Toshihiro Nakajima

Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription.

At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. Although cytotoxicities of expanded polyQ stretches are implicated, the molecular mechanisms of neurodegeneration remain unclear. We found that expanded polyQ stretches preferentially bind to TAFII130, a coactivator involved in cAMP-responsive element binding protein (CREB)-dependent transcriptional activation, and strongly suppress CREB-dependent transcriptional activation. The suppression of CREB-dependent transcription and the cell death induced by polyQ stretches were restored by the co-expression of TAFII130. Our results indicate that interference of transcription by the binding of TAFII130 with expanded polyQ stretches is involved in the pathogenetic mechanisms underlying neurodegeneration.

Regulation of Lef-mediated Transcription and p53-dependent Pathway by Associating β-Catenin with CBP/p300

CBP and its homologue p300 play significant roles in cell differentiation, cell cycle, and anti-oncogenesis. We demonstrated that β-catenin, recently known as a potent oncogene, and CBP/p300 are associated through its CH3 region, which is a primary target of adenoviral oncoprotein E1A and various nuclear proteins, such as p53, cyclin E, and AP-1, and both are colocalized in the nuclear bodies. CBP/p300 potentiated Lef-mediated transactivation of β-catenin, and E1A, a potent inhibitor of CBP/p300, repressed its transactivation. Furthermore, overexpression of stable β-catenin mutant competitively suppressed the p53-dependent pathway. These may be a key mechanism of β-catenin involved in oncogenic events underlying disruption of tumor suppressor function through CBP/p300.

Cooperative Interaction of EWS with CREB-binding Protein Selectively Activates Hepatocyte Nuclear Factor 4-mediated Transcription

The EWS gene when fused to transcription factors such as the ETS familyATF-1, Wilms tumor-1, and nuclear orphan receptors upon chromosomal translocation is thought to contribute the development of Ewing sarcoma and several malignant tumors. Although EWS is predicted to be an RNA-binding protein, an inherent EWS nuclear function has not yet been elucidated. In this study, we found that EWS associates with a transcriptional co-activator CREB-binding protein (CBP) and the hypophosphorylated RNA polymerase II, which are included preferentially in the transcription preinitiation complex. These interactions suggest the potential involvement of EWS in gene transcription, leading to the hypothesis that EWS may function as a co-activator of CBP-dependent transcription factors. Based on this hypothesis, we investigated the effect of EWS on the activation of nuclear receptors that are activated by CBP. Of nuclear receptors examined, hepatocyte nuclear factor 4-dependent transcription was selectively enhanced by EWS but not by an EWS mutant defective for CBP binding. These results suggest that EWS as a co-activator requires CBP for hepatocyte nuclear factor 4-mediated transcriptional activation.

Dual roles of RNA helicase A in CREB-dependent transcription.

ABSTRACT RNA helicase A (RHA) is a member of an ATPase/DNA and RNA helicase family and is a homologue of Drosophila maleless protein (MLE), which regulates X-linked gene expression. RHA is also a component of holo-RNA polymerase II (Pol II) complexes and recruits Pol II to the CREB binding protein (CBP). The ATPase and/or helicase activity of RHA is required for CREB-dependent transcription. To further understand the role of RHA on gene expression, we have identified a 50-amino-acid transactivation domain that interacts with Pol II and termed it the minimal transactivation domain (MTAD). The protein sequence of this region contains six hydrophobic residues and is unique to RHA homologues and well conserved. A mutant with this region deleted from full-length RHA decreased transcriptional activity in CREB-dependent transcription. In addition, mutational analyses revealed that several tryptophan residues in MTAD are important for the interaction with Pol II and transactivation. These mutants had ATP binding and ATPase activities comparable to those of wild-type RHA. A mutant lacking ATP binding activity was still able to interact with Pol II. In CREB-dependent transcription, the transcriptional activity of each of these mutants was less than that of wild-type RHA. The activity of the double mutant lacking both functions was significantly lower than that of each mutant alone, and the double mutant had a dominant negative effect. These results suggest that RHA could independently regulate CREB-dependent transcription either through recruitment of Pol II or by ATP-dependent mechanisms.

Role of notch-1 intracellular domain in activation of rheumatoid synoviocytes

OBJECTIVEnNotch family proteins are transmembrane receptors that control cell fate and proliferation. Rheumatoid arthritis (RA) is characterized by activation and abnormal proliferation/differentiation of synoviocytes. We examined the expression of Notch-1 and its role in the activation of RA synoviocytes.nnnMETHODSnThe expression of Notch-1 protein was detected by a specific antibody raised against the Notch-1 intracellular domain. Notch-1 messenger RNA (mRNA) expression in synoviocytes was analyzed by Northern blotting. Notch-1 protein expression was confirmed by Western blotting with anti-Notch-1 antibody. To analyze the role of Notch-1 in synoviocyte proliferation, we examined the effects of antisense Notch-1 oligonucleotides (ODNs) and MW167, a gamma-secretase inhibitor.nnnRESULTSnNotch-1 protein and mRNA were detected in synovium from all study subjects. The nucleus of RA synoviocytes showed strong staining with anti-Notch-1 antibody, whereas there was predominantly cytoplasmic staining of normal and osteoarthritis (OA) synoviocytes. Western blotting showed a distinct approximately 63-kd protein detected by anti-Notch-1 antibody in nuclear extracts from RA synoviocytes, indicating that nuclear staining of RA synovium and synoviocytes is likely to be the result of nuclear localization of Notch-1 intracellular domain (NICD). Furthermore, tumor necrosis factor alpha (TNFalpha) increased NICD nuclear translocation in a dose-dependent manner. Antisense Notch-1 ODNs partially blocked the proliferation of RA synoviocytes and inhibited TNFalpha-induced proliferation in both OA and RA synoviocytes. In addition, gamma-secretase inhibitor, which blocks the production of NICD, also inhibited TNFalpha-induced proliferation of RA synoviocytes.nnnCONCLUSIONnOur results demonstrate the expression of Notch-1 in synoviocytes and the presence of Notch-1 fragment in the nuclei of RA synoviocytes and suggest the involvement of Notch-1 signaling in the TNFalpha-induced proliferation of RA synoviocytes.

Expression of notch homologues in the synovium of rheumatoid arthritis and osteoarthritis patients.

Abstract. Notch is known as a receptor that controls differentiation or proliferation in various cells and is associated with several diseases. The objective of the present study was to clarify whether human Notch homologues Notch-1, -2, -3, and -4 are expressed in synovium and synoviocytes from rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Immunohistochemical staining showed that Notch-1, -2, and -3 were clearly expressed in the synovium from both RA and OA, whereas Notch-4 was only slightly detected. We further performed Western blotting with the same antibodies used in immunohistochemical staining. Notch-1 and -2 were strongly detected in both RA and OA, and the expression of Notch-3 was slightly detected, while there was no Notch-4 expression in both RA and OA synoviocytes. In contrast, all Notch homologues were strongly expressed in the synovium at the developmental stage obtained from the infant. These results indicate that the expression pattern of Notch among synovium from OA and RA patients differed from that of normal subjects.

Cell type-dependent transactivation or repression of mesoderm-restricted basic helix-loop-helix protein, POD-1/Capsulin.

A family of basic-helix-loop-helix (bHLH) nuclear factors play important roles in controlling cell growth and differentiation as critical regulatory components in transcription. Here we describe molecular characterization of mesoderm-specific bHLH protein, POD-1/Capsulin. Transactivation property of POD-1/Capsulin was analyzed by the Gal4 fusion system in six mammalian cell lines. The results indicated that an activation property was shown in HT1080 and HeLa cells, but a repression activity in HepG2 cells. Mapping analysis for the transactivation and repression activities revealed that the C-terminal domain of POD-1/Capsulin is essential for the transactivation and both the N-terminal and C-terminal domains are contributed to the repression activities. Furthermore, in order to identify possible interactants of the POD-1/Capsulin, we performed yeast two-hybrid screen in a human kidney cDNA library, and identified a class A bHLH protein, ITF-2 as potential heterodimeric partner of the bHLH protein.

Identification of N-terminal minimal transactivation domain of CBP, p300 and caenorhabditis elegans homologues.

CBP/p300 is a multidomain transcriptional cofactor that acts in junction with other factors to regulate transcription. To elucidate the domain function of CBP, we fused its dissected fragments to Ga14 DNA-binding domain and transfected the deletion mutants into several cell lines. First, we found that the minimal transactivation domain (MTD) at the N-terminal portion maps to between 344 and 451 aa, and shows activity in a cell-type dependent manner. Second, we cloned C. elegans homologues corresponding to the MTD by RT-PCR and identified the three related products, two of which exhibited weak transcriptional activity. Finally, by means of the yeast two hybrid screening using MTD as a bait, we cloned hypoxia-inducible factor (HIF) 1 alpha and Stat2 cDNAs. These results suggested a functional role of MTD located at the N-terminal region of CBP/p300 in connecting to transcriptional factors.

Potential role of HOXD9 in synoviocyte proliferation.

OBJECTIVEnTo investigate the role of HOXD9 in the proliferation activity of cultured synoviocytes as well as the mechanisms that regulate HOXD9 transcription.nnnMETHODSnSynoviocytes from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were transfected with HOXD9 complementary DNA to establish stable transformants that overexpressed HOXD9. HOXD9 expression was detected by Western blotting with anti-HOXD9 antibody. The growth properties of the transformants were investigated by proliferation and colony formation assays. The expression of basic fibroblast growth factor (bFGF), tumor necrosis factor alpha (TNFalpha), interleukin-1beta, c-Fos, and c-Myc was examined by Western blotting. Transcriptional regulation of HOXD9 was examined by transient cotransfection.nnnRESULTSnHOXD9 protein was highly expressed in RA synoviocytes, but there was no expression in OA synoviocytes. HOXD9 transfection induced stable HOXD9 protein expression in synoviocytes and showed an increased proliferation rate under both normal and serum-starved conditions, as well as an enhanced capacity to proliferate anchorage independently to form colonies in soft agar cultures, compared with control transfectants. Higher levels of bFGF and c-Fos were detected in HOXD9 transformants than in controls. Transient cotransfection assays of NIH3T3 fibroblasts and synoviocytes showed that HOXD9 activated the luciferase reporter construct containing the highly conserved region (HCR), an autoregulatory element of HOXD9 promoter. This activation was significantly increased by bFGF, suppressed by TNFalpha, and unchanged by transforming growth factor beta in synoviocytes. Human T lymphotropic virus type I tax also activated the luciferase reporter construct containing the HCR and had a synergistic effect with HOXD9 on HCR promoter activation.nnnCONCLUSIONnOur data suggest that HOXD9 plays a potential role in synovial proliferation. In addition, they suggest that the involvement of HOXD9 in the regulation of cellular growth might be mediated, at least in part, by up-regulation of growth-related factors such as bFGF and c-Fos and/or might result from increased transcription activity by its regulators.