Identification of transcriptional and metabolic programs related to mammalian cell size

SummaryBackgroundRegulation of cell size requires coordination of growth and proliferation. Conditional loss of cyclin-dependent kinase 1 in mice permits hepatocyte growth without cell division, allowing us to study cell size in vivo using transcriptomics and metabolomics.ResultsLarger cells displayed increased expression of cytoskeletal genes but unexpectedly repressed expression of many genes involved in mitochondrial functions. This effect appears to be cell autonomous because cultured Drosophila cells induced to increase cell size displayed a similar gene-expression pattern. Larger hepatocytes also displayed a reduction in the expression of lipogenic transcription factors, especially sterol-regulatory element binding proteins. Inhibition of mitochondrial functions and lipid biosynthesis, which is dependent on mitochondrial metabolism, increased the cell size with reciprocal effects on cell proliferation in several cell lines.ConclusionsWe uncover that large cell-size increase is accompanied by downregulation of mitochondrial gene expression, similar to that observed in diabetic individuals. Mitochondrial metabolism and lipid synthesis are used to couple cell size and cell proliferation. This regulatory mechanism may provide a possible mechanism for sensing metazoan cell size

Cell cycle regulation in NAFLD: when imbalanced metabolism limits cell division : when imbalanced metabolism limits cell division

Cell division is essential for organismal growth and tissue homeostasis. It is exceptionally significant in tissues chronically exposed to intrinsic and external damage, like the liver. After decades of studying the regulation of cell cycle by extracellular signals, there are still gaps in our knowledge on how these two interact with metabolic pathways in vivo. Studying the cross-talk of these pathways has direct clinical implications as defects in cell division, signaling pathways, and metabolic homeostasis are frequently observed in liver diseases. In this review, we will focus on recent reports which describe various functions of cell cycle regulators in hepatic homeostasis. We will describe the interplay between the cell cycle and metabolism during liver regeneration after acute and chronic damage. We will focus our attention on non-alcoholic fatty liver disease, especially non-alcoholic steatohepatitis. The global incidence of non-alcoholic fatty liver disease is increasing exponentially. Therefore, understanding the interplay between cell cycle regulators and metabolism may lead to the discovery of novel therapeutic targets amenable to intervention

Therapeutic targeting of the mitochondrial one-carbon pathway: perspectives, pitfalls, and potential

Most of the drugs currently prescribed for cancer treatment are riddled with substantial side effects. In order to develop more effective and specific strategies to treat cancer, it is of importance to understand the biology of drug targets, particularly the newly emerging ones. A comprehensive evaluation of these targets will benefit drug development with increased likelihood for success in clinical trials. The folate-mediated one-carbon (1C) metabolism pathway has drawn renewed attention as it is often hyperactivated in cancer and inhibition of this pathway displays promise in developing anticancer treatment with fewer side effects. Here, we systematically review individual enzymes in the 1C pathway and their compartmentalization to mitochondria and cytosol. Based on these insight, we conclude that (1) except the known 1C targets (DHFR, GART, and TYMS), MTHFD2 emerges as good drug target, especially for treating hematopoietic cancers such as CLL, AML, and T-cell lymphoma; (2) SHMT2 and MTHFD1L are potential drug targets; and (3) MTHFD2L and ALDH1L2 should not be considered as drug targets. We highlight MTHFD2 as an excellent therapeutic target and SHMT2 as a complementary target based on structural/biochemical considerations and up-to-date inhibitor development, which underscores the perspectives of their therapeutic potential

Protective functions of ZO-2/Tjp2 expressed in hepatocytes and cholangiocytes against liver injury and cholestasis

BACKGROUND & AIMS: Liver tight junctions (TJs) establish tissue barriers that isolate bile from the blood circulation. TJP2/ZO-2-inactivating mutations cause progressive cholestatic liver disease in humans. Since the underlying mechanisms remain elusive, we characterized mice with liver-specific inactivation of Tjp2.METHODS: Tjp2 was deleted in hepatocytes, cholangiocytes, or both. Effects on the liver were assessed by biochemical analyses of plasma, liver and bile and .by EM, histology and immunostaining. TJ barrier permeability was evaluated using FITC-Dextran (4kDa). Cholic acid (CA) diet was used to assess susceptibility to liver injury.RESULTS: Liver-specific deletion of Tjp2 resulted in lower Cldn1 protein levels, minor changes to the TJ, dilated canaliculi, lower microvilli density and aberrant Radixin and BSEP distribution, without an overt increase in TJ permeability. Hepatic Tjp2-defcient mice presented with mild progressive cholestasis with lower expression levels of bile acid (BA) transporter Abcb11/Bsep and detoxification enzyme Cyp2b10. A CA-diet tolerated by control mice caused severe cholestasis and liver necrosis in Tjp2-deficient animals. TCPOBOP ameliorated CA-induced injury by enhancing Cyp2b10 expression and ursodeoxycholic acid provided partial improvement. Inactivating Tjp2 separately in hepatocytes or cholangiocytes only showed mild CA-induced liver injury.CONCLUSION: Tjp2 is required for normal cortical distribution of Radixin, canalicular volume regulation and microvilli density, Its inactivation deregulated expression of Cldn1 and key BA transporters and detoxification enzymes. The mice provide a novel animal model for cholestatic liver disease caused by TJP2-inactivating mutations in humans

Kinetochore localization of Mad1 in MEFs in early and late mitosis.

<p>(A) Primary MEFs were treated as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006310#pgen.1006310.g004" target="_blank">Fig 4</a> to induce mitotic arrest and were fixed. Where indicated, cells were treated with 200nM OKA for one hour before fixation. Immunofluorescence analysis was performed using ACA and antibodies against Mad1. Different mitotic phases were determined with early mitotic cells determined by lightly condensed chromosomes that were scattered in the cytoplasm just after NEBD and late mitotic cells displaying a highly condensed chromosome mass typically caused by nocodazole treatment. Insets represent the boxed areas. Scale bar 5 μm. Quantification of kinetochore-localized Mad1 in Mastl^FLOX and Mastl^NULL cells in early prophase (NEBD) (B) or late prometaphase-like state by nocodazole treatment (C) [N>20 cells per each condition, ± standard deviation] as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006310#pgen.1006310.g003" target="_blank">Fig 3B</a> except using kinetochore-localized Mad1. To determine the statistical significance, a Student’s <i>t</i>-test was performed. (n.s. not significance; ***<i>p</i><0.0001, Student’s <i>t</i>-test, unpaired).</p

Mastl is required for phosphorylation and full activity of MPS1.

<p>(A) Phospho-proteomic analysis of mitotic arrested MEFs was performed using SILAC. The log2 SILAC ratio of phospho-peptides in each mass spectrometry run is plotted as the forward experiment against the reverse experiment. Data points colored in blue denote down-regulated phosphorylation sites in Mastl^NULL cells with at least 1.5 fold change. Black points indicate peptides with unchanged phosphorylation status. (B) MPS1 S820 phospho-peptide MS spectrum: the intensities of SILAC peptide pairs (heavy signal corresponding to Mastl^NULL cells and light signal to Mastl^FLOX) are shown. (C) Immortalized MEFs expressing lentivirally transduced HA-tagged MPS1 (human) were synchronized and released as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006310#pgen.1006310.g002" target="_blank">Fig 2D</a>. Cells were treated with or without 500ng/ml nocodazole between hours 24–28 after release. Whole cell lysate (WCL) for each condition were prepared and subjected to immunoblot analysis for Mastl and HSP90. HA-MPS1 was immunoprecipitated (IP) using anti-HA antibodies and probed for phosphorylated or total MPS1. (D, F, G) GST-tagged recombinant wild type (WT) or non-phosphorylatable mutant (S820A) MPS1 peptide fusion protein was purified and subjected to <i>in vitro</i> kinase assay with recombinant Cdk1/cyclin B1 complexes (D; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006310#sec008" target="_blank">Methods</a>; MPS1^S820A 3x designated an amount of three times of the substrates from lane 4). For F and G, phosphorylated GST-MPS1^WT peptide fusion protein was subjected to <i>in vitro</i> phosphatase assay using immunoprecipitated HA-tagged B55 or B56 complexes in presence (+) or absence (-) of 200nM OKA. A representative image for each experiment is shown and representative results for quantification of ³²P intensity signals of phosphorylated GST-MPS1 peptide fusion protein are shown as percentage derived from lane 6 for D, lane 2 for F-G. For F-G, levels of the PP2A A scaffold and PP2A C catalytic subunits co-immunoprecipitated are shown. For G, <i>in vitro</i> phosphatase assay was performed using immunoprecipitated PP2A complexes from increasing amounts of whole cell lysates from HA-B55 overexpressing 293T cells (25, 50, 100, 250 and 500μg) and phosphorylated GST-MPS1 peptide fusing protein as a substrate. Western blots for HA-B55 (middle panel) and the PP2A A scaffold subunit (bottom panel) are shown. (E) Immortalized MEFs synchronously released into full growth medium were transfected with plasmids encoding Myc-tagged MPS1 (human). Cells were arrested in mitosis by treating with 500ng/ml nocodazole for 4 hours between hours 24–28 after serum release. Cells were additionally treated with 200nM OKA (+) or DMSO (-) between hours 27–28 as indicated. Myc-MPS1 was immunoprecipitated using anti-Myc antibodies and subjected to <i>in vitro</i> kinase assay using 5μg MBP as a substrate and recombinant Cdk1/cyclin B1 as kinase (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006310#sec008" target="_blank">Methods</a>). A representative experiment is shown and representative results for quantification of ³²P intensity signals of phosphorylated MBP are shown as percentage derived from lane 2.</p

Model of SAC regulation by the Greatwall /Mastl kinase.

<p>Triangular regulation that controls the meta-to-anaphase transition whereby Cdk1 activates APC/C^Cdc20 at the same time as it does SAC, which in turn inhibits APC/C^Cdc20. Once APC/C^Cdc20 is active, it will degrade cyclin B leading to inactivation of Cdk1 but this can only happen after SAC is silenced. Here we show that Mastl through PP2A/B55 regulates MPS1 and potentially other unidentified substrates (X) to control SAC activity. These intricate feedback loops may help to fine-tune the timing of meta-to-anaphase transition. How Mastl activity decreases during this transition to allow PP2A activity remains unknown, but this could involve phosphatases. Green and red colors indicate a positive and negative regulation, respectively.</p

Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint

<div><p>The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (Mastl^NULL) MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC) maintenance since the duration of mitotic arrest caused by microtubule poisons in Mastl^NULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, Mastl^NULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of Mastl^NULL cells with the phosphatase inhibitor okadaic acid (OKA) rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using <i>in vitro</i> dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing.</p></div

Growth analysis of Mastl^NULL MEFs.

<p>(A) Three MEF lines isolated from different embryos were treated with 4-OHT or DMSO to induce Mastl knockout (Mastl^NULL) and their proliferative potential was monitored by Alamar Blue proliferation assays for 8 days. AFU, arbitrary fluorescence units. (B) MEFs were synchronized and recombination in the Mastl and Cdk1 loci were induced as described in the Methods section. Cells were arrested in mitosis for 4 hours using 5μM Eg5 inhibitor S-Trityl-L-cysteine (STLC) starting from 20 hours after release into full growth medium. Still pictures were taken using phase-contrast microscopy. Scale bar 100μm. (C) MEFs were fixed 24 hours after release into full growth medium and stained with anti-phospho-histone H3 Ser10 antibodies (pH3) to quantify mitotic cells using FACS analysis. (D) MEFs expressing the histone H2B-YFP fusion protein were analyzed by time-lapse microscopy. Still images of a dividing Mastl^NULL cell were acquired every 5 minutes. Scale bar 10μm. (E) Quantification of appearance of anaphase bridges in Mastl^FLOX and Mastl^NULL MEFs expressing the H2B-YFP. (F) Mastl^NULL cells were fixed and stained with DAPI 72 hours after synchronization and release into full growth medium. Scale bar 20μm.</p