MED1 Deficiency in Macrophages Aggravates Isoproterenol-Induced Cardiac Fibrosis in Mice.

Mediator 1 (MED1), a key subunit of the mediator complex, interacts with various nuclear receptors and functions in lipid metabolism and energy homeostasis. Dilated cardiomyopathy-related ventricular dilatation and heart failure have been reported in mice with cardiomyocyte-specific Med1 deficiency. However, the contribution of macrophage-specific MED1 in cardiac remodeling remains unclear. In this study, macrophage-specific Med1 knockout (Med1ΔMac) mice were generated and exposed to isoproterenol (ISO) to induce cardiac fibrosis; these mice showed aggravated cardiac fibrosis compared with Med1fl/fl mice. The levels of expression of marker genes for myofibroblast transdifferentiation [α-smooth muscle actin (SMA)] and of profibrotic genes, including Col1a1, Col3a1, Postn, Mmp2, Timp1, and Fn1, were significantly increased in the cardiac tissues of Med1ΔMac mice with ISO-induced myocardial fibrosis. In particular, the transforming growth factor (TGF)-ß-Smad2/3 signaling pathway was activated. In bone marrow-derived and peritoneal macrophages, Med1 deficiency was also associated with elevated levels of expression of proinflammatory genes, including Il6, Tnfa, and Il1b. These findings indicate that macrophage-specific MED1 deficiency may aggravate ISO-induced cardiac fibrosis via the regulation of the TGF-ß-SMAD2/3 pathway, and the underlying mechanism may involve MED1 deficiency triggering the activation of inflammatory cytokines in macrophages, which in turn may stimulate phenotypic switch of cardiac fibroblasts and accelerate cardiac fibrosis. Thus, MED1 is a potential therapeutic target for cardiac fibrosis.

MED1 Deficiency in Macrophages Accelerates Intimal Hyperplasia via ROS Generation and Inflammation.

Mediator complex subunit 1 (MED1) is a component of the mediator complex and functions as a coactivator involved in the regulated transcription of nearly all RNA polymerase II-dependent genes. Previously, we showed that MED1 in macrophages has a protective effect on atherosclerosis; however, the effect of MED1 on intimal hyperplasia and mechanisms regulating proinflammatory cytokine production after macrophage MED1 deletion are still unknown. In this study, we report that MED1 macrophage-specific knockout (MED1 ΔMac) mice showed aggravated neointimal hyperplasia, vascular smooth muscle cells (VSMCs), and macrophage accumulation in injured arteries. Moreover, MED1 ΔMac mice showed increased proinflammatory cytokine production after an injury to the artery. After lipopolysaccharide (LPS) treatment, MED1 ΔMac macrophages showed increased generation of reactive oxygen species (ROS) and reduced expression of peroxisome proliferative activated receptor gamma coactivator-1α (PGC1α) and antioxidant enzymes, including catalase and glutathione reductase. The overexpression of PGC1α attenuated the effects of MED1 deficiency in macrophages. In vitro, conditioned media from MED1 ΔMac macrophages induced more proliferation and migration of VSMCs. To explore the potential mechanisms by which MED1 affects inflammation, macrophages were treated with BAY11-7082 before LPS treatment, and the results showed that MED1 ΔMac macrophages exhibited increased expression of phosphorylated-p65 and phosphorylated signal transducer and activator of transcription 1 (p-STAT1) compared with the control macrophages, suggesting the enhanced activation of NF-κB and STAT1. In summary, these data showed that MED1 deficiency enhanced inflammation and the proliferation and migration of VSMCs in injured vascular tissue, which may result from the activation of NF-κB and STAT1 due to the accumulation of ROS.

Med1 deficiency alters the somatic hypermutation spectrum in murine B cells.

The Mediator complex is known to orchestrate transcription. Here we show that B cell conditional deficient mice for the Med1 subunit display robust somatic hypermutation. Nevertheless, the mutation frequency at A residues is decreased and the expected A/T ratio is abolished, implicating Mediator in the second phase of somatic hypermutation.

Mediator 1 Is Atherosclerosis Protective by Regulating Macrophage Polarization.

OBJECTIVE: MED1 (mediator 1) interacts with transcription factors to regulate transcriptional machinery. The role of MED1 in macrophage biology and the relevant disease state remains to be investigated. APPROACH AND RESULTS: To study the molecular mechanism by which MED1 regulates the M1/M2 phenotype switch of macrophage and the effect on atherosclerosis, we generated MED1/apolipoprotein E (ApoE) double-deficient (MED1ΔMac/ApoE-/-) mice and found that atherosclerosis was greater in MED1ΔMac/ApoE-/- mice than in MED1fl/fl/ApoE-/- littermates. The gene expression of M1 markers was increased and that of M2 markers decreased in both aortic wall and peritoneal macrophages from MED1ΔMac/ApoE-/- mice, whereas MED1 overexpression rectified the changes in M1/M2 expression. Moreover, LDLR (low-density lipoprotein receptor)-deficient mice received bone marrow from MED1ΔMac mice showed greater atherosclerosis. Mechanistically, MED1 ablation decreased the binding of PPARγ (peroxisome proliferator-activated receptor γ) and enrichment of H3K4me1 and H3K27ac to upstream region of M2 marker genes. Furthermore, interleukin 4 induction of PPARγ and MED1 increased the binding of PPARγ or MED1 to the PPAR response elements of M2 marker genes. CONCLUSIONS: Our data suggest that MED1 is required for the PPARγ-mediated M2 phenotype switch, with M2 marker genes induced but M1 marker genes suppressed. MED1 in macrophages has an antiatherosclerotic role via PPARγ-regulated transactivation.

Cardiomyocyte-Specific Ablation of Med1 Subunit of the Mediator Complex Causes Lethal Dilated Cardiomyopathy in Mice.

Mediator, an evolutionarily conserved multi-protein complex consisting of about 30 subunits, is a key component of the polymerase II mediated gene transcription. Germline deletion of the Mediator subunit 1 (Med1) of the Mediator in mice results in mid-gestational embryonic lethality with developmental impairment of multiple organs including heart. Here we show that cardiomyocyte-specific deletion of Med1 in mice (csMed1-/-) during late gestational and early postnatal development by intercrossing Med1fl/fl mice to α-MyHC-Cre transgenic mice results in lethality within 10 days after weaning due to dilated cardiomyopathy-related ventricular dilation and heart failure. The csMed1-/- mouse heart manifests mitochondrial damage, increased apoptosis and interstitial fibrosis. Global gene expression analysis revealed that loss of Med1 in heart down-regulates more than 200 genes including Acadm, Cacna1s, Atp2a2, Ryr2, Pde1c, Pln, PGC1α, and PGC1ß that are critical for calcium signaling, cardiac muscle contraction, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy and peroxisome proliferator-activated receptor regulated energy metabolism. Many genes essential for oxidative phosphorylation and proper mitochondrial function such as genes coding for the succinate dehydrogenase subunits of the mitochondrial complex II are also down-regulated in csMed1-/- heart contributing to myocardial injury. Data also showed up-regulation of about 180 genes including Tgfb2, Ace, Atf3, Ctgf, Angpt14, Col9a2, Wisp2, Nppa, Nppb, and Actn1 that are linked to cardiac muscle contraction, cardiac hypertrophy, cardiac fibrosis and myocardial injury. Furthermore, we demonstrate that cardiac specific deletion of Med1 in adult mice using tamoxifen-inducible Cre approach (TmcsMed1-/-), results in rapid development of cardiomyopathy and death within 4 weeks. We found that the key findings of the csMed1-/- studies described above are highly reproducible in TmcsMed1-/- mouse heart. Collectively, these observations suggest that Med1 plays a critical role in the maintenance of heart function impacting on multiple metabolic, compensatory and reparative pathways with a likely therapeutic potential in the management of heart failure.

Loss of MED1 triggers mitochondrial biogenesis in C2C12 cells.

Under stress conditions transcription factors, including their coactivators, play major roles in mitochondrial biogenesis and oxidative phosphorylation. MED1 (Mediator complex subunit 1) functions as a coactivator of several transcription factors and is implicated in adipogenesis of the lipid and glucose metabolism. This suggests that MED1 may play a role in mitochondrial function. In this study, we found that both the mtDNA content and mitochondrial mass were markedly increased and cell proliferation markedly suppressed in MED1-deficient cells. Upon MED1 loss, Nrf1 and its downstream target genes involved in mitochondrial biogenesis (Tfam, Plormt, Tfb1m), were up-regulated as were those genes in the OXPHOS pathway. Moreover, the knockdown of MED1 resulted in significant changes in the profile of mitochondrial respiration, accompanied by a prominent decrease in the generation of ATP. Collectively, these observations strongly suggest that MED1 has an important affect on mitochondrial function. This further elucidates the role of MED1, particularly its role in the energy metabolism.

Coactivator MED1 ablation in keratinocytes results in hair-cycling defects and epidermal alterations.

The transcriptional coactivator complex Mediator (MED) facilitates transcription of nuclear hormone receptors and other transcription factors. We have previously isolated the MED complex from primary keratinocytes (KCs) as the vitamin D receptor-interacting protein complex. We identified a role for MED in KC proliferation and differentiation in cultured KCs. Here, we investigated the in vivo role of MED by generating a conditional null mice model in which a critical subunit of the MED complex, MED1, is deleted from their KCs. The MED1 ablation resulted in aberrant hair differentiation and cycling, leading to hair loss. During the first hair follicle (HF) cycle, MED1 deletion resulted in a rapid regression of the HFs. Hair differentiation was reduced, and ß-catenin/vitamin D receptor (VDR)-regulated gene expression was markedly decreased. In the subsequent adult hair cycle, MED1 ablation activated the initiation of HF cycling. Shh signaling was increased, but terminal differentiation was not sufficient. Deletion of MED1 also caused hyperproliferation of interfollicular epidermal KCs, and increased the expression of epidermal differentiation markers. These results indicate that MED1 has a critical role in regulating hair/epidermal proliferation and differentiation.

Essential role of Mediator subunit Med1 in invariant natural killer T-cell development.

CD1d-restricted invariant NKT (iNKT) cells are a unique lineage of T lymphocytes that regulate both innate and adaptive immunity. The Mediator complex forms the bridge between transcriptional activators and the general transcription machinery. Med1/TRAP220 (also called DRIP205) is a key component of Mediator that interacts with ligand-bound hormone receptors, such as the vitamin D receptor. Here, we show that T-cell-specific Med1 deficiency results in a specific block in iNKT cell development but the development of conventional αß T cells remains grossly normal. The defect is cell-intrinsic and depends neither on apoptosis, cell-cycle control, nor on CD1d expression of CD4(+)CD8(+) double-positive thymocytes. Surprisingly, ectopic expression of a Vα14-Jα18 T-cell receptor transgene completely rescues the defect caused by Med1 deficiency. At the molecular level, thymic iNKT cells in Med1(-/-) animals display reduced levels of IL-2Rß and T-bet expression and could not complete terminal maturation. Thus, Med1 is essential for a complete intrathymic development of iNKT cells.

Transcription coactivator mediator subunit MED1 is required for the development of fatty liver in the mouse.

UNLABELLED: Peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor, when overexpressed in liver stimulates the induction of adipocyte-specific and lipogenesis-related genes and causes hepatic steatosis. We report here that Mediator 1 (MED1; also known as PBP or TRAP220), a key subunit of the Mediator complex, is required for high-fat diet-induced hepatic steatosis as well as PPARγ-stimulated adipogenic hepatic steatosis. Mediator forms the bridge between transcriptional activators and RNA polymerase II. MED1 interacts with nuclear receptors such as PPARγ and other transcriptional activators. Liver-specific MED1 knockout (MED1(ΔLiv) ) mice, when fed a high-fat (60% kcal fat) diet for up to 4 months failed to develop fatty liver. Similarly, MED1(ΔLiv) mice injected with adenovirus-PPARγ (Ad/PPARγ) by tail vein also did not develop fatty liver, whereas mice with MED1 (MED1(fl/fl) ) fed a high-fat diet or injected with Ad/PPARγ developed severe hepatic steatosis. Gene expression profiling and northern blot analyses of Ad/PPARγ-injected mouse livers showed impaired induction in MED1(ΔLiv) mouse liver of adipogenic markers, such as aP2, adipsin, adiponectin, and lipid droplet-associated genes, including caveolin-1, CideA, S3-12, and others. These adipocyte-specific and lipogenesis-related genes are strongly induced in MED1(fl/fl) mouse liver in response to Ad/PPARγ. Re-expression of MED1 using adenovirally-driven MED1 (Ad/MED1) in MED1(ΔLiv) mouse liver restored PPARγ-stimulated hepatic adipogenic response. These studies also demonstrate that disruption of genes encoding other coactivators such as SRC-1, PRIC285, PRIP, and PIMT had no effect on hepatic adipogenesis induced by PPARγ overexpression. CONCLUSION: We conclude that transcription coactivator MED1 is required for high-fat diet-induced and PPARγ-stimulated fatty liver development, which suggests that MED1 may be considered a potential therapeutic target for hepatic steatosis. (HEPATOLOGY 2011;).

The transcriptional mediator subunit MED1/TRAP220 in stromal cells is involved in hematopoietic stem/progenitor cell support through osteopontin expression.

MED1/TRAP220, a subunit of the transcriptional Mediator/TRAP complex, is crucial for various biological events through its interaction with distinct activators, such as nuclear receptors and GATA family activators. In hematopoiesis, MED1 plays a pivotal role in optimal nuclear receptor-mediated myelomonopoiesis and GATA-1-induced erythropoiesis. In this study, we present evidence that MED1 in stromal cells is involved in supporting hematopoietic stem and/or progenitor cells (HSPCs) through osteopontin (OPN) expression. We found that the proliferation of bone marrow (BM) cells cocultured with MED1 knockout (Med1(-/-)) mouse embryonic fibroblasts (MEFs) was significantly suppressed compared to the control. Furthermore, the number of long-term culture-initiating cells (LTC-ICs) was attenuated for BM cells cocultured with Med1(-/-) MEFs. The vitamin D receptor (VDR)- and Runx2-mediated expression of OPN, as well as Mediator recruitment to the Opn promoter, was specifically attenuated in the Med1(-/-) MEFs. Addition of OPN to these MEFs restored the growth of cocultured BM cells and the number of LTC-ICs, both of which were attenuated by the addition of the anti-OPN antibody to Med1(+/+) MEFs and to BM stromal cells. Consequently, MED1 in niche appears to play an important role in supporting HSPCs by upregulating VDR- and Runx2-mediated transcription on the Opn promoter.

A muscle-specific knockout implicates nuclear receptor coactivator MED1 in the regulation of glucose and energy metabolism.

As conventional transcriptional factors that are activated in diverse signaling pathways, nuclear receptors play important roles in many physiological processes that include energy homeostasis. The MED1 subunit of the Mediator coactivator complex plays a broad role in nuclear receptor-mediated transcription by anchoring the Mediator complex to diverse promoter-bound nuclear receptors. Given the significant role of skeletal muscle, in part through the action of nuclear receptors, in glucose and fatty acid metabolism, we generated skeletal muscle-specific Med1 knockout mice. Importantly, these mice show enhanced insulin sensitivity and improved glucose tolerance as well as resistance to high-fat diet-induced obesity. Furthermore, the white muscle of these mice exhibits increased mitochondrial density and expression of genes specific to type I and type IIA fibers, indicating a fast-to-slow fiber switch, as well as markedly increased expression of the brown adipose tissue-specific UCP-1 and Cidea genes that are involved in respiratory uncoupling. These dramatic results implicate MED1 as a powerful suppressor in skeletal muscle of genetic programs implicated in energy expenditure and raise the significant possibility of therapeutical approaches for metabolic syndromes and muscle diseases through modulation of MED1-nuclear receptor interactions.

Peroxisome-proliferator-activated receptor-binding protein (PBP) is essential for the growth of active Notch4-immortalized mammary epithelial cells by activating SOX10 expression.

PBP (peroxisome-proliferator-activated receptor-binding protein) [Med1 (mediator 1)/TRAP220 (thyroid-hormone-receptor-associated protein 220)] is essential for mammary gland development. We established a mammary epithelial cell line with a genotype of PBPLoxP/LoxP by expressing an active form of Notch4. Null mutation of PBP caused severe growth inhibition of the Notch4-immortalized mammary cells. We found that truncated PBP without the two LXXLL motifs could reverse the growth inhibition due to the deficiency of endogenous PBP, indicating that signalling through nuclear receptors is unlikely to be responsible for the growth inhibition as the result of PBP deficiency. Loss of PBP expression was shown to completely ablate the expression of SOX10 [Sry-related HMG (high-mobility group) box gene 10]. The re-expression of SOX10 was capable of reversing the growth inhibition due to PBP deficiency, whereas suppressed expression of SOX10 inhibited the growth of Notch4-immortalized mammary cells. Further studies revealed PBP is directly recruited to the enhancer of the SOX10 gene, indicating that SOX10 is a direct target gene of PBP. We conclude that PBP is essential for the growth of Notch4-immortalized mammary cells by activating SOX10 expression, providing a potential molecular mechanism through which PBP regulates the growth of mammary stem/progenitor cells.