ABSTRACT
Steroid hormone receptors act directly in the nucleus on the chromatin organization and transcriptional activity of several promoters. Furthermore, they have an indirect effect on cytoplasmic signal transduction pathways, including MAPK, impacting ultimately on gene expression. We are interested in distinguishing between the two modes of action of progesterone receptor (PR) on the control of gene expression and cell proliferation. For this, we have stably expressed, in PR-negative breast cancer cells, tagged forms of the PR isoform B mutated at regions involved either in DNA binding (DNA-binding domain) or in its ability to interact with the estrogen receptor and to activate the c-Src/MAPK/Erk/Msk cascade (estrogen receptor-interacting domain). Both mutants impair PR-mediated activation of a well-understood model promoter in response to progestin, as well as hormone-induced cell proliferation. Additional mutants affecting transactivation activity of PR (activation function 2) or a zinc-finger implicated in dimerization (D-box) have also been tested. Microarrays and gene expression experiments on these cell lines define the subsets of hormone-responsive genes regulated by different modes of action of PR isoform B, as well as genes in which the nuclear and nongenomic pathways cooperate. Correlation between CCND1 expression in the different cell lines and their ability to support cell proliferation confirms CCND1 as a key controller gene.
Subject(s)
Gene Expression Regulation, Neoplastic , Receptors, Progesterone/chemistry , Receptors, Progesterone/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin D1/genetics , Cyclin D1/metabolism , DNA Mutational Analysis , Enzyme Activation/drug effects , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Humans , Kinetics , Models, Genetic , Mutant Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Progestins/pharmacology , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Protein Kinases/metabolism , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Time FactorsABSTRACT
Steroid hormone receptors regulate gene expression, interacting with target DNA sequences but also activating cytoplasmic signaling pathways. Using the human 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) gene as a model, we have investigated the contributions of both effects on a human progesterone-responsive promoter in breast cancer cells. Chromatin immunoprecipitation has identified two different mechanisms of hormone-induced progesterone receptor (PR) recruitment to the 11beta-HSD2 promoter: (i) direct PR binding to DNA at the proximal promoter, abrogated when PR contains a mutated DNA binding domain (DBD), and (ii) STAT5A (signal transducer and activator of transcription 5A)-mediated recruitment of PR to an upstream distal region, impaired by AG490, a JAK/STAT pathway inhibitor. The JAK/STAT inhibitor, as well as expression of dominant-negative STAT5A, impairs hormone induction of 11beta-HSD2. On the other hand, the DBD-mutated PR fully supports 11beta-HSD2 expression. These results, along with data from a deletion analysis, indicate that the distal region is crucial for hormone regulation of 11beta-HSD2. We show active RNA polymerase II tracking from the distal region upon PR and STAT5A binding, concomitant with synthesis of noncoding, hormone-dependent RNAs, suggesting that this region works as a hormone-dependent transcriptional enhancer. In conclusion, coordination of PR transcriptional effects and cytoplasmic signaling activation, in particular the JAK/STAT pathway, are critical in regulating progestin-induced endogenous 11beta-HSD2 gene expression in breast cancer cells. This is not unique to this promoter, as AG490 also alters the expression of other progesterone-regulated genes.
Subject(s)
11-beta-Hydroxysteroid Dehydrogenase Type 2/genetics , Breast Neoplasms/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Progesterone/metabolism , Receptors, Progesterone/metabolism , STAT5 Transcription Factor/metabolism , Tumor Suppressor Proteins/metabolism , Cell Line, Tumor , DNA Mutational Analysis , Enhancer Elements, Genetic , Humans , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/metabolism , Progesterone/pharmacology , Progestins/pharmacology , Promoter Regions, Genetic , RNA Polymerase II/metabolism , STAT5 Transcription Factor/antagonists & inhibitors , Sequence Deletion , Transcription, Genetic/drug effects , Tumor Suppressor Proteins/antagonists & inhibitorsABSTRACT
Gene regulation by steroid hormones involves genomic and non-genomic signaling pathways and the relationship between these two pathways is unknown. Genomic actions are often mediated by binding of the ligand-activated hormone receptors to hormone responsive elements (HREs) followed by recruitment of co-regulators, remodeling of chromatin and formation of the transcription initiation complex. The non-genomic effects of steroid hormones involve the rapid and transient activation of several kinase cascades often mediated by a subpopulation of "nuclear" receptors located in the cytoplasmic side of the cell membrane. The progesterone effect on breast cancer cell proliferation involves activation of the Src/Ras/Erk cascade mediated by a specific interaction between two domains of the N-terminal half of PR and the ligand-binding domain of ERalpha. Unexpectedly, selective inhibition of Erk, or its target kinase Msk1, interferes with chromatin remodeling and blocks MMTV transcriptional activation. A complex of activated PR, Erk and Msk1 is recruited to promoter already 5 min after hormone treatment and phosphorylates histone H3 at serine 10, leading to displacement of HP1gamma, as a requisite for recruitment of Src1, chromatin remodeling complexes (hSnf2h and Brg1) and RNA polymerase II. Thus, activation of signaling cascades in the cytoplasm is essential for chromatin remodeling and transcriptional activation of a subset of steroid hormone target genes.
Subject(s)
Chromatin/genetics , Genome/genetics , Hormones/metabolism , Signal Transduction , Betaretrovirus/genetics , Cell Line, Tumor , Enzyme Activation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Glycogen Synthase Kinase 3/metabolism , Histones/metabolism , Hormones/pharmacology , Humans , Nucleosomes/metabolism , Phosphorylation/drug effects , Promoter Regions, Genetic/genetics , Protein Binding , Receptors, Progesterone/metabolism , Signal Transduction/drug effectsABSTRACT
How genes are regulated in the context of chromatin is a central question of biology. Steroid hormones control gene expression via interaction of their receptors with target sequences on DNA but can also activate cytoplasmic signaling cascades. Here we report that rapid Erk activation by progestins participates in induction of target genes by preparing the chromatin for transcription. Five minutes after hormone treatment, Erk activation leads to phosphorylation of the progesterone receptor (PR), activation of Msk1, and recruitment of a complex of the three proteins to a nucleosome on the MMTV promoter. Msk1 phosphorylates histone H3, leading to displacement of HP1gamma and recruitment of Brg1 and RNA polymerase II. Cell-free experiments show a direct interaction between PR, Erk, and Msk1 and support the importance of H3 phosphorylation for nucleosome remodeling. Inhibition of Msk1 activation blocks recruitment of the kinase complex, H3 phosphorylation, and HP1gamma displacement, thus precluding remodeling and induction of the promoter.
