Tomoko Kakizawa

Inhibition of Nuclear Receptor Signalling by Poly(ADP-Ribose) Polymerase

ABSTRACT Mammalian poly(ADP-ribose) polymerase (PARP) is a nuclear chromatin-associated protein with a molecular mass of 114 kDa that catalyzes the transfer of ADP-ribose units from NAD+ to nuclear proteins that are located within chromatin. We report here the identification of a novel property of PARP as a modulator of nuclear receptor signalling. PARP bound directly to retinoid X receptors (RXR) and repressed ligand-dependent transcriptional activities mediated by heterodimers of RXR and thyroid hormone receptor (TR). The interacting surface is located in the DNA binding domain of RXRα. Gel shift assays demonstrated that PARP bound to TR-RXR heterodimers on the response element. Overexpression of wild-type PARP selectively blocked nuclear receptor function in transient transfection experiments, while enzyme-defective mutant PARP did not show significant inhibition, suggesting that the essential role of poly(ADP-ribosyl) enzymatic activity is in gene regulation by nuclear receptors. Furthermore, PARP fused to the Gal4 DNA binding domain suppressed the transcriptional activity of the promoter harboring the Gal4 binding site. Thus, PARP has transcriptional repressor activity when recruited to the promoter. These results indicates that poly(ADP-ribosyl)ation is a negative cofactor in gene transcription, regulating a member of the nuclear receptor superfamily.

Inhibition of Peroxisome Proliferator Signaling Pathways by Thyroid Hormone Receptor COMPETITIVE BINDING TO THE RESPONSE ELEMENT

Peroxisome proliferators (e.g. clofibric acid) and thyroid hormone play an important role in the metabolism of lipids. These effectors display their action through their own nuclear receptors, peroxisome proliferator-activated receptor (PPAR) and thyroid hormone receptor (TR). PPAR and TR are ligand-dependent, DNA binding, trans-acting transcriptional factors belonging to the erbA-related nuclear receptor superfamily. The present study focused on the convergence of the effectors on the peroxisome proliferator response element (PPRE). Transcriptional activation induced by PPAR through a PPRE was significantly suppressed by cotransfection of TR in transient transfection assays. The inhibition, however, was not affected by adding 3,5,3′-triiodo-L-thyronine (T3). Furthermore, the inhibition was not observed in cells cotransfected with retinoic acid receptor or vitamin D3 receptor. The inhibitory action by TR was lost by introducing a mutation in the DNA binding domain of TR, indicating that competition for DNA binding is involved in the molecular basis of this functional interaction. Gel shift assays revealed that TRs, expressed in insect cells, specifically bound to the 32P-labeled PPRE as heterodimers with the retinoid X receptor (RXR). Both PPAR and TR bind to PPRE, although only PPAR mediates transcriptional activation via PPRE. TR·RXR heterodimers are potential competitors with PPAR·RXR for binding to PPREs. It is concluded that PPAR-mediated gene expression is negatively controlled by TR at the level of PPAR binding to PPRE. We report here the novel action of thyroid hormone receptor in controlling gene expression through PPREs.

Functional Interaction between Oct-1 and Retinoid X Receptor

The retinoid X receptor (RXR) is a member of the nuclear hormone receptor superfamily and heterodimerizes with a variety of other family members such as the thyroid hormone receptor (TR),1 retinoic acid receptor, vitamin D receptor, and peroxisome proliferator-activated receptor. Therefore, RXR is supposed to play a key role in a ligand-dependent regulation of gene transcription by nuclear receptors. In this study, we have identified the octamer-binding transcription factor-1 (Oct-1) as a novel interaction factor of RXR. In vitro pull-down assays using RXR deletion mutants showed that the interaction surfaces were located in the region encompassing the DNA binding domain (C domain) and the hinge domain (D domain) of RXR. We also showed that RXR interacted with the POU homeodomain but not with the POU-specific domain of Oct-1. Gel shift analysis revealed that Oct-1 reduced the binding of TR/RXR heterodimers to the thyroid hormone response element (TRE). In transient transfection assays using COS1 cells, Oct-1 repressed the T3-dependent transcriptional activity of TR/RXR heterodimers, consistent with in vitro DNA binding data; however, transcriptional activation by Gal4-TR(LBD) (LBD, ligand binding domain), which lacks its own DNA binding domain but retains responsiveness to T3, was not influenced by Oct-1. These results suggest that Oct-1 functionally interacts with RXR and negatively regulates the nuclear receptor signaling pathway by altering the DNA binding ability of the receptors.

The role of hinge domain in heterodimerization and specific DNA recognition by nuclear receptors.

Four structural domains are characteristic of the members of the nuclear receptor superfamily. The hinge (D) domain which is located between the DNA binding (C) domain and the ligand binding (EF) domain, is less conserved among the nuclear receptors. In this study, we investigated the effects of the D domain on receptor function with regard to ligand binding, protein-protein interaction and DNA recognition. We found that EF domain of TR lacked T3 binding activity and additional D domain was required for its ligand binding. Using pull down assays and two-hybrid assays, we also demonstrated that the EF domain of TR did not dimerize with TR or RXR in solution, while the DEF domain was able to homo-and heterodimerize with RXR. In contrast, the RXR EF domain alone was able to heterodimerize with TR. The D domain of TR is required but that of RXR is not necessary for the interaction. We further demonstrated that the D domain was required for receptor specific DNA recognition. The ABC domain of vitamin D receptor (VDR) and TR(DEF) chimeric receptor could not bind to VDR response element (VDRE). Addition of own D domain of VDR to the ABC domain enables the chimeric receptor to bind VDRE and transactivate. The D domain of TR cannot substitute for that of VDR in context of specific DNA recognition. These data suggest that the D domain is important to maintain the integrity of the functional structure of the nuclear receptors.

A tandemly repeated thyroglobulin core promoter has potential to enhance efficacy for tissue-specific gene therapy for thyroid carcinomas

Recombinant adenoviruses, carrying herpes simplex virus thymidine kinase (HSVtk) genes, were developed to evaluate the possibility of tissue-specific gene therapy for thyroid carcinomas. The HSVtk gene was driven by a minimal thyroglobulin (TG) promoter (AdTGtk) and a tandemly repeated minimal TG promoter (Ad2×TGtk) to obtain thyroid-specific cell killing ability. The transduction of HSVtk genes by infection with Ad2×TGtk followed by ganciclovir (GCV) treatment showed more powerful cytotoxicity for TG-producing FRTL5 cells, a rat normal thyroid cell line, and FTC-133 cells, a human follicular thyroid carcinoma cell line, than when infected with AdTGtk in vitro. The cell killing ability of Ad2×TGtk was 10- to 30-fold higher than that of AdTGtk and similar to that of AdCMVtk, which carries HSVtk under the control of CMV promoter. Whereas after treatment with adenovirus/GCV to non–TG-producing cell lines (undifferentiated thyroid carcinoma cell lines and carcinoma cell lines from other tissues), Ad2×TGtk and AdTGtk needed more than 100-fold concentrated GCV to reach IC50 compared to AdCMVtk. We confirmed the enhanced efficacy of Ad2×TGtk for tissue-specific cytotoxicity in vivo. After adenovirus/GCV treatment for FTC-133 tumor-bearing nude mice, Ad2×TGtk enhanced tumor growth inhibition and survival rates compared to AdTGtk. Tumor growth inhibition and survival rates by Ad2×TGtk were similar to that by AdCMVtk. Moreover, any toxic effect for rat normal tissues was not revealed after intravenous injections with Ad2×TGtk and intraperitoneal administrations with GCV in vivo, whereas severe liver damages were observed after treatment with AdCMVtk/GCV. These data indicate a beneficial effect of Ad2×TGtk for tissue-specific gene therapy for TG-producing thyroid carcinomas without toxicity for normal tissues.

Biphasic function of focal adhesion kinase in endothelial tube formation induced by fibril-forming collagens

Migration and tube formation of endothelial cells are important in angiogenesis and require a coordinated response to the extra-cellular matrix (ECM) and growth factor. Since focal adhesion kinase (FAK) integrates signals from both ECM and growth factor, we investigated its role in angiogenesis. Type I and II collagens are fibril-forming collagens and stimulate human umbilical vein endothelial cells (HUVECs) to form tube structure. Although knockdown of FAK restrained cell motility and resulted in inhibition of tube formation, FAK degradation and tube formation occurred simultaneously after incubation with fibril-forming collagens. The compensation for the FAK degradation by a calpain inhibitor or transient over-expression of FAK resulted in disturbance of tube formation. These phenomena are specific to fibril-forming collagens and mediated via alpha2beta1 integrin. In conclusion, our data indicate that FAK is functioning in cell migration, but fibril-forming collagen-induced FAK degradation is necessary for endothelial tube formation.

Characterization of Two Novel Retinoic Acid‐resistant Cell Lines Derived from HL‐60 Cells Following Long‐term Culture with all‐trans‐Retinoic Acid

Either all‐trans‐retinoic acid (RA) or vitamin D3 (VD) induces differentiation of the myeloid leukemia cell line HL‐60. RA is available for the treatment of acute promyeloleukemia, although the development of resistance to the agent is a serious problem for differentiation‐inducing therapy. To approach the mechanisms of resistance to RA, we developed two novel cell lines, HL‐60–R2 and R9, which were subcloned after exposure to increasing concentrations of RA. The growth rate of HL‐60–R2 cells was significantly increased by RA treatment, whereas the growth rate of HL‐60–R9 was not affected. RA induces apoptosis in the parental HL‐60 cells. The number of apoptotic cells, however, was not increased and nitroblue tetrazolium (NBT) reduction was not altered by 1 μM RA in either of the cloned cell lines. Treatment with VD induced monocytic differentiation and increased the expression of CD11b in HL‐60 and HL‐60–R9 cells, but not in HL‐60–R2 cells. Flow cytometric and G‐banding analysis demonstrated that R2 cells were near‐triploid. The sequencing analysis revealed a deletion of three nucleotides in the sequence of the RARα gene in HL‐60–R9 cells, resulting in deletion of codon 286. No mutation was found in HL‐60–R2 cells. Taken together, these data indicate that the resistance to RA is caused by the mutation in RARα of HL‐60–R9, but by other factor(s), which also affect the VD‐response pathways, in HL‐60–R2. The abnormal response to VD may be associated with the abnormal ploidy of the R2 cells.

Efficacy of Combination Therapy With Sitagliptin and Low-Dose Glimepiride in Japanese Patients With Type 2 Diabetes

Background We examined the effects of combination therapy with 50 mg/day of sitagliptin and low-dose glimepiride (1 mg/day) in patients with type 2 diabetes. Methods Twenty-six patients with poorly controlled type 2 diabetes currently taking high-dose glimepiride (≥ 2 mg/day) were enrolled in the study. The dose of glimepiride was reduced to 1 mg/day and 50 mg/day of sitagliptin was added without changing the doses of any other antihyperglycemic agents. The patients were divided into two groups: the low-dose group (2 or 3 mg glimepiride decreased to 1 mg: n = 15) and the high-dose group (4 or 6 mg glimepiride decreased to 1 mg: n = 11). Results Combination therapy significantly lowered HbA1c after 24 weeks of treatment in both groups. In the low-dose group, 8.1 ± 0.2% decreased to 7.0 ± 0.1%; in the high-dose group, 8.4 ± 0.1% decreased to 7.3 ± 0.2%. The time course of the degree of HbA1c reduction in the high-dose group was almost superimposable on that in the low-dose group. There were no changes in body weight and no hypoglycemia and in either group during the study period. In conclusion, our results suggested that the combination therapy used in the study is both well tolerated and effective. Conclusion This study indicated the usefulness of dipeptidyl peptidase (DPP)-4 inhibitors in Japanese patients with type 2 diabetes, and also reinforces the importance of low doses of sulfonylurea for effective glycemic management.

Novel deletional mutation of the MEN1 gene in a kindred with multiple endocrine neoplasia type 1

MEN1 gene mutation in a Japanese kindred with multiple endocrine neoplasia type 1 was examined. A heterozygous deletion involving 29 base pairs in exon 10 (1606del29) was identified in the proband, and the same deletion was found in the affected family members. Most previously reported germline MEN1 gene mutations are nucleotide substitutions and small insertions/deletions, and a large deletion is rare. The hairpin structure mediated by an incomplete palindromic sequence at deletion termini is the most likely mechanism to be associated with the deletion in the present family.