ABSTRACT
To directly activate specific gene expression, the estrogen receptor (ER) must bind to estrogen receptor response elements (EREs) in the context of nucleosomes. In order to investigate the interaction of the ER with mononucleosomes, we developed a mononucleosome gel shift assay. A 164 bp high specific activity [(32)P]probe DNA (32 bp consensus ERE with flanking regions separated by 23 nucleotides from an artificial nucleosome positioning sequence) was prepared. Nuclear extracts from MCF-7 cells or recombinant human ERalpha were incubated with the labeled ERE +/- excess ERE. A retarded band was seen which was completely obliterated with excess ERE, confirming the specificity of binding. This probe was then used to make reconstituted mononucleosomes by sequential dilution of a high salt histone preparation. The nucleosomes were purified by sucrose density gradients and footprinting analysis was performed to demonstrate that the mononucleosomes were rotationally phased as seen by a periodic digestion pattern (10 bp) of the nucleosomes versus ERE. Nucleosomes were incubated with nuclear extracts containing ER or recombinant ERalpha. Dose dependence in the shift of the mononucleosomes with increasing concentrations of ER was observed. Specificity was demonstrated in experiments with excess ERE and anti-ER antibody. Footprinting analysis was also performed. We also determined that addition of high mobility group protein-2 (HMGB-2, a protein closely related to HMGB-1) with the ER increased the interaction of ER with mononucleosomes. These studies will allow us to address the interactions of ER with core histones containing a multiplicity of variants and modifications in nucleosomal structure.
Subject(s)
Nucleosomes/metabolism , Protein Footprinting/methods , Receptors, Estrogen/metabolism , Cell Line, Tumor , DNA Probes , Electrophoretic Mobility Shift Assay/methods , Female , HMGB2 Protein/pharmacology , Humans , Protein Binding/drug effects , Response ElementsABSTRACT
The signaling pathways that modulate IL-1beta expression in human keratinocytes have not been well defined. We have previously shown that TCDD-stimulated AhR-dependent IL-1beta expression in human keratinocytes is due to posttranscriptional regulation involving mRNA stabilization. Since TCDD activates a variety of cellular signaling pathways such as PKC, JNK, and ERK, we investigated these pathways to determine their roles in TCDD-stimulated IL-1beta expression in the human keratinocyte cell line SCC-12F. In this study, we used specific signaling inhibitors to show that ERK and JNK, but not transglutaminase, PKC, or p38, signaling modulate IL-1beta expression. In addition, we show that ERK is constitutively active and unaffected by TCDD treatment and differentiation, while the JNK signaling pathway is modulated by TCDD in an AhR-dependent manner. Thus, both the ERK and JNK MAPK pathways are necessary for IL-1beta expression in TCDD-stimulated human keratinocytes, however, they act at different levels to modulate IL-1beta expression.
Subject(s)
Interleukin-1/biosynthesis , Keratinocytes/metabolism , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinases/metabolism , Cell Line, Transformed , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , JNK Mitogen-Activated Protein Kinases , Keratinocytes/enzymology , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Polychlorinated Dibenzodioxins/metabolism , Polychlorinated Dibenzodioxins/toxicity , Protein Kinase C/metabolism , RNA, Messenger/biosynthesis , Receptors, Aryl Hydrocarbon/drug effects , Receptors, Aryl Hydrocarbon/metabolismABSTRACT
TCDD stimulated IL-1beta gene expression in differentiating human keratinocyte cell lines in a time- and dose-dependent manner. Increases in prointerleukin-1beta (pIL-1beta) protein and IL-1beta steady state mRNA levels were observed in both SCC-12F and HaCaT cells following TCDD treatment. When pretreated with alpha-naphthoflavone, an AhR antagonist, TCDD-mediated increases in IL-1beta gene expression were attenuated, demonstrating for the first time that the environmental toxin, TCDD, can stimulate cytokine (IL-1beta) gene expression in an AhR-dependent manner. Nuclear run-on experiments were performed in SCC-12 cells to determine if the AhR-dependent increases in IL-1beta expression were due to transcriptional activation of the IL-1beta gene. Results showed high constitutive levels of IL-1beta transcriptional activity, however, TCDD treatment, which stimulated IL-1beta steady state mRNA levels, failed to potentiate IL-1beta transcription. Taken together, these results demonstrate that AhR-mediated IL-1beta regulation is occurring posttranscriptionally.
