AMP-activated Protein Kinase Up-regulates Mitogen-activated Protein (MAP) Kinase-interacting Serine/Threonine Kinase 1a-dependent Phosphorylation of Eukaryotic Translation Initiation Factor 4E.

AMP-activated protein kinase (AMPK) is a molecular energy sensor that acts to sustain cellular energy balance. Although AMPK is implicated in the regulation of a multitude of ATP-dependent cellular processes, exactly how these processes are controlled by AMPK as well as the identity of AMPK targets and pathways continues to evolve. Here we identify MAP kinase-interacting serine/threonine protein kinase 1a (MNK1a) as a novel AMPK target. Specifically, we show AMPK-dependent Ser(353) phosphorylation of the human MNK1a isoform in cell-free and cellular systems. We show that AMPK and MNK1a physically interact and that in vivo MNK1a-Ser(353) phosphorylation requires T-loop phosphorylation, in good agreement with a recently proposed structural regulatory model of MNK1a. Our data suggest a physiological role for MNK1a-Ser(353) phosphorylation in regulation of the MNK1a kinase, which correlates with increased eIF4E phosphorylation in vitro and in vivo.

The interaction between AMPKß2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content.

AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKß at Thr-148, which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPKß1 binds to R6 (PPP1R3D), a glycogen-targeting subunit of protein phosphatase type 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. In the present study, we further investigated the interaction of R6 with AMPKß and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid (Y2H) analyses and co-immunoprecipitation (IP) of the overexpressed proteins in human embryonic kidney (HEK) 293T) cells revealed that both AMPKß1 and AMPK-ß2 wild-type (WT) isoforms bind to R6. The AMPKß-R6 interaction was stronger with the muscle-specific AMPKß2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPKß2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPKß2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced the AMPKß2-R6 interaction in conjunction with increased Thr-148 phosphorylation in cells grown in low-glucose medium. These data are in accordance with R6 binding directly to AMPKß2 when both proteins detach from the diminishing glycogen particle, which is simultaneous with increased AMPKß2 Thr-148 autophosphorylation. Such a model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle.

Quantitative analysis of ChIP-seq data uncovers dynamic and sustained H3K4me3 and H3K27me3 modulation in cancer cells under hypoxia.

BACKGROUND: A comprehensive assessment of the epigenetic dynamics in cancer cells is the key to understanding the molecular mechanisms underlying cancer and to improving cancer diagnostics, prognostics and treatment. By combining genome-wide ChIP-seq epigenomics and microarray transcriptomics, we studied the effects of oxygen deprivation and subsequent reoxygenation on histone 3 trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3) in a breast cancer cell line, serving as a model for abnormal oxygenation in solid tumors. A priori, epigenetic markings and gene expression levels not only are expected to vary greatly between hypoxic and normoxic conditions, but also display a large degree of heterogeneity across the cell population. Where traditionally ChIP-seq data are often treated as dichotomous data, the model and experiment here necessitate a quantitative, data-driven analysis of both datasets. RESULTS: We first identified genomic regions with sustained epigenetic markings, which provided a sample-specific reference enabling quantitative ChIP-seq data analysis. Sustained H3K27me3 marking was located around centromeres and intergenic regions, while sustained H3K4me3 marking is associated with genes involved in RNA binding, translation and protein transport and localization. Dynamic marking with both H3K4me3 and H3K27me3 (hypoxia-induced bivalency) was found in CpG-rich regions at loci encoding factors that control developmental processes, congruent with observations in embryonic stem cells. CONCLUSIONS: In silico-identified epigenetically sustained and dynamic genomic regions were confirmed through ChIP-PCR in vitro, and obtained results are corroborated by published data and current insights regarding epigenetic regulation.

Hypoxia increases genome-wide bivalent epigenetic marking by specific gain of H3K27me3.

BACKGROUND: Trimethylation at histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3) controls gene activity during development and differentiation. Whether H3K4me3 and H3K27me3 changes dynamically in response to altered microenvironmental conditions, including low-oxygen conditions commonly present in solid tumors, is relatively unknown. Demethylation of H3K4me3 and H3K27me3 is mediated by oxygen and 2-oxoglutarate dioxygenases enzymes, suggesting that oxygen deprivation (hypoxia) may influence histone trimethylation. Using the MCF7 breast epithelial adenocarcinoma cell model, we have determined the relationship between epigenomic and transcriptomic reprogramming as a function of fluctuating oxygen tension. RESULTS: We find that in MCF7, H3K4me3 and H3K27me3 marks rapidly increase at specific locations throughout the genome and are largely reversed upon reoxygenation. Whereas dynamic changes are relatively highest for H3K27me3 marking under hypoxic conditions, H3K4me3 occupation is identified as the defining epigenetic marker of transcriptional control. In agreement with the global increase of H3K27 trimethylation, we provide direct evidence that the histone H3K27me3 demethylase KDM6B/JMJD3 is inactivated by limited oxygen. In situ immunohistochemical analysis confirms a marked rise of histone trimethylation in hypoxic tumor areas. Acquisition of H3K27me3 at H3K4me3-marked loci results in a striking increase in "bivalent" epigenetic marking. Hypoxia-induced bivalency substantially overlaps with embryonal stem cell-associated genic bivalency and is retained at numerous loci upon reoxygenation. Transcriptional activity is selectively and progressively dampened at bivalently marked loci upon repeated exposure to hypoxia, indicating that this subset of genes uniquely maintains the potential for epigenetic regulation by KDM activity. CONCLUSIONS: These data suggest that dynamic regulation of the epigenetic state within the tumor environment may have important consequences for tumor plasticity and biology.

Contribution of fatty acids released from lipolysis of plasma triglycerides to total plasma fatty acid flux and tissue-specific fatty acid uptake.

There is controversy over the extent to which fatty acids (FAs) derived from plasma free FAs (FFAs) or from hydrolysis of plasma triglycerides (TGFAs) form communal or separate pools and what the contribution of each FA source is to cellular FA metabolism. Chylomicrons and lipid emulsions were labeled with [(3)H]triolein, injected into mice, and appearance in plasma of [(3)H]oleic acid was estimated, either through a steady-state approach or by compartmental modeling. [(14)C]oleic acid was included to trace plasma FFA. Eighty to 90% of triglyceride (TG) label was recovered in plasma, irrespective of tracer method or TG source. The contribution of TG lipolysis to total plasma FA turnover was 10-20%. After infusion of [(3)H]TG and [(14)C]FA, the retention of these labels varied substantially among liver, adipose tissue, and skeletal and heart muscle. Retention of TG label changed during fasting in the same direction as lipoprotein lipase (LPL) activity is regulated. We propose a model that reconciles the paradoxical 80-90% loss of TG label into plasma with LPL-directed differential uptake of TGFA in tissues. In this model, TGFAs mix locally at the capillaries with plasma FFAs, where they would lead to an increase in the local FA concentration, and hence, FA uptake. Our data indicate that a distinction between TG-derived FA and plasma FFA cannot be made.

MK3 modulation affects BMI1-dependent and independent cell cycle check-points.

Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others. The relevance of MK3 for oncogenesis is currently not clear. We recently reported that MK3 controls ERK activity via a negative feedback mechanism. This prompted us to investigate a potential role for MK3 in cell proliferation. We here show that overexpression of MK3 induces a proliferative arrest in normal diploid human fibroblasts, characterized by enhanced expression of replication stress- and senescence-associated markers. Surprisingly, MK3 depletion evokes similar senescence characteristics in the fibroblast model. We previously identified MK3 as a binding partner of Polycomb Repressive Complex 1 (PRC1) proteins. In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus. In agreement with this, the PRC1 oncoprotein BMI1, but not the PCR2 protein EZH2, bypasses MK3-induced senescence in fibroblasts and suppresses P16INK4A expression. In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function. Notably, MK3 ablation enhances proliferation in two different cancer cells. Finally, the fibroblast model was used to evaluate the effect of potential tumorigenic MK3 driver-mutations on cell proliferation and M/SAPK signaling imbalance. Taken together, our findings support a role for MK3 in control of proliferation and replicative life-span, in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/SAPK signaling and, ultimately, proliferation, is cell context-dependent.

HOE 402 lowers serum cholesterol levels by reducing VLDL-lipid production, and not by induction of the LDL receptor, and reduces atherosclerosis in wild-type and LDL receptor-deficient mice.

Previous rodent studies suggested that the potent hypolipidemic agent 4-amino-2-(4,4-dimethyl-2-oxo-1-imidazolidinyl)pyrimidine-5-N-(trifluoromethyl-phenyl) carboxamide monohydrochloride (HOE 402) is an inducer of the LDL receptor (LDLR). Using wild-type and heterozygous and homozygous LDLR-deficient (LDLR+/0 and LDLR0/0) mice, fed a low or high cholesterol diet, we investigated whether HOE 402 specifically induces the LDLR and whether other pathways are affected. Upon treatment with 0.05% (w/w) HOE 402, the serum cholesterol levels of wild-type, LDLR+/0 and LDLR0/0 mice, were maximally reduced by 53, 56, and 73%, respectively (P<0.05), by reducing levels in very low density-lipoprotein (VLDL), intermediate density-lipoprotein (IDL), and low density-lipoprotein (LDL) cholesterol, whereas high density-lipoprotein (HDL) cholesterol levels were increased. The observations that HOE 402 exhibited no effect on in vivo clearance of 125I-labeled LDL in wild-type mice, and clearly reduced serum cholesterol levels in LDLR0/0 mice, indicate that the LDLR is not the main target for the compound. In wild-type mice, production of VLDL-TG, and cholesterol were reduced by more than 50% by HOE 402 (P<0.05), whereas VLDL apolipoprotein B (ApoB) secretion was unaffected, indicating that HOE 402 treatment changes the size, rather than the number of the secreted VLDL particles. The reduced VLDL production was accompanied by a 22% decreased hepatic cholesterol ester concentration (P<0.05). Additionally, HOE 402 treatment strongly reduced the aortic content of atherosclerotic lesions by 90 and 72% in LDLR+/0 and LDLR0/0 mice, respectively (P<0.01). In conclusion, HOE 402 is a potent cholesterol-lowering compound, which inhibits VLDL production, and consequently attenuates atherosclerosis development.

A low-fat diet has a higher potential than energy restriction to improve high-fat diet-induced insulin resistance in mice.

Previous studies have shown that energy restriction (ER) or low-fat (LF) diets have beneficial effects on high-fat (HF) diet-induced obesity and non-insulin-dependent diabetes. However, comparison between ER and low-fat diet regarding the effect on insulin resistance and lipid metabolism has not been reported. After inducing insulin resistance by HF feeding for 20 weeks, male C57BL/6J mice were divided into 3 groups. For a period of 12 weeks, group 1 received energy restriction (70% of ad libitum, HF diet), group 2 LF diet, and group 3 maintained on HF diet. Body weight and energy intake were reduced equally in ER and LF feeding. Plasma insulin levels were decreased on LF feeding, but were unchanged on ER, when compared with HF feeding. Glucose tolerance and insulin sensitivity tests revealed that insulin sensitivity was improved more efficiently by LF feeding than on ER. Plasma triglyceride (TG) levels were lower on LF feeding compared with ER and HF feeding. Measurement of hepatic very low-density lipoprotein (VLDL)-TG production revealed a lower production after LF diet feeding or ER compared with HF diet feeding. In summary, our data show that LF diet has a higher potential than ER to improve HF diet-induced insulin resistance, and that there is an association between improvement of insulin resistance and decrease of TG levels.

The immediate early gene product EGR1 and polycomb group proteins interact in epigenetic programming during chondrogenesis.

Initiation of and progression through chondrogenesis is driven by changes in the cellular microenvironment. At the onset of chondrogenesis, resting mesenchymal stem cells are mobilized in vivo and a complex, step-wise chondrogenic differentiation program is initiated. Differentiation requires coordinated transcriptomic reprogramming and increased progenitor proliferation; both processes require chromatin remodeling. The nature of early molecular responses that relay differentiation signals to chromatin is poorly understood. We here show that immediate early genes are rapidly and transiently induced in response to differentiation stimuli in vitro. Functional ablation of the immediate early factor EGR1 severely deregulates expression of key chondrogenic control genes at the onset of differentiation. In addition, differentiating cells accumulate DNA damage, activate a DNA damage response and undergo a cell cycle arrest and prevent differentiation associated hyper-proliferation. Failed differentiation in the absence of EGR1 affects global acetylation and terminates in overall histone hypermethylation. We report novel molecular connections between EGR1 and Polycomb Group function: Polycomb associated histone H3 lysine27 trimethylation (H3K27me3) blocks chromatin access of EGR1. In addition, EGR1 ablation results in abnormal Ezh2 and Bmi1 expression. Consistent with this functional interaction, we identify a number of co-regulated targets genes in a chondrogenic gene network. We here describe an important role for EGR1 in early chondrogenic epigenetic programming to accommodate early gene-environment interactions in chondrogenesis.

MK3 controls Polycomb target gene expression via negative feedback on ERK.

BACKGROUND: Gene-environment interactions are mediated by epigenetic mechanisms. Polycomb Group proteins constitute part of an epigenetic cellular transcriptional memory system that is subject to dynamic modulation during differentiation. Molecular insight in processes that control dynamic chromatin association and dissociation of Polycomb repressive complexes during and beyond development is limited. We recently showed that MK3 interacts with Polycomb repressive complex 1 (PRC1). The functional relevance of this interaction, however, remained poorly understood. MK3 is activated downstream of mitogen- and stress-activated protein kinases (M/SAPKs), all of which fulfill crucial roles during development. We here use activation of the immediate-early response gene ATF3, a bona fide PRC1 target gene, as a model to study how MK3 and its effector kinases MAPK/ERK and SAPK/P38 are involved in regulation of PRC1-dependent ATF3 transcription. RESULTS: Our current data show that mitogenic signaling through ERK, P38 and MK3 regulates ATF3 expression by PRC1/chromatin dissociation and epigenetic modulation. Mitogenic stimulation results in transient P38-dependent H3S28 phosphorylation and ERK-driven PRC1/chromatin dissociation at PRC1 targets. H3S28 phosphorylation by itself appears not sufficient to induce PRC1/chromatin dissociation, nor ATF3 transcription, as inhibition of MEK/ERK signaling blocks BMI1/chromatin dissociation and ATF3 expression, despite induced H3S28 phosphorylation. In addition, we establish that concomitant loss of local H3K27me3 promoter marking is not required for ATF3 activation. We identify pERK as a novel signaling-induced binding partner of PRC1, and provide evidence that MK3 controls ATF3 expression in cultured cells via negative regulatory feedback on M/SAPKs. Dramatically increased ectopic wing vein formation in the absence of Drosophila MK in a Drosophila ERK gain-of-function wing vein patterning model, supports the existence of MK-mediated negative feedback regulation on pERK. CONCLUSION: We here identify and characterize important actors in a PRC1-dependent epigenetic signal/response mechanism, some of which appear to be nonspecific global responses, whereas others provide modular specificity. Our findings provide novel insight into a Polycomb-mediated epigenetic mechanism that dynamically controls gene transcription and support a direct link between PRC1 and cellular responses to changes in the microenvironment.

Post-natal myogenic and adipogenic developmental: defects and metabolic impairment upon loss of A-type lamins.

A-type lamins are a major component of the nuclear lamina. Mutations in the LMNA gene, which encodes the A-type lamins A and C, cause a set of phenotypically diverse diseases collectively called laminopathies. While adult LMNA null mice show various symptoms typically associated with laminopathies, the effect of loss of lamin A/C on early post-natal development is poorly understood. Here we developed a novel LMNA null mouse (LMNA(GT-/-)) based on genetrap technology and analyzed its early post-natal development. We detect LMNA transcripts in heart, the outflow tract, dorsal aorta, liver and somites during early embryonic development. Loss of A-type lamins results in severe growth retardation and developmental defects of the heart, including impaired myocyte hypertrophy, skeletal muscle hypotrophy, decreased amounts of subcutaneous adipose tissue and impaired ex vivo adipogenic differentiation. These defects cause death at 2 to 3 weeks post partum associated with muscle weakness and metabolic complications, but without the occurrence of dilated cardiomyopathy or an obvious progeroid phenotype. Our results indicate that defective early post-natal development critically contributes to the disease phenotypes in adult laminopathies.

Defective intercellular adhesion complex in myocardium predisposes to infarct rupture in humans.

OBJECTIVES: Our goal was to evaluate intercellular adhesion complex proteins in myocardium in human infarct rupture. BACKGROUND: Infarct rupture, a fatal complication of myocardial infarction (MI), has been attributed to a defective cell adhesion complex in a transgenic mouse model. METHODS: Heart samples were collected from autopsies from infarct rupture and control (nonrupture) MI patients. Both infarcted and remote areas were included. Cell adhesion proteins including alphaE-catenin, beta-catenin, gamma-catenin, and N-cadherin were characterized by immunohistochemistry and immunoblotting. Genetic analysis was undertaken to evaluate mutations and polymorphisms in the alphaE-catenin gene. In addition, infarct rupture was studied in transgenic mice heterozygous for alphaE-catenin C-terminal deficiency, mimicking the situation in human infarct rupture patients. RESULTS: No alphaE-catenin was detected in 70% of remote samples of infarct rupture hearts compared with 20% in control MI by immunohistochemistry. The immunoblot analysis confirmed a significant reduction in remote areas, and complete absence of alphaE-catenin in infarct areas from infarct rupture patients. No mutation or polymorphism of the alphaE-catenin gene was discovered. Other cell adhesion proteins were not significantly affected in remote areas of infarct rupture hearts. Three-fourths of the heterozygous alphaE-catenin C-terminal truncated mice died of infarct rupture, compared with one-fourth of the wild-type littermates. CONCLUSIONS: The data show a reduced expression and defective localization of alphaE-catenin in the intercalated disc region in patients dying of infarct rupture. The mechanism of lower expression of alphaE-catenin remains to be elucidated.

Apolipoprotein C-I binds free fatty acids and reduces their intracellular esterification.

Mice that overexpress human apolipoprotein C-I (apoC-I) homozygously (APOC1(+/+) mice) are protected against obesity and show cutaneous abnormalities. Although these effects can result from our previous observation that apoC-I inhibits FFA generation by LPL, we have also found that apoC-I impairs the uptake of a FFA analog in adipose tissue. In this study, we tested the hypothesis that apoC-I interferes with cellular FFA uptake independent of LPL activity. The cutaneous abnormalities of APOC1(+/+) mice were not affected after transplantation to wild-type mice, indicating that locally produced apoC-I prevents lipid entry into the skin. Subsequent in vitro studies with apoC-I-deficient versus wild-type macrophages revealed that apoC-I reduced the cell association and subsequent esterification of [(3)H]oleic acid by approximately 35% (P < 0.05). We speculated that apoC-I binds FFA extracellularly, thereby preventing cell association of FFA. We showed that apoC-I was indeed able to mediate the binding of oleic acid to otherwise protein-free VLDL-like emulsion particles involving electrostatic interaction. We conclude that apoC-I binds FFA in the circulation, thereby reducing the availability of FFA for uptake by cells. This mechanism can serve as an additional mechanism behind the resistance to obesity and the cutaneous abnormalities of APOC1(+/+) mice.

MAPKAP kinase 3pK phosphorylates and regulates chromatin association of the polycomb group protein Bmi1.

Polycomb group (PcG) proteins form chromatin-associated, transcriptionally repressive complexes, which are critically involved in the control of cell proliferation and differentiation. Although the mechanisms involved in PcG-mediated repression are beginning to unravel, little is known about the regulation of PcG function. We showed previously that PcG complexes are phosphorylated in vivo, which regulates their association with chromatin. The nature of the responsible PcG kinases remained unknown. Here we present the novel finding that the PcG protein Bmi1 is phosphorylated by 3pK (MAPKAP kinase 3), a convergence point downstream of activated ERK and p38 signaling pathways and implicated in differentiation and developmental processes. We identified 3pK as an interaction partner of PcG proteins, in vitro and in vivo, by yeast two-hybrid interaction and co-immunoprecipitation, respectively. Activation or overexpression of 3pK resulted in phosphorylation of Bmi1 and other PcG members and their dissociation from chromatin. Phosphorylation and subsequent chromatin dissociation of PcG complexes were expected to result in de-repression of targets. One such reported Bmi1 target is the Cdkn2a/INK4A locus. Cells overexpressing 3pK showed PcG complex/chromatin dissociation and concomitant de-repression of p14(ARF), which was encoded by the Cdkn2a/INK4A locus. Thus, 3pK is a candidate regulator of phosphorylation-dependent PcG/chromatin interaction. We speculate that phosphorylation may not only affect chromatin association but, in addition, the function of individual complex members. Our findings linked for the first time MAPK signaling pathways to the Polycomb transcriptional memory system. This suggests a novel mechanism by which a silenced gene status can be modulated and implicates PcG-mediated repression as a dynamically controlled process.

Intestinal lipid absorption is not affected in CD36 deficient mice.

Increasing evidence has implicated the membrane protein CD36 (or fatty acid translocase, FAT) to be involved in high affinity fatty acid uptake. CD36 is expressed in tissues active in fatty acid metabolism, like adipose tissue and skeletal and cardiac muscle, but also in intestine. CD36 is localized in the intestine mainly in the jejunal villi, where it is confined to enterocyte apical membrane. The aim was to determine the role of CD36 in intestinal lipid absorption. Lipid absorption was determined by administering 3H-labeled triolein and 14C-labeled palmitic acid as an olive oil bolus by intragastric gavage and determine appearance of 3H and 14C label in plasma, after blocking lipolysis by i.v. injections of Triton WR 1339. Surprisingly, no differences in plasma appearance of 3H-label or 14C-label were observed in CD36(-/-) mice compared to wild type controls. These results suggest that CD36 does not play a role in intestinal lipid absorption after an acute lipid load.

CD36 deficiency increases insulin sensitivity in muscle, but induces insulin resistance in the liver in mice.

CD36 (fatty acid translocase) is involved in high-affinity peripheral fatty acid uptake. Mice lacking CD36 exhibit increased plasma free fatty acid and triglyceride (TG) levels and decreased glucose levels. Studies in spontaneous hypertensive rats lacking functional CD36 link CD36 to the insulin-resistance syndrome. To clarify the relationship between CD36 and insulin sensitivity in more detail, we determined insulin-mediated whole-body and tissue-specific glucose uptake in CD36-deficient (CD36-/-) mice. Insulin-mediated whole-body and tissue-specific glucose uptake was measured by d-[3H]glucose and 2-deoxy-d-[1-3H]glucose during hyperinsulinemic clamp in CD36-/- and wild-type control littermates (CD36+/+) mice. Whole-body and muscle-specific insulin-mediated glucose uptake was significantly higher in CD36-/- compared with CD36+/+ mice. In contrast, insulin completely failed to suppress endogenous glucose production in CD36-/- mice compared with a 40% reduction in CD36+/+ mice. This insulin-resistant state of the liver was associated with increased hepatic TG content in CD36-/- mice compared with CD36+/+ mice (110.9 +/- 12.0 and 68.9 +/- 13.6 microg TG/mg protein, respectively). Moreover, hepatic activation of protein kinase B by insulin, measured by Western blot, was reduced by 54%. Our results show a dissociation between increased muscle and decreased liver insulin sensitivity in CD36-/- mice.