Makoto Hiromura

Inhibition of Akt Kinase Activity by a Peptide Spanning the βA Strand of the Proto-oncogene TCL1

Akt plays a central role in the regulation of cellular anti-apoptosis underlying various human neoplastic diseases. We have demonstrated previously that TCL1 (a proto-oncogene underlying human T cell prolymphocytic leukemia) interacts with Akt and functions as an Akt kinase co-activator. With the aim to develop an Akt kinase inhibitor, we hypothesized that a peptide, which spans the Akt-binding site, binds to Akt and modulates Akt kinase activity and its downstream biological responses. Indeed, we demonstrated that a peptide, named “Akt-in” (Akt inhibitor, NH2-AVTDHPDRLWAWEKF-COOH, encompassing the βA strand of human TCL1), interacted with Akt and specifically inhibited its kinase activity. Nuclear magnetic resonance studies suggested that interaction of Akt-in with the pleckstrin homology domain (PH) of Akt caused conformational changes on the variable loop 1 of Akt, the locus mediating phosphoinositide binding. Consistently, interaction of Akt-in with the Akt PH domain prevented phosphoinositide binding and hence inhibited membrane translocation and activation of Akt. Moreover, Akt-in inhibited not only cellular proliferation and anti-apoptosis in vitro but also in vivo tumor growth without any adverse effect. The roles of Akt, which possesses a PH domain, in intracellular signaling were well established. Hence, Akt inhibitors create an attractive target for anticancer therapy. However, no effective inhibitors specific for Akt have been developed. Akt-in, which inhibits association of phosphatidylinositol with Akt, is the first molecule to demonstrate specific Akt kinase inhibition potency. This observation will facilitate the design of specific inhibitors for Akt, a core intracellular survival factor underlying various human neoplastic diseases.

Transcription factor IIB acetylates itself to regulate transcription.

Acetylation is a well-known regulatory post-translational modification, but a biological function for acetylation in regulating basal transcription factors has not been reported. Here we show that the general transcription factor TFIIB, which is required for the initiation of eukaryotic polymerase II transcription, is acetylated. TFIIB is also an autoacetyltransferase, although it shares no sequence homology with any known acetyltransferases. In the absence of other enzymes, it binds acetyl-coenzyme A (acetyl-CoA), and catalyses the transfer of the acetyl group onto a specific lysine residue (K238). Both recombinant and cellular TFIIB can autoacetylate, markedly stabilizing the interaction between TFIIB and transcription factor TFIIF and activating transcription in vitro and in cells. A K238A mutant, which cannot be autoacetylated, does not show this activation of transcription. Our findings suggest that there is a regulatory pathway controlling acetylation of TFIIB, and they link acetyl-CoA with basal gene transcription.

Action mechanism of bis(allixinato)oxovanadium(IV) as a novel potent insulin-mimetic complex: regulation of GLUT4 translocation and FoxO1 transcription factor

Bis(allixinato)oxovanadium(IV), VO(alx)2 (alx is 3-hydroxy-5-methoxy-6-methyl-2-pentyl-4-pyrone), has been reported to act as an antidiabetic agent in streptozotocin-induced type-1-like and obesity-linked KKAy type 2 diabetic model mice. VO(alx)2 is also proposed as a candidate agent for treating metabolic syndromes in animals. However, its functional mechanism is yet to be clarified. In this study, we examined whether VO(alx)2 contributes to both the activation of the insulin signaling cascade that activates glucose transporter 4 (GLUT4) translocation and the regulation of the forkhead box O1 (FoxO1) transcription factor that controls the gene transcription of gluconeogenesis genes. The following three important results were obtained: (1) intracellular vanadium concentration in 3T3-L1 adipocytes is higher after treatment with VO(alx)2 than with VOSO4; (2) VO(alx)2 stimulates the translocation of GLUT4 to the plasma membrane following activation of the tyrosine phosphorylation of the insulin receptor β-subunit (IRβ) and insulin receptor substrate (IRS) as well as Akt kinase in 3T3-L1 adipocytes; and (3) the mechanism of inhibition of glucose-6-phosphatase (G6Pase) catalytic subunit gene expression by vanadium is due to disruption of FoxO1 binding with the G6Pase promoter, which indicates that FoxO1 is phosphorylated by VO(alx)2-stimulated Akt in HepG2 cells. On the basis of these results, we propose that the critical functions of VO(alx)2 involve the activation of phosphatidylinositol 3-kinase–Akt signaling through the enhancement of tyrosine phosphorylation of IRβ and IRS, which in turn transmits the signal to activate GLUT4 translocation, and the regulation of the DNA binding activity of the FoxO1 transcription factor.

Essential Role of the Zinc Transporter ZIP9/SLC39A9 in Regulating the Activations of Akt and Erk in B-Cell Receptor Signaling Pathway in DT40 Cells

The essential trace element zinc is important for all living organisms. Zinc functions not only as a nutritional factor, but also as a second messenger. However, the effects of intracellular zinc on the B cell-receptor (BCR) signaling pathway remain poorly understood. Here, we present data indicating that the increase in intracellular zinc level induced by ZIP9/SLC39A9 (a ZIP Zrt-/Irt-like protein) plays an important role in the activation of Akt and Erk in response to BCR activation. In DT40 cells, the enhancement of Akt and Erk phosphorylation following BCR activation requires intracellular zinc. To clarify this event, we used chicken ZnT5/6/7-gene-triple-knockout DT40 (TKO) cells and chicken Zip9-knockout DT40 (cZip9KO) cells. The levels of Akt and ERK phosphorylation significantly decreased in cZip9KO cells. In addition, the enzymatic activity of protein tyrosine phosphatase (PTPase) increased in cZip9KO cells. These biochemical events were restored by overexpressing the human Zip9 (hZip9) gene. Moreover, we found that the increase in intracellular zinc level depends on the expression of ZIP9. This observation is in agreement with the increased levels of Akt and Erk phosphorylation and the inhibition of total PTPase activity. We concluded that ZIP9 regulates cytosolic zinc level, resulting in the enhancement of Akt and Erk phosphorylation. Our observations provide new mechanistic insights into the BCR signaling pathway underlying the regulation of intracellular zinc level by ZIP9 in response to the BCR activation.

Tissue Nonspecific Alkaline Phosphatase Is Activated via a Two-step Mechanism by Zinc Transport Complexes in the Early Secretory Pathway

A number of enzymes become functional by binding to zinc during their journey through the early secretory pathway. The zinc transporters (ZnTs) located there play important roles in this step. We have previously shown that two zinc transport complexes, ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers, are required for the activation of alkaline phosphatases, by converting them from the apo- to the holo-form. Here, we investigated the molecular mechanisms of this activation. ZnT1 and ZnT4 expressed in chicken DT40 cells did not contribute to the activation of tissue nonspecific alkaline phosphatase (TNAP). The reduced activity of TNAP in DT40 cells deficient in both ZnT complexes was not restored by zinc supplementation nor by exogenous expression of other ZnTs that increase the zinc content in the secretory pathway. Moreover, we showed that expression of ZnT5/ZnT6 heterodimers reconstituted with zinc transport-incompetent ZnT5 mutant failed to restore TNAP activity but could stabilize the TNAP protein as the apo-form, regardless of zinc status. These findings demonstrate that TNAP is activated not simply by passive zinc binding but by an elaborate two-step mechanism via protein stabilization followed by enzyme conversion from the apo- to the holo-form with zinc loaded by ZnT complexes in the early secretory pathway.

Intrinsic ADP-ATP Exchange Activity Is a Novel Function of the Molecular Chaperone, Hsp70

Hsp70 is a multifunctional molecular chaperone whose interactions with protein substrates are regulated by ATP hydrolysis and ADP-ATP exchange. We show here that, in addition to ATPase activity, purified Hsp70 free from nucleoside-diphosphate (NDP) kinase exhibits intrinsic ADP-ATP exchange activity. The rate constants for ATP hydrolysis and ATP synthesis were in a similar range at the optimum pH of 7.5–8.5 in the presence of 5 mm ATP and 0.5 mm ADP. Hsp70 exhibited a considerably strict preference for ATP as a phosphate donor, and a biased substrate specificity, unlike NDP kinase; ADP, UDP, CDP > dTDP, dCDP > GDP, dGDP. During the reaction, Hsp70 formed an acid-labile autophosphorylated intermediate, and nucleoside diphosphate-dependent dephosphorylation of the latter then occurred. These properties of Hsp70 are not identical but similar to those of NDP kinase, but are not similar to those of adenylate kinase and ATP synthase.

64Cu-DOTA-anti-CTLA-4 mAb enabled PET visualization of CTLA-4 on the T-cell infiltrating tumor tissues.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) targeted therapy by anti-CTLA-4 monoclonal antibody (mAb) is highly effective in cancer patients. However, it is extremely expensive and potentially produces autoimmune-related adverse effects. Therefore, the development of a method to evaluate CTLA-4 expression prior to CTLA-4-targeted therapy is expected to open doors to evidence-based and cost-efficient medical care and to avoid adverse effects brought about by ineffective therapy. In this study, we aimed to develop a molecular imaging probe for CTLA-4 visualization in tumor. First, we examined CTLA-4 expression in normal colon tissues, cultured CT26 cells, and CT26 tumor tissues from tumor-bearing BALB/c mice and BALB/c nude mice by reverse transcription polymerase chain reaction (RT-PCR) analysis and confirmed whether CTLA-4 is strongly expressed in CT26 tumor tissues. Second, we newly synthesized 64Cu-1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid-anti-mouse CTLA-4 mAb (64Cu-DOTA-anti-CTLA-4 mAb) and evaluated its usefulness in positron emission tomography (PET) and ex-vivo biodistribution analysis in CT26-bearing BALB/c mice. High CTLA-4 expression was confirmed in the CT26 tumor tissues of tumor-bearing BALB/c mice. However, CTLA-4 expression was extremely low in the cultured CT26 cells and the CT26 tumor tissues of tumor-bearing BALB/c nude mice. The results suggested that T cells were responsible for the high CTLA-4 expression. Furthermore, 64Cu-DOTA-anti-CTLA-4 mAb displayed significantly high accumulation in the CT26 tumor, thereby realizing non-invasive CTLA-4 visualization in the tumor. Together, the results indicate that 64Cu-DOTA-anti-CTLA-4 mAb would be useful for the evaluation of CTLA-4 expression in tumor.

Glucose lowering activity by oral administration of bis(allixinato)oxidovanadium(IV) complex in streptozotocin-induced diabetic mice and gene expression profiling in their skeletal muscles

Vanadyl(IV) complexes are anti-diabetogenic agents. Intra-peritoneal administration of bis(allixinato)oxidovanadium(IV) [VO(alx)2] lowers high blood glucose levels in animal models of type 1 and type 2 diabetes. We have examined whether oral administration of VO(alx)2 restores impaired activation in signaling cascades related to glucose metabolism and insulin action, and alters gene expression in the skeletal muscles of streptozotocin (STZ)-induced diabetic mice (STZ-diabetic mice). We report here that daily oral administration of VO(alx)2 lowered high blood glucose levels in the STZ-diabetic mice. The oral administration of VO(alx)2 enhanced phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β), located downstream of the insulin receptor cascade in the skeletal muscles. We analyzed gene expression in the muscles of the diabetic mice before and after insulin or VO(alx)2 treatment. Treating the diabetic mice with insulin or VO(alx)2 normalized the gene expression levels of 152 down-regulated and 11 up-regulated genes, and especially the up-regulation of Cyp2E1 and FoxO1 in the muscles of the diabetic mice. The insulin-mimetic effects of VO(alx)2 in the STZ-induced diabetic mice may be due to the enhancement of protein phosphorylation leading to the activation or inactivation of the transcriptional machinery. Our findings suggest that the insulin-mimetic effects of VO(alx)2 in diabetes may be due to changes in the protein phosphorylations and their gene expression levels.

Demonstration of in-vivo Multi-Probe Tracker Based on a Si/CdTe Semiconductor Compton Camera

By using a prototype Compton camera consisting of silicon (Si) and cadmium telluride (CdTe) semiconductor detectors, originally developed for the ASTRO-H satellite mission, an experiment involving imaging multiple radiopharmaceuticals injected into a living mouse was conducted to study its feasibility for medical imaging. The accumulation of both iodinated (131I) methylnorcholestenol and 85Sr into the mouses organs was simultaneously imaged by the prototype. This result implies that the Compton camera is expected to become a multi-probe tracker available in nuclear medicine and small animal imaging.

Essential Roles of Raf/Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase Pathway, YY1, and Ca2+ Influx in Growth Arrest of Human Vascular Smooth Muscle Cells by Bilirubin

Background: Bilirubin circulates throughout the human cardiovascular system. Its interaction with the vascular wall is not well known. Results: Bilirubin alters Raf/ERK/MAPK pathway, cellular transcription factor YY1 location, and calcium-dependent YY1 proteolysis in human vascular cells. Conclusion: At high physiological levels bilirubin, inhibits cell growth, inhibits proliferation, and does not cause apoptosis. Significance: The observations provide opportunities for prevention and treatment of cardiovascular diseases. The biological effects of bilirubin, still poorly understood, are concentration-dependent ranging from cell protection to toxicity. Here we present data that at high nontoxic physiological concentrations, bilirubin inhibits growth of proliferating human coronary artery smooth muscle cells by three events. It impairs the activation of Raf/ERK/MAPK pathway and the cellular Raf and cyclin D1 content that results in retinoblastoma protein hypophosphorylation on amino acids S608 and S780. These events impede the release of YY1 to the nuclei and its availability to regulate the expression of genes and to support cellular proliferation. Moreover, altered calcium influx and calpain II protease activation leads to proteolytical degradation of transcription factor YY1. We conclude that in the serum-stimulated human vascular smooth muscle primary cell cultures, bilirubin favors growth arrest, and we propose that this activity is regulated by its interaction with the Raf/ERK/MAPK pathway, effect on cyclin D1 and Raf content, altered retinoblastoma protein profile of hypophosphorylation, calcium influx, and YY1 proteolysis. We propose that these activities together culminate in diminished 5 S and 45 S ribosomal RNA synthesis and cell growth arrest. The observations provide important mechanistic insight into the molecular mechanisms underlying the transition of human vascular smooth muscle cells from proliferative to contractile phenotype and the role of bilirubin in this transition.