Kenji Ohba

In Vivo and In Vitro Studies Suggest a Possible Involvement of HPV Infection in the Early Stage of Breast Carcinogenesis via APOBEC3B Induction

High prevalence of infection with high-risk human papilloma virus (HPV) ranging from 25 to 100% (average 31%) was observed in breast cancer (BC) patients in Singapore using novel DNA chip technology. Early stage of BC demonstrated higher HPV positivity, and BC positive for estrogen receptor (ER) showed significantly higher HPV infection rate. This unique association of HPV with BC in vivo prompted us to investigate a possible involvement of HPV in early stages of breast carcinogenesis. Using normal breast epithelial cells stably transfected with HPV-18, we showed apparent upregulation of mRNA for the cytidine deaminase, APOBEC3B (A3B) which is reported to be a source of mutations in BC. HPV-induced A3B overexpression caused significant γH2AX focus formation, and DNA breaks which were cancelled by shRNA to HPV18 E6, E7 and A3B. These results strongly suggest an active involvement of HPV in the early stage of BC carcinogenesis via A3B induction.

Thyroid hormone induction of mitochondrial activity is coupled to mitophagy via ROS-AMPK-ULK1 signaling

Currently, there is limited understanding about hormonal regulation of mitochondrial turnover. Thyroid hormone (T3) increases oxidative phosphorylation (OXPHOS), which generates reactive oxygen species (ROS) that damage mitochondria. However, the mechanism for maintenance of mitochondrial activity and quality control by this hormone is not known. Here, we used both in vitro and in vivo hepatic cell models to demonstrate that induction of mitophagy by T3 is coupled to oxidative phosphorylation and ROS production. We show that T3 induction of ROS activates CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, β) mediated phosphorylation of PRKAA1/AMPK (5′ AMP-activated protein kinase), which in turn phosphorylates ULK1 (unc-51 like autophagy activating kinase 1) leading to its mitochondrial recruitment and initiation of mitophagy. Furthermore, loss of ULK1 in T3-treated cells impairs both mitophagy as well as OXPHOS without affecting T3 induced general autophagy/lipophagy. These findings demonstrate a novel ROS-AMPK-ULK1 mechanism that couples T3-induced mitochondrial turnover with activity, wherein mitophagy is necessary not only for removing damaged mitochondria but also for sustaining efficient OXPHOS.

Fluorophore Labeling Enables Imaging and Evaluation of Specific CXCR4−Ligand Interaction at the Cell Membrane for Fluorescence-Based Screening

Development of CXCR4-specific ligands is an important issue in chemotherapy of HIV infection, cancer metastasis, and rheumatoid arthritis, and numerous potential ligands have been developed to date. However, it is difficult to assess their binding mode and specificity because of uncertainties in the structure of the CXCR4-ligand complexes. To address this problem, we have synthesized fluorophore labeled Ac-TZ14011, which is derived from T140, a powerful CXCR4 antagonist. Binding of Ac-TZ14011 to CXCR4 on the cell membrane was observed by fluorescence microscope, and analysis of the binding data produced IC 50 values of several ligands comparable to those obtained in RI-based assays. This fluorescence-based assay is applicable to explore new pharmacophores of CXCR4-specific ligands with high-throughput screening and also to screening of the other GPCR binding ligands.

Functions of PIT1 in GATA2-dependent transactivation of the thyrotropin β promoter

Thyrotropin (TSH) is a heterodimer consisting of alpha and beta chains, and the beta chain (TSHbeta) is specific to TSH. The coexistence of two transcription factors, PIT1 and GATA2, is known to be essential for TSHbeta expression. Using kidney-derived CV1 cells, we investigated the role of PIT1 in the expression of Tshb gene. GATA2 Zn finger domain, which is known to recognize GATA-responsive elements (GATA-REs), is essential for cooperation by PIT1. Transactivation of TSHbeta promoter requires PIT1-binding site upstream to GATA-REs (PIT1-US), and the spacing between PIT1-US and GATA-REs strictly determines the cooperation between PIT1 and GATA2. Moreover, truncation of the sequence downstream to GATA-REs enabled GATA2 to transactivate the TSHbeta promoter without PIT1. The deleted region (nt -82/-52) designated as a suppressor region (SR) was considered to inhibit transactivation by GATA2. The cooperation of PIT1 with GATA2 was not conventional synergism but rather counteracted SR-induced suppression (derepression). The minimal sequence for SR was mapped to the 9 bp sequence downstream to GATA-REs. Electrophoretic mobility shift assay suggested that some nuclear factor exists in CV1 cells, which binds with SR and this interaction was blocked by recombinant PIT1. Our study indicates that major activator for the TSHbeta promoter is GATA2 and that PIT1 protects the function of GATA2 from the inhibition by SR-binding protein.

Remodeling of Dynamic Structures of HIV-1 Envelope Proteins Leads to Synthetic Antigen Molecules Inducing Neutralizing Antibodies

A synthetic antigen targeting membrane-fusion mechanism of HIV-1 has a newly designed template with C3-symmetric linkers mimicking N36 trimeric form. The antiserum produced by immunization of the N36 trimeric form antigen showed structural preference in binding to N36 trimer and stronger inhibitory activity against HIV-1 infection than the N36 monomer. Our results suggest an effective strategy of HIV vaccine design based on a relationship to the native structure of proteins involved in HIV fusion mechanisms.

Inappropriate Elevation of Serum Thyrotropin Levels in Patients Treated with Axitinib

BACKGROUND Although anticancer treatment with the tyrosine kinase inhibitor (TKI) axitinib frequently causes thyroid dysfunction, the associated mechanism and clinical features have not been elucidated. METHODS Six patients were treated with axitinib for metastatic renal cell carcinoma at the Hamamatsu University School of Medicine between 2008 and 2010. We reviewed their thyroid function results and compared them to those of patients treated with two other TKIs, sunitinib or sorafenib, and to those of subjects with normal hypothalamic-pituitary-thyroid (HPT) function. RESULTS Axitinib-induced thyroid dysfunction was observed in all patients, and two patterns were observed: increased serum thyrotropin (TSH) levels within one month after administration occurred in five patients and transient thyrotoxicosis due to destructive thyroiditis occurred in five patients within 7 months of treatment. Four patients exhibited both. When the relationship between the serum TSH and thyroid hormones was evaluated using plots of TSH versus both free thyroxine and free triiodothyronine, four patients showed an inappropriate elevation of serum TSH during administration of axitinib. Their values apparently shifted against the regression line compared to data from patients with a normal HPT function. A similar tendency, though weaker, was observed in some patients treated with sunitinib or sorafenib. CONCLUSION This is the first study to report an inappropriate elevation of serum TSH levels in patients treated with axitinib.

GATA2 mediates thyrotropin-releasing hormone-induced transcriptional activation of the thyrotropin β gene

Thyrotropin-releasing hormone (TRH) activates not only the secretion of thyrotropin (TSH) but also the transcription of TSHβ and α-glycoprotein (αGSU) subunit genes. TSHβ expression is maintained by two transcription factors, Pit1 and GATA2, and is negatively regulated by thyroid hormone (T3). Our prior studies suggest that the main activator of the TSHβ gene is GATA2, not Pit1 or unliganded T3 receptor (TR). In previous studies on the mechanism of TRH-induced activation of the TSHβ gene, the involvements of Pit1 and TR have been investigated, but the role of GATA2 has not been clarified. Using kidney-derived CV1 cells and pituitary-derived GH3 and TαT1 cells, we demonstrate here that TRH signaling enhances GATA2-dependent activation of the TSHβ promoter and that TRH-induced activity is abolished by amino acid substitution in the GATA2-Zn finger domain or mutation of GATA-responsive element in the TSHβ gene. In CV1 cells transfected with TRH receptor expression plasmid, GATA2-dependent transactivation of αGSU and endothelin-1 promoters was enhanced by TRH. In the gel shift assay, TRH signal potentiated the DNA-binding capacity of GATA2. While inhibition by T3 is dominant over TRH-induced activation, unliganded TR or the putative negative T3-responsive element are not required for TRH-induced stimulation. Studies using GH3 cells showed that TRH-induced activity of the TSHβ promoter depends on protein kinase C but not the mitogen-activated protein kinase, suggesting that the signaling pathway is different from that in the prolactin gene. These results indicate that GATA2 is the principal mediator of the TRH signaling pathway in TSHβ expression.

Hepatic FOXO1 Target Genes Are Co-regulated by Thyroid Hormone via RICTOR Protein Deacetylation and MTORC2-AKT Protein Inhibition

Background: Thyroid hormone (TH) and FOXO1 share similar transcriptional networks. However, TH regulation of FOXO1 activity is not well understood. Results: TH decreased RICTOR acetylation and MTORC2/AKT activity by SIRT1 activation and reduced FOXO1 phosphorylation. Conclusion: TH co-regulated transcription of FOXO1 target genes via RICTOR deacetylation. Significance: Downstream metabolic effects by TH can post-translationally activate other transcription factors. MTORC2-AKT is a key regulator of carbohydrate metabolism and insulin signaling due to its effects on FOXO1 phosphorylation. Interestingly, both FOXO1 and thyroid hormone (TH) have similar effects on carbohydrate and energy metabolism as well as overlapping transcriptional regulation of many target genes. Currently, little is known about the regulation of MTORC2-AKT or FOXO1 by TH. Accordingly, we performed hepatic transcriptome profiling in mice after FOXO1 knockdown in the absence or presence of TH, and we compared these results with hepatic FOXO1 and THRB1 (TRβ1) ChIP-Seq data. We identified a subset of TH-stimulated FOXO1 target genes that required co-regulation by FOXO1 and TH. TH activation of FOXO1 was directly linked to an increase in SIRT1-MTORC2 interaction and RICTOR deacetylation. This, in turn, led to decreased AKT and FOXO1 phosphorylation. Moreover, TH increased FOXO1 nuclear localization, DNA binding, and target gene transcription by reducing AKT-dependent FOXO1 phosphorylation in a THRB1-dependent manner. These events were associated with TH-mediated oxidative phosphorylation and NAD+ production and suggested that downstream metabolic effects by TH can post-translationally activate other transcription factors. Our results showed that RICTOR/MTORC2-AKT can integrate convergent hormonal and metabolic signals to provide coordinated and sensitive regulation of hepatic FOXO1-target gene expression.

Inhibition of GATA2-dependent transactivation of the TSHβ gene by ligand-bound estrogen receptor α

Transcriptional repression of the TSH-specific beta subunit (TSHbeta) gene has been regarded to be specific to thyroid hormone (tri-iodothyronine, T(3)) and its receptors (TRs) in physiological conditions. However, TSHbeta mRNA levels in the pituitary were reported to decrease in the administration of pharmacologic doses of estrogen (17-beta-estradiol, E(2)) and increase in E(2) receptor (ER)-alpha null mice. Here, we investigated the molecular mechanism of inhibition of the TSHbeta gene expression by E(2)-bound E(2)-estrogen receptor 1 (E(2)-ERalpha). In kidney-derived CV1 cells, transcriptional activity of the TSHbeta promoter was stimulated by GATA2 and suppressed by THRBs and ERalpha in a ligand-dependent fashion. Overexpression of PIT1 diminished the E(2)-ERalpha-induced inhibition, suggesting that PIT1 may protect GATA2 from E(2)-ERalpha targeting by forming a stable complex with GATA2. Interacting surfaces between ERalpha and GATA2 were mapped to the DNA-binding domain (DBD) of ERalpha and the Zn finger domain of GATA2. E(2)-dependent inhibition requires the ERalpha amino-terminal domain but not the tertiary structure of the second Zn finger motif in E(2)-ERalpha-DBD. In the thyrotroph cell line, TalphaT1, E(2) treatment reduced TSHbeta mRNA levels measured by the reverse transcription PCR. In the human study, despite similar free thyroxine levels, the serum TSH level was small but significantly higher in post- than premenopausal women who possessed no anti-thyroid antibodies (1.90 microU/ml+/-0.13 S.E.M. vs 1.47 microU/ml+/-0.12 S.E.M., P<0.05). Our findings indicate redundancy between T(3)-TR and E(2)-ERalpha signaling exists in negative regulation of the TSHbeta gene.

Desensitization and Incomplete Recovery of Hepatic Target Genes After Chronic Thyroid Hormone Treatment and Withdrawal in Male Adult Mice

Clinical symptoms may vary and not necessarily reflect serum thyroid hormone (TH) levels during acute and chronic hyperthyroidism as well as recovery from hyperthyroidism. We thus examined changes in hepatic gene expression and serum TH/TSH levels in adult male mice treated either with a single T3 (20 μg per 100 g body weight) injection (acute T3) or daily injections for 14 days (chronic T3) followed by 10 days of withdrawal. Gene expression arrays from livers harvested at these time points showed that among positively-regulated target genes, 320 were stimulated acutely and 429 chronically by T3. Surprisingly, only 69 of 680 genes (10.1%) were induced during both periods, suggesting desensitization of the majority of acutely stimulated target genes. About 90% of positively regulated target genes returned to baseline expression levels after 10 days of withdrawal; however, 67 of 680 (9.9%) did not return to baseline despite normalization of serum TH/TSH levels. Similar findings also were observed for negatively regulated target genes. Chromatin immunoprecipitation analysis of representative positively regulated target genes suggested that acetylation of H3K9/K14 was associated with acute stimulation, whereas trimethylation of H3K4 was associated with chronic stimulation. In an in vivo model of chronic intrahepatic hyperthyroidism since birth, adult male monocarboxylate transporter-8 knockout mice also demonstrated desensitization of most acutely stimulated target genes that were examined. In summary, we have identified transcriptional desensitization and incomplete recovery of gene expression during chronic hyperthyroidism and recovery. Our findings may be a potential reason for discordance between clinical symptoms and serum TH levels observed in these conditions.