ABSTRACT
Heat shock protein 70 (HSP70) is induced by various stresses. Since HSP70 has a protein refolding activity and an anti-inflammatory activity, the HSP70 induction will help cells from harmful acute stresses. Feeding a diet containing concentrated brewed rice vinegar Kurozu (CK) diet for 5 wk resulted in an increase of HSP70 in the brains of mice. In the present study, we evaluated whether oral feeding of 25 µL CK induces HSP70 mRNA in brain and other tissues. HSP70 mRNA was significantly increased in the esophagus, small intestine, liver, and brown adipose tissue within 1 h after the oral administration of CK. A weaker induction of HSP70 mRNA was demonstrated in the stomach, large intestine, and brain. HSP70 mRNA induction returned to basal levels within 3 h after feeding. We doubted that the induction of HSP70 mRNA was caused by manual restraint of the mice during CK administration. Manual restraint of the mice did not influence HSP70 mRNA expression in intestine 1 h after these treatments. Our results suggest that transient HSP70 mRNA induction by oral feeding of CK was not caused by retention stress. There are some compounds in CK that increase HSP70 mRNA in various tissues.
Subject(s)
Acetic Acid , HSP70 Heat-Shock Proteins , Oryza , Acetic Acid/pharmacology , Administration, Oral , Animals , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Proteins , MiceABSTRACT
Anchorage loss elicits a set of responses in cells, such as transcriptional changes, in order to prevent inappropriate cell growth in ectopic environments. However, the mechanisms underlying these responses are poorly understood. In this study, we investigated the transcriptional up-regulation of cyclin-dependent kinase inhibitor p21(Cip1) during anchorage loss, which is important for cell cycle arrest of nonadherent cells in the G1 phase. Up-regulation was mediated by an upstream element, designated as the detachment-responsive element (DRE), that contained Kruppel-like factor 4 (KLF4) and runt-related transcription factor 1 (RUNX1) recognition sites; both of these together were necessary for transactivation, as individually they were insufficient. RNAi experiments revealed that KLF4 and a multidomain adaptor protein, hydrogen peroxide-inducible clone 5 (HIC-5), were critically involved in DRE transactivation. The role of HIC-5 in this mechanism was to tether KLF4 to DNA sites in response to cellular detachment. In addition, further analysis suggested that oligomerization and subsequent nuclear matrix localization of HIC-5, which was accelerated spontaneously in cells during anchorage loss, was assumed to potentiate the scaffolding function of HIC-5 in the nucleus and consequently regulate p21(Cip1) transcription in a manner responding to anchorage loss. At the RUNX1 site, a LIM-only protein, CRP2, imposed negative regulation on transcription, which appeared to be removed by anchorage loss and contributed to increased transcriptional activity of DRE together with regulation at the KLF4 sites. In conclusion, this study revealed a novel transcriptional mechanism that regulated gene expression in a detachment-dependent manner, thereby contributing to anchorage-dependent cell growth.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cytoskeletal Proteins/metabolism , DNA-Binding Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Kruppel-Like Transcription Factors/metabolism , LIM Domain Proteins/metabolism , Transcriptional Activation , Animals , Binding Sites , Cell Adhesion , DNA/chemistry , Fibroblasts/metabolism , Gene Expression Regulation , HEK293 Cells , Humans , Kruppel-Like Factor 4 , Mice , Mice, Inbred C3H , Models, Biological , Protein Binding , Subcellular FractionsABSTRACT
Mitochondrial dysfunction, in particular, interference in the respiratory chain, is often responsible for the toxicogenic effects of xenobiotics. In this study, changes in gene expression resulting from pharmacological inhibition of the respiratory chain were studied by DNA microarray analysis using cells treated with rotenone or antimycin A, which inhibit complexes I and III of the electron transport system, respectively. Forty-eight genes were either up- or down-regulated more than 3-fold. These included stress- and/or metabolic-related effector genes and several transcriptional regulators represented by CHOP-10. Further study using siRNA showed that among the four genes studied, up-regulation of three was dependent on CHOP-10. C/EBPbeta, a dimerizing partner of CHOP-10, was also involved in two of the three genes including Trib3, implying that CHOP-10, heterodimerizing with C/EBPbeta or another partner played a key role in the expression of a set of genes under stress. Although CHOP-10 and Trib3 were both ER-stress response genes, signal inducing Trib3 during mitochondrial stress was distinct from that during ER stress. Cytotoxicity caused by inhibition of the respiratory chain was attenuated by treatment with siRNA for CHOP-10. This study demonstrated the importance of CHOP-10 in coordinating individual gene expression in response to the mitochondrial stress.
