Katarina Luciakova

ON THE ROLE OF THE GENERAL TRANSCRIPTION FACTOR SP1 IN THE ACTIVATION AND REPRESSION OF DIVERSE MAMMALIAN OXIDATIVE PHOSPHORYLATION GENES

To gain insight into the role of the general transcription factor,Sp1, in the expression of nuclear genes involved in mitochondrial biogenesis,we investigated Sp1 activation of the adenine nucleotide translocator 2,cytochrome c1, F1-ATPase β subunit, and themitochondria transcription factor (mtTFA) promoters transfected intoDrosophila cell lines. The numbers and organization of GC elementsvary in the four promoters, but the magnitude of activation by coexpressedhuman Sp1 was similar. A feature common to the four promoters is the presenceof multiple, proximal Sp1-activating elements that account for 50% ormore of the transcription activation by Sp1. The distribution and function ofindividual distal Sp1 elements is less defined and appear to be morepromoter-specific. Finally, data from transfected Drosophila cellsprovide the first direct proof for the involvement of Sp1 in the negativeregulation of the ANT2 promoter and as a possible participant in repressionof the β-subunit promoter. The role of Sp1 in both the positive andnegative regulation of OXPHOS promoters is unique.

The role of thyroid hormone and promoter diversity in the regulation of nuclear encoded mitochondrial proteins

Thyroid hormone regulates the in vivo expression of a selected set of rat nuclear genes encoding mitochondrial inner membrane proteins. Certain mRNAs, such as that for cytochrome c1, are increased as much as 20-50-fold, while others, such as core protein 1 of Complex III and the F1-ATPase beta-subunit do not respond. The promoter region of human cytochrome c1 also supports thyroid hormone induction of a reporter gene in transient transfection experiments. Thus, thyroid hormone regulates only selected genes, even for subunits within the same complex and in widely varying species. By contrast, growth activation of quiescent NIH3T3 cells, a second paradigm used for stimulating mitochondrial biogenesis, does not increase cytochrome c1 mRNA but does increase F1-ATPase beta-subunit mRNA. These findings suggest that nuclear OXPHOS genes are not necessarily expressed in a coordinated manner, and that multiple regulatory circuits might exist which are linked to different physiological stimuli. Analysis of the promoters of several OXPHOS genes reveals a great diversity and heterogeneity of transfactor binding elements. No single regulatory feature exists which could account for a coordinated expression of all OXPHOS genes. The potential diversity for regulating expression of nuclear OXPHOS genes raises the possibility for the existence of disease states linked to regulatory defects.

Increased content of natural ATPase inhibitor in tumor mitochondria

The ATPase activity of Zajdela hepatoma and Yoshida sarcoma submitochondrial particles was several times lower than the enzyme activity in rat heart and rat liver submitochondrial particles. The content of F1‐ATPase in the tumor mitochondria was found not to be very different from that in mitochondria of rat liver. Immunochemical determination of the amount of the natural ATPase inhibitor revealed that the tumor mitochondria contain 2–3‐times more ATPase inhibitor than control mitochondria. It is concluded that the low ATPase activity of the tumor mitochondria results from the inhibition of the enzyme activity by the natural ATPase inhibitor.

Amino acid incorporation by isolated rat liver mitochondria into two protein components of mitochondrial ATPase complex: Lack of incorporation into dicyclohexylcarbodiimide binding proteolipid

The role of products of mitochondrial protein synthesis in the biogenesis of the mitochondrial ATPase complex of yeast and fungi is well documented. In Saccharomyces cerevisiae four [ 1 ] and in Neurospora crassa two [2] protein subunits of the complex are synthesized intramitochondrially. Special attention has been paid to the elucidation of the site of the synthesis of DCCD binding proteolipid of the mitochondrial ATPase complex. In yeast [3] this component is synthesized within mitochondria and in fungi [4,5] outside the organelles. Less information exists on the participation of mitochondrial translation products in the formation of mitochondrial ATPase complex and on the site of the synthesis of DCCD binding proteolipid of the complex in animal cells. The intramitochondrial formation of three protein components of mitochondrial ATPase complex in oocytes [6] and in regenerating rat liver [7] has been indicated by selective invivo inhibition of cytoplasmic or mitochondrial protein synthesis. Incorporation of labeled amino acids by isolated rat [8] and mouse [9] liver mitochondria into DCCD binding proteolipid has been described and it was concluded that the DCCD binding component of rat [8] and mouse [9] liver mitochondrial ATPase complex represents a product of mitochondrial protein synthesis. On the other hand, selective in vivo inhibition of mitochondrial protein synthesis in regenerating rat liver did not affect significantly amino

TGF-β/NF1/Smad4-mediated suppression of ANT2 contributes to oxidative stress in cellular senescence

Oxidative stress and persistent activation of DNA damage response (DDR) are causally involved in the development of cellular senescence, a phenomenon implicated in fundamental (patho)physiological processes such as aging, fetal development and tumorigenesis. Here, we report that adenine nucleotide translocase-2 (ANT2) is consistently down-regulated in all three major forms of cellular senescence: replicative, oncogene-induced and drug-induced, in both normal and cancerous human cells. We previously reported formation of novel NF1/Smad transcription repressor complexes in growth-arrested fibroblasts. Here we show that such complexes form in senescent cells. Mechanistically, binding of the NF1/Smad complexes to the NF1-dependent repressor elements in the ANT2 gene promoter repressed ANT2 expression. Etoposide-induced formation of these complexes and repression of ANT2 were relatively late events co-incident with production and secretion of, and dependent on, TGF-β. siRNA-mediated knock-down of ANT2 in proliferating cells resulted in increased levels of reactive oxygen species (ROS) and activation of the DDR. Knock-down of ANT2, together with etoposide treatment, further intensified ROS production and DNA damage signaling, leading to enhanced apoptosis. Together, our data show that TGF-β-mediated suppression of ANT2 through NF1/Smad4 complexes contributes to oxidative stress and DNA damage during induction of cellular senescence.

Immunochemical analysis of the membrane proteins of rat liver and Zajdela hepatoma mitochondria.

The contents of mitochondrial inner membrane protein complexes were compared in normal liver and in Zajdela hepatoma mitochondria by the immunotransfer technique. Antibodies against core proteins 1 and 2, cytochrome c1, the iron-sulfur protein of Complex III, subunits I and II of cytochrome oxidase, and the alpha and beta subunits of the F1-ATPase were used. In addition, antibodies against a primary dehydrogenase, beta-hydroxybutyrate dehydrogenase, as well as the outer membrane pore protein were used. The results indicate that the components of the cytochrome chain and porin are greatly enriched in hepatoma mitochondria compared to normal rat liver mitochondria. This enrichment was also reflected in the rates of respiration in tumor mitochondria using a variety of substrates. Enrichment of porin may partially account for increased hexokinase binding to tumor mitochondria. In contrast to the respiratory chain components, the F1-ATPase and F0 (measured by DCCD binding) were not increased in tumor mitochondria. Thus, Zajdela hepatoma mitochondria components are nonstoichiometric, being enriched in oxidative capacity but relatively deficient in ATP synthesizing capacity. Finally, beta-hydroxybutyrate dehydrogenase, which is often decreased in hepatoma mitochondria, was shown here by immunological methods to be decreased by only 40%, whereas enzyme activity was less than 5% of that in normal rat liver.

Growth-dependent repression of human adenine nucleotide translocator-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex

NF1 (nuclear factor 1) binds to two upstream elements of the human ANT2 (adenine nucleotide translocator-2) promoter and actively represses expression of the gene in growth-arrested diploid skin fibroblasts [Luciakova, Barath, Poliakova, Persson and Nelson (2003) J. Biol. Chem. 278, 30624-30633]. ChIP (chromatin immunoprecipitation) and co-immunoprecipitation analyses of nuclear extracts from growth-arrested and growth-activated diploid cells demonstrate that NF1, when acting as a repressor, is part of a multimeric complex that also includes Smad and Sp-family proteins. This complex appears to be anchored to both the upstream NF1-repressor elements and the proximal promoter, Sp1-dependent activation elements in growth-arrested cells. In growth-activated cells, the repressor complex dissociates and NF1 leaves the promoter. As revealed by co-immunoprecipitation experiments, NF1-Smad4-Sp3 complexes are present in nuclear extracts only from growth-inhibited cells, suggesting that the growth-state-dependent formation of these complexes is not an ANT2 promoter-specific event. Consistent with the role of Smad proteins in the repression complex, TGF-beta (transforming growth factor-beta) can fully repress ANT2 transcription in normally growing fibroblasts. Finally, pull-down experiments of in vitro transcribed/translated NF1 isoforms by GST (glutathione transferase)-Smad and GST-Smad MH fusion proteins indicate direct physical interactions between members of the two families. These findings suggest a possible functional relationship between the NF1 and Smad proteins that has not been previously observed.

Quantification of mitochondrial proteins in cultured cells by immuno-flow cytometry.

Immuno-flow cytometry was tested as a tool to estimate the cellular concentration of mitochondrial proteins in cultured cells, using cytochrome c oxidase as a model enzyme. Cells labelled with antibodies against cytochrome c oxidase, in which the amount of the enzyme was reduced by various extents, showed a linear relationship between the size of the signal obtained by immuno-flow cytometry and the amount of the enzyme. The determination by immuno-flow cytometry resulted in data comparable to the results obtained by immunoprecipitation and activity measurements. Since immuno-flow cytometry requires only limited numbers of cells, the method could especially be of value for diagnostic purposes. This is illustrated by the results obtained by comparing activity measurements and immuno-flow cytometry in the initial screening of cell lines derived from patients with deficiencies in the activity of cytochrome c oxidase.

Sustained induction of cytochrome P4501A1 in human hepatoma cells by co-exposure to benzo[a]pyrene and 7H-dibenzo[c,g]carbazole underlies the synergistic effects on DNA adduct formation.

To gain a deeper insight into the potential interactions between individual aromatic hydrocarbons in a mixture, several benzo[a]pyrene (B[a]P) and 7H-dibenzo[c,g]carbazole (DBC) binary mixtures were studied. The biological activity of the binary mixtures was investigated in the HepG2 and WB-F344 liver cell lines and the Chinese hamster V79 cell line that stably expresses the human cytochrome P4501A1 (hCYP1A1). In the V79 cells, binary mixtures, in contrast to individual carcinogens, caused a significant decrease in the levels of micronuclei, DNA adducts and gene mutations, but not in cell survival. Similarly, a lower frequency of micronuclei and levels of DNA adducts were found in rat liver WB-F344 cells treated with a binary mixture, regardless of the exposure time. The observed antagonism between B[a]P and DBC may be due to an inhibition of Cyp1a1 expression because cells exposed to B[a]P:DBC showed a decrease in Cyp1a1 mRNA levels. In human liver HepG2 cells exposed to binary mixtures for 2h, a reduction in micronuclei frequency was also found. However, after a 24h treatment, synergism between B[a]P and DBC was determined based on DNA adduct formation. Accordingly, the up-regulation of CYP1A1 expression was detected in HepG2 cells exposed to B[a]P:DBC. Our results show significant differences in the response of human and rat cells to B[a]P:DBC mixtures and stress the need to use multiple experimental systems when evaluating the potential risk of environmental pollutants. Our data also indicate that an increased expression of CYP1A1 results in a synergistic effect of B[a]P and DBC in human cells. As humans are exposed to a plethora of noxious chemicals, our results have important implications for human carcinogenesis.

TGF-β signals the formation of a unique NF1/Smad4-dependent transcription repressor-complex in human diploid fibroblasts.

We earlier reported the formation of a unique nuclear NF1/Smad complex in serum-restricted fibroblasts that acts as an NF1-dependent repressor of the human adenine nucleotide translocase-2 gene (ANT2) [K. Luciakova, G. Kollarovic, P. Barath, B.D. Nelson, Growth-dependent repression of human adenine nucleotide translocator-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex, Biochem. J. 412 (2008) 123-130]. In the present study, we show that TGF-β, like serum-restriction: (a) induces the formation of NF1/Smad repressor complexes, (b) increases binding of the complexes to the repressor elements (Go elements) in the ANT2 promoter, and (c) inhibits ANT2 expression. Repression of ANT2 by TGF-β is eliminated by mutating the NF1 binding sites in the Go repressor elements. All of the above responses to TGF-β are prevented by inhibitors of TGF-β RI and MAPK p38. These inhibitors also prevent NF1/Smad4 repressor complex formation and repression of ANT2 expression in serum-restricted cells, suggesting that similar signaling pathways are initiated by TGF-β and serum-restriction. The present finding that NF1/Smad4 repressor complexes are formed through TGF-β signaling pathways suggests a new, but much broader, role for these complexes in the initiation or maintenance of the growth-inhibited state.