ABSTRACT
The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor primarily known for its toxicological functions. Recent studies have established its importance in many physiological processes including female reproduction, although there is limited data about the precise mechanisms how Ahr itself is regulated during ovarian follicle maturation. This study describes the expression of Ahr in ovarian granulosa cells (GCs) of immature mice in a gonadotropin-dependent manner. We show that Ahr upregulation in vivo requires both follicle stimulating hormone (FSH) and luteinizing hormone (LH) activities. FSH alone increased Ahr mRNA, but had no effect on Ahr protein level, implicating a possible LH-dependent post-transcriptional regulation. Also, the increase in Ahr protein is specific to large antral follicles in induced follicle maturation. We show that Ahr expression in GCs of mid-phase follicular maturation is downregulated by protein kinase A (PKA) signaling and activation of Ahr promoter is regulated by chromatin remodeling.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Epigenesis, Genetic , Follicle Stimulating Hormone/metabolism , Gene Expression Regulation , Granulosa Cells/metabolism , Luteinizing Hormone/metabolism , Receptors, Aryl Hydrocarbon/metabolism , Signal Transduction , Animals , Biomarkers , Chromatin/genetics , Chromatin/metabolism , Female , Mice , Ovarian Follicle/metabolism , Receptors, Aryl Hydrocarbon/genetics , Transcription, GeneticABSTRACT
Recent advances in establishing the role of the aryl hydrocarbon receptor (Ahr) in normophysiology have discovered its fundamental role, amongst others, in female reproduction. Considering previous studies suggesting the hormonal modulation of Ahr, we aimed to investigate whether in murine granulosa cells (GCs) the gonadotropins regulate Ahr expression and how this is mechanistically implemented. We found that the FSH-like substance--pregnant mare serum gonadotropin--led to stimulation of Ahr expression. More importantly hCG produced relatively rapid reduction of Ahr mRNA in GCs of preovulatory follicles. We show for the first time that LHCGR signaling in regulating the Ahr message involves protein kinase A pathway and is attributable to decreased transcription rate. Finally, we found that Ahr promoter accessibility was decreased by hCG, implicating chromatin remodeling in Ahr gene regulation by LH.
Subject(s)
Chromatin/metabolism , Granulosa Cells/metabolism , Receptors, Aryl Hydrocarbon/genetics , Receptors, LH/metabolism , Repressor Proteins/metabolism , Transcription, Genetic , Animals , Chorionic Gonadotropin/pharmacology , Chromatin Assembly and Disassembly/drug effects , Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme Activation/drug effects , Female , Follicular Phase/drug effects , Follicular Phase/genetics , Gene Expression Regulation/drug effects , Granulosa Cells/drug effects , Horses , Humans , Mice , Models, Biological , Pregnancy , Protein Biosynthesis/drug effects , RNA Stability/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Aryl Hydrocarbon/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Transcription, Genetic/drug effectsABSTRACT
Mast cells are the principal effectors of IgE-mediated immune responses, including allergic reactions. Tribbles homolog 3 (Trib3) encodes a pseudokinase implicated in the cellular stress response and has been linked to inflammation in certain situations. Here we report the role of Trib3 in mouse bone marrow-derived mast cells (BMMCs). Our results show that Trib3 mRNA expression in BMMCs is positively regulated by the growth factor interleukin (IL)-3. BMMCs originating from Trib3 knockout mice demonstrate unaltered differentiation kinetics and cell surface expression of mast cell markers. When challenged with transient IL-3 deprivation, Trib3-deficient BMMCs display delayed recovery, and during prolonged IL-3 starvation, cell death is accelerated in Trib3-null cultures. IgE-dependent and pharmacologically induced degranulation is impaired in Trib3-deficient BMMCs, as is activation-induced cytokine mRNA expression. Thus, Trib3 contributes to the survival and activity of primary cultured mast cells, which suggests a role for Trib3 in the modulation of the immune response.
Subject(s)
Cell Cycle Proteins/physiology , Gene Expression Regulation/drug effects , Interleukin-3/pharmacology , Mast Cells/drug effects , Animals , Bone Marrow Cells/cytology , Cell Cycle Proteins/biosynthesis , Cell Cycle Proteins/deficiency , Cell Cycle Proteins/genetics , Cell Degranulation/drug effects , Cell Degranulation/physiology , Cell Differentiation/drug effects , Cells, Cultured/drug effects , Cells, Cultured/metabolism , G1 Phase/drug effects , Immunoglobulin E/immunology , Interleukin-3/deficiency , Interleukin-3/physiology , Mast Cells/immunology , Mast Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Passive Cutaneous Anaphylaxis , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Real-Time Polymerase Chain ReactionABSTRACT
The aryl hydrocarbon receptor (AHR) mediates the toxicity of a variety of environmental chemicals. Apart from this, an understanding is emerging that the AHR has a fundamental role in female reproduction. Evidence suggests that AHR participates in regulation of follicle-stimulating hormone receptor (Fshr) transcript level in mouse ovary by binding to the promoter of this gene in vivo. The purpose of this study was to demonstrate the molecular interplay of the Fshr promoter involved in the transactivation by AHR in mouse granulosa cells. We found that AHR activates the Fshr promoter through the region from -209 to -99 bp. In this region, the importance of the E-box motif was revealed by site-directed mutagenesis followed by promoter analysis. By focusing on the DNA/protein interactions, we defined the fact that the intact E-box but not upstream transcription factor 1 (USF1), which is known to bind this motif, is necessary for AHR binding to mouse Fshr promoter. Furthermore, by constructing AHR mutants defective in DNA interaction, we confirmed the importance of DNA binding for AHR's ability to bind to and activate Fshr promoter. Collectively, the present study demonstrates that AHR modulates Fshr transactivation by its direct association through an E-box and not by recruitment via interaction with USFs. These observations suggest that although AHR and USF may respond to different signals, they compete on binding to the same E-box. Our data, together with that from one prior study suggesting involvement of E-box motif in AHR-mediated transcription, provide novel understanding of the way in which AHR may regulate its target genes through E-box sites.
Subject(s)
E-Box Elements/physiology , Promoter Regions, Genetic/physiology , Receptors, Aryl Hydrocarbon/physiology , Receptors, FSH/physiology , Transcriptional Activation/physiology , Animals , Base Sequence , Female , Mice , Mice, Inbred C57BL , Models, Animal , Molecular Sequence Data , Mutation/genetics , Protein Binding/physiology , Receptors, Aryl Hydrocarbon/genetics , Upstream Stimulatory Factors/physiologySubject(s)
Autoantibodies/immunology , Blood Coagulation Factor Inhibitors/immunology , Factor VIII/immunology , Factor VIII/pharmacology , Hemophilia A/immunology , von Willebrand Factor/immunology , Animals , Disease Models, Animal , Factor VIII/therapeutic use , Hemophilia A/drug therapy , Mice , Mice, Mutant Strains , von Willebrand Factor/pharmacology , von Willebrand Factor/therapeutic useABSTRACT
In up to 25% haemophilia A patients, the administration of coagulation factor VIII (FVIII) preparations for treatment of haemorrhages results in production of factor VIII specific antibodies. Plasma-derived FVIII preparations contain other plasma proteins, which may modulate the immune response to FVIII. We used FVIII-deficient mice to assess the role of von Willebrand factor (VWF) and cytokine transforming growth factor beta-1 (TGF-beta1) in the immune response against FVIII. Using the FVIII and FVIII in complex with VWF purified from the plasma-derived FVIII preparation, we demonstrated that a lower concentration of FVIII antibody was induced in FVIII-VWF-treated mice compared to FVIII-treated mice (p<0.05). The addition of recombinant latent TGF-beta1 to FVIII decreased the antibody response against FVIII compared to FVIII treatment alone (p<0.01). The obtained results suggest that VWF and latent TGF-beta1 present in plasma-derived FVIII preparations reduce the immune response against FVIII. However, we cannot exlude possible modulatory effects of other plasma proteins.
