SCFFbl12 Increases p21Waf1/Cip1 Expression Level through Atypical Ubiquitin Chain Synthesis.

The cyclin-dependent kinase (CDK) inhibitor p21 is an unstructured protein regulated by multiple turnover pathways. p21 abundance is tightly regulated, and its defect causes tumor development. However, the mechanisms that underlie the control of p21 level are not fully understood. Here, we report a novel mechanism by which a component of the SCF ubiquitin ligase, Fbl12, augments p21 via the formation of atypical ubiquitin chains. We found that Fbl12 binds and ubiquitinates p21. Unexpectedly, Fbl12 increases the expression level of p21 by enhancing the mixed-type ubiquitination, including not only K48- but also K63-linked ubiquitin chains, followed by promotion of binding between p21 and CDK2. We also found that proteasome activator PA28γ attenuates p21 ubiquitination by interacting with Fbl12. In addition, UV irradiation induces a dissociation of p21 from Fbl12 and decreases K63-linked ubiquitination, leading to p21 degradation. These data suggest that Fbl12 is a key factor that maintains adequate intracellular concentration of p21 under normal conditions. Our finding may provide a novel possibility that p21's fate is governed by diverse ubiquitin chains.

TSLP expression induced via Toll-like receptor pathways in human keratinocytes.

The skin epidermis and mucosal epithelia (airway, ocular tissues, gut, and so on) are located at the interface between the body and environment and have critical roles in the response to various stimuli. Thymic stromal lymphopoietin (TSLP), a cytokine expressed mainly by epidermal keratinocytes (KCs) and mucosal epithelial cells, is a critical factor linking the innate response at barrier surfaces to Th2-skewed acquired immune response. TSLP is highly expressed in skin lesions of atopic dermatitis patients. Here, we describe on Toll-like receptor (TLR)-mediated induction of TSLP expression in primary cultured human KCs, placing emphasis on experimental methods used in our studies. Double-stranded RNA (TLR3 ligand), flagellin (TLR5 ligand), and diacylated lipopeptide (TLR2-TLR6 ligand) stimulated human KCs to express TSLP and Staphylococcus aureus membranes did so via the TLR2-TLR6 pathway. Atopic cytokine milieu upregulated the TLR-mediated induction of TSLP. Culturing in the absence of glucocorticoid before stimulation enhanced the TSLP expression. Extracellular double-stranded RNA induced TSLP via endosomal acidification- and NF-κB-dependent pathway. Specific measurement of the long TSLP transcript, which contributes to the production of the TSLP protein, rather than total or the short transcript is useful for accurate detection of functional human TSLP gene expression. The results suggest that environment-, infection-, and/or self-derived TLR ligands contribute to the initiation and/or amplification of Th2-type skin inflammation including atopic dermatitis and atopic march through the induction of TSLP expression in KCs and include information helpful for understanding the role of the gene-environment interaction relevant in allergic diseases.

Expression and functional characterization of retinoic acid-inducible gene-I-like receptors of mast cells in response to viral infection.

To investigate the precise mechanisms of virus recognition by mast cells, the expression and functional characteristics of virus recognition receptors that lead to mast cell activation were investigated. Our results suggest that mast cells are partly responsible for the early in vivo production of antiviral cytokines and chemokines upon vesicular stomatitis virus (VSV) infection. Analysis of the expression of double-stranded RNA (dsRNA) recognition receptors in murine bone marrow-derived mast cells (BMMCs) revealed that BMMCs express melanoma differentiation-associated gene 5 (MDA5), protein kinase RNA-activated, retinoic acid-inducible gene-I (RIG-I) and Toll-like receptor 3. The expression levels of these receptors were found to increase upon stimulation of mast cells with VSV as well as synthetic dsRNA: polyinosinic-polycytidylic acid. Moreover, small interfering RNA analysis to identify the receptors responsible for mast cell activation by VSV revealed that both RIG-I and MDA5 were involved in cytokine production but not in the degranulation of mast cells. Our findings suggest that mast cells produce cytokines and chemokines in the early infection stage after recognizing viruses via RIG-I and MDA5, and may contribute to antiviral responses. These data provide additional novel information that improves our understanding of antiviral innate responses that involve mast cells.

The rest repression of the neurosecretory phenotype is negatively modulated by BHC80, a protein of the BRAF/HDAC complex.

Expression of neurosecretion by nerve cells requires the levels of the transcription repressor element-1 silencing transcription factor (REST) to be very low. However, when high-REST clones of PC12 cells, defective of neurosecretion, were fused to other high-REST, non-neurosecretory cells, some neurosecretion was recovered. To clarify the mechanism of this recovery, we fused defective PC12 cells with human lymphocytes. A cytogenetic analysis revealed all hybrid clones that recovered neurosecretion to contain a fragment of chromosome 11 including the gene encoding BHC80, a protein of one of the complexes that mediate REST repression. In these clones, REST levels were as high as in defective PC12, whereas BHC80, localized in the nucleus, was 4- to 5-fold higher. Transient transfection of defective PC12 with various amounts of BHC80 cDNA induced (1) in defective PC12, the reexpression of only neurosecretion mRNAs; (2) in defective PC12 cotransfected with the REST negative construct DNA-binding domain (to attenuate gene repression), the recovery of a weak, but complete neurosecretory phenotype, including dense-core granules and their regulated exocytosis. Chromatin immunoprecipitation and immunodepletion analyses revealed the extensive BHC80 association with REST at the genes of two neurosecretion proteins, chromograninB and SNAP25, however only in the low-REST PC12, whereas in high-REST defective PC12 no association was appreciable. In defective PC12 transfected with BHC80 some association was reestablished. Therefore, the recovery of neurosecretion observed after fusion/transfection of defective PC12 depends on the reciprocal level of BHC80 and REST, with BHC80 working as a negative modulator of REST repression. This role appears of possible cell physiological and pathological importance.

Selective stabilization of mammalian microRNAs by 3' adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2.

The steady-state levels of microRNAs (miRNAs) and their activities are regulated by the post-transcriptional processes. It is known that 3' ends of several miRNAs undergo post-dicing adenylation or uridylation. We isolated the liver-specific miR-122 from human hepatocytes and mouse livers. Direct analysis by mass spectrometry revealed that one variant of miR-122 has a 3'-terminal adenosine that is introduced after processing by Dicer. We identified GLD-2, which is a regulatory cytoplasmic poly(A) polymerase, as responsible for the 3'-terminal adenylation of miR-122 after unwinding of the miR-122/miR-122* duplex. In livers from GLD-2-null mice, the steady-state level of the mature form of miR-122 was specifically lower than in heterozygous mice, whereas no reduction of pre-miR-122 was observed, demonstrating that 3'-terminal adenylation by GLD-2 is required for the selective stabilization of miR-122 in the liver.

Unique ethylene-regulated touch responses of Arabidopsis thaliana roots to physical hardness.

Although touch responses of plant roots are an important adaptive behavior, the molecular mechanism remains unclear. We have developed a bioassay for measuring root-bending responses to physical hardness in Arabidopsis thaliana seedlings. Our test requires a two-layer solid medium. Primary roots growing downward through an upper layer of 0.3% phytagel either penetrate the lower layer or bend along an interface between the upper and lower layers with different concentrations (0.2-0.5%, corresponding to 1.57-6.79 gw mm(-2) in hardness). In proportion to increasing hardness of the lower layer, we found that the percentage of bending roots increased and ethylene production decreased, suggesting an inverse relationship between the root-bending response and ethylene production. Studies with ethylene biosynthesis modulators and mutants also suggested that bending and non-bending responses of roots to medium hardness depend, respectively, on decreased and increased ethylene biosynthesis. In addition, the degrees of root-tip softening and differential root-cell growth, both possible factors determining root-bending response, were enhanced and attenuated by decreased and increased amounts of ethylene, respectively--also in bending roots and non-bending roots. Our findings indicate that ethylene regulates root touch responses, probably through a combination of root-tip softening (or hardening) and differential root-cell growth.

Birth of normal offspring from mouse eggs activated by a phospholipase Czeta protein lacking three EF-hand domains.

Sperm-specific phospholipase C, PLCzeta, is a candidate for the Ca(2+) oscillation-inducing factor that is introduced into the ooplasm upon sperm-egg fusion. In addition to the 647-residue full-length PLCzeta, s-PLCzeta lacking the N-terminal 110 amino acids is known to be present in the mouse testis. In this study, we attempted to obtain full-term offspring from s-PLCzeta-activated eggs by round spermatid injection. Metaphase II-arrested eggs injected with a high RNA concentration of s-PLCzeta RNA normally developed to blastocysts. When the round spermatid nucleus was injected into telophase II-stage eggs previously activated by s-PLCzeta RNA, three live offspring were successfully obtained by transfer of the developed 4-cell embryos to pseudopregnant mice. These three offspring all grew to be normal adults and reproduced healthy second-generation mice.

Centromeric DNA hypomethylation as an epigenetic signature discriminates between germ and somatic cell lineages.

Germ cells have unique features strikingly distinguishable from somatic cells. The functional divergence between these two cell lineages has been postulated to result from epigenetic mechanisms. Here we show that the chromosomal centric and pericentric (C/P) regions in male and female germline cells are specifically DNA-hypomethylated, despite the hypermethylation status in somatic cells. In multipotent germline stem cells, the C/P region was initially hypomethylated and then shifted to the hypermethylation status during differentiation into somatic lineage in vitro. Moreover, the somatic-type hypermethylation pattern was maintained in the somatic cell-derived nuclear transfer embryos throughout preimplantation development. These results imply that the identity of germ cell lineage may be warranted by the hypomethylation status of the C/P region as an epigenetic signature.

A component of BRAF-HDAC complex, BHC80, is required for neonatal survival in mice.

BHC80 is a component of BRAF-HDAC complex (BHC) involved in transcriptional repression of neuron-specific genes in non-neuronal cells. However, BHC80 is present in both neuronal and non-neuronal cells. To explore the physiological importance of BHC80 in vivo, and the precise mechanism underlying neuron-specific gene repression by BHC80, we have produced mutant mice lacking Bhc80. The loss of Bhc80 resulted in neonatal lethality without sucking mother's breast milk sufficiently. Although Bhc80-deficient mice showed no developmental defect in the neuronal and non-neuronal tissues, Bhc80 is indispensable for the survival of neonatal pups.

A specific CpG site demethylation in the human interleukin 2 gene promoter is an epigenetic memory.

DNA demethylation plays a critical role in transcriptional regulation in differentiated somatic cells. However, there is no experimental evidence that CpG methylation in a small region of a genome restricts gene expression. Here, we show that the anti-CD3repsilon/CD28 antibody stimulation of human CD4+ T cells induces IL2 expression following epigenetic changes, including active demethylation of a specific CpG site, recruitment of Oct-1, and changes in histone modifications. When the stimulatory signal is withdrawn, Oct-1 remains on the enhancer region as a stable marker of the stimulation, causing the second induction to be faster and stronger. Our observations indicate that Oct-1-binding followed by CpG demethylation are key events in the epigenetic regulation of IL2 expression and may act as a memory of the regulatory event.

Regulation of LSD1 histone demethylase activity by its associated factors.

LSD1 is a recently identified human lysine (K)-specific histone demethylase. LSD1 is associated with HDAC1/2; CoREST, a SANT domain-containing corepressor; and BHC80, a PHD domain-containing protein, among others. We show that CoREST endows LSD1 with the ability to demethylate nucleosomal substrates and that it protects LSD1 from proteasomal degradation in vivo. We find hyperacetylated nucleosomes less susceptible to CoREST/LSD1-mediated demethylation, suggesting that hypoacetylated nucleosomes may be the preferred physiological substrates. This raises the possibility that histone deacetylases and LSD1 may collaborate to generate a repressive chromatin environment. Consistent with this model, TSA treatment results in derepression of LSD1 target genes. While CoREST positively regulates LSD1 function, BHC80 inhibits CoREST/LSD1-mediated demethylation in vitro and may therefore confer negative regulation. Taken together, these findings suggest that LSD1-mediated histone demethylation is regulated dynamically in vivo. This is expected to have profound effects on gene expression under both physiological and pathological conditions.

Survey of captive cynomolgus macaque colonies for SRV/D infection using polymerase chain reaction assays.

The exogenous simian type D retroviruses (SRV/Ds) are prevalent in macaque monkeys and sometimes cause immunodeficiency with anemia, weight loss, and persistent unresponsive diarrhea. SRV/D isolates are classified as subtypes 1 to 6, and the entire sequences of the gag region of SRV/D-1, -2, and -3 and SRV/D-Tsukuba (SRV/D-T) have been determined. We designed specific primers in the gag region of SRV/D-T that enabled us to directly detect by polymerase chain reaction (PCR) SRV/D-T proviral DNA sequences in DNA extracted from whole blood. Using this assay and another PCR assay that detects multiple SRV/D subtypes, we performed a survey for SRV/D infection in our specific pathogen-free (SPF) and conventional colonies at Tsukuba Primate Center (TPC). In the SPF colony, no SRV/D signal was detected in any animal. On the other hand, SRV/D-T was detected in 11 of 49 animals (22.5%) in the conventional colony. SRV/D-T was the only SRV/D subtype detected. Consequently, SRV/D-T is the major SRV/D subtype present in cynomolgus monkeys at TPC.

Isolation and characterization of a new simian retrovirus type D subtype from monkeys at the Tsukuba Primate Center, Japan.

Exogenous type D simian retroviruses (SRV/D) are prevalent in captive and feral populations of various macaque monkeys. Thus far, five subtypes of SRV/Ds have been reported, three of which (SRV-1, -2 and -3) have been molecularly characterized. Two SRV/D strains (N27 and T150) were isolated from seropositive cynomolgus macaques at the Tsukuba Primate Center (TPC) in Japan, showing clinical signs of SRV/D infection, including anemia and persistent unresponsive diarrhea. Electron microscopy demonstrated that both SRV/D isolates have a virion morphology typical of type D retrovirus. The SRV/D N27 and T150 isolates were essentially the same based on sequence analysis. From homology analysis of the entire gag sequence, the N27 isolate is closely related to the other known SRV/Ds but is distinct from the three molecularly characterized SRV/Ds. Thus, we have tentatively designated the N27 and T150 viruses isolated from TPC cynomolgus macaques as SRV/D-Tsukuba (SRV/D-T).

Ligand-dependent switching of ubiquitin-proteasome pathways for estrogen receptor.

Recent evidence indicates that the transactivation of estrogen receptor alpha (ERalpha) requires estrogen-dependent receptor ubiquitination and degradation. Here we show that estrogen-unbound (unliganded) ERalpha is also ubiquitinated and degraded through a ubiquitin-proteasome pathway. To investigate this ubiquitin-proteasome pathway, we purified the ubiquitin ligase complex for unliganded ERalpha and identified a protein complex containing the carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP preferentially bound to misfolded ERalpha and ubiquitinated it to induce degradation. Ligand binding to the receptor induced the dissociation of CHIP from ERalpha. In CHIP-/- cells, the degradation of unliganded ERalpha was abrogated; however, estrogen-induced degradation was observed to the same extent as in CHIP+/+ cells. Our findings suggest that ERalpha is regulated by two independent ubiquitin-proteasome pathways, which are switched by ligand binding to ERalpha. One pathway is necessary for the transactivation of the receptor and the other is involved in the quality control of the receptor.

Characterization of BHC80 in BRAF-HDAC complex, involved in neuron-specific gene repression.

BRAF-HDAC complex (BHC) has been shown to contain six components, including BHC80, and to mediate REST-dependent transcriptional repression of neuron-specific genes in non-neuronal cells. In this study, we have examined the functional role(s) of BHC80 in mouse tissues and human cultured cells. Two isoforms of mouse BHC80 were predominantly present in the central nervous system and spermatogenic cells. Human cultured cells also contained two isoforms of BHC80. Immunohistochemical analysis showed the presence of mouse BHC80 in the nucleus of neuronal cells in the hippocampus and cerebellum. The C-terminal region of human BHC80 containing PHD zinc-finger domain was capable of binding directly to each of five other components of BHC, and of organizing BHC mediating transcriptional repression. Moreover, two isoforms of human BHC80 were distinguished from each other by reduced binding to HDAC1 and HDAC2, despite the presence of the PHD finger domain. These results suggest that BHC80 presumably serves as a scaffold protein in BHC in neuronal as well as non-neuronal cells. A possible role of BHC80 in spermatogenesis is also suggested.

Testicular cytochrome P450scc and LHR as possible targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the mouse.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in adult animals has been reported to perturb the regulation of steroidogenesis in the testis, possibly by arylhydrocarbon receptor (AhR). To clarify how AhR is involved in the testicular steroidogenesis, we carried out comparative experiments using wild-type and AhR-null male mice that were intraperitoneally administered TCDD. The TCDD administration to wild-type mice showed significant reduction of P450scc and LHR in the testis, whereas the levels in the AhR-null mouse testis were unchanged. To compare anti-androgenic properties on hypothalamo-pituitary-gonadal (HPG) axis, estradiol-3-benzoate (EB), a synthetic estrogen agonist, was administered to mice, the expression of the LHalpha/FSHalpha, LHbeta, FSHbeta and GnRHR genes was severely impaired in the pituitary gland, in contrast to no observed effects in the TCDD-treated mice. In addition, the expression of the LHR gene was increased in the testis of the EB-treated mice. These observations suggest that the target of TCDD is different from that of EB on HPG axis and that TCDD treatment suppresses the P450scc and LHR genes in the testis in an AhR-dependent manner.

An RNA-dependent protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer's disease.

Various types of stress, such as disruption of calcium homeostasis, inhibition of protein glycosylation and reduction of disulfide bonds, result in accumulation of misfolded proteins in the endoplasmic reticulum (ER). The initial cellular response involves removal of such proteins by the ER, but excessive and/or long-term stress results in apoptosis. In this study, we used a randomized ribozyme library and ER stress-mediated apoptosis (tunicamycin-induced apoptosis) in SK-N-SH human neuroblastoma cells as a selective phenotype to identify factors involved in this process. We identified a double-stranded RNA-dependent protein kinase (PKR) as one of the participants in this process. The level of nuclear PKR was elevated, but the level of cytoplasmic PKR barely changed in tunicamycin-treated SK-N-SH cells. Furthermore, tunicamycin also raised levels of phosphorylated PKR in the nucleus. We also detected the accumulation of phosphorylated PKR in the nuclei of autopsied brain tissues in Alzheimer's disease. Thus, PKR might play a role in ER stress-induced apoptosis and in Alzheimer's disease.

Analysis of a mitochondrial apoptotic pathway using Bid-targeted ribozymes in human MCF7 cells in the absence of a caspase-3-dependent pathway.

In normal cells, tumor necrosis factor-alpha (TNF-alpha) activates caspase 8 in both mitochondrion-dependent and mitochondrion-independent apoptotic pathways. It is believed that these two pathways converge, with resultant activation of effector caspases, such as caspase 6 and caspase 7. However, the precise mechanism of the activation of caspases 6 and 7 remains unknown. In this study, in order to focus on the mitochondrion-dependent pathway, we employed MCF7 human breast carcinoma cells, which do not have a functional mitochondrion-independent (caspase 3-dependent) pathway. We specifically targeted the transcript of Bid, a proapoptotic facilitator that is a substrate of caspase 8 in the mitochondrial pathway. In the TNF-alpha-treated MCF7 cells that expressed Bid-targeted ribozymes, the release of cytochrome c and the activation of caspase 9, but not of caspase 8, was delayed. Furthermore, the proteolysis of procaspase 7 was also delayed in Bid ribozyme-expressing cells. Because MCF7 cells are caspase 3 deficient, the direct cross-talk between caspase 8 and caspase 3 does not take place. Therefore, it became clear for the first time that caspase 9 by itself can activate caspase 7 in the absence of the caspase 3-dependent pathway in TNF-alpha-induced apoptosis by the use of specific ribozymes.

Full activation of estrogen receptor alpha activation function-1 induces proliferation of breast cancer cells.

The effects of estrogen and anti-estrogen are mediated through the estrogen receptors (ER) alpha and beta, which function as ligand-induced transcriptional factors. Recently, one of the phthalate esters, n-butylbenzyl phthalate (BBP), has been shown to induce estrogen receptor-mediated responses. By using the truncated types of ER mutants, we revealed that activation function-1 (AF-1) activity was necessary for the BBP-dependent transactivation function of ERalpha. AF-1 is also known to be responsible for the partial agonistic activity of tamoxifen. Whereas tamoxifen exhibits an anti-estrogenic effect on proliferation of the MCF-7 breast cancer cell line, BBP showed an estrogenic effect on MCF-7 to stimulate proliferation. In vivo and in vitro binding assays revealed that whereas 4-hydroxytamoxifen (OHT) induced binding of ERalpha to both an AF-1 coactivator complex (p68/p72 and p300) and corepressor complexes (N-CoR/SMRT), BBP selectively enhanced the binding to the AF-1 coactivators. We also showed that the transcriptional activity of OHT-bound ERalpha was modulated by the ratio between the AF-1 coactivator and corepressor complexes. Expression of a dominant-negative type of N-CoR inhibited the interaction between OHT-bound ERalpha and N-CoR/SMRT and enhanced the transcriptional activity of OHT-bound ERalpha. Furthermore, the cell growth of MCF-7 stably expressing the dominant-negative type of N-CoR was enhanced by the addition of OHT. These results indicated that fully activated AF-1 induces the stimulation of breast cancer growth and that the ratio between AF-1 coactivators and corepressors plays a key role to prevent proliferation of tumor by tamoxifen.

Confirmation by FRET in individual living cells of the absence of significant amyloid beta -mediated caspase 8 activation.

When cells are exposed to death-inducing molecules such as tumor necrosis factor-alpha or Fas, caspase 8 is activated and cleaves an apoptotic facilitator, Bid, that is a member of the Bcl-2 family. After additional modification, the C-terminal moiety of Bid is translocated to the mitochondria and induces the release of cytochrome c into the cytoplasm. In an attempt to directly observe the cleavage of Bid and the following events in living cells, we constructed a vector that encoded Bid fused with yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) (YFP-Bid-CFP). On expression of YFP-Bid-CFP in mammalian cells, we were able to observe the efficient transfer of energy from excited CFP to YFP within the YFP-Bid-CFP molecule and, importantly, the fusion protein YFP-Bid-CFP was fully functional in cells. When YFP-Bid-CFP was cleaved by caspase 8, on activation by anti-Fas Abs but not by Abeta or tunicamycin, no such transfer of energy was detected. To our knowledge, this is the first report of (i) visualization of the activation of Bid by proteolytic cleavage, with direct observation of the cleavage of YFP-Bid-CFP in the cytoplasm and subsequent translocation of the cleaved Bid to mitochondria and (ii) the absence of Abeta- or tunicamycin-mediated significant activation of caspase 8 in individual living cells.