Yusaku Nakabeppu

DNA binding activities of three murine Jun proteins: Stimulation by Fos

Three members of the Jun/AP-1 family have been identified in mouse cDNA libraries: c-Jun, Jun-B, and Jun-D. We have compared the DNA binding properties of the Jun proteins by using in vitro translation products in gel retardation assays. Each protein was able to bind to the consensus AP-1 site (TGACTCA) and, with lower affinity, to related sequences, including the cyclic AMP response element TGACGTCA. The relative binding to the oligonucleotides tested was similar for the different proteins. The Jun proteins formed homodimers and heterodimers with other members of the family, and they were bound to the AP-1 site as dimers. When Fos translation product was present, DNA binding by Jun increased markedly, and the DNA complex contained Fos. The C-terminal homology region of Jun was sufficient for DNA binding, dimer formation, and interaction with Fos. Our general conclusion is that c-Jun, Jun-B, and Jun-D are similar in their DNA binding properties and in their interaction with Fos. If there are functional differences between them, they are likely to involve other activities of the Jun proteins.

Induction of a long-lasting AP-1 complex composed of altered Fos-like proteins in brain by chronic cocaine and other chronic treatments

Following chronic cocaine treatment, we have found a long-lasting increase in AP-1 binding in the rat nucleus accumbens and striatum, two important targets of the behavioral effects of cocaine. This increase develops gradually over several days and remains at 50% of maximal levels 7 days after the last cocaine exposure. Supershift experiments, along with one- and two-dimensional Western blots, indicate that this chronic AP-1 complex contains at least four Fos-related antigens (FRAs), some of which display delta FosB-like immunoreactivity, that are induced selectively by chronic, but not acute, cocaine treatment. The same chronic FRAs were also induced by several different types of chronic treatments in a region-specific manner in the brain. Thus, the chronic FRAs and associated chronic AP-1 complex could mediate some of the long-term changes in gene expression unique to the chronic-treated state as opposed to the acute-treated and normal states.

Galectin-1 is essential in tumor angiogenesis and is a target for antiangiogenesis therapy

We describe that galectin-1 (gal-1) is a receptor for the angiogenesis inhibitor anginex, and that the protein is crucial for tumor angiogenesis. gal-1 is overexpressed in endothelial cells of different human tumors. Expression knockdown in cultured endothelial cells inhibits cell proliferation and migration. The importance of gal-1 in angiogenesis is illustrated in the zebrafish model, where expression knockdown results in impaired vascular guidance and growth of dysfunctional vessels. The role of gal-1 in tumor angiogenesis is demonstrated in gal-1-null mice, in which tumor growth is markedly impaired because of insufficient tumor angiogenesis. Furthermore, tumor growth in gal-1-null mice no longer responds to antiangiogenesis treatment by anginex. Thus, gal-1 regulates tumor angiogenesis and is a target for angiostatic cancer therapy.

A naturally occurring truncated form of FosB that inhibits Fos/Jun transcriptional activity

Fos and Jun transcription factors are induced by a variety of extracellular signaling agents. We describe here an unusual member of the Fos family that is also induced, namely, a truncated form of FosB (delta FosB) missing the C-terminal 101 amino acids of FosB. delta FosB retains the dimerization and DNA-binding activities of FosB but has lost the ability in transfection assays to activate a promoter with an AP-1 site and to repress the c-fos promoter. Rather, delta FosB inhibits gene activation by Jun or Jun + Fos and inhibits repression of the c-fos promoter by FosB or c-Fos, presumably by competing with full-length Fos proteins at the steps of dimerization with Jun and binding to DNA. In stimulated cells delta FosB may act to limit the transcriptional effects of Fos and Jun proteins.

Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase

Oxygen radicals, which can be produced through normal cellular metabolism, are thought to play an important role in mutagenesis and tumorigenesis. Among various classes of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is most important because of its abundance and mutagenicity. The MTH1 gene encodes an enzyme that hydrolyzes 8-oxo-dGTP to monophosphate in the nucleotide pool, thereby preventing occurrence of transversion mutations. By means of gene targeting, we have established MTH1 gene-knockout cell lines and mice. When examined 18 months after birth, a greater number of tumors were formed in the lungs, livers, and stomachs of MTH1-deficient mice, as compared with wild-type mice. The MTH1-deficient mouse will provide a useful model for investigating the role of the MTH1 protein in normal conditions and under oxidative stress.

JSAP1, a novel Jun N-terminal protein kinase (JNK)-binding protein that functions as a scaffold factor in the JNK signaling pathway

ABSTRACT The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38α), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.

Mutagenesis and carcinogenesis caused by the oxidation of nucleic acids.

Abstract Genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are generated both as byproducts of oxygen respiration or molecular executors in the host defense, and by environmental exposure to ionizing radiation and chemicals. To counteract such oxidative damage in nucleic acids, mammalian cells are equipped with three distinct enzymes. MTH1 protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-2′-deoxyguanosine triphosphate and 2-hydroxy-2′-deoxyadenosine triphosphate (2-OH-dATP), to the corresponding monophosphates. We observed increased susceptibility to spontaneous carcinogenesis in MTH1-null mice, which exhibit an increased occurrence of A:T→C:G and G:C→T:A transversion mutations. 8-Oxoguanine (8-oxoG) DNA glycosylase, encoded by the OGG1 gene, and adenine DNA glycosylase, encoded by the MUTYH gene, are responsible for the suppression of G:C to T:A transversions caused by the accumulation of 8-oxoG in the genome. Deficiency of these enzymes leads to increased tumorigenesis in the lung and intestinal tract in mice, respectively. MUTYH deficiency may also increase G:C to T:A transversions through the misincorporation of 2-OH-dATP, especially in the intestinal tract, since MUTYH can excise 2-hydroxyadenine opposite guanine in genomic DNA and the repair activity is selectively impaired by a mutation found in patients with autosomal recessive colorectal adenomatous polyposis.

Chronic Alterations in Dopaminergic Neurotransmission Produce a Persistent Elevation of ΔFosB-like Protein(s) in both the Rodent and Primate Striatum

Using an antibody that recognizes the products of all known members of the fos family of immediate early genes, it was demonstrated that destruction of the nigrostriatal pathway by 6‐hydroxydopamine (6‐OHDA) lesions of the medial forebrain bundle produces a prolonged (>3 months) elevation of Fos‐like immunoreactivity in the striatum. Using retrograde tract tracing techniques, we have previously shown that this increase in Foslike immunoreactivity is located predominantly in striatal neurons that project to the globus pallidus. In the present study, Western blots were performed on nuclear extracts from the intact and denervated striatum of 6‐OHDA‐lesioned rats to determine the nature of Fos‐immunoreactive protein(s) responsible for this increase. Approximately 6 weeks after the 6‐OHDA lesion, expression of two Fos‐related antigens with apparent molecular masses of 43 and 45 kDa was enhanced in the denervated striatum. Chronic haloperidol administration also selectively elevated expression of these Fos‐related antigens, suggesting that their induction after dopaminergic denervation is mediated by reduced activation of D2‐like dopamine receptors. Western blot immunostaining using an antibody which recognizes the N‐terminus of FosB indicated that the 43 and 45 kDa Fos‐related antigens induced by dopaminergic denervation and chronic haloperidol administration may be related to a truncated from of FosB known as ΔFosB. Consistent with this proposal, retrograde tracing experiments confirmed that ΔFosB‐like immunoreactivity in the deafferented striatum was located predominantly in striatopallidal neurons. Gel shift experiments demonstrated that elevated AP‐1 binding activity in denervated striata contained FosB‐like protein(s), suggesting that enhanced ΔFosB levels may mediate some of the effects of prolonged dopamine depletion on AP‐1‐regulated genes in striatopallidal neurons. In contrast, chronic administration of the D1‐like receptor agonist CY 208–243 to 6‐OHDA‐lesioned rats dramatically enhanced ΔFosB‐like immunoreactivity in striatal neurons projecting to the substantia nigra. Western blot immunostaining revealed that ΔFosB and, to a lesser extent, FosB are elevated by chronic D1‐like agonist administration. Both the quantitative reverse transcriptase‐polymerase chain reaction and the ribonuclease protection assay demonstrated that Δfos B mRNA levels were substantially enhanced in the denervated striatum by chronic D1‐like agonist administration. Lastly, we examined the effects of chronic administration of D1‐like and D2‐like dopamine receptor agonists on striatal ΔFosB expression in the 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP) primate model of Parkinsons disease. In monkeys rendered Parkinsonian by MPTP, there was a modest increase in ΔFosB‐like protein(s), while the development of dyskinesia produced by chronic D1‐like agonist administration was accompanied by large increases in ΔFosB‐like protein(s). In contrast, administration of the long‐acting D2‐like agonist cabergoline, which alleviated Parkinsonian symptoms without producing dyskinesia reduced ΔFosB levels to near normal. Taken together, these results demonstrate that chronic alterations in dopaminergic neurotransmission produce a persistent elevation of ΔFosB‐like protein(s) in both the rodent and primate striatum.

The Oxidized Forms of dATP Are Substrates for the Human MutT Homologue, the hMTH1 Protein

The possibility that Escherichia coliMutT and human MTH1 (hMTH1) hydrolyze oxidized DNA precursors other than 8-hydroxy-dGTP (8-OH-dGTP) was investigated. We report here that hMTH1 hydrolyzed 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dATP (8-OH-dATP), oxidized forms of dATP, but not (R)-8,5′-cyclo-dATP, 5-hydroxy-dCTP, and 5-formyl-dUTP. The kinetic parameters indicated that 2-OH-dATP was hydrolyzed more efficiently and with higher affinity than 8-OH-dGTP. 8-OH-dATP was hydrolyzed as efficiently as 8-OH-dGTP. The preferential hydrolysis of 2-OH-dATP over 8-OH-dGTP was observed at all of the pH values tested (pH 7.2 to pH 8.8). In particular, a 5-fold difference in the hydrolysis efficiencies for 2-OH-dATP over 8-OH-dGTP was found at pH 7.2. However, E. coli MutT had no hydrolysis activity for either 2-OH-dATP or 8-OH-dATP. Thus, E. coli MutT is an imperfect counterpart for hMTH1. Furthermore, we found that 2-hydroxy-dADP and 8-hydroxy-dGDP competitively inhibited both the 2-OH-dATP hydrolase and 8-OH-dGTP hydrolase activities of hMTH1. The inhibitory effects of 2-hydroxy-dADP were 3-fold stronger than those of 8-hydroxy-dGDP. These results suggest that the three damaged nucleotides share the same recognition site of hMTH1 and that it is a more important sanitization enzyme than expected thus far.

Oxidative damage in nucleic acids and Parkinson's disease

Oxidative DNA lesions, such as 8‐oxoguanine (8‐oxoG), accumulate in nuclear and mitochondrial genomes during aging, and such accumulation can increase dramatically in patients with Parkinsons disease (PD). To counteract oxidative damage to nucleic acids, human and rodents are equipped with three distinct enzymes. One of these, MTH1, hydrolyzes oxidized purine nucleoside triphosphates, such as 8‐oxo‐2′‐deoxyguanosine triphosphate and 2‐hydroxy‐2′‐deoxyadenosine triphosphate, to their monophosphate forms. The other two enzymes are 8‐oxoG DNA glycosylase encoded by the OGG1 gene and adenine/2‐hydroxyadenine DNA glycosylase encoded by the MUTYH gene. We have shown a significant increase in 8‐oxoG in mitochondrial DNA as well as an elevated expression of MTH1, OGG1, and MUTYH in nigrostriatal dopaminergic neurons of PD patients, suggesting that the buildup of these lesions may cause dopamine neuron loss. We established MTH1‐null mice and found that MTH1‐null fibroblasts were highly susceptible to cell death caused by H2O2 characterized by pyknosis and electron‐dense deposits in the mitochondria, and that this was accompanied by an ongoing accumulation of 8‐oxoG in nuclear and mitochondrial DNA. We also showed that MTH1‐null mice exhibited an increased accumulation of 8‐oxoG in striatal mitochondrial DNA, followed by more extreme neuronal dysfunction after 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine administration than that of wild‐type mice. In conclusion, oxidative damage in nucleic acids is likely to be a major risk factor for Parkinsons disease, indicating that a solid understanding of the defense mechanisms involved will enable us to develop new strategies for protecting the brain against oxidative stress.