMT and HDAC inhibitors reduce ER expression by altering this interaction. Biotin pull-down assays using a biotinylated GATA3 RNA probe confirmed that HuR also binds to the GATA3 3'UTR. Inhibition of HuR using siRNA probes decreased GATA3 mRNA, mRNA stability and protein expression, indicating that HuR plays a role in regulating GATA3 expression. Inhibition of either HuR or GATA3 reduced cell growth of MCF7 cells. Based on our findings, it is clear that coordinate regulation of ER and GATA3 occurs, however differences do exist. These findings may aid in identification of new targets that control cell growth of breast cancer cells.
Subject(s)
Antigens, Surface/physiology , Breast Neoplasms/genetics , GATA3 Transcription Factor/genetics , Gene Expression Regulation, Neoplastic , RNA Stability/physiology , RNA, Messenger/metabolism , RNA, Neoplasm/metabolism , RNA-Binding Proteins/physiology , 3' Untranslated Regions , Base Sequence , Binding Sites , Biotinylation , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor/drug effects , Cell Line, Tumor/metabolism , Consensus Sequence , ELAV Proteins , ELAV-Like Protein 1 , Epigenesis, Genetic , Estrogen Receptor alpha/biosynthesis , Estrogen Receptor alpha/genetics , Female , GATA3 Transcription Factor/biosynthesis , GATA3 Transcription Factor/physiology , Histone Deacetylase Inhibitors/pharmacology , Humans , Molecular Sequence Data , Protein Binding , RNA, Small Interfering/pharmacologyABSTRACT
Restoration of estrogen receptor (ER) expression using epigenetic inhibitors re-establishes expression of the estrogen receptor (ER) and restores tamoxifen sensitivity in ER negative breast cancer cells. We tested if order of administration of the DNMT (5-aza 2' deoxycytidine/AZA) or HDAC (trichostatin A/TSA) inhibitors and tamoxifen affected ER re-expression and tamoxifen sensitivity. Treatment with AZA followed by co-administration of TSA plus tamoxifen resulted in the greatest ER re-expression and tamoxifen sensitivity, although sensitivity was not increased as robustly as expected. This could be due to increased cytoplasmic levels of HuR, suggesting that cytoplasmic HuR levels are central to tamoxifen responsiveness.
Subject(s)
Azacitidine/analogs & derivatives , Estrogen Receptor Modulators/administration & dosage , Hydroxamic Acids/administration & dosage , RNA, Messenger/genetics , Receptors, Estrogen/genetics , Tamoxifen/administration & dosage , Azacitidine/administration & dosage , Azacitidine/pharmacology , Cell Line, Tumor , Decitabine , Drug Administration Schedule , Estrogen Receptor Modulators/pharmacology , Humans , Hydroxamic Acids/pharmacology , Tamoxifen/pharmacologyABSTRACT
Zebrafish are an attractive model for studying the earliest cellular defects occurring during renal cyst formation because its kidney (the pronephros) is simple and genes that cause cystic kidney diseases (CKD) in humans, cause pronephric dilations in zebrafish. By comparing phenotypes in three different mutants, locke, swt and kurly, we find that dilations occur prior to 48 hpf in the medial tubules, a location similar to where cysts form in some mammalian diseases. We demonstrate that the first observable phenotypes associated with dilation include cilia motility and luminal remodeling defects. Importantly, we show that some phenotypes common to human CKD, such as an increased number of cells, are secondary consequences of dilation. Despite having differences in cilia motility, locke, swt and kurly share similar cystic phenotypes, suggesting that they function in a common pathway. To begin to understand the molecular mechanisms involved in cyst formation, we have cloned the swt mutation and find that it encodes a novel leucine rich repeat containing protein (LRRC50), which is thought to function in correct dynein assembly in cilia. Finally, we show that knock-down of polycystic kidney disease 2 (pkd2) specifically causes glomerular cysts and does not affect cilia motility, suggesting multiple mechanisms exist for cyst formation.
Subject(s)
Cilia/physiology , Mutation , Zebrafish Proteins/genetics , Zebrafish/physiology , Animals , Cloning, Molecular , Embryo, Nonmammalian/physiology , Kidney Glomerulus/physiology , Kidney Tubules/physiology , Microscopy, Video , Mutagenesis , Nephrons/embryology , Nephrons/physiology , Nephrons/physiopathology , Phenotype , Zebrafish/geneticsABSTRACT
The embryonic kidney of the zebrafish is the pronephros. The ease of genetic analysis and experimentation in zebrafish, coupled with the simplicity of the pronephros, make the zebrafish an ideal model system for studying kidney development and function. Several mutations have been isolated in zebrafish genetic screens that result in cyst formation in the pronephros. Cloning and characterization of these mutations will provide insight into kidney development but may also provide understanding of the molecular basis of cystic kidney diseases. In this review, we focus on the zebrafish as a model for understanding cystic kidney disease and the links between cystic kidney disease and left-right patterning.
