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The discovery of a novel series of β-methyltryptophan (β MeTrp) derivatives as selective and orally active non-peptide somatostatin receptor 2 (SSTR2) agonists for the treatment of Type 2 diabetes is described. In our previous research, Compound A, β-MeTrp derivative with highly potent and selective SSTR2 agonistic activity IC50 (SSTR2/SSTR5)=0.3/>100 (nM), was identified asa drug candidate for treatment of Type 2 diabetes which lowers significantly plasma glucose level in Wistar fatty rats in its oral administrations. However, as serious increase in AUC and phospholipidosis (PLsis) were observed in its toxicological studies in rats, follow-up compounds were searched to avoid risk of PLsis with reference to their in vitro PLsis potentials evaluated on the basis of accumulation of phospholipids in HepG2 cells exposed to the compounds. It has been found that introduction of a carbonyl group onto the piperidine and piperazine or aniline moiety of compounds A and B reduced markedly the in vitro PLsis potentials. And further modification of the compounds and their evaluation led to a discovery of compounds 3k with lower in vitro PLsis potentials exhibiting lowering effect of hypoglycemia-induced glucagon secretion in SD rats (ED50=1.1mg/kg) and glucose excursion in meal tolerance test in Wistar fatty diabetic rats (MED=3.0mg/kg) in oral administrations. Compound 3k was selected asa new drug candidate of selective and orally active non-peptide SSTR2 agonists for treatment of Type 2 diabetes with low in vivo PLsis potential.

The smooth muscle myosin heavy chain (MHC) gene and its isoforms are excellent molecular markers that reflect smooth muscle phenotypes. The SMemb/Nonmuscle Myosin Heavy Chain B (NMHC-B) is a distinct MHC gene expressed predominantly in phenotypically modulated SMCs (synthetic-type SMC). To dissect the molecular mechanisms governing phenotypic modulation of SMCs, we analyzed the transcriptional regulatory mechanisms underlying expression of the SMemb gene. We previously reported two transcription factors, BTEB2/IKLF and Hex, which transactivate the SMemb gene promoter based on the transient reporter transfection assays. BTEB2/IKLF is a zinc finger transcription factor, whereas Hex is a homeobox protein. BTEB2/IKLF expression in SMCs is downregulated with vascular development in vivo but upregulated in cultured SMCs and in neointima in response to vascular injury after balloon angioplasty. BTEB2/IKLF and Hex activate not only the SMemb gene but also other genes activated in synthetic SMCs including plasminogen activator inhibitor-1 (PAI-1), iNOS, PDGF-A, Egr-1, and VEGF receptors. Mitogenic stimulation activates BTEB2/IKLF gene expression through MEK1 and Egr-1. Elevation of intracellular cAMP is also important in phenotypic modulation of SMCs, because the SMemb promoter is activated under cooperatively by cAMP-response element binding protein (CREB) and Hex.

Regulation of chromatin in eukaryotic transcription requires histone-modifying enzymes, nucleosome remodeling complexes, and histone chaperones. Specific regulation of histone incorporation/eviction by histone chaperones on the promoter (e.g., region specific) is still poorly understood. In the present study, we show that direct and functional interaction of histone chaperone and DNA-binding transcription factor leads to promoter region-specific histone incorporation and inhibition of histone acetylation. We report here that the DNA-binding transcription factor Krüppel-like factor 5 (KLF5) interacts with the novel histone chaperone acidic nuclear phosphoprotein 32B (ANP32B), leading to transcriptional repression of a KLF5-downstream gene. We further show that recruitment of ANP32B onto the promoter region requires KLF5 and results in promoter region-specific histone incorporation and inhibition of histone acetylation by ANP32B. Extracellular stimulus (e.g., phorbol ester) regulates this mechanism in the cell. Collectively, we have identified a novel histone chaperone, ANP32B, and through analysis of the actions of this factor show a new mechanism of promoter region-specific transcriptional regulation at the chromatin level as mediated by the functional interaction between histone chaperone and DNA-binding transcription factor.

Phenotypic modulation of smooth muscle from the contractile to the synthetic type underlies the pathogenesis of atherosclerosis. We previously isolated a transcription factor, KLF5 (also known as BTEB2 and IKLF), to activate various gene promoters that are activated in phenotypically modulated smooth muscle cells, such as embryonic smooth muscle myosin heavy chain gene (SMemb), plasminogen activator inhibitor-1 (PAI-1), iNOS, PDGF-A, Egr-1 and VEGF receptors. KLF5 is downregulated with vascular development but upregulated in neointima. Chromatin immunoprecipitation assay showed KLF5 to be induced and to bind the promoter of the PDGF-A gene in response to angiotensin II stimulation. To define the role of KLF5 in cardiovascular remodeling, we targeted the KLF5 gene in mice. In response to external stress, arteries of heterozygotes exhibited diminished levels of smooth muscle and adventitial cell activation. We also found that RARα binds KLF5, and that Am80, a potent synthetic RAR agonist, inhibits angiotensin II-induced cardiac hypertrophy. These results indicate that KLF5 is an essential transcription factor that causes not only smooth muscle phenotypic modulation but also cardiac hypertrophy and fibrosis.

We recently isolated a Krüppel-like zinc-finger transcription factor 5 (KLF5; also known as BTEB2 and IKLF), which is markedly induced in activated vascular smooth-muscle cells and fibroblasts. Here we describe our analysis of the in vivo function of KLF5 using heterozygous KLF5-knockout mice (Klf5(+/-)). In response to external stress, Klf5(+/-) mice showed diminished levels of arterial-wall thickening, angiogenesis, cardiac hypertrophy and interstitial fibrosis. Also, angiotensin II induced expression of KLF5, which in turn activated platelet-derived growth factor-A (PDGF-A) and transforming growth factor-beta (TGF-beta) expression. In addition, we determined that KLF5 interacted with the retinoic-acid receptor (RAR), that synthetic RAR ligands modulated KLF5 transcriptional activity, and that in vivo administration of RAR ligands affected stress responses in the cardiovascular system in a KLF5-dependent manner. KLF5 thus seems to be a key element linking external stress and cardiovascular remodeling.

Drug candidates under development by industry frequently show phospholipidosis as a side-effect in pre-clinical toxicity studies. This study sets up a cell-based assay for drug-induced phospholipidosis (PLD) and its performance was evaluated based on the in vivo PLD potential of compounds in 2-week toxicity studies in rats. When HepG2 cells were exposed simultaneously to PLD-inducing chemicals and a phospholipid having a fluorophore, an accumulation of phospholipids was detected as an increasing fluorescent intensity. Amiodarone, amitriptyline, fluoxetine, AY-9944, and perhexiline, which are common PLD-inducing chemicals, increased the fluorescent intensity, but acetaminophen, ampicillin, cimetidine, famotidine, or valproic acid, which are non-PLD-inducing chemicals, did not. The fluorescent intensity showed concordance with the pathological observations of phospholipid lamellar bodies in the cells. Then to confirm the predictive performance of the in vitro PLD assay, the 32 proprietary compounds characterized in 2-week toxicity studies in rats were evaluated with this in vitro assay. Because this in vitro assay was vulnerable to cytotoxicity, the innate PLD potential was calculated for each compound. A statistically significant increase in the in vitro PLD potential was seen for the compounds having in vivo PLD-inducing potential in the rat toxicity studies. The results suggest that the in vitro PLD potential could be appropriate to detect the appearance of PLD as a side effect in pre-clinical toxicity studies in rats.

Although moderate hypothermia is one of the most robust and effective techniques available for reducing ischemic injury, its key mechanism still remains unclear. Our proteomic analysis of the brains of rats treated with a 2-h middle cerebral artery occlusion showed that postischemic hypothermia markedly potentiated a sustained increase in heat-shock protein 70 (Hsp70). The elevated Hsp70 level was confirmed by enzyme-linked immunosorbent assay, western blot analysis, and immunohistochemical staining. Expression of other Hsp proteins was unaffected by hypothermia. Interestingly, hypothermia did not increased, even decreased, the upregulation of hsp70 mRNA expression by ischemia, suggesting that Hsp70 abundance is controlled by an unknown posttranscriptional regulation. As Hsp70 exerts a protective role against ischemic damage, the specific increase in Hsp70 production may contribute to the neuroprotective effect of hypothermia.

Transcription involves molecular interactions between general and regulatory transcription factors with further regulation by protein-protein interactions (e.g. transcriptional cofactors). Here we describe functional interaction between DNA-binding transcription factor and histone chaperone. Affinity purification of factors interacting with the DNA-binding domain of the transcription factor Sp1 showed Sp1 to interact with the histone chaperone TAF-I, both alpha and beta isoforms. This interaction was specific as Sp1 did not interact with another histone chaperone CIA nor did other tested DNA-binding regulatory factors (MyoD, NFkappaB, p53) interact with TAF-I. Interaction of Sp1 and TAF-I occurs both in vitro and in vivo. Interaction with TAF-I results in inhibition of DNA-binding, and also likely as a result of such, inhibition of promoter activation by Sp1. Collectively, we describe interaction between DNA-binding transcription factor and histone chaperone which results in negative regulation of the former. This novel regulatory interaction advances our understanding of the mechanisms of eukaryotic transcription through DNA-binding regulatory transcription factors by protein-protein interactions, and also shows the DNA-binding domain to mediate important regulatory interactions.

KLF5/BTEB2 is a Kruppel-like zinc-finger type transcription factor of a non-muscle type myosin heavy chain gene SMemb, which is markedly induced in phenotypically modulated smooth muscle (SMC). KLF5/BTEB2 expression in SMC is downregulated with vascular development in vivo but upregulated in neointima that is produced in response to vascular injury. KLF5/BTEB2 activates the promoter of not only SMemb gene, but also of other genes which are actvated in synthetic SMC, including plasminogen activator inhibitor-1 (PAI-1), iNOS, PDGF-A, Egr-1 and VEGF receptors. Mitogenic stimulation activates KLF5/BTEB2 gene expression through MEK1 and Egr-1. KLF5/BTEB2 +/- mice showed a marked reduction in neointimal formation, angiogenesis and cardiac fibrosis induced by various kinds of stress. We suggest that KLF5/BTEB2 is a crucial transcription factor involved in smooth muscle phenotypic modulation as well as mesenchymal cell activation in cardiovascular disease.

The purpose of this study was to investigate the cause of polycythemia occurring in hepatocellular carcinoma-bearing control male B6C3F, mice from 2-yr carcinogenicity studies. Erythrocyte counts and plasma levels of erythropoietin in mice with hepatocellular carcinomas were significantly increased compared with the values in non-tumor-bearing mice. Erythropoietin mRNA in 4 of 5 nontumor-bearing mice was detected in the kidney, but no visible signals for hepatic erythropoietin mRNA in 5 of 5 non-tumor-bearing mice were detected by the reverse transcriptase competitive polymerase chain reaction method. Erythropoietin mRNA was expressed in neoplastic hepatocytes from 8 of 9 hepatocellular carcinoma-bearing mice, and this expression was accompanied by decreased expression of erythropoietin mRNA in the kidneys from these mice. The present findings show that polycythemia in hepatocellular carcinoma-bearing mice occurs secondary to excess synthesis of erythropoietin mRNA by neoplastic hepatocytes. Keyvords. Erythropoietin messenger RNA; hepatocellular carcinoma; kidney; polycythemia; RT-PCR; B6C3F, mice

Excretion of putrescine from Escherichia coli was assessed by measuring its uptake into inside-out membrane vesicles. The vesicles were prepared from wild-type E. coli or E. coli transformed with plasmids containing one of the three polyamine transport systems. The results indicate that excretion of putrescine is catalyzed by the putrescine transport protein, encoded by the potE gene located at 16 min on the E. coli chromosome. Loading of ornithine (or lysine) inside the vesicles was essential for the uptake of putrescine, indicating that the protein exchanges putrescine and ornithine (or lysine) by an antiport mechanism. The Km and Vmax values for the putrescine uptake by inside-out membrane vesicles were 73 microM and 0.82 nmol/min per mg of protein, respectively. The antiport protein (potE protein) also catalyzed putrescine-putrescine and ornithine-ornithine exchange. The transport activity was not disturbed by inhibitors of energy production such as KCN and carbonyl cyanide m-chlorophenylhydrazone. When intact E. coli was used instead of the inside-out membrane vesicles, excretion of putrescine was also catalyzed by the antiport protein in the presence of ornithine in the medium.