Thyroid Hormone Signaling In Vivo Requires a Balance between Coactivators and Corepressors

Resistance to thyroid hormone (RTH), a human syndrome, is characterized by high thyroid hormone (TH) and thyroid-stimulating hormone (TSH) levels. Mice with mutations in the thyroid hormone receptor beta (TRβ) gene that cannot bind steroid receptor coactivator 1 (SRC-1) and Src-1^−/− mice both have phenotypes similar to that of RTH. Conversely, mice expressing a mutant nuclear corepressor 1 (Ncor1) allele that cannot interact with TRβ, termed NCoRΔID, have low TH levels and normal TSH. We hypothesized that Src-1^−/− mice have RTH due to unopposed corepressor action. To test this, we crossed NCoRΔID and Src-1^−/− mice to create mice deficient for coregulator action in all cell types. Remarkably, NCoR^ΔID/ΔID Src-1^−/− mice have normal TH and TSH levels and are triiodothryonine (T₃) sensitive at the level of the pituitary. Although absence of SRC-1 prevented T₃ activation of key hepatic gene targets, NCoR^ΔID/ΔID Src-1^−/− mice reacquired hepatic T₃ sensitivity. Using in vivo chromatin immunoprecipitation assays (ChIP) for the related coactivator SRC-2, we found enhanced SRC-2 recruitment to TR-binding regions of genes in NCoR^ΔID/ΔID Src-1^−/− mice, suggesting that SRC-2 is responsible for T₃ sensitivity in the absence of NCoR1 and SRC-1. Thus, T₃ targets require a critical balance between NCoR1 and SRC-1. Furthermore, replacement of NCoR1 with NCoRΔID corrects RTH in Src-1^−/− mice through increased SRC-2 recruitment to T₃ target genes.     

Thyroid Hormone Receptor Sumoylation Is Required for Preadipocyte Differentiation and Proliferation

Thyroid hormone and thyroid hormone receptor (TR) play an essential role in metabolic regulation. However, the role of TR in adipogenesis has not been established. We reported previously that TR sumoylation is essential for TR-mediated gene regulation and that mutation of either of the two sites in TRα or any of the three sites in TRβ reduces TR sumoylation. Here, we transfected TR sumoylation site mutants into human primary preadiocytes and the mouse 3T3L1 preadipocyte cell line to determine the role of TR sumoylation in adipogenesis. Reduced sumoylation of TRα or TRβ resulted in fewer and smaller lipid droplets and reduced proliferation of preadipocytes. TR sumoylation mutations, compared with wild-type TR, results in reduced C/EBP expression and reduced PPARγ₂ mRNA and protein levels. TR sumoylation mutants recruited NCoR and disrupted PPARγ-mediated perilipin1 (Plin1) gene expression, associated with impaired lipid droplet formation. Expression of NCoRΔID, a mutant NCoR lacking the TR interaction domain, partially “rescued” the delayed adipogenesis and restored Plin1 gene expression and adipogenesis. TR sumoylation site mutants impaired Wnt/β-catenin signaling pathways and the proliferation of primary human preadipocytes. Expression of the TRβ K146Q sumoylation site mutant down-regulated the essential genes required for canonical Wnt signal-mediated proliferation, including Wnt ligands, Fzds, β-catenin, LEF1, and CCND1. Additionally, the TRβ K146Q mutant enhanced the canonical Wnt signaling inhibitor Dickkopf-related protein 1 (DKK1). Our data demonstrate that TR sumoylation is required for activation of the Wnt canonical signaling pathway during preadipocyte proliferation and enhances the PPARγ signaling that promotes differentiation.     

Identification of thyroid hormone response elements in?vivo using mice expressing a tagged thyroid hormone receptor α1

TRα1 (thyroid hormone receptor α1) is well recognized for its importance in brain development. However, due to the difficulties in predicting TREs (thyroid hormone response elements) in silico and the lack of suitable antibodies against TRα1 for ChIP (chromatin immunoprecipitation), only a few direct TRα1 target genes have been identified in the brain. Here we demonstrate that mice expressing a TRα1–GFP (green fluorescent protein) fusion protein from the endogenous TRα locus provide a valuable animal model to identify TRα1 target genes. To this end, we analysed DNA–TRα1 interactions in vivo using ChIP with an anti-GFP antibody. We validated our system using established TREs from neurogranin and hairless, and by verifying additional TREs from known TRα1 target genes in brain and heart. Moreover, our model system enabled the identification of novel TRα1 target genes such as RNF166 (ring finger protein 166). Our results demonstrate that transgenic mice expressing a tagged nuclear receptor constitute a feasible approach to study receptor–DNA interactions in vivo, circumventing the need for specific antibodies. Models like the TRα1–GFP mice may thus pave the way for genome-wide mapping of nuclear receptor-binding sites, and advance the identification of novel target genes in vivo.     

Expression of Dominant-Negative Thyroid Hormone Receptor Alpha1 in Leydig and Sertoli Cells Demonstrates No Additional Defect Compared with Expression in Sertoli Cells Only

Background

In the testis, thyroid hormone (T3) regulates the number of gametes produced through its action on Sertoli cell proliferation. However, the role of T3 in the regulation of steroidogenesis is still controversial.

Methods

The TRα^AMI knock-in allele allows the generation of transgenic mice expressing a dominant-negative TRα1 (thyroid receptor α1) isoform restricted to specific target cells after Cre-loxP recombination. Here, we introduced this mutant allele in both Sertoli and Leydig cells using a novel aromatase-iCre (ARO-iCre) line that expresses Cre recombinase under control of the human Cyp19(IIa)/aromatase promoter.

Findings

We showed that loxP recombination induced by this ARO-iCre is restricted to male and female gonads, and is effective in Sertoli and Leydig cells, but not in germ cells. We compared this model with the previous introduction of TRα^AMI specifically in Sertoli cells in order to investigate T3 regulation of steroidogenesis. We demonstrated that TRα^AMI-ARO males exhibited increased testis weight, increased sperm reserve in adulthood correlated to an increased proliferative index at P3 in vivo, and a loss of T3-response in vitro. Nevertheless, TRα^AMI-ARO males showed normal fertility. This phenotype is similar to TRα^AMI-SC males. Importantly, plasma testosterone and luteinizing hormone levels, as well as mRNA levels of steroidogenesis enzymes StAR, Cyp11a1 and Cyp17a1 were not affected in TRα^AMI-ARO.

Conclusions/Significance

We concluded that the presence of a mutant TRα^AMI allele in both Leydig and Sertoli cells does not accentuate the phenotype in comparison with its presence in Sertoli cells only. This suggests that direct T3 regulation of steroidogenesis through TRα1 is moderate in Leydig cells, and that Sertoli cells are the main target of T3 action in the testis.     

Correlation of Perfusion MRI and 18F-FDG PET Imaging Biomarkers for Monitoring Regorafenib Therapy in Experimental Colon Carcinomas with Immunohistochemical Validation

ObjectivesTo investigate a multimodal, multiparametric perfusion MRI / ¹⁸F-fluoro-deoxyglucose-(¹⁸F-FDG)-PET imaging protocol for monitoring regorafenib therapy effects on experimental colorectal adenocarcinomas in rats with immunohistochemical validation.ResultsRegorafenib significantly (p<0.01) suppressed PF (81.1±7.5 to 50.6±16.0 mL/100mL/min), PV (12.1±3.6 to 7.5±1.6%) and PS (13.6±3.2 to 7.9±2.3 mL/100mL/min) as well as TTB (3.4±0.6 to 1.9±1.1) between baseline and day 7. Immunohistochemistry revealed significantly (p<0.03) lower tumor microvascular density (CD-31, 7.0±2.4 vs. 16.1±5.9) and tumor cell proliferation (Ki-67, 434.0 ± 62.9 vs. 663.0 ± 98.3) in the therapy group. Perfusion MRI parameters ΔPF, ΔPV and ΔPS showed strong and significant (r = 0.67-0.78; p<0.01) correlations to the PET parameter ΔTTB and significant correlations (r = 0.57-0.67; p<0.03) to immunohistochemical Ki-67 as well as to CD-31-stainings (r = 0.49-0.55; p<0.05).ConclusionsA multimodal, multiparametric perfusion MRI/PET imaging protocol allowed for non-invasive monitoring of regorafenib therapy effects on experimental colorectal adenocarcinomas in vivo with significant correlations between perfusion MRI parameters and ¹⁸F-FDG-PET validated by immunohistochemistry.     

Aryl Hydrocarbon Receptors in Osteoclast Lineage Cells Are a Negative Regulator of Bone Mass

Aryl hydrocarbon receptors (AhRs) play a critical role in various pathological and physiological processes. Although recent research has identified AhRs as a key contributor to bone metabolism following studies in systemic AhR knockout (KO) or transgenic mice, the cellular and molecular mechanism(s) in this process remain unclear. In this study, we explored the function of AhR in bone metabolism using AhR^{RANKΔOc/ΔOc} (RANK^Cre/+;AhR^flox/flox) mice. We observed enhanced bone mass together with decreased resorption in both male and female 12 and 24-week-old AhR^{RANKΔOc/ΔOc} mice. Control mice treated with 3-methylcholanthrene (3MC), an AhR agonist, exhibited decreased bone mass and increased bone resorption, whereas AhR^{CtskΔOc/ΔOc} (Ctsk^Cre/+;AhR^flox/flox) mice injected with 3MC appeared to have a normal bone phenotype. In vitro, bone marrow-derived macrophages (BMDMs) from AhR^{RANKΔOc/ΔOc} mice exhibited impaired osteoclastogenesis and repressed differentiation with downregulated expression of B lymphocyte-induced maturation protein 1 (Blimp1), and cytochrome P450 genes Cyp1b1 and Cyp1a2. Collectively, our results not only demonstrated that AhR in osteoclast lineage cells is a physiologically relevant regulator of bone resorption, but also highlighted the need for further studies on the skeletal actions of AhR inhibitors in osteoclast lineage cells commonly associated with bone diseases, especially diseases linked to environmental pollutants known to induce bone loss.     

Caspase-8 auto-cleavage regulates programmed cell death and collaborates with RIPK3/MLKL to prevent lymphopenia

Caspase-8 is an initiator of death receptor-induced apoptosis and an inhibitor of RIPK3-MLKL-dependent necroptosis. In addition, caspase-8 has been implicated in diseases such as lymphoproliferation, immunodeficiency, and autoimmunity in humans. Although auto-cleavage is indispensable for caspase-8 activation, its physiological functions remain poorly understood. Here, we generated a caspase-8 mutant lacking E385 in auto-cleavage site knock-in mouse (Casp8^{ΔE385/ΔE385}). Casp8^{ΔE385/ΔE385} cells were expectedly resistant to Fas-induced apoptosis, however, Casp8^{ΔE385/ΔE385} cells could switch TNF-α-induced apoptosis to necroptosis by attenuating RIPK1 cleavage. More importantly, CASP8(ΔE385) sensitized cells to RIPK3-MLKL-dependent necroptosis through promoting complex II formation and RIPK1-RIPK3 activation. Notably, Casp8^{ΔE385/ΔE385}Ripk3^−/− mice partially rescued the perinatal death of Ripk1^−/− mice by blocking apoptosis and necroptosis. In contrast to the Casp8^−/−Ripk3^−/− and Casp8^−/−Mlkl^−/− mice appearing autoimmune lymphoproliferative syndrome (ALPS), both Casp8^{ΔE385/ΔE385}Ripk3^−/− and Casp8^{ΔE385/ΔE385}Mlkl^−/− mice developed transplantable lymphopenia that could be significantly reversed by RIPK1 heterozygosity, but not by RIPK1 kinase dead mutation. Collectively, these results demonstrate previously unappreciated roles for caspase-8 auto-cleavage in regulating necroptosis and maintaining lymphocytes homeostasis.Subject terms: Cell death and immune response, Immune cell death     

Mouse-Hamster Chimeric Prion Protein (PrP) Devoid of N-Terminal Residues 23-88 Restores Susceptibility to 22L Prions,but Not to RML Prions in PrP-Knockout Mice

Prion infection induces conformational conversion of the normal prion protein PrP^C, into the pathogenic isoform PrP^Sc, in prion diseases. It has been shown that PrP-knockout (Prnp^0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp^0/0 mice, neither developed the disease nor accumulated MHM2^ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice developed the disease with abundant accumulation of MHM2^ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2^ScΔ23-88, and that the co-expressing wild-type PrP^C can stimulate the conversion of MHM2Δ23-88 to MHM2^ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp^0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp^0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2^ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2^ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice. However, wild-type PrP^Sc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice, compared with RML- and 22L-inoculated Prnp^0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2^ScΔ23-88 without the help of the trans-acting PrP^C, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrP^C stimulates the conversion of MHM2Δ23-88 into MHM2^ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrP^C into PrP^Sc.     

Deficiency of thyroid hormone receptor protects retinal pigment epithelium and photoreceptors from cell death in a mouse model of age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of vision loss in the elderly. Progressive dystrophy of the retinal pigment epithelium (RPE) and photoreceptors is the characteristic of dry AMD, and oxidative stress/damage plays a central role in the pathogenic lesion of the disease. Thyroid hormone (TH) regulates cell growth, differentiation, and metabolism, and regulates development/function of photoreceptors and RPE in the retina. Population-/patient-based studies suggest an association of high free-serum TH levels with increased risk of AMD. We recently showed that suppressing TH signaling by antithyroid treatment reduces cell damage/death of the RPE and photoreceptors in an oxidative-stress/sodium iodate (NaIO₃)-induced mouse model of AMD. This work investigated the effects of TH receptor (THR) deficiency on cell damage/death of the RPE and photoreceptors and the contribution of the receptor subtypes. Treatment with NaIO₃ induced RPE and photoreceptor cell death/necroptosis, destruction, and oxidative damage. The phenotypes were significantly diminished in Thrα1^−/−, Thrb^−/−, and Thrb2^−/− mice, compared with that in the wild-type (C57BL/6 J) mice. The involvement of the receptor subtypes varies in the RPE and retina. Deletion of Thrα1 or Thrb protected RPE, rods, and cones, whereas deletion of Thrb2 protected RPE and cones but not rods. Gene-expression analysis showed that deletion of Thrα1 or Thrb abolished/suppressed the NaIO₃-induced upregulation of the genes involved in cellular oxidative-stress responses, necroptosis/apoptosis signaling, and inflammatory responses. In addition, THR antagonist effectively protected ARPE-19 cells and hRPE cells from NaIO₃-induced cell death. This work demonstrates the involvement of THR signaling in cell damage/death of the RPE and photoreceptors after oxidative-stress challenge and the receptor-subtype contribution. Findings from this work support a role of THR signaling in the pathogenesis of AMD and the strategy of suppressing THR signaling locally in the retina for protection of the RPE/retina in dry AMD.Subject terms: Necroptosis, Cell biology     

Block of Death-Receptor Apoptosis Protects Mouse Cytomegalovirus from Macrophages and Is a Determinant of Virulence in Immunodeficient Hosts

The inhibition of death-receptor apoptosis is a conserved viral function. The murine cytomegalovirus (MCMV) gene M36 is a sequence and functional homologue of the human cytomegalovirus gene UL36, and it encodes an inhibitor of apoptosis that binds to caspase-8, blocks downstream signaling and thus contributes to viral fitness in macrophages and in vivo. Here we show a direct link between the inability of mutants lacking the M36 gene (ΔM36) to inhibit apoptosis, poor viral growth in macrophage cell cultures and viral in vivo fitness and virulence. ΔM36 grew poorly in RAG1 knockout mice and in RAG/IL-2-receptor common gamma chain double knockout mice (RAGγC^−/−), but the depletion of macrophages in either mouse strain rescued the growth of ΔM36 to almost wild-type levels. This was consistent with the observation that activated macrophages were sufficient to impair ΔM36 growth in vitro. Namely, spiking fibroblast cell cultures with activated macrophages had a suppressive effect on ΔM36 growth, which could be reverted by z-VAD-fmk, a chemical apoptosis inhibitor. TNFα from activated macrophages synergized with IFNγ in target cells to inhibit ΔM36 growth. Hence, our data show that poor ΔM36 growth in macrophages does not reflect a defect in tropism, but rather a defect in the suppression of antiviral mediators secreted by macrophages. To the best of our knowledge, this shows for the first time an immune evasion mechanism that protects MCMV selectively from the antiviral activity of macrophages, and thus critically contributes to viral pathogenicity in the immunocompromised host devoid of the adaptive immune system.