Ectopic expression of AINTEGUMENTA in Arabidopsis plants results in increased growth of floral organs.

AINTEGUMENTA (ANT) was previously shown to be involved in floral organ initiation and growth in Arabidopsis. ant flowers have fewer and smaller floral organs and possess ovules that lack integuments and a functional embryo sac. The present work shows that young floral meristems of ant plants are smaller than those in wild type. Failure to initiate the full number of organ primordia in ant flowers may result from insufficient numbers of meristematic cells. The decreased size of ant floral organs appears to be a consequence of decreased cell division within organ primordia. Ectopic expression of ANT under the control of the constitutive 35S promoter results in the development of larger floral organs. The number and shape of these organs is not altered and the size of vegetative organs is normal. Microscopic and molecular analyses indicate that the increased size of 35S::ANT sepals is the result of increased cell division, whereas the increased sizes of 35S::ANT petals, stamens, and carpels are primarily attributable to increased cell expansion. In addition, 35S::ANT ovules often exhibit increased growth of the nucellus and the funiculus. These results suggest that ANT stimulates cell growth in floral organs.     

Impact of segmental chromosomal duplications on leaf size in the grandifolia-D mutants of Arabidopsis thaliana

The number of cells in an organ is a major factor that specifies its size. However, the genetic basis of cell number determination is not well understood. To obtain insight into this genetic basis, three grandifolia-D ( gra-D ) mutants of Arabidopsis thaliana were characterized that developed huge leaves with two to three times more cells than the wild-type. Genetic and microarray analyses showed that a large segmental duplication had occurred in all the gra-D mutants, consisting of the lower part of chromosome 4. In the duplications, genes were found that encode AINTEGUMENTA (ANT), a factor that extends the duration of cell proliferation, and CYCD3;1, a G₁/S cyclin. The expression levels of both genes increased and the duration of cell proliferation in the leaf primordia was extended in the gra-D mutants. Data obtained by RNAi-mediated knockdown of ANT expression suggested that ANT contributed to the huge-leaf phenotype, but that it was not the sole factor. Introduction of an extra genomic copy of CYCD3;1 into the wild-type partially mimicked the gra-D phenotype. Furthermore, combined elevated expression of ANT and CYCD3;1 enhanced cell proliferation in a cumulative fashion. These results indicate that the duration of cell proliferation in leaves is determined in part by the interaction of ANT and CYCD3;1 , and also demonstrate the potential usefulness of duplication mutants in the elucidation of genetic relationships that are difficult to uncover by standard single-gene mutations or gain-of-function analysis. We also discuss the potential effect of chromosomal duplication on evolution of organ size.     

Enhanced cytokinin degradation in leaf primordia of transgenic Arabidopsis plants reduces leaf size and shoot organ primordia formation

The plant hormone cytokinin is a key morphogenic factor controlling cell division and differentiation, and thus the formation and growth rate of organs during a plant's life cycle. In order to explore the relevance of cytokinin during the initial phase of leaf primordia formation and its impact on subsequent leaf development, we increased cytokinin degradation in young shoot organ primordia of Arabidopsis thaliana by expressing a cytokinin oxidase/dehydrogenase (CKX) gene under control of the AINTEGUMENTA (ANT) promoter. The final leaf size in ANT:CKX3 plants was reduced to ∼27% of the wild-type size and the number of epidermal cells was reduced to ∼12% of the wild type. Kinematic analysis revealed that cell proliferation ceased earlier and cell expansion was accelerated in ANT:CKX3 leaves, demonstrating that cytokinin controls the duration of the proliferation phase by delaying the onset of cell differentiation. The reduction of the cell number was partially compensated by an increased cell expansion. Interestingly, ANT:CKX3 leaf cells became about 60% larger than those of 35S:CKX3 leaves, indicating that cytokinin has an important function during cell expansion as well. Furthermore, ANT:CKX3 expression significantly reduced the capacity of both the vegetative as well as the generative shoot apical meristem to initiate the formation of new leaves and flowers, respectively. We therefore hypothesize that the cytokinin content in organ primordia is important for regulating the activity of the shoot meristem in a non-autonomous fashion.     

The fourth isoform of the adenine nucleotide translocator inhibits mitochondrial apoptosis in cancer cells

The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1–4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANT1 and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondrial network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli.     

IL-4-induced upregulation of adenine nucleotide translocase 3 and its role in Th cell survival from apoptosis

We have identified mitochondrial adenine nucleotide translocase (ANT)3 as a novel target up-regulated by IL-4 in human T cells. The IL-4-induced ANT3 expression is dependent on tyrosine kinase, NF-kappaB, PI3K/Akt, and Erk pathways. In fact, IL-4 induced specific activation of NF-kappaB, Akt, and Erk in Jurkat T cells and partially rescued these cells from dexamethasone-induced apoptosis. The IL-4-mediated T cell survival was blocked by inhibitors of tyrosine kinase, NF-kappaB, PI3K/Akt, and Erk. During the IL-4-induced T cell rescue, there was a concomitant increase in ANT3, nuclear NF-kappaB, and Bcl-2 and a decrease in ANT1, I-kappaB, and mitochondrial Bax-alpha levels. Importantly, overexpression of ANT3 effectively protected T cells from dexamethasone-induced apoptosis, while forced expression of ANT1 caused apoptosis. In contrast, siRNA knock-out of ANT3 expression induced T cell apoptosis and blocked the IL-4-mediated cell survival. Together these results suggest that ANT3 has a potential role in Th cell survival and immune cell homeostasis.     

Expression of human and mouse adenine nucleotide translocase (ANT) isoform genes in adipogenesis

Adenine nucleotide translocases (ANTs) are mitochondrial proteins encoded by nuclear DNA that catalyze the exchange of ATP generated in the mitochondria for ADP produced in cytosol. There are four ANT isoforms in humans (hANT1-4) and three in mice (mANT1, mANT2 and mANT4), all encoded by distinct genes. The aim of this study was to quantify expression of ANT isoform genes during the adipogenesis of mouse 3T3-L1 and human Simpson–Golabi–Behmel syndrome (SGBS)-derived preadipocytes. We also studied the effects of the adipogenesis regulators, insulin and rosiglitazone, on ANT isoform expression in differentiated adipocytes and examined the expression of ANT isoforms in subcutaneous and visceral white adipose tissue (WAT) from mice and humans. We found that adipogenesis was associated with an increase in the expression of ANT isoforms, specifically mANT2 in mouse 3T3-L1 cells and hANT3 in human SGBS cells. These changes could be involved in the increases in oxidative metabolism and decreases in lactate production observed during differentiation. Insulin and rosiglitazone induced mANT2 gene expression in mature 3T3-L1 cells and hANT2 and hANT3 gene expression in SGBS adipocytes. Furthermore, human WAT expressed greater amounts of hANT3 than hANT2, and the expression of both of these isoforms was greater in subcutaneous WAT than in visceral WAT. Finally, inhibition of ANT activity by atractyloside or bongkrekic acid impaired proper adipocyte differentiation. These results suggest that changes in the expression of ANT isoforms may be involved in adipogenesis in both human and mouse WAT.     

红莲型细胞质雄性不育（HL-CMS）水稻不育系与保持系中3个ANTs基因表达模式的比较

探讨红莲型细胞质雄性不育（HL-CMS）水稻不育系‘粤泰A’（‘YTA’）和保持系‘粤泰B’（‘YTB’）中3个腺苷酸转位酶（ANT）基因在三叶期根、茎、叶以及不同发育时期的幼穗中的表达模式。结果表明：ANT1和ANT2在‘YTA’和‘YTB’三叶期的根、茎、叶中的表达量都较高,而在生殖生长不同时期的幼穗中表达量较低。‘YTA’中ANT1在不同时期的幼穗中表达量都较低,而ANT2的表达量到穗生长期II的幼穗才较低。‘YTB’中ANT1在生殖生长的穗生长期III的幼穗有很高的表达。ANT3的表达水平在研究的各组织中的表达都较低,在‘YTB’穗生长期V的幼穗中表达最高。相对于‘YTA’,‘YTB’中只有ANT3在穗生长期III和穗生长期V的幼穗中呈现明显的优势表达;而相对于‘YTB’,‘YTA’中ANT2和ANT3在穗生长期I、ANT2在穗生长期VI幼穗具有明显的优势表达。3个ANTs基因在HL-CMS不育系‘YTA’和保持系‘YTB’不同组织及发育时期的表达模式的差异,暗示它们可能与HL-CMS水稻不育系和保持系的发育调控有关。     

The hepatoprotective effects of adenine nucleotide translocator-2 against aging and oxidative stress

Mitochondrial adenine nucleotide translocator (ANT) plays important roles in the regulation of mitochondrial permeability transition and cell bioenergetics. The mouse has three ANT isoforms (1, 2 and 4) showing tissue-specific expression patterns. Although ANT1 is known to have a pro-apoptotic property, the specific functions of ANT2 have not been well determined. In the present study, ANT2 expression was significantly lower in the aged rat liver and in a liver fibrosis model. To explore the protective role of ANT2 in the liver, we established a hepa1c1c7 cell line overexpressing ANT2. Overexpression of ANT2 caused hepa1c1c7 cells to be more resistant to oxidative stress, and mitochondrial membrane potential (MMP, ?Ψm) was relatively intact in ANT2-overexpressing cells under oxidative stress. In addition, ANT2 was found to increase ATP production by influencing mitochondrial bioenergetics. These results imply that the hepatoprotective effect of ANT2 is due to the stabilization of MMP and enhanced ATP production, and thus, maintaining ANT2 levels in the liver might be important to enhance resistance to aging and oxidative stress.     

Adenine nucleotide translocase 2 is a key mitochondrial protein in cancer metabolism

Adenine nucleotide translocase (ANT), a mitochondrial protein that facilitates the exchange of ADP and ATP across the mitochondrial inner membrane, plays an essential role in cellular energy metabolism. Human ANT presents four isoforms (ANT1-4), each with a specific expression depending on the nature of the tissue, cell type, developmental stage and status of cell proliferation. Thus, ANT1 is specific to muscle and brain tissues; ANT2 occurs mainly in proliferative, undifferentiated cells; ANT3 is ubiquitous; and ANT4 is found in germ cells. ANT1 and ANT3 export the ATP produced by oxidative phosphorylation (OxPhos) from the mitochondria into the cytosol while importing ADP. In contrast, the expression of ANT2, which is linked to the rate of glycolytic metabolism, is an important indicator of carcinogenesis. In fact, cancers are characterized by major metabolic changes that switch cells from the normally dual oxidative and glycolytic metabolisms to an almost exclusively glycolytic metabolism. When OxPhos activity is impaired, ANT2 imports glycolytically produced ATP into the mitochondria. In the mitochondrial matrix, the F1F0-ATPase complex hydrolyzes the ATP, pumping out a proton into the intermembrane space. The reverse operations of ANT2 and F1F0-ATPase under glycolytic conditions contribute to maintaining the mitochondrial membrane potential, ensuring cell survival and proliferation. Unlike the ANT1 and ANT3 isoforms, ANT2 is not pro-apoptotic and may therefore contribute to carcinogenesis. Since the expression of ANT2 is closely linked to the mitochondrial bioenergetics of tumors, it should be taken into account for individualizing cancer treatments and for the development of anticancer strategies.     

Mitochondrial adenine nucleotide translocator 3 is regulated by IL-4 and IFN-gamma via STAT-dependent pathways

IL-4 and IFN-γ are prototypical Th2 and Th1 cytokines, respectively. They reciprocally regulate a number of genes involved in Th1 vs Th2 immune balance. Using DD-PCR analysis, adenine nucleotide translocase (ANT) 3, an enzyme which exchanges ATP and ADP through mitochondrial membrane, has been identified as a novel target counter-regulated by IL-4 and IFN-γ. We have observed that IL-4 and IFN-γ each up-regulates ANT3 in T cells both at mRNA and protein levels, while cotreatment of IL-4 and IFN-γ counter-regulates ANT3 expression. In contrast, other isoforms of ANT were not affected by IL-4 or IFN-γ. Emplyoing transfection and overexpression of STAT6 and STAT1 in STAT-deficient cells, we demonstrate that induction of ANT3 by IL-4 and IFN-γ proceeds via pathways involving STAT6 and STAT1, respectively. Furthermore, regulation of ANT3 expression by IL-4 and IFN-γ correlated with the modulation T cell survival by these cytokines from dex-induced apoptosis. Considering the critical role of mitochondrial ANTs in energy metabolism and apoptosis, ANT3 regulation by IL-4 and IFN-γ may have a functional implication in cytokine-mediated T cell survival.     

A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling

Oxidative stress and mitochondrial dysfunction are associated with disease and aging. Oxidative stress results from overproduction of reactive oxygen species (ROS), often leading to peroxidation of membrane phospholipids and production of reactive aldehydes, particularly 4-hydroxy-2-nonenal. Mild uncoupling of oxidative phosphorylation protects by decreasing mitochondrial ROS production. We find that hydroxynonenal and structurally related compounds (such as trans-retinoic acid, trans-retinal and other 2-alkenals) specifically induce uncoupling of mitochondria through the uncoupling proteins UCP1, UCP2 and UCP3 and the adenine nucleotide translocase (ANT). Hydroxynonenal-induced uncoupling was inhibited by potent inhibitors of ANT (carboxyatractylate and bongkrekate) and UCP (GDP). The GDP-sensitive proton conductance induced by hydroxynonenal correlated with tissue expression of UCPs, appeared in yeast mitochondria expressing UCP1 and was absent in skeletal muscle mitochondria from UCP3 knockout mice. The carboxyatractylate-sensitive hydroxynonenal stimulation correlated with ANT content in mitochondria from Drosophila melanogaster expressing different amounts of ANT. Our findings indicate that hydroxynonenal is not merely toxic, but may be a biological signal to induce uncoupling through UCPs and ANT and thus decrease mitochondrial ROS production.