Mieko Kumagai

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.

3,5,3′-Triiodo-l-Thyronine Potentiates All-Trans-Retinoic Acid-Induced Apoptosis during Differentiation of the Promyeloleukemic Cell HL-60

Although the programmed cell death mediated by thyroid hormone is not well evaluated in mammalian cells, thyroid hormone plays a crucial role in differentiation of the cells during the metamorphosis of Xenopus, suggesting that thyroid hormone has the potential ability to induce the apoptosis. To investigate the thyroid hormone-inducible apoptosis, we cultured HL-60 cells with various amounts of all-trans-retinoic acid (RA) and l-T3. T3 alone did not induce the apoptosis of the cells. T3, however, suppressed the proliferation of cells in the presence of RA. DNA ladder and microscopical examination showed that the reduction of cell number was due to the apoptosis induced by RA. These findings suggested that T3 affects the apoptotic process during the differentiation of HL-60 cells by RA. T3-inducible apoptosis may require the factors augmented by RA in HL-60 cells.

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.