Hirofumi Jono

Vinpocetine inhibits NF-κB–dependent inflammation via an IKK-dependent but PDE-independent mechanism

Inflammation is a hallmark of many diseases, such as atherosclerosis, chronic obstructive pulmonary disease, arthritis, infectious diseases, and cancer. Although steroids and cyclooxygenase inhibitors are effective antiinflammatory therapeutical agents, they may cause serious side effects. Therefore, developing unique antiinflammatory agents without significant adverse effects is urgently needed. Vinpocetine, a derivative of the alkaloid vincamine, has long been used for cerebrovascular disorders and cognitive impairment. Its role in inhibiting inflammation, however, remains unexplored. Here, we show that vinpocetine acts as an antiinflammatory agent in vitro and in vivo. In particular, vinpocetine inhibits TNF-α–induced NF-κB activation and the subsequent induction of proinflammatory mediators in multiple cell types, including vascular smooth muscle cells, endothelial cells, macrophages, and epithelial cells. We also show that vinpocetine inhibits monocyte adhesion and chemotaxis, which are critical processes during inflammation. Moreover, vinpocetine potently inhibits TNF-α- or LPS-induced up-regulation of proinflammatory mediators, including TNF-α, IL-1β, and macrophage inflammatory protein-2, and decreases interstitial infiltration of polymorphonuclear leukocytes in a mouse model of TNF-α- or LPS-induced lung inflammation. Interestingly, vinpocetine inhibits NF-κB–dependent inflammatory responses by directly targeting IKK, independent of its well-known inhibitory effects on phosphodiesterase and Ca2+ regulation. These studies thus identify vinpocetine as a unique antiinflammatory agent that may be repositioned for the treatment of many inflammatory diseases.

Humanized Anti-Interleukin-6 Receptor Antibody Suppresses Tumor Angiogenesis and In vivo Growth of Human Oral Squamous Cell Carcinoma

Purpose: The biological effect of interleukin-6 (IL-6) signaling in oral squamous cell carcinoma (OSCC) and whether IL-6 receptor (IL-6R)-mediated signaling can be a therapeutic target for OSCC are unclear. The aim of this study was to investigate the effects of inhibition of IL-6R–mediated signaling on OSCC progression and to evaluate the availability of tocilizumab, a humanized antihuman IL-6R antibody, as a therapeutic agent for OSCC. Experimental Design: We evaluated expression levels of IL-6 and IL-6R in 58 OSCC tissues and 4 OSCC cell lines by real-time quantitative reverse transcription-PCR and/or immunohistochemstry. We investigated the effects of tocilizumab on OSCC growth in vitro and in xenografts. Xenografts were analyzed by immunohistochemistry for phosphorylated signal transducer and activator of transcription 3 (pSTAT3), Ki-67, and CD31, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay was done. Results: Expression levels of IL-6 at both mRNA and protein levels in OSCC tissues were significantly higher than those in normal mucosal tissues. In addition, OSCC cell lines expressed higher levels of both IL-6 and IL-6R mRNA than did HaCaT keratinocytes. Tocilizumab significantly reduced in vivo growth of SAS cells with a drastic reduction of STAT3 phosphorylation in tumor cells in mice. Inhibition of IL-6 signaling significantly decreased vascular endothelial growth factor mRNA expression in SAS, and microvessel density and vessel diameter in SAS tumors in tocilizumab-treated mice. Conclusions: Therapeutic approaches targeting IL-6R by tocilizumab may be effective for OSCC treatment by at least inhibiting angiogenesis. (Clin Cancer Res 2009;15(17):5426–34)

TGF-β induces p65 acetylation to enhance bacteria-induced NF-κB activation

Transforming growth factor‐β (TGF‐β) family members are multifunctional growth factors involved in regulating diverse biological processes. Despite the critical role for TGF‐β in regulating cell proliferation, differentiation, migration and development, its role in regulating NF‐κB‐dependent inflammatory response still remains unclear. Here, we show that TGF‐β1 induces acetylation of NF‐κB p65 subunit to synergistically enhance bacterium nontypeable Haemophilus influenzae‐induced NF‐κB activation and inflammatory response in vitro and in vivo. The TGF‐β1‐induced acetylation of p65 is mediated via a Smad3/4‐PKA‐p300‐dependent signaling pathway. Acetylation of p65 at lysine 221 by TGF‐β1 is critical for synergistic enhancement of bacteria‐induced DNA‐binding activity, NF‐κB activation, NF‐κB‐dependent transcription of TNF‐α and IL‐1β and interstitial polymorphonuclear neutrophil infiltration in vitro and in vivo. These studies provide new insights into the novel regulation of NF‐κB by TGF‐β signaling.

CYLD negatively regulates transforming growth factor-β-signalling via deubiquitinating Akt.

Lung injury, whether induced by infection or caustic chemicals, initiates a series of complex wound-healing responses. If uncontrolled, these responses may lead to fibrotic lung diseases and loss of function. Thus, resolution of lung injury must be tightly regulated. The key regulatory proteins required for tightly controlling the resolution of lung injury have yet to be identified. Here we show that loss of deubiquitinase CYLD led to the development of lung fibrosis in mice after infection with Streptococcus pneumoniae. CYLD inhibited transforming growth factor-β-signalling and prevented lung fibrosis by decreasing the stability of Smad3 in an E3 ligase carboxy terminus of Hsc70-interacting protein-dependent manner. Moreover, CYLD decreases Smad3 stability by deubiquitinating K63-polyubiquitinated Akt. Together, our results unveil a role for CYLD in tightly regulating the resolution of lung injury and preventing fibrosis by deubiquitinating Akt. These studies may help develop new therapeutic strategies for preventing lung fibrosis.

In Vitro and In Vivo gene delivery mediated by Lactosylated Dendrimer/α-Cyclodextrin Conjugates (G2) into Hepatocytes

The purpose of this study is to evaluate in vitro and in vivo gene delivery efficiency of polyamidoamine (PAMAM) starburst dendrimer (generation 2, G2) conjugates with alpha-cyclodextrin (alpha-CDE (G2)) bearing lactose (Lac-alpha-CDE) with various degrees of substitution of the lactose moiety (DSL) as a novel hepatocyte-selective carrier in hepatocytes. Lac-alpha-CDE (DSL 2.6) was found to have much higher gene transfer activity than dendrimer, alpha-CDE, Lac-alpha-CDE (DSL 1.2, 4.6, 6.2 and 10.2) and lactosylated dendrimer (Lac-dendrimer, DSL 2.4) in HepG2 cells, which are dependent on the expression of cell-surface asialoglycoprotein receptor (ASGP-R), reflecting the cellular association of the plasmid DNA (pDNA) complexes. The physicochemical properties of pDNA complex with Lac-alpha-CDE (DSL 2.6) were almost comparable to that with alpha-CDE. Lac-alpha-CDE (DSL 2.6) provided negligible cytotoxicity up to a charge ratio of 150 in HepG2 cells. Lac-alpha-CDE (DSL 2.6) provided gene transfer activity higher than jetPEI-Hepatocyte to hepatocytes with much less changes of blood chemistry values 12h after intravenous administration in mice. These results suggest the potential use of Lac-alpha-CDE (DSL 2.6) as a non-viral vector for gene delivery toward hepatocytes.

SELDI-TOF Mass Spectrometry Evaluation of Variant Transthyretins for Diagnosis and Pathogenesis of Familial Amyloidotic Polyneuropathy

BACKGROUND Mass spectrometric analyses are valuable for detection of transthyretin (TTR) variants, which cause familial amyloidotic polyneuropathy (FAP). However, those methods require an immunoprecipitation step with an anti-TTR antibody and are not suitable for quantitative detection. We investigated the usefulness of SELDI-TOF mass spectrometry (MS) without an immunoprecipitation step. METHODS We used ProteinChips with chromatographic capture formats to detect TTRs. We attempted to correlate the intensity of mixed samples of amyloidogenic TTR (ATTR) V30M to wild-type (WT) TTR. We analyzed the proportion of ATTR V30M in amyloid-laden cardiac tissues from FAP patients, and also evaluated samples from FAP patients with 16 other TTR mutations. RESULTS Detection of ATTR required only 3 h of SELDI-TOF MS analysis. We determined that SELDI-TOF MS was suitable for quantitative detection of ATTR V30M and demonstrated that the proportion of ATTR V30M to WT TTR was 46.6% in amyloid-laden cardiac tissue from an FAP patient who died 10 years after liver transplantation. With this method, we identified 12 of 17 TTR variants. Small mass shifts and low concentrations of variants prevented ATTR detection. By changing the analytical conditions, we achieved detection of low concentrations of ATTR Y114C in serum. CONCLUSIONS SELDI-TOF MS is a reliable tool for quantitative evaluation of TTR variants, in both tissue amyloid deposits and body fluids. This method is useful for the diagnosis and investigation of the pathogenesis of FAP.

Tumor Suppressor Cylindromatosis Acts as a Negative Regulator for Streptococcus pneumoniae-induced NFAT Signaling

Gram-positive bacterium Streptococcus pneumoniae is an important human pathogen that colonizes the upper respiratory tract and is also the major cause of morbidity and mortality worldwide. S. pneumoniae causes invasive diseases such as pneumonia, meningitis, and otitis media. Despite the importance of pneumococcal diseases, little is known about the molecular mechanisms by which S. pneumoniae-induced inflammation is regulated, especially the negative regulatory mechanisms. Here we show that S. pneumoniae activates nuclear factor of activated T cells (NFAT) signaling pathway and the subsequent up-regulation of inflammatory mediators via a key pneumococcal virulence factor, pneumolysin. We also demonstrate that S. pneumoniae activates NFAT transcription factor independently of Toll-like receptors 2 and 4. Moreover, S. pneumoniae induces NFAT activation via both Ca2+-calcineurin and transforming growth factor-β-activated kinase 1 (TAK1)-mitogen-activated protein kinase kinase (MKK) 3/6-p38α/β-dependent signaling pathways. Interestingly, we found for the first time that tumor suppressor cylindromatosis (CYLD) acts as a negative regulator for S. pneumoniae-induced NFAT signaling pathway via a deubiquitination-dependent mechanism. Finally, we showed that CYLD interacts with and deubiquitinates TAK1 to negatively regulate the activation of the downstream MKK3/6-p38α/β pathway. Our studies thus bring new insights into the molecular pathogenesis of S. pneumoniae infections through the NFAT-dependent mechanism and further identify CYLD as a negative regulator for NFAT signaling, thereby opening up new therapeutic targets for these diseases.

Midkine as a prognostic biomarker in oral squamous cell carcinoma

The aim of this study was to evaluate serum midkine (S-MK) concentrations as a prognostic tumour marker in oral squamous cell carcinoma (OSCC). We measured S-MK concentrations in patients with OSCC and healthy volunteers. In addition, we performed real-time quantitative reverse transcription–PCR analysis and immunohistochemistry with fresh tumour samples. To determine whether S-MK concentrations have prognostic value, we performed survival analyses with clinical information by using the log-rank test. Serum midkine concentrations were significantly higher in patients with OSCC than in healthy controls (P<0.001). Serum midkine concentrations were also significantly increased in early-stage OSCC compared with those of healthy individuals (P<0.001). In addition, immunohistochemistry allowed identification of overexpressed MK protein in OSCC tissues. MK mRNA showed higher expression in OSCC samples compared with normal mucosal samples. Patients in high S-MK groups showed a significantly lower 5-year survival rate compared with patients in low S-MK groups (P<0.05). The increased S-MK concentrations in early-stage OSCC were strongly associated with poor survival. Serum midkine concentrations may thus be a useful marker not only for cancer screening but also for predicting prognosis of OSCC patients.

Wild-type transthyretin-derived amyloidosis in various ligaments and tendons ☆

Transthyretin-derived amyloid deposition is commonly found in intercarpal ligaments of patients with senile systemic amyloidosis. However, the frequency of transthyretin-derived amyloid deposits in ligaments of other tissues remains to be elucidated. This study aimed to determine the frequency of amyloid deposition and the precursor proteins of amyloid found in orthopedic disorders. We studied 111 specimens from patients with carpal tunnel syndrome (flexor tenosynovium specimens), rotator cuff tears (rotator cuff tendon specimens), and lumbar canal stenosis (yellow ligament specimens). To identify amyloid precursor proteins, we used immunohistochemical staining with antibodies that react with transthyretin, immunoglobulin light chain, amyloid A protein, and β(2)-microglobulin. By means of Congo red staining, we identified 47 (42.3%) amyloid-positive samples, 39 of which contained transthyretin-derived amyloid (18 flexor tenosynovium specimens, 5 rotator cuff tendon specimens, and 16 yellow ligament specimens). Genetic testing and/or clinical findings suggested that all patients with transthyretin amyloid deposits did not have familial amyloidotic polyneuropathy. The occurrence of amyloid deposition in those tissues depended on age. These results suggest that transthyretin-derived amyloid deposits may occur more frequently in various ligaments and tendons than originally expected. In the future, such amyloid deposits may aid determination of the pathogenesis of ligament and tendon disorders in older patients.

Synergistic induction of nuclear factor-κB by transforming growth factor-β and tumour necrosis factor-α is mediated by protein kinase A-dependent RelA acetylation

The TGF-beta (transforming growth factor-beta) pathway represents an important signalling pathway involved in regulating diverse biological processes, including cell proliferation, differentiation and inflammation. Despite the critical role for TGF-beta in inflammatory responses, its role in regulating NF-kappaB (nuclear factor-kappaB)-dependent inflammatory responses still remains unknown. In the present study we show that TGF-beta1 synergizes with proinflammatory cytokine TNF-alpha (tumour necrosis factor-alpha) to induce NF-kappaB activation and the resultant inflammatory response in vitro and in vivo. TGF-beta1 synergistically enhances TNF-alpha-induced NF-kappaB DNA binding activity via induction of RelA acetylation. Moreover, synergistic enhancement of TNF-alpha-induced RelA acetylation and DNA-binding activity by TGF-beta1 is mediated by PKA (protein kinase A). Thus the present study reveals a novel role for TGF-beta in inflammatory responses and provides new insight into the regulation of NF-kappaB by TGF-beta signalling.