A proteolytic fragment of histone deacetylase 4 protects the heart from failure by regulating the hexosamine biosynthetic pathway.

The stress-responsive epigenetic repressor histone deacetylase 4 (HDAC4) regulates cardiac gene expression. Here we show that the levels of an N-terminal proteolytically derived fragment of HDAC4, termed HDAC4-NT, are lower in failing mouse hearts than in healthy control hearts. Virus-mediated transfer of the portion of the Hdac4 gene encoding HDAC4-NT into the mouse myocardium protected the heart from remodeling and failure; this was associated with decreased expression of Nr4a1, which encodes a nuclear orphan receptor, and decreased NR4A1-dependent activation of the hexosamine biosynthetic pathway (HBP). Conversely, exercise enhanced HDAC4-NT levels, and mice with a cardiomyocyte-specific deletion of Hdac4 show reduced exercise capacity, which was characterized by cardiac fatigue and increased expression of Nr4a1. Mechanistically, we found that NR4A1 negatively regulated contractile function in a manner that depended on the HBP and the calcium sensor STIM1. Our work describes a new regulatory axis in which epigenetic regulation of a metabolic pathway affects calcium handling. Activation of this axis during intermittent physiological stress promotes cardiac function, whereas its impairment in sustained pathological cardiac stress leads to heart failure.

An AMP-activated protein kinase-stabilizing peptide ameliorates adipose tissue wasting in cancer cachexia in mice.

Cachexia represents a fatal energy-wasting syndrome in a large number of patients with cancer that mostly results in a pathological loss of skeletal muscle and adipose tissue. Here we show that tumor cell exposure and tumor growth in mice triggered a futile energy-wasting cycle in cultured white adipocytes and white adipose tissue (WAT), respectively. Although uncoupling protein 1 (Ucp1)-dependent thermogenesis was dispensable for tumor-induced body wasting, WAT from cachectic mice and tumor-cell-supernatant-treated adipocytes were consistently characterized by the simultaneous induction of both lipolytic and lipogenic pathways. Paradoxically, this was accompanied by an inactivated AMP-activated protein kinase (Ampk), which is normally activated in peripheral tissues during states of low cellular energy. Ampk inactivation correlated with its degradation and with upregulation of the Ampk-interacting protein Cidea. Therefore, we developed an Ampk-stabilizing peptide, ACIP, which was able to ameliorate WAT wasting in vitro and in vivo by shielding the Cidea-targeted interaction surface on Ampk. Thus, our data establish the Ucp1-independent remodeling of adipocyte lipid homeostasis as a key event in tumor-induced WAT wasting, and we propose the ACIP-dependent preservation of Ampk integrity in the WAT as a concept in future therapies for cachexia.

Ataxin-10 is part of a cachexokine cocktail triggering cardiac metabolic dysfunction in cancer cachexia.

OBJECTIVES: Cancer cachexia affects the majority of tumor patients and significantly contributes to high mortality rates in these subjects. Despite its clinical importance, the identity of tumor-borne signals and their impact on specific peripheral organ systems, particularly the heart, remain mostly unknown. METHODS AND RESULTS: By combining differential colon cancer cell secretome profiling with large-scale cardiomyocyte phenotyping, we identified a signature panel of seven "cachexokines", including Bridging integrator 1, Syntaxin 7, Multiple inositol-polyphosphate phosphatase 1, Glucosidase alpha acid, Chemokine ligand 2, Adamts like 4, and Ataxin-10, which were both sufficient and necessary to trigger cardiac atrophy and aberrant fatty acid metabolism in cardiomyocytes. As a prototypical example, engineered secretion of Ataxin-10 from non-cachexia-inducing cells was sufficient to induce cachexia phenotypes in cardiomyocytes, correlating with elevated Ataxin-10 serum levels in murine and human cancer cachexia models. CONCLUSIONS: As Ataxin-10 serum levels were also found to be elevated in human cachectic cancer patients, the identification of Ataxin-10 as part of a cachexokine cocktail now provides a rational approach towards personalized predictive, diagnostic and therapeutic measures in cancer cachexia.

Hepatic transforming growth factor-ß 1 stimulated clone-22 D1 controls systemic cholesterol metabolism.

Disturbances in lipid homeostasis are hallmarks of severe metabolic disorders and their long-term complications, including obesity, diabetes, and atherosclerosis. Whereas elevation of triglyceride (TG)-rich very-low-density lipoproteins (VLDL) has been identified as a risk factor for cardiovascular complications, high-density lipoprotein (HDL)-associated cholesterol confers atheroprotection under obese and/or diabetic conditions. Here we show that hepatocyte-specific deficiency of transcription factor transforming growth factor ß 1-stimulated clone (TSC) 22 D1 led to a substantial reduction in HDL levels in both wild-type and obese mice, mediated through the transcriptional down-regulation of the HDL formation pathway in liver. Indeed, overexpression of TSC22D1 promoted high levels of HDL cholesterol in healthy animals, and hepatic expression of TSC22D1 was found to be aberrantly regulated in disease models of opposing energy availability. The hepatic TSC22D1 transcription factor complex may thus represent an attractive target in HDL raising strategies in obesity/diabetes-related dyslipidemia and atheroprotection.

TSC22D4 is a molecular output of hepatic wasting metabolism.

In mammals, proper storage and distribution of lipids in and between tissues is essential for the maintenance of energy homeostasis. Here, we show that tumour growth triggers hepatic metabolic dysfunction as part of the cancer cachectic phenotype, particularly by reduced hepatic very-low-density-lipoprotein (VLDL) secretion and hypobetalipoproteinemia. As a molecular cachexia output pathway, hepatic levels of the transcription factor transforming growth factor beta 1-stimulated clone (TSC) 22 D4 were increased in cancer cachexia. Mimicking high cachectic levels of TSC22D4 in healthy livers led to the inhibition of hepatic VLDL release and lipogenic genes, and diminished systemic VLDL levels under both normal and high fat dietary conditions. Liver-specific ablation of TSC22D4 triggered hypertriglyceridemia through the induction of hepatic VLDL secretion. Furthermore, hepatic TSC22D4 expression levels were correlated with the degree of body weight loss and VLDL hypo-secretion in cancer cachexia, and TSC22D4 deficiency rescued tumour cell-induced metabolic dysfunction in hepatocytes. Therefore, hepatic TSC22D4 activity may represent a molecular rationale for peripheral energy deprivation in subjects with metabolic wasting diseases, including cancer cachexia.

Immature and maturation-resistant human dendritic cells generated from bone marrow require two stimulations to induce T cell anergy in vitro.

Immature dendritic cells (DC) represent potential clinical tools for tolerogenic cellular immunotherapy in both transplantation and autoimmunity. A major drawback in vivo is their potential to mature during infections or inflammation, which would convert their tolerogenicity into immunogenicity. The generation of immature DC from human bone marrow (BM) by low doses of GM-CSF (lowGM) in the absence of IL-4 under GMP conditions create DC resistant to maturation, detected by surface marker expression and primary stimulation by allogeneic T cells. This resistence could not be observed for BM-derived DC generated with high doses of GM-CSF plus IL-4 (highGM/4), although both DC types induced primary allogeneic T cell anergy in vitro. The estabishment of the anergic state requires two subsequent stimulations by immature DC. Anergy induction was more profound with lowGM-DC due to their maturation resistance. Together, we show the generation of immature, maturation-resistant lowGM-DC for potential clinical use in transplant rejection and propose a two-step-model of T cell anergy induction by immature DC.

An improved method to generate equine dendritic cells from peripheral blood mononuclear cells: divergent maturation programs by IL-4 and LPS.

Equine dendritic cells (eqDC) can be generated from peripheral blood monocytes by propagation in GM-CSF and IL-4. Despite similarities with the generation of human DC, we found significant improvements for eqDC generation and functional influences on eqDC maturation. The fractionation of peripheral blood mononuclear cells (PBMC) by two subsequent gradients at densities of 1.090 and 1.077 as well as an adherence step in AIM V((R)) medium on dishes coated with extracellular matrix components (Primaria) improved the purity and yield of DC. After 3 days, eqDC cultures with GM-CSF alone developed into three subsets of (i) MHC II(neg) cells, (ii) MHC II(low) immature, endocytic cells and (iii) MHC II(high) spontaneously mature, non-endocytic DC. The immature DC fraction of the GM-CSF cultures matured, as detected by MHC II up-regulation, upon LPS exposure overnight. DC cultures in GM-CSF plus IL-4 resulted in higher cell yields, a loss of the immature MHC II(low) population but increased mature MHC II(high) DC, suggesting maturation. However, the MHC II(high) DC fraction was still endocytically active and did not lose their endocytic function after LPS treatment. They marginally up-regulated MHC II expression but this did not result in an enhanced stimulation of an allogeneic mixed lymphocyte reaction. However, LPS treatment clearly induced mRNA for IL-12p35 and p40, which was not observed by addition of IL-4 alone. Together our data indicate that IL-4 and LPS induce two different maturation programs. IL-4 induces a semi-maturation where the cells are still endocytic, which can be further matured to secrete cytokines in a second step by LPS.

PKCtheta and PKA are antagonistic partners in the NF-AT transactivation pathway of primary mouse CD3+ T lymphocytes.

We here investigate the crosstalk of PKC and PKA signaling during primary CD3(+) T-lymphocyte activation using pharmacologic inhibitors and activators in combination with our established panel of PKC isotype-deficient mouse T cells in vitro. PKCtheta and PKA inversely affect the CD3/CD28-induced IL-2 expression, whereas other PKC isotypes are dispensable in this signaling pathway. Gene ablation of PKCtheta selectively results in a profound reduction of IL-2 production; however, complete abrogation of IL-2 production in these PKCtheta(-/-) T cells was achieved only by simultaneous coactivation of the cAMP/PKA pathway in CD3(+) T cells. Conversely, the reduced IL-2 production in PKC inhibitor-treated T cells can be rescued by inhibition of the cAMP/PKA pathway in wild-type but not in PKCtheta(-/-) T cells. Mechanistically, the cAMP/PKA and PKCtheta pathways converge at the level of NF-AT, as shown by DNA binding analysis. The combined increase in PKA and decrease in PKCtheta activity leads to an enhanced inhibition of nuclear NF-AT translocation. This PKCtheta/PKA crosstalk significantly affects neither the NF-kappaB, the AP-1, nor the CREB pathways. Taken together, this opposite effect between the positive PKCtheta and the negative cAMP/PKA signaling pathways appears rate limiting for NF-AT transactivation and IL-2 secretion responses of CD3(+) T lymphocytes.