ABSTRACT
RecQ-mediated genome instability 1 (RMI1) has been identified as a novel energy homeostasis-related molecule. While recent studies have suggested that change in RMI1 expression levels in adipose tissue may affect the body's energy balance, no reports have identified the mechanism behind this expression regulation. In the present study, we found that RMI1 expression increased on differentiation of 3T3-L1 fibroblasts to adipocytes. In addition, glucose stimulation induced RMI1 expression to approximately eight times the baseline level. Further, knockdown of either E2F5 or E2F8 mRNA using siRNA suppressed this glucose-induced up-regulation of RMI1 expression. These results suggest that RMI1 expression may be regulated by glucose, at least in part, via E2F expression.
Subject(s)
Adipose Tissue/metabolism , Carrier Proteins/metabolism , E2F Transcription Factors/metabolism , Glucose/pharmacology , Nuclear Proteins/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , 3T3-L1 Cells , Adipocytes/cytology , Adipocytes/drug effects , Adipocytes/metabolism , Adipose Tissue/cytology , Adipose Tissue/drug effects , Animals , Cell Differentiation/physiology , DNA-Binding Proteins , E2F Transcription Factors/drug effects , E2F Transcription Factors/genetics , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Mice , Models, Animal , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacologyABSTRACT
In spite of the fact that cyclin-dependent kinase (cdk) inhibiting drugs are potent transcriptional repressors, we discover that p57 (Kip2, CDKN1C) transcription is significantly upregulated by three small molecule cdk inhibitors, including BMS-387032. Treatment of MDA-MB-231 breast cancer cells with BMS-387032 led to a stabilization of the E2F1 protein that was accompanied by significant increases in the p57 mRNA and protein. This increase did not occur in an E2F1-deficient cell line. An E2F1-estrogen receptor fusion protein activated the endogenous p57 promoter in response to hydroxytamoxifen treatment in the presence of cycloheximide. Luciferase constructs driven by the p57 promoter verified that upregulation of p57 mRNA by BMS-387032 is transcriptional and dependent on E2F-binding sites in the promoter. Expression of exogenous p57 significantly decreased the fraction of cells in S phase. Furthermore, p57-deficient MDA-MB-231 cell lines were significantly more sensitive to BMS-387032-induced apoptosis than controls. The results presented in this manuscript demonstrate that small molecule cdk inhibitors transcriptionally activate p57 dependent upon E2F1 and that this activation in turn serves to limit E2F1's death-inducing activity.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p57/genetics , Cyclin-Dependent Kinases/antagonists & inhibitors , E2F Transcription Factors/metabolism , Enzyme Inhibitors/pharmacology , Feedback, Physiological , Oxazoles/pharmacology , Thiazoles/pharmacology , Binding Sites , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Line , Cyclin-Dependent Kinase Inhibitor p57/drug effects , Cyclin-Dependent Kinase Inhibitor p57/metabolism , E2F Transcription Factors/drug effects , E2F1 Transcription Factor/drug effects , E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/metabolism , Gene Expression Regulation/drug effects , Humans , Promoter Regions, Genetic , Transcription, Genetic , Up-RegulationABSTRACT
UVB radiation-induced DNA damage in skin activates cellular pathways involved in DNA repair, cell cycle regulation, and apoptosis, important events that prevent conversion of damaged skin cells into cancer. We reported recently the efficacy of silibinin against photocarcinogenesis along with altered molecular events in tumors (Cancer Research, 64:6349-56, 2004). The molecular and biological events modulated by silibinin in chronically UVB-irradiated skin leading to cancer prevention, however, are not known. Herein, we describe effect of silibinin on skin 15 and 25 weeks after UVB exposure and compared them with molecular alterations in skin tumors. UVB decreased E2F1 but increased E2F2 and E2F3 protein levels in skin, and these were reversed by silibinin treatment. Silibinin-induced E2F1 was accompanied by an inhibition of apoptosis and decreases in p53 and cyclin-dependent kinase inhibitors. Silibinin-caused decrease in E2F2 and E2F3 was accompanied by reduced levels of cyclin-dependent kinases, cyclins, CDC25C, and mitogen-activated protein kinases and Akt signaling and inhibition of cell proliferation. In tumorigenesis protocols, topical or dietary silibinin significantly inhibited tumor appearance and growth. As opposed to UVB-exposed skin, UVB-induced tumors showed elevated levels of E2F1, but these were reduced in silibinin-treated tumors without any effect on E2F2 and E2F3. Contrary to the inhibition of apoptosis and p53 expression in UVB-exposed skin cells, silibinin increased these variables in tumors. These differential effects of silibinin on E2F1 versus E2F2 and E2F3 and their associated molecular alterations and biological effects in chronic UVB-exposed skin suggest their role in silibinin interference with photocarcinogenesis.
