18 research outputs found
Regulation of type 1 iodothyronine deiodinase by LXRα
<div><p>The iodothyronine deiodinases are selenoenzymes that regulate the activity of thyroid hormone via specific inner- or outer-ring deiodination. In humans, type 1 deiodinase (D1) is highly expressed in the liver, but the mechanism by which its gene expression is regulated remains to be elucidated. Liver X receptor α (LXRα), a transcription factor of the nuclear receptor superfamily, is highly expressed in the liver, where it functions as a sensor for excess intracellular oxysterols. LXRα interacts with other nuclear receptors on promoters of genes that contain a binding core sequence for nuclear receptors. In addition, it is reported that the promoter of the gene encoding human D1 (<i>hDIO1</i>) contains the core sequence for one of nuclear receptors, thyroid hormone receptor (TR). We investigated the involvement of LXRα in the regulation of <i>hDIO1</i>, in the liver. We performed <i>hDIO1</i> promoter–reporter assays using a synthetic LXR agonist, T0901317, and compared promoter activity between a human liver carcinoma cell line, HepG2, and a clone of human embryonic kidney cells, TSA201. We defined the region between nucleotides −131 and −114, especially nucleotides −126 and −125, of the <i>hDIO1</i> promoter as critical for basal and LXRα-mediated specific transcriptional activation in HepG2 cells. An increase in <i>hDIO1</i> expression was observed in LXRα-stimulated cells, but absent in cycloheximide-treated cells, indicating that new protein synthesis is required for LXRα-mediated regulation of <i>hDIO1</i>. On the other hand, electrophoretic mobility shift assays revealed that LXRα and RXRα bound to the <i>hDIO1</i> promoter. We also demonstrated that LXRα and TRβ compete with each other on this specific region of the promoter. In conclusion, our results indicated that LXRα plays a specific and important role in activation of TH by regulating D1, and that LXRα binds to and regulates the <i>hDIO1</i> promoter, competing with TRβ on specific sequences within the promoter.</p></div
Mutational analysis of activation of the <i>hDIO1</i> promoter by T0901317 (TO).
<p>A. The nucleotide sequence of the 5´-flanking region of the <i>hDIO1</i> promoter used in the analysis, along with the positional relationship among mutated oligonucleotides, thyroid hormone response element (TRE), and the putative activator protein 1 (AP-1) site. † and black horizontal bars represent the site-specific mutation. B and C. A series of mutated <i>hDIO1</i> promoter constructs was transiently transfected into HepG2 cells along with expression vectors for LXRα/RXRα, with or without 10<sup>−7</sup> M TO. Schematic diagram to the left of the figure representing mutant (No. 1–5) and wild-type (No. 6) <i>hDIO1</i> promoters, which were introduced upstream of the luciferase gene. No. 7 represents the promoterless pGL 4.10 construct. Promoter activity was normalized against <i>Renilla</i> luciferase activity, and is expressed relative to that of promoterless pGL 4.10 in the absence of TO. Values are expressed as means ± SEM. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; N.S., not significant. B. Basal luciferase activity of each construct. Statistical analysis was performed on comparisons between all constructs. Because most pairs exhibited significance, only non-significant pairs are presented. C. Luciferase activities of each construct with and without 10<sup>−7</sup> M TO. TO induction indicates ratios of promoter activity with TO to the activity without TO. Statistical analysis was performed on comparisons of TO induction of each construct with that of promoterless pGL 4.10, and significant differences are presented.</p
Sequences of double-stranded oligonucleotides used in EMSA.
<p>Sequences of double-stranded oligonucleotides used in EMSA.</p
Wild-type and mutated sequence of constructs used in mutational analysis.
<p>Wild-type and mutated sequence of constructs used in mutational analysis.</p
Binding of LXRα on the <i>hDIO1</i> promoter.
<p>A. EMSA with oligonucleotide containing the wild-type sequence of the region between nucleotides −141 and −112 of the <i>hDIO1</i> promoter (Wt1) with nuclear extracts from vehicle-treated (V), T0901317 (TO)-treated (T), or TO-treated and LXRα/RXRα-overexpressing (T+LXRα/RXRα) HepG2 cells; also shown is a supershift assay with an antibody against LXRα. Specific DNA/protein complexes are indicated by arrows. In lane 1, as a control, only biotin-labeled Wt1 oligonucleotide was present. In lanes 2, 3, and 4, biotin-labeled Wt1 oligonucleotide was incubated with nuclear extracts from V, T, and T+LXRα/RXRα HepG2 cells without competitor, respectively. In lane 5, biotin-labeled Wt1 oligonucleotide was incubated with nuclear extracts from T+LXRα/RXRα HepG2 cells with excess unlabeled Wt1 oligonucleotides as competitor. The results of the supershift assay are shown with normal mouse IgG as a control in lane 6 and with an antibody against LXRα in lane 7. B. EMSA with Wt1 oligonucleotides with nuclear extracts from T+LXRα/RXRα HepG2 cells and a supershift assay with antibodies against TRβ and RXRα. Specific DNA/protein complexes are indicated by arrows. In lane 1, as a control, only biotin-labeled Wt1 oligonucleotide was present. Biotin-labeled Wt1 oligonucleotide was incubated with the nuclear extracts in lane 2 and with the nuclear extracts and excess unlabeled Wt1 oligonucleotides as competitor in lane 3. The results of the supershift assay are shown with normal mouse IgG as a control in lane 4, with an antibody against TRβ in lane 5, and with an antibody against RXRα in lane 6. C. EMSA was performed with oligonucleotides containing the wild-type sequence of the region between nucleotides −131 and −104 of the <i>hDIO1</i> promoter (Wt2) with nuclear extracts from T+LXRα/RXRα HepG2 cells; also shown is a supershift assay with antibodies against LXRα, TRβ, and RXRα. A specific DNA/protein complex is indicated by an arrow. In lane 1, as a control, only biotin-labeled Wt2 was present. Biotin-labeled Wt2 was incubated with nuclear extracts in lane 2, with nuclear extracts and excess unlabeled Wt2 oligonucleotides as competitor in lane 3, and with excess unlabeled −126/−125 mut2 as competitor in lane 4. The results of the supershift assay are shown with normal mouse IgG as a control in lane 5, with an antibody against LXRα in lane 6, with an antibody against TRβ in lane 7, and with an antibody against RXRα in lane 8.</p
HepG2-specific regulation of the <i>hDIO1</i> promoter by T0901317 (TO).
<p>A series of 5´-deletion constructs of the <i>hDIO1</i> promoter were transiently transfected into HepG2 and TSA201 cells along with expression vectors for human LXRα and human RXRα (LXRα/RXRα) with and without 10<sup>−7</sup> M TO. Promoter activity was normalized against <i>Renilla</i> luciferase activity, and the normalized value is expressed relative to that of promoterless pGL 4.10 in the absence of TO. Results are expressed as means ± SEM. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01. A. Basal luciferase activity of each construct. Statistical analysis was performed on pairwise comparisons of constructs, and significant pairs are presented. B. Luciferase activities of each construct with and without 10<sup>−7</sup> M TO. TO induction indicates ratio of promoter activity with TO to the activity without TO. Statistical analysis was performed to compare TO induction of each construct with that of promoterless pGL 4.10, and significant differences are presented.</p
Specific binding of transcription factors to the region between nucleotides −131 and −114 of the <i>hDIO1</i> promoter.
<p>A. EMSA with oligonucleotide containing the wild-type sequence of the region between nucleotides −141 and −112 of the <i>hDIO1</i> promoter (Wt1) and nuclear extracts from un-treated HepG2 cells and TSA201 cells. In lane 1, as a control, only the biotin-labeled Wt1 oligonucleotide was present. Biotin-labeled Wt1 oligonucleotide was incubated with nuclear extracts from HepG2 or TSA201 cells without competitors in lanes 2 and 4, respectively, and with 25-fold molar excesses of unlabeled Wt1 oligonucleotides in lanes 3 and 5, respectively. The specific DNA/protein complexes formed are indicated by arrows. B. EMSA with mutant oligonucleotides and nuclear extracts from HepG2 cells. Biotin-labeled Wt1 oligonucleotide was incubated with nuclear extracts from HepG2 cells without competitors in lane 2, and with 25-fold molar excesses of unlabeled Wt1, −124mut1, and −126/−125mut1 oligonucleotides in lanes 3, 4, and 5, respectively. The specific DNA/protein complexes formed are indicated by arrows.</p
Image_1_Double somatic mutations in CTNNB1 and GNA11 in an aldosterone-producing adenoma.pdf
Double somatic mutations in CTNNB1 and GNA11/Q have recently been identified in a small subset of aldosterone-producing adenomas (APAs). As a possible pathogenesis of APA due to these mutations, an association with pregnancy, menopause, or puberty has been proposed. However, because of its rarity, characteristics of APA with these mutations have not been well characterized. A 46-year-old Japanese woman presented with hypertension and hypokalemia. She had two pregnancies in the past but had no history of pregnancy-induced hypertension. She had regular menstrual cycle at presentation and was diagnosed as having primary aldosteronism after endocrinologic examinations. Computed tomography revealed a 2 cm right adrenal mass. Adrenal venous sampling demonstrated excess aldosterone production from the right adrenal gland. She underwent right laparoscopic adrenalectomy. The resected right adrenal tumor was histologically diagnosed as adrenocortical adenoma and subsequent immunohistochemistry (IHC) revealed diffuse immunoreactivity of aldosterone synthase (CYP11B2) and visinin like 1, a marker of the zona glomerulosa (ZG), whereas 11β-hydroxylase, a steroidogenic enzyme for cortisol biosynthesis, was mostly negative. CYP11B2 IHC-guided targeted next-generation sequencing identified somatic CTNNB1 (p.D32Y) and GNA11 (p.Q209H) mutations. Immunofluorescence staining of the tumor also revealed the presence of activated β-catenin, consistent with features of the normal ZG. The expression patterns of steroidogenic enzymes and related proteins indicated ZG features of the tumor cells. PA was clinically and biochemically cured after surgery. In conclusion, our study indicated that CTNNB1 and GNA11-mutated APA has characteristics of the ZG. The disease could occur in adults with no clear association with pregnancy or menopause.</p
Rats deficient C-type natriuretic peptide suffer from impaired skeletal growth without early death
<div><p>We have previously investigated the physiological role of C-type natriuretic peptide (CNP) on endochondral bone growth, mainly with mutant mouse models deficient in CNP, and reported that CNP is indispensable for physiological endochondral bone growth in mice. However, the survival rate of CNP knockout (KO) mice fell to as low as about 70% until 10 weeks after birth, and we could not sufficiently analyze the phenotype at the adult stage. Herein, we generated CNP KO rats by using zinc-finger nuclease-mediated genome editing technology. We established two lines of mutant rats completely deficient in CNP (CNP KO rats) that exhibited a phenotype identical to that observed in mice deficient in CNP, namely, a short stature with severely impaired endochondral bone growth. Histological analysis revealed that the width of the growth plate, especially that of the hypertrophic chondrocyte layer, was markedly lower and the proliferation of growth plate chondrocytes tended to be reduced in CNP KO rats. Notably, CNP KO rats did not have malocclusions and survived for over one year after birth. At 33 weeks of age, CNP KO rats persisted significantly shorter than wild-type rats, with closed growth plates of the femur in all samples, which were not observed in wild-type rats. Histologically, CNP deficiency affected only bones among all body tissues studied. Thus, CNP KO rats survive over one year, and exhibit a deficit in endochondral bone growth and growth retardation throughout life.</p></div
Typical examples of H&E-stained specimens of the heart of female WT (left) and CNP KO (homozygous Δ11 mutant, right) rats.
<p>Scale bar shows 1 mm.</p