Project description:Progesterone receptors (PRs) are critical context-dependent transcription factors required for normal uterine (PR-A) and mammary gland (PR-B) development. Progesterone is proliferative in the breast, where PR-target genes include paracrine factors that mediate mammary stem cell self-renewal. In the context of altered signal transduction that typifies breast tumorigenesis, dysregulated (i.e. hyper-phosphorylated) PRs likely contribute to tumor progression by promoting cancer cell pro-survival and proliferation. Notably, in breast cancer cells, progestin-bound PRs induce rapid MAPK activation leading to selective regulation of growth-promoting genes by phosphorylated PR species. Functional domains within PR that interact with c-Src and estrogen receptors (ER) have been identified as indirect routes to MAPK activation. Herein, we describe a common docking (CD) domain located within the PR-B N-terminus, a motif first described in MAPKs that facilitates direct interactions between MAPKs and MEK1 or MAPK-phosphatases (MKPs). Mutation of negatively-charged amino acids, previously determined to be critical for CD domain function in MAPKs, within PR-B (mCD PR) did not alter MEK-binding or progestin-induced rapid signaling (i.e. MAPK activation) and PR transcriptional activity as measured by PRE-luciferase (reporter) assays. Microarray gene-expression analysis revealed that endogenous genes regulated by wt PR, but not mCD PR, are involved in critical cellular pathways regulating growth, proliferation, survival, and cancer. mCD PR failed to undergo ligand-induced phosphorylation on Ser81, a ck2-dependent site required for progestin-regulation of select growth-promoting genes (BIRC3, HSD11β2, HbEGF). Progestin-induced PR Ser81 phosphorylation mapped to CD domain-dependent binding of PR-B to MKP3, but did not require phosphatase activity. Receptors containing either mutant CD domains (mCD PR) or point mutations of Ser81 (S79/81A PR) failed to upregulate STAT5 and Wnt1, key PR-target gene products that act as critical mediators of mammary stem cell expansion. Inhibition of JAK/STAT signaling blocked progestin-induced STAT5 and Wnt1 expression. ChIP assays demonstrated that wt, but not phospho-mutant (S79/81A), PR-B was co-recruited to a PRE-containing enhancer region of the Wnt1 gene along with MKP3, ck2 and STAT5. Our studies reveal a novel scaffolding action of MKP3 mediated by interaction with the PR CD domain and required for ck2-dependent PR Ser81 phosphorylation. Co-regulation of select target genes by phospho-Ser81 PR and phospho-STAT5 is likely a global mechanism required for the activation of growth promoting programs active during normal mammary gland development and relevant to mechanisms of breast cancer progression. The study contains 6 different sample groups measured in triplicate, for a total of 18 individual samples (18 arrays). From parental T47D-Y human breast cancer cell lines, we created three stable clones expressing (1) an empty vector (pSG5), (2) the wild type progesterone receptor isoform B (pSG5-PR-B), or (3) a mutant mutant CD domain PR-B. These cell lines were treated with either (1) vehicle control (ethanol) or (2) R5020 10e-8 M for 6 hours before total RNA harvest. Thus, the experiment contains three cell lines, and two treatments (6 sample groups) treated and analyzed in triplicate (18 microarrays). Standard Illumina HT-12v4 chip controls were used during hybridization.

Project description:Progesterone receptors (PRs) are critical context-dependent transcription factors required for normal uterine (PR-A) and mammary gland (PR-B) development. Progesterone is proliferative in the breast, where PR-target genes include paracrine factors that mediate mammary stem cell self-renewal. In the context of altered signal transduction that typifies breast tumorigenesis, dysregulated (i.e. hyper-phosphorylated) PRs likely contribute to tumor progression by promoting cancer cell pro-survival and proliferation. Notably, in breast cancer cells, progestin-bound PRs induce rapid MAPK activation leading to selective regulation of growth-promoting genes by phosphorylated PR species. Functional domains within PR that interact with c-Src and estrogen receptors (ER) have been identified as indirect routes to MAPK activation. Herein, we describe a common docking (CD) domain located within the PR-B N-terminus, a motif first described in MAPKs that facilitates direct interactions between MAPKs and MEK1 or MAPK-phosphatases (MKPs). Mutation of negatively-charged amino acids, previously determined to be critical for CD domain function in MAPKs, within PR-B (mCD PR) did not alter MEK-binding or progestin-induced rapid signaling (i.e. MAPK activation) and PR transcriptional activity as measured by PRE-luciferase (reporter) assays. Microarray gene-expression analysis revealed that endogenous genes regulated by wt PR, but not mCD PR, are involved in critical cellular pathways regulating growth, proliferation, survival, and cancer. mCD PR failed to undergo ligand-induced phosphorylation on Ser81, a ck2-dependent site required for progestin-regulation of select growth-promoting genes (BIRC3, HSD11β2, HbEGF). Progestin-induced PR Ser81 phosphorylation mapped to CD domain-dependent binding of PR-B to MKP3, but did not require phosphatase activity. Receptors containing either mutant CD domains (mCD PR) or point mutations of Ser81 (S79/81A PR) failed to upregulate STAT5 and Wnt1, key PR-target gene products that act as critical mediators of mammary stem cell expansion. Inhibition of JAK/STAT signaling blocked progestin-induced STAT5 and Wnt1 expression. ChIP assays demonstrated that wt, but not phospho-mutant (S79/81A), PR-B was co-recruited to a PRE-containing enhancer region of the Wnt1 gene along with MKP3, ck2 and STAT5. Our studies reveal a novel scaffolding action of MKP3 mediated by interaction with the PR CD domain and required for ck2-dependent PR Ser81 phosphorylation. Co-regulation of select target genes by phospho-Ser81 PR and phospho-STAT5 is likely a global mechanism required for the activation of growth promoting programs active during normal mammary gland development and relevant to mechanisms of breast cancer progression.

Project description:Progesterone receptor (PR) and its co-activators are direct targets of activated cyclin dependent kinases (CDKs) in response to peptide growth factors, progesterone, and deregulation of cell cycle inhibitors. Herein, using the T47D breast cancer model, we probed mechanisms of cell cycle-dependent PR action. In the absence of exogenous progestin, PR is specifically phosphorylated during the G2/M phase. Accordingly, numerous PR target genes are cell cycle regulated, including HSPB8, a heat-shock protein whose high expression is associated with tamoxifen-resistance. Progestin-induced HSPB8 expression required cyclin D1 and was insensitive to anti-estrogens, but blocked by anti-progestins or inhibition of specificity factor 1 (SP1). HSPB8 expression increased with or without ligand when cells were G2/M synchronized or contained high levels of cyclin D1. Knock-down of PR abrogated ligand-independent HSPB8 expression in synchronized cells. Notably, PR and cyclin D1 co-purified in whole cell lysates of transiently transfected COS-1 cells and in PR-positive T47D breast cancer cells expressing endogenous cyclin D1. PR, cyclin D1, and SP1 were recruited to the HSPB8 promoter in progestin-treated T47D breast cancer cells. Mutation of PR Ser345 to Ala (S345A) or inhibition of CDK2 activity using roscovitine disrupted PR/cyclin D1 interactions with DNA and blocked HSPB8 mRNA expression. Interaction of phosphorylated PRs with SP1 and cyclin D1 provides a mechanism for targeting transcriptionally active PRs to selected gene promoters relevant to breast cancer progression. Understanding the functional linkage between PR and cell cycle regulatory proteins will provide keys to targeting novel PR/cyclin D1 cross-talk in both hormone-responsive and HSPB8-high refractory disease. The study contains 4 different sample groups measured in triplicate, for a total of 12 individual samples (12 arrays). In T47D human breast cancer cell lines stably expressing PR-B, cells were synchronized (or not synchronized) before G2/M phase using nocodazole. These cell lines (synchronized or not synchronized) were treated with either (1) vehicle control (ethanol) or (2) PR ligand R5020 10e-8 M for 6 hours before total RNA harvest. Thus, the experiment contains two cell lines, and two treatments (4 sample groups) treated and analyzed in triplicate (12 microarrays). Standard Illumina HT-12v4 chip controls were used during hybridization.

Project description:Properly regulated transcriptional responses to environmental perturbations are critical for the fitness of fish. Although gene expression profiles in the tissues of common carp upon cold stress were previously characterized, the transcriptional programs underlying cold acclimation are still not well known. In this study, the ability of three common carp strains including Hebao red carp (HB), Songpu mirror carp (SPM) and Yellow river carp (YR) to establish cold resistance after acclimation to a mild hypothermia stress at 18°C for 24 h was confirmed by measurements of the critical thermal minimums (CTMin). The gene expression profiles of the brain and the heart from these strains under both control and cold-acclimated conditions were characterized with RNA-sequencing. The data of the three common carp strains with different genetic background were combined in the differential gene expression analyses to balance the effects of genetic diversity on gene expression. Marked effects of tissue origins on the cold-induced transcriptional responses were revealed by comparing the differentially expressed gene (DEG) lists of the two tissues. Functional categories including spliceosome and RNA splicing were highly enriched in the DEGs of both tissues. However, steroid biosynthesis was specifically enriched in DEGs of the brain and response to unfolded protein was solely enriched in DEGs of the heart. Consistent with the up-regulation of the genes involved in cholesterol biosynthesis, total cholesterol content of the brain was significantly increased upon cold stress. Moreover, cold-induced alternative splicing (AS) events were explored and AS of the rbmx (RNA-binding motif protein, X chromosome) gene was confirmed by real-time quantitative PCR. Finally, a core set of cold responsive genes (CRGs) were defined by comparative transcriptomic analyses. Our data provide insights into the transcriptional programs underlying cold acclimation of common carp and offer valuable clues for further investigating the genetic determinants for cold resistance of farmed fish.

Dataset Information

A Common Docking Domain in Progesterone Receptor-B links DUSP6 and CK2 signaling to proliferative transcriptional programs in breast cancer cells.

Publications

A Common Docking Domain in Progesterone Receptor-B links DUSP6 and CK2 signaling to proliferative transcriptional programs in breast cancer cells.

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