RPAP3 splicing variant isoform 1 interacts with PIH1D1 to compose R2TP complex for cell survival.

We previously characterized RNA polymerase II-associated protein 3 (RPAP3) as a cell death enhancer. Here we report the identification and characterization of splicing isoform of RPAP3, isoform 1 and 2. We investigated the interaction between RPAP3 and PIH1 domain containing protein 1 (PIH1D1), and found that RPAP3 isoform 1, but not isoform 2, interacted with PIH1D1. Furthermore, knockdown of RPAP3 isoform 1 by small interfering RNA down-regulated PIH1D1 protein level without affecting PIH1D1 mRNA. RPAP3 isoform 2 potentiated doxorubicin-induced cell death in human breast cancer T-47 cells although isoform 1 showed no effect. These results suggest that R2TP complex is composed of RPAP3 isoform 1 for its stabilization, and that RPAP3 isoform 2 may have a dominant negative effect on the survival potency of R2TP complex.

Critical role of leukotriene B4 receptor signaling in mouse 3T3-L1 preadipocyte differentiation.

BACKGROUND: Various inflammatory mediators related to obesity might be closely related to insulin resistance. Leukotrienes (LTs) are involved in inflammatory reactions. However, there are few reports regarding the role of LTs in adipocyte differentiation. Therefore, we investigated the role of leukotriene B4 (LTB4)-leukotriene receptor (BLT) signaling in mouse 3T3-L1 fibroblastic preadipocyte differentiation to mature adipocytes. METHODS: Mouse 3T3-L1 preadipocytes were treated with lipoxygenase (LOX) inhibitors, BLT antagonist, and small interfering RNA (siRNA) for BLT1 and BLT2 to block the LTB4-BLT signaling pathway, then the adipocyte differentiation such as lipid accumulation and the increase in triglyceride was evaluated. RESULTS: Blockade of BLT signaling by treatment with a LOX inhibitor or a BLT antagonist suppressed preadipocyte differentiation into mature adipocytes. In addition, knockdown of BLT1 and BLT2 by siRNAs dramatically inhibited differentiation. These results indicate the LTB4-BLT signaling pathway may positively regulate preadipocyte differentiation and be a rate-limiting system to control adipocyte differentiation. CONCLUSIONS: The LTB4-BLT signaling pathway provides a potent regulatory signal that accelerates the differentiation of mouse 3T3-L1 preadipocytes. Further investigations are necessary to confirm the exact role of LTB4 and BLTs signaling pathways in preadipocyte differentiation.

Role of leukotriene B4 receptor signaling in human preadipocyte differentiation.

We investigated the role of leukotriene B(4) (LTB(4))-leukotriene receptor (BLT) signaling in preadipocyte differentiation into mature adipocytes. Blockade of BLT signaling by treatment with lipoxygenase inhibitors, a BLT antagonist, and small interfering RNAs for BLTs in human and mouse preadipocytes isolated from adipose tissues showed acceleration of differentiation into mature adipocytes. DNA microarray analysis revealed regulation of transforming growth factor, beta-induced 68 kDa (TGFBI) expression through the BLT signaling pathway during adipocyte differentiation. Knockdown of TGFBI also showed acceleration of preadipocyte differentiation. The LTB(4)-BLT signaling pathway may negatively regulate preadipocyte differentiation via induction of TGFBI expression as a rate-limiting system to control adipocyte differentiation.

Involvement of a periodontal pathogen, Porphyromonas gingivalis on the pathogenesis of non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome that is closely associated with multiple factors such as obesity, hyperlipidemia and type 2 diabetes mellitus. However, other risk factors for the development of NAFLD are unclear. With the association between periodontal disease and the development of systemic diseases receiving increasing attention recently, we conducted this study to investigate the relationship between NAFLD and infection with Porphyromonas gingivalis (P. gingivalis), a major causative agent of periodontitis. METHODS: The detection frequencies of periodontal bacteria in oral samples collected from 150 biopsy-proven NAFLD patients (102 with non-alcoholic steatohepatitis (NASH) and 48 with non-alcoholic fatty liver (NAFL) patients) and 60 non-NAFLD control subjects were determined. Detection of P. gingivalis and other periodontopathic bacteria were detected by PCR assay. In addition, effect of P. gingivalis-infection on mouse NAFLD model was investigated. To clarify the exact contribution of P. gingivalis-induced periodontitis, non-surgical periodontal treatments were also undertaken for 3 months in 10 NAFLD patients with periodontitis. RESULTS: The detection frequency of P. gingivalis in NAFLD patients was significantly higher than that in the non-NAFLD control subjects (46.7% vs. 21.7%, odds ratio: 3.16). In addition, the detection frequency of P. gingivalis in NASH patients was markedly higher than that in the non-NAFLD subjects (52.0%, odds ratio: 3.91). Most of the P. gingivalis fimbria detected in the NAFLD patients was of invasive genotypes, especially type II (50.0%). Infection of type II P. gingivalis on NAFLD model of mice accelerated the NAFLD progression. The non-surgical periodontal treatments on NAFLD patients carried out for 3 months ameliorated the liver function parameters, such as the serum levels of AST and ALT. CONCLUSIONS: Infection with high-virulence P. gingivalis might be an additional risk factor for the development/progression of NAFLD/NASH.

Critical role of aquaporin 3 on growth of human esophageal and oral squamous cell carcinoma.

Aquaporins (AQP) play important roles in water and glycerol transport. We examined whether AQP3 is expressed in primary squamous cell carcinoma (SCC) such as esophageal and oral cancer and lymph node metastasis, and whether AQP3 is a potential target for tumor therapy. A high level expression of AQP3 was observed in tumor areas of human primary SCC such as esophageal and lingual cancers, and lymph node metastasis, but was not observed in normal areas. Treatment with pan-AQP inhibitor caused apoptotic cell death on the SCC cell lines in a concentration-dependent manner. Small interfering RNA (siRNA) specific for AQP3 also inhibited cell adhesion and growth of SCC, but not those of adenocarcinoma cell lines and fibroblasts. Expression of integrin α5 and ß1, counter adhesion molecules for fibronectin, was inhibited by treatment with AQP3-siRNA. The phosphorylation of focal adhesion kinase (FAK) was decreased by treatment with AQP3-siRNA, which then caused decreases in phosphorylation of Erk and MAPK. These results indicate that the decreases in integrins and the inhibition of cell adhesion might cause inhibition of the FAK signaling pathways. Combination of AQP3-siRNA with cisplatin, a major anti-cancer drug, strongly inhibited the growth of SCC. Cell death caused by the inhibition of AQP3 was a result of direct interference with cell adhesion involving intracellular FAK-MAPK signaling pathways. These results imply a potentially important and novel role for the inhibition of AQP3 function via the use of specific siRNA in the treatment of SCC.

RPAP3 enhances cytotoxicity of doxorubicin by impairing NF-kappa B pathway.

Activation of anti-apoptotic gene transcription by NF-κB (nuclear factor-kappa B) has been reported to be linked with a resistance of cancer cells against chemotherapy. NEMO (NF-κB essential modulator) interacts with a number of proteins and modulates the activity of NF-κB pathway. In this study, we revealed that RPAP3 (RNA polymerase II-associated protein 3) possesses an activity to bind with NEMO and to inhibit the ubiquitination of NEMO and that RPAP3 enhances doxorubicin-induced cell death in breast cancer cell line T-47D through the marked impairment of NF-κB pathway. These results indicate that RPAP3 may be a novel modulator of NF-κB pathway in apoptosis induced by anti-cancer chemotherapeutic agents.

Methamphetamine induces endoplasmic reticulum stress related gene CHOP/Gadd153/ddit3 in dopaminergic cells.

We examined the toxicity of methamphetamine and dopamine in CATH.a cells, which were derived from mouse dopamine-producing neural cells in the central nervous system. Use of the quantitative real-time polymerase chain reaction revealed that transcripts of the endoplasmic reticulum stress related gene (CHOP/Gadd153/ddit3) were considerably induced at 24-48 h after methamphetamine administration (but only under apoptotic conditions), whereas dopamine slightly induced CHOP/Gadd153/ddit3 transcripts at an early stage. We also found that dopamine and methamphetamine weakly induced transcripts for the glucose-regulated protein 78 gene (Grp78/Bip) at the early stage. Analysis by immunofluorescence microscopy demonstrated an increase of　CHOP/Gadd153/ddit3 and Grp78/Bip proteins at 24 h after methamphetamine administration. Treatment of CATH.a cells with methamphetamine caused a re-distribution of dopamine inside the cells, which mimicked the presynaptic activity of neurons with cell bodies located in the ventral tegmental area or the substantia nigra. Thus, we have demonstrated the existence of endoplasmic reticulum stress in a model of presynaptic dopaminergic neurons for the first time. Together with the recent evidence suggesting the importance of presynaptic toxicity, our findings provide new insights into the mechanisms of dopamine toxicity, which might represent one of the most important mechanisms of methamphetamine toxicity and addiction.

PIH1D1, a subunit of R2TP complex, inhibits doxorubicin-induced apoptosis.

We have previously reported that the two components of R2TP complex, RNA polymerase II-associated protein 3 (RPAP3), and Reptin, regulate apoptosis. Here we characterize another component of the complex, PIH1 domain containing protein 1 (PIH1D1). PIH1D1 interacts with both RPAP3 and Monad in HEK293 or U2OS cells. PIH1D1 transcripts were abundant in lung, leukocyte, and placenta. The reduction in endogenous PIH1D1 by siRNA enhanced apoptosis and caspase-3 activation induced by doxorubicin in U2OS cells. These results suggest that PIH1D1 may also function as a novel modulator of apoptosis pathway.

Roles of oral bacteria in cardiovascular diseases--from molecular mechanisms to clinical cases: Involvement of Porphyromonas gingivalis in the development of human aortic aneurysm.

Accumulating evidence suggests the involvement of Porphyromonas gingivalis (P. gingivalis), a periodontal pathogen, in cardiovascular diseases. Clinical specimens of aneurysmal tissue and dental plaque collected from patients infected with or without P. gingivalis were analyzed. The number of aneurysms in the distal aorta in the P. gingivalis-infected group was significantly higher than that in the non-infected group. Cellular accumulation of adipocytes in aneurysms was less frequently identified in the infected group. The expression of embryonic myosin heavy chain isoform, a phenotypic marker for proliferative smooth muscle cells, was higher in the P. gingivalis-infected group than the non-infected group. Clinical and histopathological features of aortic aneurysms associated with P. gingivalis infection are different from those present in non-infected patients. The major characteristic of P. gingivalis infection associated with aneurysms is smooth muscle cell proliferation in the distal aorta.

[Anti-inflammatory effect of PPARgamma agonists: basics and clinical applications].

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays critical roles on insulin sensitivity and adipocyte differentiation, and therefore, several agonists such as pioglitazone and rosiglitazone are used as anti-diabetic drugs. In addition to the original use, clinical applications for the therapy of several specific inflammatory diseases such as inflammatory bowel disease and rheumatoid arthritis are expected, because many reports indicated the anti-inflammatory effects of PPARgamma agonists on various animal disease models. In fact, several drugs and compounds are used or under clinical trials for the therapy of rheumatoid arthritis, ulcerative colitis, and other inflammation-related diseases. Further clinical applications for the therapy of intractable or chronic inflammatory diseases will be progressed.

RPAP3 interacts with Reptin to regulate UV-induced phosphorylation of H2AX and DNA damage.

We have previously reported that Monad, a novel WD40 repeat protein, potentiates apoptosis induced by tumor necrosis factor-alpha and cycloheximide. By affinity purification and mass spectrometry, RNA polymerase II-associated protein 3 (RPAP3) was identified as a Monad binding protein and may function with Monad as a novel modulator of apoptosis pathways. Here we report that Reptin, a highly conserved AAA + ATPase that is part of various chromatin-remodeling complexes, is also involved in the association of RPAP3 by immunoprecipitation and confocal microscopic analysis. Overexpression of RPAP3 induced HEK293 cells to death after UV-irradiation. Loss of RPAP3 by RNAi improved HeLa cell survival after UV-induced DNA damage and attenuated the phosphorylation of H2AX. Depletion of Reptin reduced cell survival and facilitated the phosphorylation on H2AX. These results suggest that RPAP3 modulates UV-induced DNA damage by regulating H2AX phosphorylation.

Molecular cloning of novel Monad binding protein containing tetratricopeptide repeat domains.

We have previously reported that Monad, a novel WD40 repeat protein, potentiates apoptosis induced by tumor necrosis factor-alpha(TNF-alpha) and cycloheximide (CHX). By affinity purification and mass spectrometry, we identified RNA polymerase II-associated protein 3 (RPAP3) as a binding protein of Monad. Overexpression of RPAP3 in HEK 293 potentiated caspase-3 activation and apoptosis induced by TNF-alpha and CHX. In addition, knockdown of RPAP3 by RNA interference resulted in a significant reduction of apoptosis induced by TNF-alpha and CHX in HEK293 and HeLa cells. These results raise the possibility that RPAP3, together with Monad, may function as a novel modulator of apoptosis pathway.

PICK1 binds to calcineurin B and modulates the NFAT activity in PC12 cells.

In the central nervous system, calcineurin has been implicated in a number of Ca2+-sensitive pathways, including the regulation of neurotransmitter release and modulation of synaptic plasticity. PDZ domain-containing proteins also play an important role in the targeting and clustering of synaptic proteins. Using a yeast two-hybrid screen, we herein identified the PDZ domain-containing protein PICK1 as a specific interactor of calcineurin B. The interaction of calcineurin B and PICK1 was confirmed by GST pull-down assay in HEK293 cells and immunoprecipitation using rat brain lysate. Calcineurin B contains the consensus C-terminal peptide sequence required for interacting with the PDZ domain. The deletion of this sequence was sufficient to abolish the interaction between calcineurin B and PICK1. In addition, the knockdown of PICK1 by RNA interference inhibited the calcineurin-dependent activation of NFAT in PC12 cells. These results suggest that PICK1 may be a positive regulator of calcineurin in the central nervous system.

Leukotriene B4 and lipoxin A4 are regulatory signals for neural stem cell proliferation and differentiation.

Leukotrienes (LTs) and lipoxins (LXs) are lipid mediators that play a key role in regulating acute inflammatory responses. Their roles in neural stem cell (NSC) functions are of interest. We showed here that LTB(4) and LXA(4) regulated proliferation and differentiation of murine NSCs that were isolated from embryo brains. Proliferation of NSCs was stimulated by LTB(4) (3 to 100 nM) and blocked by receptor antagonist (IC(50)=2.7 microM). In contrast, LXA(4), and its aspirin-triggered-15-epi-LXA(4) stable analog attenuated growth of NSCs at as little as 1 nM. Both lipoxygenase (LOX) inhibitors and LTB(4) receptor antagonists caused apoptosis and cell death. Gene chip analysis revealed that growth-related gene expressions such as epidermal growth factor (EGF) receptor, cyclin E, p27, and caspase 8 were tightly regulated by LTB(4); LXA(4) gave the opposite gene expressions. In addition to proliferation, LTB(4) induced differentiation of NSCs into neurons as monitored by neurite outgrowth and MAP2 expression. These results indicate for the first time that LTB(4) and LXA(4) directly regulate proliferation and differentiation of NSCs, suggesting these new pathways may be useful in restoring stem cells.

Critical role of peroxisome proliferator-activated receptor gamma on anoikis and invasion of squamous cell carcinoma.

PURPOSE: Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a important role in various physiological functions. We examined whether PPARgamma is expressed in primary squamous cell carcinoma and lymph node metastasis and whether PPARgamma is a potential target for tumor therapy. EXPERIMENTAL DESIGN AND RESULTS: A high-level expression of PPARgamma was observed in tumor cells of human primary squamous cell carcinoma, lymph node metastasis, and squamous cell carcinoma cell lines. Treatment with PPARgamma-specific antagonists, but not agonists, caused apoptotic cell death on squamous cell carcinoma cell lines in a concentration-dependent manner. Small interfering RNA for PPARgamma also inhibited cell adhesion and growth of squamous cell carcinomas. The phosphorylation of focal adhesion kinase (FAK) was decreased by treatment with PPARgamma antagonists, and resulted in decreases in phosphorylation of Erk and mitogen-activated protein kinase. Furthermore, PPARgamma antagonists decreased the adhesion of squamous cell carcinomas into fibronectin-coated plates, indicating the inhibition of interaction between squamous cell carcinomas and fibronectin. Expression of integrin alpha5, a counter adhesion molecule for fibronectin, was inhibited by the treatment with PPARgamma antagonists. These results indicate that the decrease in integrin alpha5 and following inhibition of cell adhesion may cause the inhibition of FAK signaling pathways. PPARgamma antagonists also strongly inhibited invasion of squamous cell carcinoma via down-regulation of CD151 expression. CONCLUSIONS: The cell death caused by the PPARgamma antagonists was a result of direct interference with cell adhesion "anoikis" involving intracellular FAK signaling pathways. These results imply a potentially important and novel role for the inhibition of PPARgamma function via the use of specific antagonists in the treatment of squamous cell carcinoma and the prevention of tumor invasion and metastasis.

Nitration of PPARgamma inhibits ligand-dependent translocation into the nucleus in a macrophage-like cell line, RAW 264.

Nitration of tyrosine residues in proteins has been observed in many inflammatory tissues of arthritis, ulcerative colitis, septic shock and ischemia-reperfusion injury. Although several studies have been carried out, it is still unclear what type of protein is nitrated and whether tyrosine nitration interferes with protein function. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor whose activation is linked to several physiological pathways including regulation of insulin sensitivity and control of inflammation. PPARgamma possesses several tyrosine residues, which might be potential targets for nitration by peroxynitrite during inflammatory responses. Here we have investigated whether PPARgamma is nitrated in macrophage-like RAW 264 cells and the effect of nitration on the translocation of PPARgamma into the nucleus. Western blot analysis showed that tumor necrosis factor-alpha, lipopolysaccharide or peroxynitrite treatment significantly increases the nitration of PPARgamma. Cell fractionation analysis and immunofluorescence coupled with confocal laser microscopy revealed that nitration of PPARgamma inhibits its ligand-dependent translocation from the cytosol into the nucleus. Together, these results indicate that nitration of PPARgamma during inflammation may be involved in a reduction in the control of inflammatory responses and also in the development of resistance to PPARgamma ligand-based therapies against inflammation.

RNA interfering approach for clarifying the PPARgamma pathway using lentiviral vector expressing short hairpin RNA.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a central role in adipocyte differentiation and insulin sensitivity. Although PPARgamma also appears to regulate diverse cellular processes in other cell types such as lymphocytes, the detailed mechanisms remain unclear. In this study, we established a lentivirus-mediated short hairpin RNA expression system and identified a potent short hairpin RNA which suppresses PPARgamma expression, resulting in marked inhibition of preadipocyte-to-adipocyte differentiation in 3T3-L1 cells. Our PPARgamma-knockdown method will serve to clarify the PPARgamma pathway in various cell types in vivo and in vitro, and will facilitate the development of therapeutic applications for a variety of diseases.

Diclofenac inhibits proliferation and differentiation of neural stem cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in clinical situations as anti-inflammatory, analgesic and antipyretic drugs. However, it is still unknown whether NSAIDs have effects on the development of the central nervous system. In the present study, we investigated the effects of NSAIDs on neural stem cell (NSC) proliferation and differentiation into neurons. In contrast to aspirin, naproxen, indomethacin and ibuprofen, treatment with diclofenac (10 microM) for 2 days induced the death of NSCs in a concentration-dependent manner. Diclofenac also inhibited the proliferation of NSCs and their differentiation into neurons. Treatment with diclofenac resulted in nuclear condensation (a morphological change due to apoptosis of NSCs) 24hr after the treatment and activated caspase-3 after 6 hr, indicating that diclofenac may cause apoptosis of neuronal cells via activation of the caspase cascade. These results suggest that diclofenac may affect the development of the central nervous system.

Formation of high molecular weight caspase-3 complex in neonatal rat brain.

Caspase-3 plays an essential role in normal brain development. Recently, a large protein complex known as apoptosome, which catalyzes the activation of caspase-3, has been reported. To investigate structural characteristics of caspase-3 in the developing brain, rat neonatal cortex extract was analysed by gel filtration chromatography. We show here the formation of high molecular complex including procaspase-3 in the extract. When the extract was activated by cytochrome c, caspase-3 recruitment to the apoptosome was not observed, although apoptotic protease activating factor-1 (Apaf-1), caspase-9, and X-linked inhibitor of apoptosis protein (XIAP) existed in the apoptosome. These results indicate that procaspase-3 exists as a high molecular weight complex during brain development.