IDH2 deficiency promotes mitochondrial dysfunction and cardiac hypertrophy in mice

Cardiac hypertrophy, a risk factor for heart failure, is associated with enhanced oxidative stress in the mitochondria, resulting from high levels of reactive oxygen species (ROS). The balance between ROS generation and ROS detoxification dictates ROS levels. As such, disruption of these processes results in either increased or decreased levels of ROS. In previous publications, we have demonstrated that one of the primary functions of mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) is to control the mitochondrial redox balance, and thereby mediate the cellular defense against oxidative damage, via the production of NADPH. To explore the association between IDH2 expression and cardiac function, we measured myocardial hypertrophy, apoptosis, and contractile dysfunction in IDH2 knockout (idh2^−/−) and wild-type (idh2^+/+) mice. As expected, mitochondria from the hearts of knockout mice lacked IDH2 activity and the hearts of IDH2-deficient mice developed accelerated heart failure, increased levels of apoptosis and hypertrophy, and exhibited mitochondrial dysfunction, which was associated with a loss of redox homeostasis. Our results suggest that IDH2 plays an important role in maintaining both baseline mitochondrial function and cardiac contractile function following pressure-overload hypertrophy, by preventing oxidative stress.     

MicroRNA-29a inhibited epididymal epithelial cell proliferation by targeting nuclear autoantigenic sperm protein (NASP)

Cell proliferation often decreases gradually during postnatal development of some organs. However, the underlying molecular mechanisms remain unclear. Epididymis, playing important roles in sperm maturation, is a typical organ of this type, which displays a decreased proliferation during postnatal development and even ceased at the adult stage. Here, epididymis was employed as a model to explore the underlying mechanisms. We profiled the microRNA and mRNA expression of newborn (1 day) and adult (90 day) rat epididymis by microarray analysis, and found that the level of miR-29a was dramatically up-regulated during postnatal development of rat epididymis. Subsequent investigations demonstrated that overexpression of miR-29a inhibited the proliferation of epididymal epithelial cells in vitro. The nuclear autoantigenic sperm protein (NASP), a novel target of miR-29a, was significantly down-regulated during postnatal development of rat epididymis. Further analysis showed that silence of NASP mimicked the anti-proliferation effect of miR-29a, whereas overexpression of this protein attenuated the effect of miR-29a. As in rat epididymis, miR-29a was up-regulated and Nasp was down-regulated during postnatal development of mouse epididymis, heart, liver, and lung. Moreover, miR-29a can also inhibit the proliferation of cancer cells by targeting Nasp. Thus, an increase of miR-29a, and hence decrease of Nasp, may contribute to inhibit cell proliferation during postnatal organ development.     

Steroid receptor coactivator-interacting protein (SIP) inhibits caspase-independent apoptosis by preventing apoptosis-inducing factor (AIF) from being released from mitochondria

Apoptosis-inducing factor (AIF) is a caspase-independent death effector. Normally residing in the mitochondrial intermembrane space, AIF is released and translocated to the nucleus in response to proapoptotic stimuli. Nuclear AIF binds to DNA and induces chromatin condensation and DNA fragmentation, characteristics of apoptosis. Until now, it remained to be clarified how the mitochondrial-nuclear translocation of AIF is regulated. Here we report that steroid receptor coactivator-interacting protein (SIP) interacts directly with AIF in mitochondria and specifically inhibits caspase-independent and AIF-dependent apoptosis. Challenging cells with apoptotic stimuli leads to rapid degradation of SIP, and subsequently AIF is liberated from mitochondria and translocated to the nucleus to induce apoptosis. Together, our data demonstrate that SIP is a novel regulator in caspase-independent and AIF-mediated apoptosis.     

Lin-28 homologue A (LIN28A) promotes cell cycle progression via regulation of cyclin-dependent kinase 2 (CDK2), cyclin D1 (CCND1), and cell division cycle 25 homolog A (CDC25A) expression in cancer

The RNA-binding protein LIN28A regulates the translation and stability of a large number of mRNAs as well as the biogenesis of certain miRNAs in embryonic stem cells and developing tissues. Increasing evidence indicates that LIN28A functions as an oncogene promoting cancer cell growth. However, little is known about its molecular mechanism of cell cycle regulation in cancer. Using tissue microarrays, we found that strong LIN28A expression was reactivated in about 10% (7.1-17.1%) of epithelial tumors (six tumor types, n = 369). Both in vitro and in vivo experiments demonstrate that LIN28A promotes cell cycle progression in cancer cells. Genome-wide RNA-IP-chip experiments indicate that LIN28A binds to thousands of mRNAs, including a large group of cell cycle regulatory mRNAs in cancer and embryonic stem cells. Furthermore, the ability of LIN28A to stimulate translation of LIN28A-binding mRNAs, such as CDK2, was validated in vitro and in vivo. Finally, using a combined gene expression microarray and bioinformatics approach, we found that LIN28A also regulates CCND1 and CDC25A expression and that this is mediated by inhibiting the biogenesis of let-7 miRNA. Taken together, these results demonstrate that LIN28A is reactivated in about 10% of epithelial tumors and promotes cell cycle progression by regulation of both mRNA translation (let-7-independent) and miRNA biogenesis (let-7-dependent).     

Involvements of mitochondrial thioredoxin reductase (TrxR2) in cell proliferation

Mammalian cells contain two forms of thioredoxin reductase (TrxR), cytosolic TrxR1 and mitochondrial TrxR2. To investigate the biological roles of TrxR2, we generated stable HeLa cell lines expressing a dominant negative form of TrxR2 (TrxR2DN) under the control of the tetracycline-off system. We observed that TrxR2DN-induced cells, following stimulation with EGF, produced more hydrogen peroxide than uninduced cells. The extent of protein tyrosine phosphorylation of many proteins including ERK was higher in TrxR2DN-induced cells than in uninduced cells when stimulated with fetal bovine serum or EGF. Induction of TrxR2DN also resulted in the increased rate of progression of G1 to S phase in cell cycle and cell proliferation and affected the expression of many proteins involved in cell cycle. These results suggest that TrxR2 participates in the regulation of protein tyrosine phosphorylation and cell growth as a component of the mitochondria specific H2O2-eliminating system that includes peroxiredoxin III and thioredoxin 2.     

Chronic high glucose induced INS‐1β cell mitochondrial dysfunction: A comparative mitochondrial proteome with SILAC

As glucose‐stimulated insulin secretion of pancreatic β cell is triggered and promoted by the metabolic messengers derived from mitochondria, mitochondria take a central stage in the normal function of β cells. β cells in diabetics were chronically exposed to hyperglycemia stimulation, which have been reported to exert deleterious effects on β‐cell mitochondria. However, the mechanism of the toxic effects of hyperglycemia on β‐cell mitochondria was not clear. In this study, we characterized the biological functional changes of rat INS‐1β cells and their mitochondria with chronic exposure to hyperglycemia and created a research model of chronic hyperglycemia‐induced dysfunctional β cells with damaged mitochondria. Then, SILAC‐based quantitative proteomic approach was used to compare the mitochondrial protein expression from high glucose treated INS‐1β cells and control cells. The expression of some mitochondrial proteins was found with significant changes. Functional classification revealed most of these proteins were related with oxidative phosphorylation, mitochondrial protein biosynthesis, substances metabolism, transport, and cell death. These results presented some useful information about the effect of glucotoxicity on the β‐cell mitochondria.     

Lamprey buccal gland secretory protein-2(BGSP-2) inhibits human T lymphocyte proliferation

Lamprey is a representative of the agnathans, the most ancient class of vertebrates. Parasitic lampreys secrete anticoagulant from their buccal glands and prevent blood coagulation of host fishes. We identified a buccal gland secretory protein-2 (BGSP-2) from a buccal gland cDNA library of Lampetra japonica. The full-length BGSP-2 gene was cloned and the recombinant BGSP-2 protein was generated. The role of BGSP-2 on lymphocyte proliferation was studied by examining its effects on human T lymphocytes. We found that lamprey BGSP-2 was able to effectively block the proliferation of T cells in vitro by inducing G_1/S cell cycle arrest. Furthermore, it inhibited the proliferation of human T lymphocytes stimulated by phytohemagglutinin (PHA) at a minimum concentration of 0.1μg/ml. Our data suggest that lamprey BGSP-2 is able to block the mitosis of human T lymphocytes at the G1/S point, and has the potential of anti-proliferative effect on PHA-activated T lymphocytes.     

Inhibition of cyclooxygenase (COX)-2 affects endothelial progenitor cell proliferation

Growing evidence indicates that inducible cyclooxygenase-2 (COX-2) is involved in the pathogenesis of inflammatory disorders and various types of cancer. Endothelial progenitor cells recruited from the bone marrow have been shown to be involved in the formation of new vessels in malignancies and discussed for being a key point in tumour progression and metastasis. However, until now, nothing is known about an interaction between COX and endothelial progenitor cells (EPC). Expression of COX-1 and COX-2 was detected by semiquantitative RT-PCR and Western blot. Proliferation kinetics, cell cycle distribution and rate of apoptosis were analysed by MTT test and FACS analysis. Further analyses revealed an implication of Akt phosphorylation and caspase-3 activation. Both COX-1 and COX-2 expression can be found in bone-marrow-derived endothelial progenitor cells in vitro. COX-2 inhibition leads to a significant reduction in proliferation of endothelial progenitor cells by an increase in apoptosis and cell cycle arrest. COX-2 inhibition leads further to an increased cleavage of caspase-3 protein and inversely to inhibition of Akt activation. Highly proliferating endothelial progenitor cells can be targeted by selective COX-2 inhibition in vitro. These results indicate that upcoming therapy strategies in cancer patients targeting COX-2 may be effective in inhibiting tumour vasculogenesis as well as angiogenic processes.     

The identification and characterization of a new GTP-binding protein (Gbp45) involved in cell proliferation and death related to mitochondrial function

We describe the identification and characterization of a GTP-binding protein with a molecular weight of 45 kD (Gbp45). Gbp45 cDNA was found to overlap with a hypothetical human protein, PTD004, the sequence of which was previously deposited in the databases. The gene for PTD004 was recently found to be one of the ATPases, hOLA1 (human Obg-like ATPase 1). The Gbp45 gene encodes a protein of 396 amino acid residues. Immunocytochemical analysis and examination with GFP-tagged protein revealed that Gbp45 is primarily located in the cytosolic compartment. Immunoblot analysis showed that the Gbp45 protein is strongly expressed in the neuronal tissues and pancreas. T43N and T56N mutations resulted in a loss of Gbp45’s ability to bind to GTP and a loss of GTPase activity. In cultured cells, the transfection of wild-type Gbp45 accelerated cell proliferation, though T43N and T56N mutations induced cell death. Down-regulating Gbp45 expression decreased the cell proliferation rate and increased the rate of cell death induced by the inhibition of mitochondrial electron transport. These findings indicate that Gbp45 plays important roles in cell proliferation and death related to mitochondrial function.     

A Universal Scaffold for Synthesis of the Fe(CN)2(CO) Moiety of [NiFe] Hydrogenase

Hydrogen-cycling [NiFe] hydrogenases harbor a dinuclear catalytic center composed of nickel and iron ions, which are coordinated by four cysteine residues. Three unusual diatomic ligands in the form of two cyanides (CN⁻) and one carbon monoxide (CO) are bound to the iron and apparently account for the complexity of the cofactor assembly process, which involves the function of at least six auxiliary proteins, designated HypA, -B, -C, -D, -E, and -F. It has been demonstrated previously that the HypC, -D, -E, and -F proteins participate in cyanide synthesis and transfer. Here, we show by infrared spectroscopic analysis that the purified HypCD complexes from Ralstonia eutropha and Escherichia coli carry in addition to both cyanides the CO ligand. We present experimental evidence that in vivo the attachment of the CN⁻ ligands is a prerequisite for subsequent CO binding. With the aid of genetic engineering and subsequent mutant analysis, the functional role of conserved cysteine residues in HypD from R. eutropha was investigated. Our results demonstrate that the HypCD complex serves as a scaffold for the assembly of the Fe(CN)₂(CO) entity of [NiFe] hydrogenase.     

CHCM1/CHCHD6, novel mitochondrial protein linked to regulation of mitofilin and mitochondrial cristae morphology

The structural integrity of mitochondrial cristae is crucial for mitochondrial functions; however, the molecular events controlling the structural integrity and biogenesis of mitochondrial cristae remain to be fully elucidated. Here, we report the functional characterization of a novel mitochondrial protein named CHCM1 (coiled coil helix cristae morphology 1)/CHCHD6. CHCM1/CHCHD6 harbors a coiled coil helix-coiled coil helix domain at its C-terminal end and predominantly localizes to mitochondrial inner membrane. CHCM1/CHCHD6 knockdown causes severe defects in mitochondrial cristae morphology. The mitochondrial cristae in CHCM1/CHCHD6-deficient cells become hollow with loss of structural definitions and reduction in electron-dense matrix. CHCM1/CHCHD6 depletion also leads to reductions in cell growth, ATP production, and oxygen consumption. CHCM1/CHCHD6 through its C-terminal end strongly and directly interacts with the mitochondrial inner membrane protein mitofilin, which is known to also control mitochondrial cristae morphology. CHCM1/CHCHD6 also interacts with other mitofilin-associated proteins, including DISC1 and CHCHD3. Knockdown of CHCM1/CHCHD6 reduces mitofilin protein levels; conversely, mitofilin knockdown leads to reduction in CHCM1 levels, suggesting coordinate regulation between these proteins. Our results further indicate that genotoxic anticancer drugs that induce DNA damage down-regulate CHCM1/CHCHD6 expression in multiple human cancer cells, whereas mitochondrial respiratory chain inhibitors do not affect CHCM1/CHCHD6 levels. CHCM1/CHCHD6 knockdown in human cancer cells enhances chemosensitivity to genotoxic anticancer drugs, whereas its overexpression increases resistance. Collectively, our results indicate that CHCM1/CHCHD6 is linked to regulation of mitochondrial cristae morphology, cell growth, ATP production, and oxygen consumption and highlight its potential as a possible target for cancer therapeutics.     

Interaction with Cyclin H/Cyclin-dependent Kinase 7 (CCNH/CDK7) Stabilizes C-terminal Binding Protein 2 (CtBP2) and Promotes Cancer Cell Migration

CtBP2 has been demonstrated to possess tumor-promoting capacities by virtue of up-regulating epithelial-mesenchymal transition (EMT) and down-regulating apoptosis in cancer cells. As a result, cellular CtBP2 levels are considered a key factor determining the outcome of oncogenic transformation. How pro-tumorigenic and anti-tumorigenic factors compete for fine-tuning CtBP2 levels is incompletely understood. Here we report that the cyclin H/cyclin-dependent kinase 7 (CCNH/CDK7) complex interacted with CtBP2 in vivo and in vitro. Depletion of either CCNH or CDK7 decreased CtBP2 protein levels by accelerating proteasome-dependent CtBP2 clearance. Further analysis revealed that CCNH/CDK7 competed with the tumor repressor HIPK2 for CtBP2 binding and consequently inhibited phosphorylation and dimerization of CtBP2. Phosphorylation-defective CtBP2 interacted more strongly with CCNH/CDK7 and was more resistant to degradation. Finally, overexpression of CtBP2 increased whereas depletion of CtBP2 dampened the invasive and migratory potential of breast cancer cells. CtBP2 promoted the invasion and migration of breast cancer cells in a CCNH-dependent manner. Taken together, our data have delineated a novel pathway that regulates CtBP2 stability, suggesting that targeting the CCNH/CDK7-CtBP2 axis may yield a viable anti-tumor strategy.     

Syndecan-2 expression enhances adhesion and proliferation of stably transfected Swiss 3T3 cells

Syndecans (heparan sulfate proteoglycans) participate in cell-cell and cell-matrix adhesion and are co- and low-affinity receptors for growth factors and enzymes, respectively. We examined the influence of stable syndecan-2 expression in Swiss 3T3 cells on cell-adhesion and proliferation. Higher syndecan-2 expression changed cell morphology and increased spreading and adhesion in these cells and proliferation induced by FCS and FGF-2. This emphasizes the role of syndecan-2 in the integration of signals from soluble and insoluble factors.     

Crystal structure of mitochondrial fission complex reveals scaffolding function for mitochondrial division 1 (Mdv1) coiled coil

The mitochondrial fission machinery is best understood in the yeast Saccharomyces cerevisiae, where Fis1, Mdv1, and Dnm1 are essential components. Fis1 is a mitochondrial outer membrane protein that recruits the dynamin-related GTPase Dnm1 during the fission process. This recruitment occurs via Mdv1, which binds both Fis1 and Dnm1 and therefore functions as a molecular adaptor linking the two molecules. Mdv1 has a modular structure, consisting of an N-terminal extension that binds Fis1, a central coiled coil for dimerization, and a C-terminal WD40 repeat region that binds Dnm1. We have solved the crystal structure of a dimeric Mdv1-Fis1 complex that contains both the N-terminal extension and coiled-coil regions of Mdv1. Consistent with previous studies, Mdv1 binds Fis1 through a U-shaped helix-loop-helix motif, and dimerization of the Mdv1-Fis1 complex is mediated by the antiparallel coiled coil of Mdv1. However, the complex is surprisingly compact and rigid due to two additional contacts mediated by the surface of the Mdv1 coiled coil. The coiled coil packs against both Fis1 and the second helix of the Mdv1 helix-loop-helix motif. Mutational analyses showed that these contacts are important for mitochondrial fission activity. These results indicate that, in addition to dimerization, the unusually long Mdv1 coiled coil serves a scaffolding function to stabilize the Mdv1-Fis1 complex.     

Panaxydol inhibits the proliferation and induces the differentiation of human hepatocarcinoma cell line HepG2

Panaxydol, a polyacetylene compound isolated from Panax ginseng, exerts anti-proliferative effects against malignant cells. No previous study, however, has been reported on its effects on hepatocellular carcinoma cells. Here, we investigated the effects of panaxydol on the proliferation and differentiation of human hepatocarcinoma cell line HepG2. We studied by electronic microscopy of morphological and ultrastructural changes induced by panaxydol. We also examined the cytotoxicities of panaxydol against HepG2 cells using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and the effect of panaxydol on cell cycle distributions by flow cytometry. We investigated the production of liver proteins in panaxydol-treated cells including alpha-fetoprotein and albumin and measured the specific activity of alkaline phosphatase and gamma-glutamyl transferase. We further investigated the effects of panaxydol on the expression of Id-1, Id-2, p21 and pRb by RT-PCR or immunoblotting analysis. We found that panaxydol inhibited the proliferation of HepG2 cells and caused morphological and ultrastructural changes in HepG2 cells resembling more mature forms of hepatocytes. Moreover, panaxydol induced a cell cycle arrest at the G₁ to S transition in HepG2 cells. It also significantly decreased the secretion of alpha-fetoprotein and the activity of gamma-glutamyl transferase. By contrast, panaxydol remarkably increased the secretion of albumin and the alkaline phosphatase activity. Furthermore, panaxydol increased the mRNA content of p21 while reducing that of Id-1 and Id-2. Panaxydol also increased the protein levels of p21, pRb and the hypophosphorylated pRb in a dose-dependent manner. These findings suggest that panaxydol is of value for further exploration as a potential anti-cancer agent.     

CB2 cannabinoid receptors promote neural progenitor cell proliferation via mTORC1 signaling

The endocannabinoid system is known to regulate neural progenitor (NP) cell proliferation and neurogenesis. In particular, CB(2) cannabinoid receptors have been shown to promote NP proliferation. As CB(2) receptors are not expressed in differentiated neurons, CB(2)-selective agonists are promising candidates to manipulate NP proliferation and indirectly neurogenesis by overcoming the undesired psychoactive effects of neuronal CB(1) cannabinoid receptor activation. Here, by using NP cells, brain organotypic cultures, and in vivo animal models, we investigated the signal transduction mechanism involved in CB(2) receptor-induced NP cell proliferation and neurogenesis. Exposure of hippocampal HiB5 NP cells to the CB(2) receptor-selective agonist HU-308 led to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway, which, by inhibiting its downstream target p27Kip1, induced NP proliferation. Experiments conducted with the CB(2) receptor-selective antagonist SR144528, inhibitors of the PI3K/Akt/mTORC1 axis, and CB(2) receptor transient-transfection vector further supported that CB(2) receptors control NP cell proliferation via activation of mTORC1 signaling. Likewise, CB(2) receptor engagement induced cell proliferation in an mTORC1-dependent manner both in embryonic cortical slices and in adult hippocampal NPs. Thus, HU-308 increased ribosomal protein S6 phosphorylation and 5-bromo-2'-deoxyuridine incorporation in wild-type but not CB(2) receptor-deficient NPs of the mouse subgranular zone. Moreover, adult hippocampal NP proliferation induced by HU-308 and excitotoxicity was blocked by the mTORC1 inhibitor rapamycin. Altogether, these findings provide a mechanism of action and a rationale for the use of nonpsychotomimetic CB(2) receptor-selective ligands as a novel strategy for the control of NP cell proliferation and neurogenesis.     

Critical parameters in the MCF-7 cell proliferation bioassay (E-Screen)

The MCF-7 cell proliferation bioassay has grown in popularity as a rapid test for detecting potentially oestrogenic compounds. Several MCF-7 cell sublines with different sensitivities to oestrogens are currently used, with maximal proliferation responses ranging from two- to 10-fold above those of hormone-free controls. In the highly responsive MCF-7 BUS cell line, we evaluated critical assay parameters for test performance, including growth conditions, initial seeding densities and differences in growth stimulation in medium containing human serum or fetal calf serum as well as appropriate solvents for oestrogen-mimicking compounds. Modifications significantly reduced the labour-intensive steps and overall assay costs without affecting the sensitivity of the assay. Using this optimized test regimen, the responsiveness of treated MCF-7 BUS cells was consistently increased up to 11-fold over hormone-free controls. The specificity was characterized by examining the effects of oestradiol-17 β, the anti-oestrogen ICI 182,780, and dieldrin, a recognized xeno-oestrogen. The improved proliferation bioassay will be a useful tool in identifying potential xeno-oestrogens.     

Critical parameters in the MCF-7 cell proliferation bioassay (E-Screen)

The MCF-7 cell proliferation bioassay has grown in popularity as a rapid test for detecting potentially oestrogenic compounds. Several MCF-7 cell sublines with different sensitivities to oestrogens are currently used, with maximal proliferation responses ranging from two- to 10-fold above those of hormone-free controls. In the highly responsive MCF-7 BUS cell line, we evaluated critical assay parameters for test performance, including growth conditions, initial seeding densities and differences in growth stimulation in medium containing human serum or fetal calf serum as well as appropriate solvents for oestrogen-mimicking compounds. Modifications significantly reduced the labour-intensive steps and overall assay costs without affecting the sensitivity of the assay. Using this optimized test regimen, the responsiveness of treated MCF-7 BUS cells was consistently increased up to 11-fold over hormone-free controls. The specificity was characterized by examining the effects of oestradiol-17β, the anti-oestrogen ICI 182,780, and dieldrin, a recognized xeno-oestrogen. The improved proliferation bioassay will be a useful tool in identifying potential xeno-oestrogens.