Expression of fatty acid binding protein 4 is involved in the cell growth of oral squamous cell carcinoma.

Fatty acid binding proteins (FABPs) are a family of small and highly conserved lipid chaperone molecules with highly varied functions. Among them, fatty acid binding protein 4 (FABP4, also known as aP2) is highly expressed by adipocytes, macrophages and dendritic cells. Although the role of FABP4 in cancer is still unclear, it has been reported to be highly expressed by human tumors such as ovarian and bladder cancers. In the present study, we investigated the expression and role of FABP4 in oral squamous cell carcinoma (SCC) and its expression in oral SCC tissues. Immunohistochemical staining revealed that FABP4 expression in the tumor tissue was much higher than that in the non-tumor area of the same specimen. In the in vitro studies, an FABP4-knockdown SCC cell line (established through FABP4-specific siRNA) showed inhibited growth, and inhibited expression and activation of mitogen-activated protein kinase (MAPK). These results indicate that expression of FABP4 plays an important role in the cell growth of oral SCC through the MAPK pathway.

PIH1D1 interacts with mTOR complex 1 and enhances ribosome RNA transcription.

PIH1D1 is the defining component of the R2TP complex. Recently, R2TP has been reported to stabilize mTOR (mammalian target of rapamycin), an important regulator of cell growth and protein synthesis. Two complexes of mTOR, mTORC1 and mTORC2, have been identified. We demonstrate that immunoprecipitation (IP) of PIH1D1 results in the co-IP of Raptor (mTORC1 specific), but not Rictor (mTORC2 specific), and that knockdown of PIH1D1 decreases mTORC1 assembly, S6 kinase phosphorylation (indicator of mTORC1 activity), and rRNA transcription without affecting mTORC2 in human breast cancer MCF-7 cells. In addition, we provide evidence that PIH1D1 is overexpressed in various breast cancer cell lines. These findings collectively suggest that PIH1D1 may have an important role in mTORC1 regulation in breast cancers.

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.

Exosome-bound WD repeat protein Monad inhibits breast cancer cell invasion by degrading amphiregulin mRNA.

Increased stabilization of mRNA coding for key cancer genes can contribute to invasiveness. This is achieved by down-regulation of exosome cofactors, which bind to 3'-UTR in cancer-related genes. Here, we identified amphiregulin, an EGFR ligand, as a target of WD repeat protein Monad, a component of R2TP/prefoldin-like complex, in MDA-MB-231 breast cancer cells. Monad specifically interacted with both the 3'-UTR of amphiregulin mRNA and the RNA degrading exosome, and enhanced decay of amphiregulin transcripts. Knockdown of Monad increased invasion and this effect was abolished with anti-amphiregulin neutralizing antibody. These results suggest that Monad could prevent amphiregulin-mediated invasion by degrading amphiregulin mRNA.

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.

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.

Differential expression of aquaporin 5 and aquaporin 3 in squamous cell carcinoma and adenoid cystic carcinoma.

Aquaporins (AQPs) are a membrane protein family involved in the selective transport of water across cell membranes. Recent studies have reported the expression of AQP5 in several tumor types such as gastric, pulmonary, ovarian, pancreatic and colorectal cancer. We have previously reported the expression on tumor cells and the important role of AQP3 on cell growth in tongue cancer. However, little is known about the expression and precise role of AQP5 on squamous cell carcinoma (SCC) of the tongue. We investigated the expression of AQP5 and AQP3 in human oral SCC and adenoid cystic carcinoma (ACC). Overexpression of both AQP5 and AQP3 were immunohistochemically observed on tumor cells in SCC, whereas ACC cells were faintly stained with those antibodies against AQPs. Treatment with pan-AQP inhibitor or specific AQP5-siRNA showed inhibition of cell growth in SCC cell lines via the inhibition of integrins and the mitogen-activated protein kinase pathway. AQPs play important roles in cell growth in SCC rather than ACC.

Infection of specific strains of Streptococcus mutans, oral bacteria, confers a risk of ulcerative colitis.

Although oral bacteria-associated systemic diseases have been reported, association between Streptococcus mutans, pathogen of dental caries, and ulcerative colitis (UC) has not been reported. We investigated the effect of various S. mutans strains on dextran sodium sulfate (DSS)-induced mouse colitis. Administration of TW295, the specific strain of S. mutans, caused aggravation of colitis; the standard strain, MT8148 did not. Localization of TW295 in hepatocytes in liver was observed. Increased expression of interferon-γ in liver was also noted, indicating that the liver is target organ for the specific strain of S. mutans-mediated aggravation of colitis. The detection frequency of the specific strains in UC patients was significantly higher than in healthy subjects. Administration of the specific strains of S. mutans isolated from patients caused aggravation of colitis. Infection with highly-virulent specific types of S. mutans might be a potential risk factor in the aggravation of UC.

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.

Role of endothelin receptor signalling in squamous cell carcinoma.

Endothelin plays important roles in various physiological functions including vascular constriction. Recent studies reported that the endothelin receptors ETA and ETB are highly expressed in lung and skin tumor tissues. In contrast, there are few reports on endothelin signalling in the proliferation of head and neck cancer. We found that both ETA and ETB endothelin receptors were overexpressed in tumor cells of tongue cancer samples by immunohistochemistry. ETA and ETB were expressed in cultured lingual and esophageal squamous cell carcinoma (SCCs) cell lines. When both cultured cell lines were treated with an ETA selective antagonist (BQ123) or an ETB selective antagonist (BQ788), inhibition of cell growth was observed. Similar results were observed when SCCs were treated with specific siRNA for the suppression of ETA or ETB. Furthermore, inhibition of the mitogen-activated protein (MAP) kinase pathway by the treatments with ET receptor antagonists and siRNA was also observed. These results indicate that endothelin signalling may, in part, play important roles in cell growth in SCCs through the MAP kinase pathway.

The collagen-binding protein of Streptococcus mutans is involved in haemorrhagic stroke.

Although several risk factors for stroke have been identified, one-third remain unexplained. Here we show that infection with Streptococcus mutans expressing collagen-binding protein (CBP) is a potential risk factor for haemorrhagic stroke. Infection with serotype k S. mutans, but not a standard strain, aggravates cerebral haemorrhage in mice. Serotype k S. mutans accumulates in the damaged, but not the contralateral hemisphere, indicating an interaction of bacteria with injured blood vessels. The most important factor for high-virulence is expression of CBP, which is a common property of most serotype k strains. The detection frequency of CBP-expressing S. mutans in haemorrhagic stroke patients is significantly higher than in control subjects. Strains isolated from haemorrhagic stroke patients aggravate haemorrhage in a mouse model, indicating that they are haemorrhagic stroke-associated. Administration of recombinant CBP causes aggravation of haemorrhage. Our data suggest that CBP of S. mutans is directly involved in haemorrhagic stroke.

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.

The small molecule harmine regulates NFATc1 and Id2 expression in osteoclast progenitor cells.

Small molecule compounds that potently affect osteoclastogenesis could be useful as chemical probes for elucidating the mechanisms of various biological phenomena and as effective therapeutic strategies against bone resorption. An osteoclast progenitor cell-based high-throughput screening system was designed to target activation of NFAT, which is a key event for osteoclastogenesis. Orphan ligand library screening using this system identified the ß-carboline derivative harmine, which is a highly potent inhibitor of dual-specificity tyrosine-phosphorylation regulated kinase 1A (DYRK1A), to be an NFAT regulator in osteoclasts. RAW264.7 cells highly expressed DYRK1A protein, and in vitro phosphorylation assay demonstrated that harmine directly inhibited the DYRK1A-mediated phosphorylation (in-activation) of NFATc1. Harmine promoted the dephosphorylation (activation) of NFATc1 in RAW264.7 cells within 24h, and it significantly increased the expression of NFATc1 in RAW264.7 cells and mouse primary bone marrow macrophages (BMMs) both in the presence and absence of RANKL stimulation. Although harmine promoted NFATc1 expression and stimulated target genes for osteoclastogenesis, cell-cell fusion and the formation of TRAP-positive multinucleated osteoclasts from RAW264.7 cells and BMMs was significantly inhibited by harmine treatment. Meanwhile, harmine remarkably promoted the expression of inhibitor of DNA binding/differentiation-2 (Id2), which is a negative regulator for osteoclastogenesis, in RAW264.7 cells and BMMs. An Id2-null-mutant showed slightly increased osteoclast formation from BMMs, and the harmine-mediated inhibition of osteoclast formation was abolished in the BMMs of Id2-null-mutant mice. These results suggest that harmine is a potent activator of NFATc1 that interferes with the function of DYRK1A in osteoclast precursors and also up-regulates Id2 protein, which may dominantly inhibit expression pathways associated with cell-cell fusion, thereby leading to the disruption of the fusion events mediating osteoclastogenesis. The small molecule harmine is therefore expected to provide an experimental tool for investigating signaling cascades in osteoclastogenesis, especially those centered on DYRK1A-mediated NFATc1 and Id2 regulation.

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.

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.

cis9, trans11-Conjugated Linoleic Acid Differentiates Mouse 3T3-L1 Preadipocytes into Mature Small Adipocytes through Induction of Peroxisome Proliferator-activated Receptor γ.

Dietary conjugated linoleic acid (CLA) has been reported to exhibit a number of therapeutic effects in animal models and patients, such as anti-hypertensive, anti-hyperlipidemic, anti-arteriosclerotic, anti-carcinogenic, and anti-diabetic effects. However, the underlying mechanism is not well-characterized. In the present study, the effects of cis(c)9, trans(t)11-CLA on the differentiation of mouse 3T3-L1 preadipocytes into mature adipocytes were examined. Treatment with c9, t11-CLA in the presence of insulin, dexamethasone, and 3-isobutyl-1-methyl-xanthine (differentiation cocktail) significantly stimulated the accumulation of triacylglycerol. The microscopic observation of cells stained by Oil Red O demonstrated that c9, t11-CLA increases the amount and proportion of small mature adipocytes secreting adiponectin, a benign adipocytokine, when compared to the differentiation cocktail alone. Furthermore, c9, t11-CLA increased bioactive peroxisome proliferator-activated receptor γ (PPARγ) levels in a nuclear extract of 3T3-L1 cells, suggesting the enhancing effect of this fatty acid on the nuclear transmission of PPARγ, a master regulator of adipocyte differentiation, in 3T3-L1 cells. These results suggest that the therapeutic effects of c9, t11-CLA on lifestyle-related diseases are partially due to the enhanced formation of small adipocytes from preadipocytes via PPARγ stimulation.

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.