Apoptosis may underlie the pathology of zinc-deficient skin

The trace element zinc is essential for the survival and function of all cells. Zinc deficiency, whether nutritional or genetic, is fatal if left untreated. The effects of zinc deficiency are particularly obvious in the skin, seen as an erythematous rash, scaly plaques, and ulcers. Electron microscopy reveals degenerative changes within keratinocytes. Despite the well-documented association between zinc deficiency and skin pathology, it is not clear which cellular processes are most sensitive to zinc deficiency and could account for the typical pathological features. We used the cultured HaCaT keratinocyte line to obtain insight into the cellular effects of zinc deficiency, as these cells show many characteristics of normal skin keratinocytes. Zinc deficiency was induced by growing cells in the presence of the zinc chelator, TPEN, or by growth in zinc-deficient medium. Growth of cells in zinc-deficient medium resulted in a 44% reduction of intracellular zinc levels and a 75% reduction in the activity of the zinc-dependent enzyme, 5'-nucleotidase, relative to the control cells. Over a period of 7 days of exposure to zinc-deficient conditions, no changes in cell viability and growth, or in the cytoskeletal and cell adhesion systems, were found in HaCaT cells. At 7 days, however, induction of apoptosis was indicated by the presence of DNA fragmentation and expression of active caspase-3 in cells. These results demonstrate that apoptosis is the earliest detectable cellular change induced by zinc deficiency in HaCaT keratinocytes. Our observations account for many of the features of zinc deficiency, including the presence of degenerate nuclei, chromatin aggregates and abnormal organization of keratin, that may represent the later stages of apoptosis. In summary, a major causal role for apoptosis in the pathology of zinc deficiency in the skin is proposed. This role is consistent with the previously unexplained diverse range of degenerative cellular changes seen at the ultrastructural level in zinc-deficient keratinocytes.

Targeted disruption of the basic Krüppel-like factor gene (Klf3) reveals a role in adipogenesis

Krüppel-like factors (KLFs) recognize CACCC and GC-rich sequences in gene regulatory elements. Here, we describe the disruption of the murine basic Krüppel-like factor gene (Bklf or Klf3). Klf3 knockout mice have less white adipose tissue, and their fat pads contain smaller and fewer cells. Adipocyte differentiation is altered in murine embryonic fibroblasts from Klf3 knockouts. Klf3 expression was studied in the 3T3-L1 cellular system. Adipocyte differentiation is accompanied by a decline in Klf3 expression, and forced overexpression of Klf3 blocks 3T3-L1 differentiation. Klf3 represses transcription by recruiting C-terminal binding protein (CtBP) corepressors. CtBPs bind NADH and may function as metabolic sensors. A Klf3 mutant that does not bind CtBP cannot block adipogenesis. Other KLFs, Klf2, Klf5, and Klf15, also regulate adipogenesis, and functional CACCC elements occur in key adipogenic genes, including in the C/ebpα promoter. We find that C/ebpα is derepressed in Klf3 and Ctbp knockout fibroblasts and adipocytes from Klf3 knockout mice. Chromatin immunoprecipitations confirm that Klf3 binds the C/ebpα promoter in vivo. These results implicate Klf3 and CtBP in controlling adipogenesis.

AMP-activated protein kinase regulates GLUT4 transcription by phosphorylating histone deacetylase 5

Objective: Insulin resistance associated with obesity and diabetes is ameliorated by specific overexpression of GLUT4 in skeletal muscle. The molecular mechanisms regulating skeletal muscle GLUT4 expression remain to be elucidated. The purpose of this study was to examine these mechanisms.

Research Design and Methods and Results: Here, we report that AMP-activated protein kinase (AMPK) regulates GLUT4 transcription through the histone deacetylase (HDAC)5 transcriptional repressor. Overexpression of HDAC5 represses GLUT4 reporter gene expression, and HDAC inhibition in human primary myotubes increases endogenous GLUT4 gene expression. In vitro kinase assays, site-directed mutagenesis, and site-specific phospho-antibodies establish AMPK as an HDAC5 kinase that targets S259 and S498. Constitutively active but not dominant-negative AMPK and 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR) treatment in human primary myotubes results in HDAC5 phosphorylation at S259 and S498, association with 14-3-3 isoforms, and H3 acetylation. This reduces HDAC5 association with the GLUT4 promoter, as assessed through chromatin immunoprecipitation assays and HDAC5 nuclear export, concomitant with increases in GLUT4 gene expression. Gene reporter assays also confirm that the HDAC5 S259 and S498 sites are required for AICAR induction of GLUT4 transcription.

Conclusions: These data reveal a signal transduction pathway linking cellular energy charge to gene transcription directed at restoring cellular and whole-body energy balance and provide new therapeutic targets for the treatment and management of insulin resistance and type 2 diabetes.

Exercise-induced histone modifications in human skeletal muscle

Skeletal muscle adaptations to exercise confer many of the health benefits of physical activity and occur partly through alterations in skeletal muscle gene expression. The exact mechanisms mediating altered skeletal muscle gene expression in response to exercise are unknown. However, in recent years, chromatin remodelling through epigenetic histone modifications has emerged as a key regulatory mechanism controlling gene expression in general. The purpose of this study was to examine the effect of exercise on global histone modifications that mediate chromatin remodelling and transcriptional activation in human skeletal muscle in response to exercise. In addition, we sought to examine the signalling mechanisms regulating these processes. Following 60 min of cycling, global histone 3 acetylation at lysine 9 and 14, a modification associated with transcriptional initiation, was unchanged from basal levels, but was increased at lysine 36, a site associated with transcriptional elongation. We examined the regulation of the class IIa histone deacetylases (HDACs), which are enzymes that suppress histone acetylation and have been implicated in the adaptations to exercise. While we found no evidence of proteasomal degradation of the class IIa HDACs, we found that HDAC4 and 5 were exported from the nucleus during exercise, thereby removing their transcriptional repressive function. We also observed activation of the AMP-activated protein kinase (AMPK) and the calcium–calmodulin-dependent protein kinase II (CaMKII) in response to exercise, which are two kinases that induce phosphorylation-dependent class IIa HDAC nuclear export. These data delineate a signalling pathway that might mediate skeletal muscle adaptations in response to exercise.

Histone modifications and skeletal muscle metabolic gene expression

1.      Skeletal muscle oxidative function and metabolic gene expression are co-ordinately downregulated in metabolic diseases such as insulin resistance, obesity and Type 2 diabetes. Altering skeletal muscle metabolic gene expression to favour enhanced energy expenditure is considered a potential therapy to combat these diseases.

2.      Histone deacetylases (HDACs) are chromatin-remodelling enzymes that repress gene expression. It has been shown that HDAC4 and 5 co-operatively regulate a number of genes involved in various aspects of metabolism. Understanding how HDACs are regulated provides insights into the mechanisms regulating skeletal muscle metabolic gene expression.

3.      Multiple kinases control phosphorylation-dependent nuclear export of HDACs, rendering them unable to repress transcription. We have found a major role for the AMP-activated protein kinase (AMPK) in response to energetic stress, yet metabolic gene expression is maintained in the absence of AMPK activity. Preliminary evidence suggests a potential role for protein kinase D, also a Class IIa HDAC kinase, in this response.

4.      The HDACs are also regulated by ubiquitin-mediated proteasomal degradation, although the exact mediators of this process have not been identified.

5.      Because HDACs appear to be critical regulators of skeletal muscle metabolic gene expression, HDAC inhibition could be an effective therapy to treat metabolic diseases.

6.      Together, these data show that HDAC4 and 5 are critical regulators of metabolic gene expression and that understanding their regulation could provide a number of points of intervention for therapies designed to treat metabolic diseases, such as insulin resistance, obesity and Type 2 diabetes.

The neuronal noradrenaline transporter, anxiety and cardiovascular disease

Panic disorder can serve as a clinical model for testing whether mental stress can cause heart disease. Potential neural mechanisms of cardiac risk are the sympathetic activation during panic attacks, continuing release of adrenaline as a co-transmitter in the cardiac sympathetic nerves, and impairment of noradrenaline neuronal reuptake, augmenting sympathetic neural respnses.

The phenotype of impaired neuronal reuptake of noradrenaline: an epigenetic mechanism? We suspect that this phenotype, in sensitizing people to heart symptom development, is a cause of panic disorder, and by magnifying the sympathetic neural signal in the heart, underlies increased cardiac risk. No loss of function mutations of the coding region of the norepinephrine transporter (NET) are evident, but we do detect hypermethylation of CpG islands in the NET gene promoter region. Chromatin immunoprecipitation methodology demonstrates binding of the inhibitory transcription factor, MeCP2, to promoter region DNA in panic disorder patients.

Cardiovascular illnesses co-morbid with panic disorder. Panic disorder commonly coexists with essential hypertension and the postural tachycardia syndrome. In both of these cardiovascular disorders the impaired neuronal noradrenaline reuptake phenotype is also present and, as with panic disorder, is associated with NET gene promoter region DNA hypermethylation. An epigenetic ‘co-morbidity’ perhaps underlies the clinical concordance.

Brain neurotransmitters. Using internal jugular venous sampling, in the absence of a panic attack we find normal norepinephrine turnover, but based on measurements of the overflow of the serotonin metabolite, 5HIAA, a marked increase (six to sevenfold) in brain serotonin turnover in patients with panic disorder. This appears to represent the underlying neurotransmitter substrate for the disorder. Whether this brain serotonergic activation is a prime mover, or consequential on other primary causes of panic disorder, including cardiac sensitization by faulty neuronal noradrenaline reuptake leading to cardiac symptoms and the enhanced vigilance which accompanies them, is unclear at present.

Spermatogenesis in the blue swimming crab, Portunus pelagicus, and evidence for histones in mature sperm nuclei

Spermatogenesis in the blue swimming crab, Portunus pelagicus, is described by light and electron microscopy. The testis is composed of anterior (AT) and posterior (PT) lobes, that are partitioned into lobules by connective tissue trabecula, and further divided into zones (germinal, transformation and evacuation), each with various stages of cellular differentiation. The vas deferens is classified into three distinct regions: anterior (AVD), median (MVD), and posterior (PVD), on the presence of spermatophores and two secretions, termed substance I and II. Based on the degree and patterns of heterochromatin, spermatogenesis is classified into 13 stages: two spermatogonia (SgA and SgB), six primary spermatocytes (leptotene, zygotene, pachytene, diplotene, diakinesis, and metaphase), a secondary spermatocyte (SSc), three spermatids (St 1–3), and a mature spermatozoon. Spermatid stages are differentiated by chromatin decondensation and the formation of an acrosomal complex, which is unique to brachyurans. Mature spermatozoa are aflagellated, and have a nuclear projection and a spherical acrosome. AUT-PAGE and Western blots show that, during chromatin decondensation, there is a reduction of most histones, with only small amounts of H2B and H3 remaining in mature spermatozoa.

Repeated social defeat selectively increases ΔFosB expression and histone H3 acetylation in the infralimbic medial prefrontal cortex

Exposure to social stress has been linked to the development and maintenance of mood-related psychopathology; however, the underlying neurobiological changes remain uncertain. In this study, we examined numbers of ΔFosB-immunoreactive cells in the forebrains of rats subjected to 12 episodes of social defeat. This was achieved using the social conflict model whereby animals are introduced into the home cage of older males (“residents”) trained to attack and defeat all such “intruders”; importantly, controls were treated identically except that the resident was absent. Our results indicated that the only region in which ΔFosB-positive cells were found in significantly higher numbers in intruders than in controls was the infralimbic medial prefrontal cortex (mPFC). This same effect was not apparent using another psychological stressor, noise stress. Cells of the infralimbic mPFC also displayed evidence of chromatin remodeling. We found that exposure to repeated episodes of social defeat increased numbers of cells immunoreactive for histone H3 acetylation, but not for histone H3 phosphoacetylation, in the infralimbic mPFC. Collectively, these findings highlight the importance of the infralimbic mPFC in responding to social stress—a finding that provides insight into the possible neurobiological alterations associated with stress-induced psychiatric illness.

The COMPASS subunit Spp1 links histone methylation to initiation of meiotic recombination

During meiosis, combinatorial associations of genetic traits arise from homologous recombination between parental chromosomes. Histone H3 lysine 4 trimethylation marks meiotic recombination hotspots in yeast and mammals, but how this ubiquitous chromatin modification relates to the initiation of double-strand breaks (DSBs) dependent on Spo11 remains unknown. Here, we show that the tethering of a PHD-containing protein, Spp1 (a component of the COMPASS complex), to recombinationally cold regions is sufficient to induce DSB formation. Furthermore, we found that Spp1 physically interacts with Mer2, a key protein of the differentiated chromosomal axis required for DSB formation. Thus, by interacting with H3K4me3 and Mer2, Spp1 promotes recruitment of potential meiotic DSB sites to the chromosomal axis, allowing Spo11 cleavage at nearby nucleosome-depleted regions.

Imprinted CDKN1C is a tumor suppressor in rhabdoid tumor and activated by restoration of SMARCB1 and histone deacetylase inhibitors

SMARCB1 is deleted in rhabdoid tumor, an aggressive paediatric malignancy affecting the kidney and CNS. We hypothesized that the oncogenic pathway in rhabdoid tumors involved epigenetic silencing of key cell cycle regulators as a consequence of altered chromatin-remodelling, attributable to loss of SMARCB1, and that this hypothesis if proven could provide a biological rationale for testing epigenetic therapies in this disease. We used an inducible expression system to show that the imprinted cell cycle inhibitor CDKN1C is a downstream target for SMARCB1 and is transcriptionally activated by increased histone H3 and H4 acetylation at the promoter. We also show that CDKN1C expression induces cell cycle arrest, CDKN1C knockdown with siRNA is associated with increased proliferation, and is able to compete against the anti-proliferative effect of restored SMARCB1 expression. The histone deacetylase inhibitor (HDACi), Romidepsin, specifically restored CDKN1C expression in rhabdoid tumor cells through promoter histone H3 and H4 acetylation, recapitulating the effect of SMARCB1 on CDKNIC allelic expression, and induced cell cycle arrest in G401 and STM91-01 rhabdoid tumor cell lines. CDKN1C expression was also shown to be generally absent in clinical specimens of rhabdoid tumor, however CDKN1A and CDKN1B expression persisted. Our observations suggest that maintenance of CDKN1C expression plays a critical role in preventing rhabdoid tumor growth. Significantly, we report for the first time, parallels between the molecular pathways of SMARCB1 restoration and Romidepsin treatment, and demonstrate a biological basis for the further exploration of histone deacetylase inhibitors as relevant therapeutic reagents in the treatment of rhabdoid tumor.

The small molecule, genistein, increases hepcidin expression in human hepatocytes

Hepcidin, a peptide hormone that decreases intestinal iron absorption and macrophage iron release, is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. Endogenous stimulants of Hepcidin transcription include bone morphogenic protein 6 (BMP6) and interleukin-6 (IL-6) by effects on mothers against decapentaplegic homolog (Smad)4 or signal transducer and activator of transcription (Stat)3, respectively. We conducted a small-scale chemical screen in zebrafish embryos to identify small molecules that modulate hepcidin expression. We found that treatment with the isoflavone, genistein, from 28-52 hours postfertilization in zebrafish embryos enhanced Hepcidin transcript levels, as assessed by whole-mount in situ hybridization and quantitative real-time reverse-transcriptase polymerase chain reaction. Genistein's stimulatory effect was conserved in human hepatocytes: Genistein treatment of HepG2 cells increased both Hepcidin transcript levels and promoter activity. We found that genistein's effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either BMP response elements or the Stat3-binding site in the Hepcidin promoter. RNA sequencing of transcripts from genistein-treated hepatocytes indicated that genistein up-regulated 68% of the transcripts that were up-regulated by BMP6; however, genistein raised levels of several transcripts involved in Stat3 signaling that were not up-regulated by BMP6. Chromatin immunoprecipitation and ELISA experiments revealed that genistein enhanced Stat3 binding to the Hepcidin promoter and increased phosphorylation of Stat3 in HepG2 cells. Conclusion: Genistein is the first small-molecule experimental drug that stimulates Hepcidin expression in vivo and in vitro. These experiments demonstrate the feasibility of identifying and characterizing small molecules that increase Hepcidin expression. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes.