Skeletal muscle is composed of heterogeneous myofibers with distinctive rates of contraction, metabolic properties, and susceptibility to fatigue. We show that class II histone deacetylase (HDAC) proteins, which function as transcriptional repressors of the myocyte enhancer factor 2 (MEF2) transcription factor, fail to accumulate in the soleus, a slow muscle, compared with fast muscles (e.g., white vastus lateralis). Accordingly, pharmacological blockade of proteasome function specifically increases expression of class II HDAC proteins in the soleus in vivo. Using gain- and loss-of-function approaches in mice, we discovered that class II HDAC proteins suppress the formation of slow twitch, oxidative myofibers through the repression of MEF2 activity. Conversely, expression of a hyperactive form of MEF2 in skeletal muscle of transgenic mice promotes the formation of slow fibers and enhances running endurance, enabling mice to run almost twice the distance of WT littermates. Thus, the selective degradation of class II HDACs in slow skeletal muscle provides a mechanism for enhancing physical performance and resistance to fatigue by augmenting the transcriptional activity of MEF2. These findings provide what we believe are new insights into the molecular basis of skeletal muscle function and have important implications for possible therapeutic interventions into muscular diseases.
Matthew J. Potthoff, Hai Wu, Michael A. Arnold, John M. Shelton, Johannes Backs, John McAnally, James A. Richardson, Rhonda Bassel-Duby, Eric N. Olson
We have previously reported that genetically increased angiotensin-converting enzyme levels, or absence of the bradykinin B2 receptor, increase kidney damage in diabetic mice. We demonstrate here that this is part of a more general phenomenon — diabetes and, to a lesser degree, absence of the B2 receptor, independently but also largely additively when combined, enhance senescence-associated phenotypes in multiple tissues. Thus, at 12 months of age, indicators of senescence (alopecia, skin atrophy, kyphosis, osteoporosis, testicular atrophy, lipofuscin accumulation in renal proximal tubule and testicular Leydig cells, and apoptosis in the testis and intestine) are virtually absent in WT mice, detectable in B2 receptor–null mice, clearly apparent in mice diabetic because of a dominant mutation (Akita) in the Ins2 gene, and most obvious in Akita diabetic plus B2 receptor–null mice. Renal expression of several genes that encode proteins associated with senescence and/or apoptosis (TGF-β1, connective tissue growth factor, p53, α-synuclein, and forkhead box O1) increases in the same progression. Concomitant increases occur in 8-hydroxy-2′-deoxyguanosine, point mutations and deletions in kidney mitochondrial DNA, and thiobarbituric acid–reactive substances in plasma, together with decreases in the reduced form of glutathione in erythrocytes. Thus, absence of the bradykinin B2 receptor increases the oxidative stress, mitochondrial DNA damage, and many senescence-associated phenotypes already present in untreated Akita diabetic mice.
Masao Kakoki, Catherine M. Kizer, Xianwen Yi, Nobuyuki Takahashi, Hyung-Suk Kim, C. Robert Bagnell, Cora-Jean S. Edgell, Nobuyo Maeda, J. Charles Jennette, Oliver Smithies
Amorphic mutations in the recombination activating genes RAG1 and RAG2 have been reported to cause T–B– SCID, whereas hypomorphic mutations led to the expansion of a few autoimmune T cell clones responsible for the Omenn syndrome phenotype. We report here a novel clinical and immunological phenotype associated with recessive RAG1 hypomorphic mutations in 4 patients from 4 different families. The immunological phenotype consists of the oligoclonal expansion of TCRγδ T cells combined with TCRαβ T cell lymphopenia. The clinical phenotype consists of severe, disseminated CMV infection and autoimmune blood cell manifestations. Repertoire studies suggest that CMV infection, in the setting of this particular T cell immunodeficiency, may have driven the TCRγδ T cell clonal expansion. This observation extends the range of clinical and immunological phenotypes associated with RAG mutations, emphasizing the role of the genetic background and microbial environment in determining disease phenotype.
Jean-Pierre de Villartay, Annick Lim, Hamoud Al-Mousa, Sophie Dupont, Julie Déchanet-Merville, Edith Coumau-Gatbois, Marie-Lise Gougeon, Arnaud Lemainque, Céline Eidenschenk, Emmanuelle Jouanguy, Laurent Abel, Jean-Laurent Casanova, Alain Fischer, Françoise Le Deist
The Ink4a/Arf locus encodes 2 tumor suppressor molecules, p16INK4a and Arf, which are principal mediators of cellular senescence. To study the links between senescence and aging in vivo, we examined Ink4a/Arf expression in rodent models of aging. We show that expression of p16INK4a and Arf markedly increases in almost all rodent tissues with advancing age, while there is little or no change in the expression of other related cell cycle inhibitors. The increase in expression is restricted to well-defined compartments within each organ studied and occurs in both epithelial and stromal cells of diverse lineages. The age-associated increase in expression of p16INK4a and Arf is attenuated in the kidney, ovary, and heart by caloric restriction, and this decrease correlates with diminished expression of an in vivo marker of senescence, as well as decreased pathology of those organs. Last, the age-related increase in Ink4a/Arf expression can be independently attributed to the expression of Ets-1, a known p16INK4a transcriptional activator, as well as unknown Ink4a/Arf coregulatory molecules. These data suggest that expression of the Ink4a/Arf tumor suppressor locus is a robust biomarker, and possible effector, of mammalian aging.
Janakiraman Krishnamurthy, Chad Torrice, Matthew R. Ramsey, Grigoriy I. Kovalev, Khalid Al-Regaiey, Lishan Su, Norman E. Sharpless
The enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) is selectively expressed in aldosterone target tissues, where it confers aldosterone selectivity for the mineralocorticoid receptor by inactivating 11β-hydroxyglucocorticoids. Variable activity of 11βHSD2 is relevant for blood pressure control and hypertension. The present investigation aimed to elucidate whether an epigenetic mechanism, DNA methylation, accounts for the rigorous control of expression of the gene encoding 11βHSD2, HSD11B2. CpG islands covering the promoter and exon 1 of HSD11B2 were found to be densely methylated in tissues and cell lines with low expression but not those with high expression of HSD11B2. Demethylation induced by 5-aza-2′-deoxycytidine and procainamide enhanced the transcription and activity of the 11βHSD2 enzyme in human cells in vitro and in rats in vivo. Methylation of HSD11B2 promoter–luciferase constructs decreased transcriptional activity. Methylation of recognition sequences of transcription factors, including those for Sp1/Sp3, Arnt, and nuclear factor 1 (NF1) diminished their DNA-binding activity. Herein NF1 was identified as a strong HSD11B2 stimulatory factor. The effect of NF1 was dependent on the position of CpGs and the combination of CpGs methylated. A methylated-CpG–binding protein complex 1 transcriptional repression interacted directly with the methylated HSD11B2 promoter. These results indicate a role for DNA methylation in HSD11B2 gene repression and suggest an epigenetic mechanism affecting this gene causally linked with hypertension.
Rasoul Alikhani-Koopaei, Fatemeh Fouladkou, Felix J. Frey, Brigitte M. Frey
Here we describe the effect of immunization with dendritic cells loaded with syngeneic tumor cells (DC/Ts) by polyethylene glycol treatment, on tumor development in adenomatous polyposis coli (APC) gene mutant mouse models, APC1309 and APCMin–/+, in which adenomatous polyps of the gastrointestinal tracts develop with a high incidence. Treatment with DC/Ts prevented the development of gastrointestinal tumors, and coadministration of DC/Ts and IL-12 caused a further reduction in tumor incidence. Splenocytes from APC1309 mice treated with DC/Ts and IL-12 showed no cytotoxic activity toward the tumor cells, but serum antibody specific to them was detected. IgG from the treated mice exhibited cytotoxic activity against the tumor cells in vitro. Predominance of Th2 cell response over Th1 response was also suggested by ELISPOT assays in the treated mice. Depletion in vivo of CD4+ T cells, not CD8+ T cells, by the intraperitoneal administration of corresponding mAb’s decreased the antitumor effect of DC/T inoculation. Immunofluorescence microscopic studies showed that Ig was attached to tumor cells in mice treated with DC/Ts and IL-12. These findings indicate that DC/T vaccination prevents tumor development through APC gene mutation and that its preventive effects are mediated by humoral antitumor immunity.
Toshio Iinuma, Sadamu Homma, Tetsuo Noda, Donald Kufe, Tsuneya Ohno, Gotaro Toda
How ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) lower plasma lipid levels is incompletely understood. We previously showed that marine ω-3 PUFAs (docosahexaenoic acid [DHA] and eicosapentaenoic acid) stimulate a novel pathway, post-ER presecretory proteolysis (PERPP), that degrades apolipoprotein B100 (ApoB100), thereby reducing lipoprotein secretion from liver cells. To identify signals stimulating PERPP, we examined known actions of ω-3 PUFA. In rat hepatoma or primary rodent hepatocytes incubated with ω-3 PUFA, cotreatment with the iron chelator desferrioxamine, an inhibitor of iron-dependent lipid peroxidation, or vitamin E, a lipid antioxidant, suppressed increases in thiobarbituric acid–reactive substances (TBARSs; a measure of lipid peroxidation products) and restored ApoB100 recovery and VLDL secretion. Moreover, ω-6 and nonmarine ω-3 PUFA, also prone to peroxidation, increased ApoB100 degradation via intracellular induction of TBARSs. Even without added fatty acids, degradation of ApoB100 in primary hepatocytes was blocked by desferrioxamine or antioxidant cotreatment. To extend these results in vivo, mice were infused with DHA, which increased hepatic TBARSs and reduced VLDL-ApoB100 secretion. These results establish a novel link between lipid peroxidation and oxidant stress with ApoB100 degradation via PERPP, and may be relevant to the hypolipidemic actions of dietary PUFAs, the basal regulation of ApoB100 secretion, and hyperlipidemias arising from ApoB100 overproduction.
Meihui Pan, Arthur I. Cederbaum, Yuan-Li Zhang, Henry N. Ginsberg, Kevin Jon Williams, Edward A. Fisher
Recent reports of tumor regression following delivery of autologous tumor antigen–pulsed DCs suggest that defective antigen presentation may play a key role in tumor escape. Here we show in two different murine tumor models, CT26 (colon adenocarcinoma) and B16 (melanoma), that the number and activation state of intratumoral DCs are critical factors in the host response to tumors. We used CCL20/macrophage inflammatory protein-3α (MIP-3α) chemokine to increase the number of tumoral DCs and intratumoral injections of CG-rich motifs (CpGs) to activate such cells. Expression of CCL20 in the tumor site attracted large numbers of circulating DCs into the tumor mass and, in the case of CT26 tumors, led to complete tumor regression. Intratumoral CpG injections, in addition to CCL20, were required to induce therapeutic immunity against B16 tumors. In this model CpG overcame tumor-mediated inhibition of DC activation and enabled tumoral DCs to cross-present tumor antigens to naive CD8 T cells. CpG activation of tumoral DCs alone was not sufficient to induce tumor regression in either tumor model, nor was systemic delivery of the DC growth factor, Flt3 ligand, which dramatically increased the number of circulating DCs but not the number of tumoral DCs. These results indicate that the number of tumoral DCs as well as the tumor milieu determines the ability of tumor-bearing hosts to mount an effective antitumor immune response. Our results also suggest that DCs can be manipulated in vivo without delivery of defined tumor antigens to induce a specific T cell–mediated antitumor response and provide the basis for the use of chemokines in DC-targeted clinical strategies.
Katsuyoshi Furumoto, Luis Soares, Edgar G. Engleman, Miriam Merad
Cellular acquisition of folate is mediated by folate receptors (FRs) in many malignant and normal human cells. Although FRs are upregulated in folate deficiency and downregulated following folate repletion, the mechanistic basis for this relationship is unclear. Previously we demonstrated that interaction of an 18-base cis-element in the 5′-untranslated region of FR mRNA and a cystolic trans-factor (heterogeneous nuclear ribonucleoprotein E1 [hnRNP E1]) is critical for FR synthesis. However, the molecular mechanisms controlling this interaction, especially within the context of FR regulation and folate status, have remained obscure. Human cervical carcinoma cells exhibited progressively increasing upregulation of FRs after shifting of folate-replete cells to low-folate media, without a proportionate rise in FR mRNA or rise in hnRNP E1. Translational FR upregulation was accompanied by a progressive accumulation of the metabolite homocysteine within cultured cells, which stimulated interaction of the FR mRNA cis-element and hnRNP E1 as well as FR biosynthesis in a dose-dependent manner. Abrupt reversal of folate deficiency also led to a rapid parallel reduction in homocysteine and FR biosynthesis to levels observed in folate-replete cells. Collectively, these results suggest that homocysteine is the key modulator of translational upregulation of FRs and establishes the linkage between perturbed folate metabolism and coordinated upregulation of FRs.
Aśok C. Antony, Ying-Sheng Tang, Rehana A. Khan, Mangatt P. Biju, Xiangli Xiao, Qing-Jun Li, Xin-Lai Sun, Hiremagalur N. Jayaram, Sally P. Stabler
Viruses can cause but can also prevent autoimmune disease. This dualism has certainly hampered attempts to establish a causal relationship between viral infections and type 1 diabetes (T1D). To develop a better mechanistic understanding of how viruses can influence the development of autoimmune disease, we exposed prediabetic mice to various viral infections. We used the well-established NOD and transgenic RIP-LCMV models of autoimmune diabetes. In both cases, infection with the lymphocytic choriomeningitis virus (LCMV) completely abrogated the diabetic process. Interestingly, such therapeutic viral infections resulted in a rapid recruitment of T lymphocytes from the islet infiltrate to the pancreatic draining lymph node, where increased apoptosis was occurring. In both models this was associated with a selective and extensive expression of the chemokine IP-10 (CXCL10), which predominantly attracts activated T lymphocytes, in the pancreatic draining lymph node, and in RIP-LCMV mice it depended on the viral antigenic load. In RIP-LCMV mice, blockade of TNF-α or IFN-γ in vivo abolished the prevention of T1D. Thus, virally induced proinflammatory cytokines and chemokines can influence the ongoing autoaggressive process beneficially at the preclinical stage, if produced at the correct location, time, and levels.
Urs Christen, Dirk Benke, Tom Wolfe, Evelyn Rodrigo, Antje Rhode, Anna C. Hughes, Michael B.A. Oldstone, Matthias G. von Herrath
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