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Human cyclin-dependent kinase inhibitor 3 (CDKN3) is a known oncogene in hepatocellular carcinoma (HCC) and its expression is promoted during tumor development. CDKN3 serves as a cell cycle regulator and its dysregulation is considered to be a causal factor for tumor progression. However, the molecular basis of the regulation of CDKN3 expression remains largely elusive. Using in silico approach, we identified CDKN3SE, a super enhancer (SE), and enhancer RNA (eRNA) candidates transcribed from this SE. Among the eRNA candidates, the expression of CDKN3eRNA was detected in the human HCC model cell line HepG2, and was found to facilitate the expression of CDKN3 without affecting the cell proliferation rate. In silico screening revealed two DNA-binding transcription factors, upstream stimulatory factor (USF) 1 and 2, involved in the regulation of CDKN3eRNA expression on CDKN3SE. A knock-down of USF1/USF2 expression in the HepG2 cells did not affect CDKN3eRNA expression, while the expression of CDKN3 was down-regulated. In a USF2 dominant negative HepG2 cell line generated by genome editing, a drastically altered cell shape and lowered cell proliferation rate were found; however, the expression of CDKN3eRNA appeared unaffected. Thus, the present study illustrated two regulators for CDKN3 expression: USF2, as a cell cycle-associated protein regulator, and CDKN3eRNA, as a cell cycle-unassociated RNA regulator.

The clinical use of androgen receptor (AR) antagonists has been successful in treating prostate cancer patients, inducing remission of androgen-dependent tumors. However, a couple of years after treatment, prostate tumors transition into an androgen-independent state with altered gene expression profiles, but the molecular basis is not understood. Since the AR antagonists trigger this transition, we assessed whether AR antagonists induce chromatin reorganization in an androgen-dependent prostate cancer cell line (LNCaP). Treatment of LNCaP cells with two clinically used AR antagonists (bicalutamide [Bic] and enzalutamide [Enz]) expectedly resulted in antagonistic effects on cell proliferation, AR transactivation, and dihydrotestosterone (DHT)-induced expression of AR target genes. Thus, the antagonists expectedly acted to antagonize the transactivation function of AR activated by androgen binding. By ChIP-qPCR assay, AR bound to Bic, but not Enz, was recruited to an endogenous consensus AR-binding site within the kallikrein-related peptidase 3 gene promoter after treatment with Bic, similar to the effect of DHT. By ATAC-seq analysis of the cells after long-term treatment for 5 days, Bic and dihydrotestosterone DHT induced different chromatin reorganization patterns and gene expression profiles, suggesting that Bic exhibited a distinct action from that by DHT. Thus, these results suggest that the action of a known AR antagonist is mediated by chromatin reorganization in a prostate cancer cell line.

Background To minimize the interval from symptom onset to treatment commencement is essential for a favorable outcome among breast cancer patients. This study examined whether provider interval (time elapsed from first consultation to treatment onset) lengthened among breast cancer patients following Japan’s 2011 earthquake, tsunami, and nuclear disaster in Fukushima. Factors associated with the length of post-disaster interval and whether interval was associated with breast cancer stage, were also investigated. Materials and Methods So-so District (study site), was an area damaged by the 2011 disasters. Data of all breast cancer patients who made their first medical consultation and received initial treatment at the core medical institutions in the area five years pre- or post-disaster, were extracted from the patients’ medical records. We employed several regression approaches to fulfill our study objectives. Results We included 263 patients (140 pre-disaster and 123 post-disaster). After adjustment for covariates, the interval did not significantly change post-disaster compared with pre-disaster. Those with 4 or 5 cohabiting family members experienced shorter interval post-disaster than those with 0 or 1 cohabiting family members (relative length 0.47, 95% confidence interval 0.28–0.78). Those with >60 days interval had lower odds of stages III or IV cancer than those with Conclusion Overall, the total interval did not lengthen post-disaster. However, those with restricted cohabiting family members might have experienced a longer total interval. Cancer stage may not necessarily reflect the influence of provider interval on the patients’ outcome. Micro Abstract This study assessed provider interval among breast cancer patients in a post-disaster setting. The participants were patients diagnosed in Fukushima, Japan, either five years before or after the 2011 disasters. Although statistically significant change in the provider interval post-disaster was not observed after adjusting for covariates, the number of cohabiting family members might be associated with longer intervals.

Objectives The authors sought to investigate the prevalence, risk factors, and mid-term mortality in Asian patients with prosthesis–patient mismatch (PPM) after transcatheter aortic valve replacement (TAVR). Background Little information is available on PPM after TAVR in Asian patients. Methods The authors included 1,558 patients enrolled in the OCEAN-TAVI (Optimized transCathEter vAlvular iNtervention) Japanese multicenter registry from October 2013 to July 2016 after excluding patients who died following TAVR before discharge. PPM was defined as moderate if ≧0.65 but ≦0.85 cm2/m2, or severe if Results Of the 1,546 patients, moderate and severe PPM were observed in 138 (8.9%) and 11 (0.7%) patients, respectively. These 149 patients were included in the PPM group. The median age and body surface area were 85 years (interquartile range [IQR]: 81 to 88 years) and 1.41 m2 (IQR: 1.30 to 1.53 m2), respectively. In our multivariate analysis, younger age, larger body surface area, smaller aortic valve area, smaller annulus area, no balloon post-dilatation, and use of Edwards Sapien 3 (Edwards Lifesciences, Irvine, California) were identified as independent predictors of PPM. The estimated cumulative all-cause mortality at 1 year using the Kaplan-Meier method was similar between the PPM and non-PPM groups (10.2% vs. 8.3%; log-rank; p = 0.41). Conclusions The low prevalence of PPM and mortality at 1 year in patients with PPM after TAVR in this Japanese cohort implies that PPM is not a risk factor for mid-term mortality in Asian patients who have undergone TAVR.

The nuclear vitamin D receptor (VDR) is generally recognized as a ligand-dependent transcription factor that mediates the actions of its natural ligand, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) on multiple target genes involved in mineral homeostasis, bone development, as well as immune reactivity. As the VDR is widely distributed in nearly all cells of the body, it implies that the vitamin D endocrine system may regulate many cell types and functions. Experiments in VDR null mice established that the VDR has intrinsically critical roles in skin and keratinocyte biology but not in immune responses. Oppositely, absence of the VDR ligand is linked to susceptibility to autoimmunity, illustrating a potential role for the unliganded VDR in the immune system. This discrepancy stimulated us to further investigate the impact of the VDR on the phenotype and function of myeloid dendritic cells (DCs) generated ex vivo from bone marrow precursors of VDR null (with a truncated VDR) and VDR ΔAF2 mice (with a mutated C-terminal activation factor 2 domain thus rendering ligand-induced gene transcription impossible). Absent or unliganded VDR did not affect bone marrow-derived myeloid DC generation. DCs obtained from VDR null and VDR ΔAF2 bone marrow cells had comparable MHC-II, and costimulatory molecule CD86, CD80 and CD40 expression than DCs from wild-type bone marrow cells. Additionally, an unliganded VDR did not affect the cytokine production nor the antigen-specific T cell stimulatory capacity of bone marrow-derived DCs. In conclusion, we showed that although clear effects of 1α,25-dihydroxyvitamin D3 are described on DC generation, absence of VDR or presence of an unliganded VDR does not affect the profile and function of ex vivo generated bone marrow-derived DCs. publisher: Elsevier articletitle: The phenotype and function of murine bone marrow-derived dendritic cells is not affected by the absence of VDR or its ability to bind 1α,25-dihydroxyvitamin D3 journaltitle: The Journal of Steroid Biochemistry and Molecular Biology articlelink: http://dx.doi.org/10.1016/j.jsbmb.2015.08.010 content_type: article copyright: © 2015 Elsevier Ltd. All rights reserved. ispartof: Journal of Steroid Biochemistry and Molecular Biology vol:164 pages:239-245 ispartof: location:NETHERLANDS, Delft status: published

Vitamin D deficiency is associated with beta-cell dysfunction and a higher risk of diabetes, but mice and humans with an absence of the vitamin D receptor (VDR) display normal glucose tolerance. Here, we investigated the direct effects of absence of VDR or absence of ligand activation of VDR on beta-cell function. For this purpose, we generated mice, with a mutation in the AF2 domain of Vdr (VDRΔAF2), preventing ligand-driven transcriptional activation of vitamin D responsive genes. VDRΔAF2 mice were compared to Vdr full knockout (VDR-/-) and wild type (WT) mice. In order to avoid hypocalcemia, which has a direct effect on beta-cell function, mice were fed a high calcium, high lactose diet yielding comparable serum calcium in all mice. While VDR-/- mice developed extensive alopecia by the age of 24 weeks, the fur of VDRΔAF2 remained normal. All VDRΔAF2 mice weighed significantly less than WT, while male but not female VDR-/- mice had a lower body weight than WT mice. Dual-energy X-ray absorptiometry showed that both VDRΔAF2 (17.2% (females) and 16.6% (males)) and VDR-/- (15.7% and 14.8%) mice have a lower percentage of body fat (vs 19.3% and 22.2% in WT). Serum 25(OH)D3 concentrations were lower for both VDRΔAF2 (-4.55 fold, P

Understanding how a disaster impact functions of healthcare facilities is important future disaster risk reduction. This study aims to synthesise the impact of the 2011 Fukushima nuclear disaster, one of the largest chemical, biological, radiation, nuclear, or explosive (CRBNE) events in world history, on the functional status of healthcare facilities. Two data sources, Medline and Nursing databases, were searched for peer-reviewed papers written in English or Japanese. CiNii and Ichi-shi Web were searched for papers in Japanese. Articles written in English or Japanese and published from March 2011 to April 2019 were included. For further information within the month of the disaster, newspaper articles from Asahi Shimbun and Yomiuri Shimbun were also searched, as well as an inquiry report published by the National Diet of Japan. After screening, 66 articles and a book were included. In the acute phase of the disaster, problems around logistics and human resources resulted in the most severe damage to healthcare facilities, despite the relatively limited structural damage. Hospital evacuation posed a large problem, and ill-planned evacuations resulted in increased mortality among evacuated patients. No literature described direct impacts of radiation exposure, or radiation screenings, to the functioning of healthcare facilities. In the chronic phase, issues around human resources including staff shortages and mental health deterioration among healthcare staff remained as major problems. In conclusion, after the Fukushima nuclear disaster and subsequent social disruption, there was prominent damage to the infrastructure and logistics of healthcare facilities, and staff shortages at local healthcare facilities continued long-term.

Disaster has a negative impact on health conditions, especially on those of temporary housing residents. Health status has a close relationship with physical activity and performance. However, few reports have assessed physical performance among residents living in temporary housing. In this study, we compared physical capabilities between the elderly who evacuated to temporary housing and those who stayed in their own homes after the Great East Japan Earthquake. Subjects were recruited from those over 65 years of age who participated in the medical check-ups for temporary housing residents (TH group) or check-ups for residents of downtown areas (control group) in Soma City, Fukushima, in 2012. The subjects underwent grip strength, one-leg standing (OLS), and timed up and go tests (TUG). In total, 1890 participants were recruited. The TH group showed significantly stronger grip strength than that of the control group. On the other hand, the TH group showed weaker standing stability, according to decreased OLS and increased TUG scores. We revealed that standing stability was impaired among elderly temporary housing residents 1.5 years after the disaster. Disaster responders should take into account the health risks associated with living in temporary housing., Highlights • Physical performance were assessed a year after the Great East Japan Earthquake. • Standing stability among residents living in temporary housing was deteriorated. • Physical inactivity was cited as a reason for the decreased standing stability. • Health risks associated with living in temporary housing should be noted.

Lipid metabolism drastically changes in response to the environmental factors in metazoans. Lipid is accumulated at the food rich condition, while mobilized in adipocyte tissue in starvation. Such lipid mobilization is also evident during the pupation of the insects. Pupation is induced by metamorphosis hormone, ecdysone via ecdysone receptor (EcR) with lipid mobilization, however, the molecular link of the EcR-mediated signal to the lipid mobilization remains elusive. To address this issue, EcR was genetically knocked-down selectively in 3rd instar larva fat body of Drosophila, corresponding to the adipocyte tissues in mammalians, that contains adipocyte-like cells. In this mutant, lipid accumulation was increased in the fat body. Lipid accumulation was also increased when knocked-down of taiman, which served as the EcR co-activator. Two lipid metabolism regulatory factor, E75B and adipose (adp) as well as cell growth factor, dMyc, were found as EcR target genes in the adipocyte-like cells, and consistently knock-down of these EcR target genes brought phenotypes in lipid accumulation supporting EcR function. These findings suggest that EcR-mediated ecdysone signal is significant in lipid metabolism in insects.

Osteoclastogenesis is a highly regulated process governed by diverse classes of regulators. Among them, nuclear factor of activated T-cells calcineurin-dependent 1 (NFATc1) is the primary osteoclastogenic transcription factor, and its expression is transcriptionally induced during early osteoclastogenesis by receptor activation of nuclear factor κB ligand (RANKL), an osteoclastogenic cytokine. Here, we report the novel enzymatic function of JMJD5, which regulates NFATc1 protein stability. Among the tested Jumonji C (JmjC) domain-containing proteins, decreased mRNA expression levels during osteoclastogenesis were found for JMJD5 in RAW264 cells stimulated by RANKL. To examine the functional role of JMJD5 in osteoclast differentiation, we established stable JMJD5 knockdown cells, and osteoclast formation was assessed. Down-regulated expression of JMJD5 led to accelerated osteoclast formation together with induction of several osteoclast-specific genes such as Ctsk and DC-STAMP, suggesting that JMJD5 is a negative regulator in osteoclast differentiation. Although JMJD5 was recently reported as a histone demethylase for histone H3K36me2, no histone demethylase activity was detected in JMJD5 in vitro or in living cells, even for other methylated histone residues. Instead, JMJD5 co-repressed transcriptional activity by destabilizing NFATc1 protein. Protein hydroxylase activity mediated by the JmjC domain in JMJD5 was required for the observed functions of JMJD5. JMJD5 induced the association of hydroxylated NFATc1 with the E3 ubiquitin ligase Von Hippel-Lindau tumor suppressor (VHL), thereby presumably facilitating proteasomal degradation of NFATc1 via ubiquitination. Taken together, the present study demonstrated that JMJD5 is a post-translational co-repressor for NFATc1 that attenuates osteoclastogenesis.

Cell cycle-dependent expression of canonical histone proteins enables newly synthesized DNA to be integrated into chromatin in replicating cells. However, the molecular basis of cell cycle-dependency in the switching of histone gene regulation remains to be uncovered. Here, we report the identification and biochemical characterization of a molecular switcher, HERS (histone gene-specific epigenetic repressor in late S phase), for nucleosomal core histone gene inactivation in Drosophila. HERS protein is phosphorylated by a cyclin-dependent kinase (Cdk) at the end of S-phase. Phosphorylated HERS binds to histone gene regulatory regions and anchors HP1 and Su(var)3-9 to induce chromatin inactivation through histone H3 lysine 9 methylation. These findings illustrate a salient molecular switch linking epigenetic gene silencing to cell cycle-dependent histone production.

Members of the nuclear steroid/thyroid hormone receptor (NR) gene superfamily are DNA-binding transcription factors that regulate target genes in a spatiotemporal manner, depending on the promoter context. In vivo observations of ligand responses in NR-mediated gene regulation led to the identification of ligand-dependent coregulators that directly interact with NRs. Functional dissection of NR coregulators revealed that their transcriptional coregulation was linked to histone acetylation. However, recent work in the fields of reversible histone modification and chromatin remodeling indicates that histone-modifying enzymes, including histone methylases and chromatin remodelers, are potential transcriptional coregulators that interact directly and indirectly with NRs.

Summary The two p160 transcriptional coregulator family members SRC-1 and TIF2 have important metabolic functions in white and brown adipose tissues as well as in the liver. To analyze TIF2 cell-autonomous functions in skeletal muscles, we generated TIF2 (i)skm−/− mice in which TIF2 was selectively ablated in skeletal muscle myofibers at adulthood. We found that increased mitochondrial uncoupling in skeletal muscle myocytes protected these mice from decreased muscle oxidative capacities induced by sedentariness, delayed the development of type 2 diabetes, and attenuated high-caloric-diet-induced obesity. Moreover, our results demonstrate that SRC-1 and TIF2 can modulate the expression of the uncoupling protein 3 (UCP3) in an antagonistic manner and that enhanced SRC-1 levels in TIF2-deficient myofibers are critically involved in the metabolic changes of TIF2 (i)skm−/− mice. Thus, modulation of the expression and/or activity of these coregulators represents an attractive way to prevent or treat metabolic disorders.

O-glycosylation has emerged as an important modification of nuclear proteins, and it appears to be involved in gene regulation. Recently, we have shown that one of the histone methyl transferases (MLL5) is activated through O-glycosylation by O-GlcNAc transferase (OGT). Addition of this monosaccharide is essential for forming a functional complex. However, in spite of the abundance of OGT in the nucleus, the impact of nuclear O-glycosylation by OGT remains largely unclear. To address this issue, the present study was undertaken to test the impact of nuclear O-glycosylation in a monocytic cell line, THP-1. Using a cytokine array, MIP-1alpha and -1beta genes were found to be regulated by nuclear O-glycosylation. Biochemical purification of the OGT interactants from THP-1 revealed that OGT is an associating partner for distinct co-regulatory complexes. OGT recruitment and protein O-glycosylation were observed at the MIP-1alpha gene promoter; however, the known OGT partner (HCF-1) was absent when the MIP-1alpha gene promoter was not activated. From these findings, we suggest that OGT could be a co-regulatory subunit shared by functionally distinct complexes supporting epigenetic regulation.

It has been well established that estrogen is involved in the pathophysiology of breast cancer. Estrogen receptor (ER) alpha appears to promote the proliferation of cancer tissues, while ERbeta can protect against the mitogenic effect of estrogen in breast tissue. The expression status of ERalpha and ERbeta may greatly influence on the development, treatment, and prognosis of breast cancer. Previous studies have indicated that the deleted in breast cancer 1 (DBC1/KIAA1967) gene product has roles in regulating functions of nuclear receptors. The gene encoding DBC1 is a candidate for tumor suppressor identified by genetic search for breast cancer. Caspase-dependent processing of DBC1 promotes apoptosis, and depletion of the endogenous DBC1 negatively regulates p53-dependent apoptosis through its specific inhibition of SIRT1. In addition, DBC1 modulates ERalpha expression and promotes breast cancer cell survival by binding to ERalpha. Here we report an ERbeta-specific repressive function of DBC1. Immunoprecipitation and immunofluorescence studies show that ERbeta and DBC1 interact in a ligand-independent manner similar to ERalpha. In vitro pull-down assays revealed a direct interaction between DBC1 amino-terminus and activation function-1/2 domain of ERbeta. Although DBC1 shows no influence on the ligand-dependent transcriptional activation function of ERalpha, the expression of DBC1 negatively regulates the ligand-dependent transcriptional activation function of ERbetain vivo, and RNA interference-mediated depletion of DBC1 stimulates the transactivation function of ERbeta. These results implicate the principal role of DBC1 in regulating ERbeta-dependent gene expressions.

Vitamin D plays a major role in mineral and skeletal homeostasis through interaction with the nuclear vitamin D receptor (VDR) of target cells. Recent reports have indicated that some cellular effects of vitamin D may occur via alternative signaling pathways, but concrete evidence for mineral homeostasis has not been shown in vivo. To investigate this issue, the actions of calcitriol (1,25D) and maxacalcitol (OCT), which were developed for treatment of uremia-induced secondary hyperparathyroidism, were analyzed in VDR knockout (VDR(-/-)) mice. The VDR(-/-) mice were fed a rescue diet immediately after weaning. 1,25D, OCT or a control solution was administered intraperitoneally to these mice three times a week for eight weeks. Biological markers and bone growth were measured and bone histomorphometric analysis of the calcein-labeled tibia was performed 24 h after the final administration. Significantly higher levels of serum Ca(2+) were observed in 1,25D- and OCT-treated mice, but the serum parathyroid hormone level was unchanged by both agents. Impaired bone growth, enlarged and distorted cartilaginous growth plates, morphological abnormalities of cancellous and cortical bones; a morbid osteoid increase, lack of calcein labeling, and thinning of cortical bone, were all significantly improved by 1,25D and OCT. The significance of these effects was confirmed by bone histomorphometrical analysis. Upregulation of the calbindin D(9k) mRNA expression level in the duodenum may explain these findings, since this protein is a major modulator of Ca transport in the small intestine. We conclude that 1,25D and OCT both at a high dose exert significant effects on Ca and skeletal homeostasis with the principal improvement of Ca status in VDR(-/-) mice, and some of these effects may occur through an alternative vitamin D signaling pathway.

Steroid hormones and their cognate nuclear receptors exert a wide spectrum of biological actions through regulation of transcriptional and posttranscriptional processes. However, the underlying molecular mechanism by which steroid hormones control posttranscriptional processes is largely unknown. We now report that estrogen receptor alpha (ERalpha) inhibits the maturation of a particular microRNA (miRNA) and thereby stabilizes the mRNA of an ERalpha target gene through the 3'UTR. Estrogen-bound ERalpha downregulated expression of a set of miRNAs in both animals and cultured cells. Activated ERalpha attenuated the processing of primary miRNAs into pre-miRNAs through estrogen-dependent association with the Drosha complex, resulting in stabilization of the transcript of an ERalpha target gene through its 3'UTR. Thus, a steroid hormone achieves posttranscriptional control by regulating the maturation of miRNA.

Vitamin D3 analogues have been developed for a mutant vitamin D receptor (VDR), Arg274Leu. The mutant VDR has a mutation at Arg274, which forms an important hydrogen bond with 1α-OH of 1α,25-dihydroxyvitamin D3 to anchor the ligand tightly in the VDR ligand binding pocket. Stereoselective synthesis of the A-ring part of the novel vitamin D analogue, 2α-(3-hydroxypropyl)-1α-methyl-25-hydroxyvitamin D3 (4), from d -galactose was accomplished with the key steps of the introduction of the methyl and allyl groups to the chiral building blocks. The new analogue 4 is ca. 7.3-fold more active than the natural hormone 1α,25-dihydroxyvitamin D3 (1).

Chondromodulin-I (Chm-I) is a glycoprotein that stimulates the growth of chondrocytes and inhibits angiogenesis in vitro. Mice lacking the Chm1 gene show abnormal bone metabolism and pathological angiogenesis in cardiac valves in the mature stage although they develop normally without aberrations in endochondral bone formation during embryogenesis or in cartilage development during growth. These findings indicate that Chm-I is critical under conditions of stress such as bone repair through endochondral ossification of a fracture callus. We carried out the present study to examine the expression and role of Chm-I in bone repair using a stabilized tibial fracture model, and compared fracture healing in Chm1 knockout (Chm1(-/-)) mice with that in wild-type mice. Chm-I mRNA and protein localized in the external cartilaginous callus in the reparative phase of fracture healing. Radiological examination showed a delayed union in Chm1(-/-) mice although the fracture site was covered with both external and internal calluses. Chm1 null mutation reduced external cartilaginous callus formation as judged by marked decrease of type X collagen alpha 1 (Col10a1) expression and the total amount of cartilage matrix. Interestingly, the majority of chondrocytes in the periosteal callus failed to differentiate into mature chondrocytes in Chm1(-/-) mice, while the hypertrophic maturation of chondrocytes between the cortices was not affected. These results suggest that Chm-I is involved in hypertrophic maturation of periosteal chondrocytes. Although a direct effect of Chm-I on bones is still unclear, bony callus formation was increased while external cartilaginous callus decreased in Chm1(-/-) mice. We conclude that in the absence of Chm1, predominant primary bone healing occurs due to an indirect effect induced by reduction of cartilaginous callus rather than to a direct effect on osteogenic function, resulting in a delayed union.

The arylhydrocarbon receptor (AhR) mediates sex steroid hormone-related actions in both normal physiology and in dioxin toxicity. In addition to regulation of direct target genes, the ligand-activated AhR associates with estrogen or androgen receptors (ERalpha or AR) to regulate transcription as a functional unit. Given that endogenous and exogenous AhR-ligands are structurally diverse, it is unclear whether cross-talk regulation of ERalpha/AR by the activated AhR is an intrinsic function of the AhR or the result of ligand-type-selective differences. To ensure uniform activity of the AhR irrespective of ligand-type-specific differences, we employed CA-AhR, which lacks the ligand-binding domain and has a constitutive activity. We found that CA-AhR, in the absence of a ligand, acted as a transcriptional co-regulator for the unliganded ERalpha/AR as well as for mutants of ERalpha/AR lacking a ligand-binding domain. CA-AhR was recruited to estrogen-/androgen-responsive promoters with endogenous ERalpha/AR. Moreover, CA-AhR had an E3 ubiquitin ligase activity and promoted proteasomal degradation of ERalpha/AR. Thus, these findings indicate that the cross-talk function of the AhR with sex hormone receptors is an intrinsic function of the AhR.

Testosterone (T) is an important factor for determining body composition in males. Abdominal obesity is inversely correlated with serum T levels in men, leading to greater mortality. Pathologically hypogonadal men also have a significantly higher fat mass, which is reversed by T administration. However, the mechanism for such anti-obesity effect of androgen has not been well clarified. Androgen receptor (AR) null male mice revealed late-onset obesity. Male ARKO mice were euphagic compared to the wild-type male controls, but also less dynamic and less oxygen consuming. Transcript profiling indicated that male ARKO mice had lower transcripts for the thermogenetic uncoupling protein 1 (UCP1). We also found enhanced secretion of adiponectin, which is insulin-sensitizing, from adipose tissue in comparison to wild type, which might partly explain why the overall insulin sensitivity of male ARKO mice remained almost intact despite their apparent obesity. In addition, decreased lipolysis rather than increased lipid synthesis was observed, which might also account for the increased adiposity in male ARKO mice. The results revealed that AR plays important roles in male metabolism by affecting the energy balance, and is negative to both adiposity and insulin sensitivity.

We designed and synthesized 1alpha- and 1beta-hydroxymethyl-2alpha-(3-hydroxypropyl)-25-hydroxyvitamin D(3) (2a,b) and related analogues 2alpha-(3-hydroxypropyl)-25-hydroxyvitamin D(3) (3), Posner's analogues of 1alpha- and 1beta-hydroxymethyl-25-hydroxyvitamin D(3) (4a,b), as well as 2alpha-(3-hydroxypropyl)-1alpha,25-dihydroxyvitamin D(3) (5), to confirm the effect of the 1alpha-hydroxy group and/or 2alpha-(3-hydroxypropyl) group of vitamin D(3) analogues with the modified A-ring moiety on the mutant vitamin D receptor, VDR(Arg274Leu). The 2alpha-(3-hydroxypropyl) group showed better effect on enhancement of the transcriptional activity through the mutant VDR than the 1alpha- and 1beta-hydroxymethyl groups.

International audience; Transcriptional activators, several different coactivators, and general transcription factors are necessary to access specific loci in the dense chromatin structure to allow precise initiation of RNA polymerase II (Pol II) transcription. Histone acetyltransferase (HAT) complexes were implicated in loosening the chromatin around promoters and thus in gene activation. Here we demonstrate that the 2 MDa GCN5 HAT-containing metazoan TFTC/STAGA complexes contain a histone H2A and H2B deubiquitinase activity. We have identified three additional subunits of TFTC/STAGA (ATXN7L3, USP22, and ENY2) that form the deubiquitination module. Importantly, we found that this module is an enhancer of position effect variegation in Drosophila. Furthermore, we demonstrate that ATXN7L3, USP22, and ENY2 are required as cofactors for the full transcriptional activity by nuclear receptors. Thus, the deubiquitinase activity of the TFTC/STAGA HAT complex is necessary to counteract heterochromatin silencing and acts as a positive cofactor for activation by nuclear receptors in vivo.

1α,25-Dihydroxyvitamin D 3 (1α,25-D 3 ) has potent antiproliferative and anti-invasive properties in vitro in cancer cells. However, the major limitation to its clinical use is that it causes hypercalcemia. Therefore, vitamin D analogs with potent cell regulatory effects but with weaker calcemic effects than 1α,25-D 3 are required. Among them, 22-oxa-1α,25-D 3 and 19-nor-1α,25-D 3 have anti-cancer effects with relatively low calcemic effects. Modifications at the C-2α position of the A-ring also produced analogs with a unique biological profile. Not only the side-chain but also the A-ring modification thus generates a unique analog with potent cell regulatory effects and low calcemic activity as well. We report here that the hybrid 1α,25-D 3 analog, synthesized via the highly regio- and stereo-selective ring opening 2α-fluorination and catalytic asymmetric carbonyl-ene cyclization, with 2α-fluoro, 19-nor, and 22-oxa modification exhibits unique cell regulatory activities against the development of metastatic lung carcinoma.

Retinoid-X-receptor alpha (RXRalpha), a member of the nuclear receptor (NR) superfamily, is a ligand-dependent transcriptional regulatory factor. It plays a crucial role in NR signalling through heterodimerization with some 15 NRs. We investigated the role of RXRalpha and its partners on mouse skin tumor formation and malignant progression upon topical DMBA/TPA treatment. In mutants selectively ablated for RXRalpha in keratinocytes, epidermal tumors increased in size and number, and frequently progressed to carcinomas. As keratinocyte-selective peroxisome proliferator-activated receptor gamma (PPARgamma) ablation had similar effects, RXRalpha/PPARgamma heterodimers most probably mediate epidermal tumor suppression. Keratinocyte-selective RXRalpha-null and vitamin-D-receptor null mice also exhibited more numerous dermal melanocytic growths (nevi) than control mice, but only nevi from RXRalpha mutant mice progressed to invasive human-melanoma-like tumors. Distinct RXRalpha-mediated molecular events appear therefore to be involved, in keratinocytes, in cell-autonomous suppression of epidermal tumorigenesis and malignant progression, and in non-cell-autonomous suppression of nevi formation and progression. Our study emphasizes the crucial role of keratinocytes in chemically induced epidermal and melanocytic tumorigenesis, and raises the possibility that they could play a similar role in UV-induced tumorigenesis, notably in nevi formation and progression to melanoma.

Nontraumatic osteonecrosis of the femoral head (ONFH) is caused by disruption of blood flow. This disease often occurs in association with steroid treatment. The pathology of steroid-induced ONFH remains unclear, although abnormalities in lipid metabolism have been reported to be involved. In this study, we examined the differences of gene polymorphism frequencies of apolipoprotein B (ApoB) and apolipoprotein A1 (ApoA1), which are important proteins for lipid transport, as well as of lipid parameters, between ONFH cases and referent patients among those who were subjected to renal transplantation.Subjects were 158 cases who had undergone renal transplant, including 34 cases that were diagnosed as ONFH after renal transplantation and 124 cases that were not. Four single nucleotide polymorphisms including C7623T and G12619A for the ApoB gene and G75A and C83T for the ApoA1 gene were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and Taqman real-time PCR chemistry. Also, serum levels of low-density lipoprotein (LDL), high-density lipoprotein (HDL), ApoB, and ApoA1 were measured. Their relationship to ONFH was statistically evaluated.A higher frequency of 7623TT or CT of the ApoB gene was observed in ONFH cases than in referent patients (P = 0.033), resulting in an elevated odds ratio that was statistically significant (adjusted odds ratio = 6.37, 95% CI = 1.53-26.5, P = 0.011). No significant relationship was observed between other genes and ONFH. Regarding lipid parameters, a higher value of ApoB/ApoA1 ratio was observed in cases (P = 0.045).For the prediction of ONFH, it is useful to analyze ApoB C7623T and plasma ApoB/ApoA1 ratio before the administration of steroids.

Vitamin D receptor (VDR) mediates a wide variety of vitamin D actions through transcriptional controls of target genes as a ligand-dependent transcription factor. The transactivation by VDR is known to associate with two co-activator complexes, DRIP/TRAP and p160/CBP, through physical interaction with DRIP205 and p160 members (TIF2) components, respectively. However, functional difference between the two co-activator complexes for VDR co-activation remains unclear. In the present study, to address this issue, a series of point mutants in VDR helix 12 were generated to test the functional association. Alanine replacement of VDR valine 418 resulted in loss of DRIP205 interaction, but it was still transcriptionally potent with ability to interact with TIF2. Surprisingly, the V421A mutant was only partially impaired in transactivation without co-activator interaction, implying presence of a putative co-activator/complex. Thus, these findings suggest that ligand-induced transcriptional controls by VDR require a number of known and unknown co-regulator complexes, that may support the tissue-specific function of VDR.

We have previously shown that the novel ATP-dependent chromatin remodeling complex WINAC is required for the ligand-bound Vitamin D receptor (VDR)-mediated transrepression of the 25(OH)D(3) 1alpha-hydroxylase [1alpha(OH)ase] gene. However, the molecular basis for VDR promoter association, which does not involve its binding to specific DNA sequences, remains unclear. To address this issue, we investigated the function of WSTF in terms of the association between WINAC and chromatin for ligand-induced transrepression by VDR. Results of in vitro experiments using chromatin templates showed that the association of unliganded VDR with the promoter required physical interactions between WSTF and both VDR and acetylated histones prior to VDR association with chromatin. The acetylated histone-interacting region of WSTF was mapped to the bromodomain, and a WSTF mutant lacking the bromodomain served as a dominant-negative mutant in terms of ligand-induced transrepression of the 1alpha(OH)ase gene. Thus, our findings indicate that WINAC associates with chromatin through a physical interaction between the WSTF bromodomain and acetylated histones, that appears to be indispensable for VDR/promoter association for ligand-induced transrepression of 1alpha(OH)ase gene expression.

CYP27B1 is a critical enzyme of Vitamin D biosynthesis that hydroxylates 25(OH)D(3) at the final step of the biosynthetic pathway. The CYP27B1 gene is expressed primarily in kidney and negatively controlled by Vitamin D receptor. We have characterized the negative vitamin D response element and its binding protein, a bHLH transcription factor. This factor directly binds to the 1alphanVDRE and activates transcription, but its transcriptional activity is suppressed by the ligand-activated Vitamin D receptor through recruitment of histone deacetylase. We have shown that histone deacetylation is a critical step for chromatin structure remodeling in suppression of the CYP27B1 gene. We have further demonstrated that, in addition to histone acetylation, this transrepression by VDR requires DNA methylation in the CYP27B1 gene promoter. Thus, transcriptional regulation of the CYP27B1 gene appears to be mediated by dual epigenetic modifications.

Estrogens are essential for normal reproductive activity in females and males and for ovarian differentiation during a critical developmental stage in many vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in the Japanese giant salamander ( Andrias japonicus ), we isolated cDNA encoding the estrogen receptor (ER) from the liver. A full-length Japanese giant salamander ER cDNA (jgsER) was obtained using 5′ and 3′ rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the jgsER showed high identity to the Xenopus ERα (ESR1) (77.7%). We have applied both the conventional ERE-luciferase reporter assay system and the GAL4-transactivation system to characterize this receptor. In two different transient transfection assay systems using mammalian cells, the jgsER protein displayed estrogen-dependent activation of transcription. The GAL4-transactivation system showed about 10-fold greater activity of the estrogen receptor by hormone when compared to the conventional ERE-luciferase reporter assay system. Tissue distribution of ERα mRNA was examined and kidney, ovary and liver exhibited expression. This is the first isolation of an estrogen receptor from a salamander and also is the first functional cDNA obtained from the Japanese giant salamander, an endangered species considered a special natural monument of Japan.

The type IIa sodium-dependent phosphate cotransporter (NPT2a) expressed in renal proximal tubules represents an important determinant in maintaining inorganic phosphate (Pi) homeostasis. In the present study, we identified two variant transcripts of the mouse NPT2a gene, Npt2a-v1 and Npt2a-v2, characterized by the presence of alternative first exons (either exon 1A or exon 1B). The chromosomal structure analysis revealed that the Npt2a gene comprises of two promoters (promoters 1 and 2) and 14 exons, and spans approximately 17 kb. Quantitative PCR analysis showed that renal mRNA levels of both the variants markedly decreased in X-linked vitamin D-resistant hypophosphatemic rickets (Hyp) mice compared to normal littermates. Interestingly, transcriptional activity of a reporter gene, containing Npt2a promoters 1 and 2, was renal cell—specifically increased by 1α, 25(OH) 2 D 3 and its analogs. The deletion analysis revealed that the CAAT box in the Npt2a promoter 2 is important for the 1α, 25(OH) 2 D 3 -dependent renal cell-specific activation of the reporter gene. These data suggested that two alternative promoters control the renal expression of Npt2a gene and both Npt2a variant transcripts are down regulated in Hyp mice.

We previously reported the three-dimensional structure of human CYP27B1 (25-hydroxyvitamin D3 1alpha-hydroxylase) constructed by homology modeling. Using the three-dimensional model we studied the docking of the substrate, 25-hydroxyvitamin D3, into the substrate binding pocket of CYP27B1. In this study, we focused on the amino acid residues whose point mutations cause vitamin D-dependent rickets type 1, especially unconserved residues among mitochondrial CYPs such as Gln65 and Thr409. Recently, we successfully overexpressed mouse CYP27B1 by using a GroEL/ES co-expression system. In a mutation study of mouse CYP27B1 that included spectroscopic analysis, we concluded that in a 1alpha-hydroxylation process, Ser408 of mouse CYP27B1 corresponding to Thr409 of human CYP27B1 forms a hydrogen bond with the 25-hydroxyl group of 25-hydroxyvitamin D3. This is the first report that shows a critical amino acid residue recognizing the 25-hydroxyl group of the vitamin D3.

TBP-free TAF II-containing-type HAT complex subclasses, which contain hGCN5 HAT and TRRAP, appear to act as common coactivator complexes for nuclear receptors. However, their physiological significance with respect to each nuclear receptor remains to be established. To address this issue, we used hepatic cell lines (HepG2) with reduced endogenous TRRAP expression through antisense RNA expression or with overexpressed TRRAP or other major coactivators. The ligand-induced transactivation function of liver X receptor alpha (LXRalpha) and farnesoid X receptor/bile acid receptor reflected TRRAP expression levels, while that of PPARgamma did not. A GST pull-down assay indicated that TRRAP contains two potential LXRalpha-interacting domains in the C-terminal and central domains. Expression of antisense TRRAP RNA in HepG2 cells abolished the ligand-induced expression of LXRalpha target genes. These results suggested that TRRAP plays an important role as a coactivator, presumably part of a complex, in lipid metabolism through regulation of the LXRalpha-mediated gene cascade in hepatic cells.

Fibroblast growth factor-23 (FGF-23), a novel phosphate-regulating factor, was elevated in hypophosphatemic patients with X-linked hypophosphatemic rickets/osteomalacia and also in patients with chronic kidney disease. These observations suggested the pathophysiological importance of FGF-23 on phosphate homeostasis. However, regulation of FGF-23 production is still unclear. We investigated effects of both dietary phosphorus and 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) on circulating FGF-23 in vivo Administration of. 1alpha,25(OH)(2)D(3) dose-dependently increased serum FGF-23 in thyroparathyroidectomized rats without correlating with serum inorganic phosphorus or serum parathyroid hormone. On the other hand, vitamin D receptor null mice had very low serum FGF-23 and did not respond to the 1alpha,25(OH)(2)D(3) administration. These observations suggested 1alpha,25(OH)(2)D(3) directly or indirectly regulates circulating FGF-23. Serum FGF-23 had a strong correlation with serum inorganic phosphorus controlled by dietary phosphorus in 5/6 nephrectomized rats. High phosphate diet elicited a 5-fold increase in serum FGF-23 compared with sham-operated rats, whereas serum FGF-23 did not correlate with serum calcium or serum creatinine in 5/6 nephrectomized rats. Administration of 1alpha,25-dihydroxyvitamin D(3) also elicited a severalfold increase in serum FGF-23 in the uremic rats. Taken together, this shows that both serum phosphorus and 1alpha,25(OH)(2)D(3) regulate circulating FGF-23 independent of each other. Therefore, we proposed there was a feedback loop existing among serum phosphorus, 1alpha,25(OH)(2)D(3), and FGF-23, in which the novel phosphate-regulating bone-kidney axis integrated with the parathyroid hormone-vitamin D(3) axis in regulating phosphate homeostasis.

The expression of mouse CYP27B1 in Escherichia coli has been dramatically enhanced by coexpression of GroEL/ES. To reveal the enzymatic properties of CYP27B1, we measured its hydroxylation activity toward vitamin D3 and 1alpha-hydroxyvitamin D3 (1alpha(OH)D3) in addition to the physiological substrate 25(OH)D3. Surprisingly, CYP27B1 converted vitamin D3 to 1alpha,25(OH)D3. Both 1alpha-hydroxylation activity toward vitamin D3, and 25-hydroxylation activity toward 1alpha(OH)D3 were observed. The Km and Vmax values for 25-hydroxylation activity toward 1alpha(OH)D3 were estimated to be 1.7 microM and 0.51 mol/min/mol P450, respectively, while those for 1alpha-hydroxylation activity toward 25(OH)D3 were 0.050 microM and 2.73 mol/min/mol P450, respectively. Note that the substrate must be fixed in the opposite direction in the substrate-binding pocket of CYP27B1 between 1alpha-hydroxylation and 25-hydroxylation. Based on these results and the fact that human CYP27A1 and Streptomyces CYP105A1 also convert vitamin D3 to 1alpha,25(OH)D3, 1alpha-hydroxylation, and 25-hydroxylation of vitamin D3 appear to be closely linked together.

Wnt and estrogen signaling represent important regulatory pathways, each controlling a wide range of biological processes. While an increasing number of observations suggest potential convergence between these pathways, no direct evidence of their functional interaction has been reported. Using human colon and breast cancer cells, we found that estrogen receptor (ER) alpha- and beta-catenin precipitated within the same immunocomplexes, reciprocally enhanced the transactivation of cognate reporter genes, and were reciprocally recruited to cognate response elements in the promoters of endogenous target genes. Using transgenic Drosophila that ectopically expressed human ERalpha alone or together with metabolically stable beta-catenin/Armadillo mutants, we demonstrated genetic interaction between these signal transducers in vivo. Thus, we present here the first direct evidence of cross-talk between Wnt and estrogen signaling pathways via functional interaction between beta-catenin and ERalpha.

Insect development is controlled by the combined actions of ecdysteroid and juvenile hormones. Transcriptional control by ecdysteroid hormones is mediated via two nuclear receptor superfamily members, ecdysone receptor (EcR) and its heterodimeric partner, ultraspiracle (USP). Although the ecdysteroid hormone 20-hydroxyecdysone acts as an EcR ligand and activates transcription through EcR/USP heterodimers, the activity of juvenile hormones, such as Juvenile hormone III (JH III), and methoprenic acid (MA) via USP remains unclear. Here, we demonstrate that juvenile hormones act as USP ligands and exhibit suppressive effects on ecdysone-dependent EcR transactivation. JH III- and MA-bound USP markedly repressed ecdysone-dependent EcR transactivation through shifting of the USP ligand-binding domain alpha-helix 12 without affecting EcR/USP heterodimerization or DNA binding. Moreover, transcriptional repression by USP ligands was attenuated by a histone deacetylation inhibitor. Our results suggested that juvenile hormones serve as USP ligands that antagonize EcR-mediated ecdysone actions through the recruitment of histone deacetylase complexes.

Most of the androgen actions are considered to be mediated by the androgen receptor (AR) of the target genes. The AR is composed of a fairly large molecule because of the long A/B domains of its N-terminal. However, the independent roles of the AR as well as those of the estrogen receptors largely remained unknown mainly due to the lack of the AR knockout (ARKO) mice line. We have succeeded in generating the ARKO mouse by means of a conditional targeting using the Cre/loxP system. The ARKO males grew healthily although they showed a typical feature of the testicular feminization mutation (Tfm) and the hormonal assay revealed significantly lower serum androgen and higher LH levels in comparison with those of the wild type (WT) males. The serum estrogen levels were, however, comparable between both the ARKO and the WT. Another hallmark of the ARKO males was a state of high bone turnover osteopenia, in which the acceleration in the bone resorption clearly exceeded the bone formation. Male-typical behaviors were disrupted in male ARKO mice. Aiming at a quick differentiation of an androgen-dependent polyQ disease such as Kennedy's disease, the authors also developed the Drosophila fly-eye model in which the wild type and the polyQ-expanded human AR (hAR) was induced in the eyes of Drosophila. When androgen was administered to the flies induced with the polyQ-expanded hAR, their optical nerves were devastated.

We have purified nuclear complexes for Vitamin D receptor (VDR), and identified one of them as a novel ATP-dependent chromatine remodeling containing Williams syndrome transcription factor (WSTF), that is supposed to be responsible for Williams syndrome. This complex (WSTF including nucleosome assembly complex (WINAC)) exhibited an ATP-dependent chromatin remodeling activity in vitro. Transient expression assays revealed that WINAC potentiates ligand-induced function of VDR in gene activation and repression. Thus, this study describes a molecular basis of the VDR function on chromosomal DNA through chromatine remodeling.

Estrogen receptors (ERs) play a central role in the diverse actions of estrogen. A number of synthetic ER ligands have been generated that can modulate various ER functions. Here we show that TAS-108, representing a novel class of synthetic ER ligands, blocked both ER transactivation functions without inhibiting DNA-binding activity. A transient expression assay showed that similar to ICI182,780, TAS-108 exhibited pure antagonistic activity as it blocked both the N-terminal AF-1 and C-terminal AF-2 transactivation functions. However, unlike ICI182,780, TAS-108 promoted the recruitment of the SMRT co-repressor that abolished ER transactivation function without inhibition of the ability of ERalpha to bind to its target DNA. Both TAS-108 and ICI182,780 acted as antagonists for the transactivation functions of the D351Y mutant, derived from tamoxifen-resistant breast cancer cells, while estrogen and known selective estrogen receptor modulators (SERMs), 4-OH tamoxifen and raloxifene, stimulated D351Y-mediated transcription. Thus, our findings indicated that TAS-108 acts as a novel estrogen antagonist that recruits co-repressors to ERs without AF-1 activation or prevention of DNA binding. Therefore, TAS-108 may be effective against tamoxifen-resistant breast cancer via a different mechanism than that for ICI182,780.

The androgen receptor (AR) has two transactivation functions that have been mapped to the N- and C-terminal domains and designated as activation function-1 (AF-1) and AF-2, respectively. While the molecular basis for AF-2 function has been well studied, little is known about AF-1 coregulators. Therefore, we attempted to identify AF-1-interacting proteins from HEK293 cells by biochemical purification followed by mass fingerprinting by matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI–TOF MS). Purified AF-1 region-interacting proteins were found to contain nuclear RNA-binding protein p54 nrb , polypyrimidine tract-binding protein-associated splicing factor (PSF), paraspeckle protein 1 (PSP1), and PSP2, which are assumed to be involved in pre-mRNA processing. p54 nrb interacted with AR via the A/B domain in a ligand-dependent manner. Reflecting the physical interaction between p54 nrb and the AR A/B domain, AR AF-1 function was potentiated by p54 nrb . Our results suggest that p54 nrb functions as a coactivator of AR that potentiates transcription, and presumably splicing as well.

Fibroblast Growth Factor (FGF) signaling is known to play an important role during cutaneous development. To elucidate the role of FGF10 during whisker formation, we examined the expression of Fgf10 in normal developing whiskers and phenotypes of Fgf10-deficient whiskers. Fgf10 is first expressed in the maxillary process, lateral and medial nasal processes, then in the mesenchymal cells underneath the future whisker placodes, and in the surrounding mesenchyme of developing whiskers. Fgf10-null whiskers exhibit a significant decrease in number and their structure is disorganized as revealed by scanning electron microscopy. Hair follicle marker genes such as Sonic hedgehog, Patched, and Patched 2 are aberrantly expressed in the mutant whiskers. Thus, FGF10 is required for proper whisker development mediated by SHH signaling in the mouse.

Initiation of transcription of protein-encoding genes by RNA polymerase II was thought to require transcription factor TFIID, a complex comprising the TATA-binding protein (TBP) and TBP-associated factors (TAFs). In the presence of TBP-free TAF complex (TFTC), initiation of polymerase II transcription can occur in the absence of TFIID. TFTC contains several subunits that have been shown to play the role of transcriptional coactivators, including the GCN5 histone acetyltransferase (HAT), which acetylates histone H3 in a nucleosomal context. Here we analyze the coactivator function of TFTC. We show direct physical interactions between TFTC and the two distinct activation regions (H1 and H2) of the VP16 activation domain, whereas the HAT-containing coactivators, p300/CBP (CREB-binding protein), interact only with the H2 subdomain of VP16. Accordingly, cell transfection experiments demonstrate the requirement of both p300 and TFTC for maximal transcriptional activation by GAL-VP16. In agreement with this finding, we show that in vitro on a chromatinized template human TFTC mediates the transcriptional activity of the VP16 activation domain in concert with p300 and in an acetyl-CoA-dependent manner. Thus, our results suggest that these two HAT-containing co-activators, p300 and TFTC, have complementary rather than redundant roles during the transcriptional activation process.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked, adult-onset, neurodegenerative disorder affecting only males and is caused by expanded polyglutamine (polyQ) stretches in the N-terminal A/B domain of human androgen receptor (hAR). Although no overt phenotype was detected in adult fly eye photoreceptor neurons expressing mutant hAR (polyQ 52), ingestion of androgen or its known antagonists caused marked neurodegeneration with nuclear localization and structural alteration of the hAR mutant. Ligand-independent toxicity was detected with a truncated polyQ-expanded A/B domain alone, which was attenuated with cytosolic trapping by coexpression of the unliganded hAR E/F ligand binding domain. Thus, our findings suggest that the full binding of androgen to the polyQ-expanded hAR mutants leads to structural alteration with nuclear translocation that eventually results in the onset of SBMA in male patients.

Cyclic adenosine monophosphate-dependent protein kinase (PKA) is involved in various biological functions in neurons. To investigate the subcellular localization of PKA, we stained cultured hippocampal neurons with anti-PKA catalytic subunit antisera. PKA catalytic subunit colocalized with microtubules (MTs) in dendrites as well as with the actin filaments (F-actin) in growth cones. After treatment with cytochalasin B, the colocalization of PKA catalytic subunits with MTs was enhanced, whereas the colocalization with F-actin was suppressed. This result indicates that PKA is anchored to the actin and MT cytoskeletons, and disruption of F-actin releases PKA to the cytoplasm, which then leads to an increase in the amount of PKA in MT domains in the neuron.