Novel role of Rac-Mid1 signaling in medial cerebellar development.

Rac signaling impacts a relatively large number of downstream targets; however, few studies have established an association between Rac pathways and pathological conditions. In the present study, we generated mice with double knockout of Rac1 and Rac3 (Atoh1-Cre;Rac1flox/flox;Rac3-/- ) in cerebellar granule neurons (CGNs). We observed impaired tangential migration at E16.5, as well as numerous apoptotic CGNs at the deepest layer of the external granule layer (EGL) in the medial cerebellum of Atoh1-Cre;Rac1flox/flox;Rac3-/- mice at P8. Atoh1-Cre;Rac1flox/flox;Rac3-/- CGNs differentiated normally until expression of p27kip1 and NeuN in the deep EGL at P5. Primary CGNs and cerebellar microexplants from Atoh1-Cre;Rac1flox/flox;Rac3-/- mice exhibited impaired neuritogenesis, which was more apparent in Map2-positive dendrites. Such findings suggest that impaired tangential migration and final differentiation of CGNs have resulted in decreased cerebellum size and agenesis of the medial internal granule layer, respectively. Furthermore, Rac depleted/deleted cells exhibited decreased levels of Mid1 and impaired mTORC1 signaling. Mid1 depletion in CGNs produced mild impairments in neuritogenesis and reductions in mTORC1 signaling. Thus, a novel Rac-signaling pathway (Rac1-Mid1-mTORC1) may be involved in medial cerebellar development.

Hearing vulnerability after noise exposure in a mouse model of reactive oxygen species overproduction.

Previous studies have convincingly argued that reactive oxygen species (ROS) contribute to the development of several major types of sensorineural hearing loss, such as noise-induced hearing loss (NIHL), drug-induced hearing loss, and age-related hearing loss. However, the underlying molecular mechanisms induced by ROS in these pathologies remain unclear. To resolve this issue, we established an in vivo model of ROS overproduction by generating a transgenic (TG) mouse line expressing the human NADPH oxidase 4 (NOX4, NOX4-TG mice), which is a constitutively active ROS-producing enzyme that does not require stimulation or an activator. Overproduction of ROS was detected at the cochlea of the inner ear in NOX4-TG mice, but they showed normal hearing function under baseline conditions. However, they demonstrated hearing function vulnerability, especially at high-frequency sounds, upon exposure to intense noise, which was accompanied by loss of cochlear outer hair cells (OHCs). The vulnerability to loss of hearing function and OHCs was rescued by treatment with the antioxidant Tempol. Additionally, we found increased protein levels of the heat-shock protein 47 (HSP47) in models using HEK293 cells, including H2 O2 treatment and cells with stable and transient expression of NOX4. Furthermore, the up-regulated levels of Hsp47 were observed in both the cochlea and heart of NOX4-TG mice. Thus, antioxidant therapy is a promising approach for the treatment of NIHL. Hsp47 may be an endogenous antioxidant factor, compensating for the chronic ROS overexposure in vivo, and counteracting ROS-related hearing loss.

Development of an Oropharyngeal Scope with an Integrated Tongue Depressor: NTOP2013 Study.

The oropharynx is examined with a light source such as an electric light, a penlight, or a forehead mirror based on an acquired visual field using a tongue depressor. However, it is extremely difficult to obtain objective and reproducible images of tissue within the pharynx required in recent years with these methods, and insufficient progress in the examination tools has been made. There is an increasing need to develop a method for display during oropharyngeal examination. We conducted the present study to develop a novel oropharyngeal endoscope as an objective observation method.

Maintenance of stereocilia and apical junctional complexes by Cdc42 in cochlear hair cells.

Cdc42 is a key regulator of dynamic actin organization. However, little is known about how Cdc42-dependent actin regulation influences steady-state actin structures in differentiated epithelia. We employed inner ear hair-cell-specific conditional knockout to analyze the role of Cdc42 in hair cells possessing highly elaborate stable actin protrusions (stereocilia). Hair cells of Atoh1-Cre;Cdc42(flox/flox) mice developed normally but progressively degenerated after maturation, resulting in progressive hearing loss particularly at high frequencies. Cochlear hair cell degeneration was more robust in inner hair cells than in outer hair cells, and began as stereocilia fusion and depletion, accompanied by a thinning and waving circumferential actin belt at apical junctional complexes (AJCs). Adenovirus-encoded GFP-Cdc42 expression in hair cells and fluorescence resonance energy transfer (FRET) imaging of hair cells from transgenic mice expressing a Cdc42-FRET biosensor indicated Cdc42 presence and activation at stereociliary membranes and AJCs in cochlear hair cells. Cdc42-knockdown in MDCK cells produced phenotypes similar to those of Cdc42-deleted hair cells, including abnormal microvilli and disrupted AJCs, and downregulated actin turnover represented by enhanced levels of phosphorylated cofilin. Thus, Cdc42 influenced the maintenance of stable actin structures through elaborate tuning of actin turnover, and maintained function and viability of cochlear hair cells.

Role of the retrotrapezoid nucleus/parafacial respiratory group in coughing and swallowing in guinea pigs.

The retrotrapezoid/parafacial respiratory group (RTN/pFRG) located ventral to the facial nucleus plays a key role in regulating breathing, especially enhanced expiratory activity during hypercapnic conditions. To clarify the roles of the RTN/pFRG region in evoking coughing, during which reflexive enhanced expiration is produced, and in swallowing, during which the expiratory activity is consistently halted, we recorded extracellular activity from RTN/pFRG neurons during these fictive behaviors in decerebrate, paralyzed, and artificially ventilated guinea pigs. The activity of the majority of recorded respiratory neurons was changed in synchrony with coughing and swallowing. To further evaluate the contribution of RTN/pFRG neurons to these nonrespiratory behaviors, the motor output patterns during breathing, coughing, and swallowing were compared before and after brain stem transection at the caudal margin of RTN/pFRG region. In addition, the effects of transection at its rostral margin were also investigated to evaluate pontine contribution to these behaviors. During respiration, transection at the rostral margin attenuated the postinspiratory activity of the recurrent laryngeal nerve. Meanwhile, the late expiratory activity of the abdominal nerve was abolished after caudal transection. The caudal transection also decreased the amplitude of the coughing-related abdominal nerve discharge but did not abolish the activity. Swallowing could be elicited even after the caudal end transection. These findings raise the prospect that the RTN/pFRG contributes to expiratory regulation during normal respiration, although this region is not an essential element of the neuronal networks involved in coughing and swallowing.

Computational model of a circulation current that controls electrochemical properties in the mammalian cochlea.

Sound-evoked mechanical stimuli permit endolymphatic K(+) to enter sensory hair cells. This transduction is sensitized by an endocochlear potential (EP) of +80 mV in endolymph. After depolarizing the cells, K(+) leaves hair cells in perilymph, and it is then circulated back to endolymph across the lateral cochlear wall. In theory, this process entails a continuous and unidirectional current carried by apical K(+) channels and basolateral K(+) uptake transporters in both the marginal cell and syncytial layers of the lateral wall. The transporters regulate intracellular and extracellular [K(+)], allowing the channels to form K(+) diffusion potentials across each of the two layers. These diffusion potentials govern the EP. What remains uncertain is whether these transport mechanisms accumulating across diverse cell layers make up a continuous circulation current in the lateral wall and how this current might affect the characteristics of the endolymph. To address this question, we developed an electrophysiological model that incorporates channels and transporters of the lateral wall and channels of hair cells that derive a circulation current. The simulation replicated normal experimental EP values and reproduced experimentally measured changes in the EP and intra- and extracellular [K(+)] in the lateral wall when different transporters and channels were blocked. The model predicts that, under these different conditions, the circulation current's contribution to the EP arises from different sources. Finally, our model also accurately simulated EP loss in a mouse model of a chloride channelopathy associated with deafness.

The mechanism underlying maintenance of the endocochlear potential by the K+ transport system in fibrocytes of the inner ear.

The endocochlear potential (EP) of +80 mV in the scala media, which is indispensable for audition, is controlled by K+ transport across the lateral cochlear wall. This wall includes two epithelial barriers, the syncytium and the marginal cells. The former contains multiple cell types, such as fibrocytes, which are exposed to perilymph on their basolateral surfaces. The apical surfaces of the marginal cells face endolymph. Between the two barriers lies the intrastrial space (IS), an extracellular space with a low K+ concentration ([K+]) and a potential similar to the EP. This intrastrial potential (ISP) dominates the EP and represents the sum of the diffusion potential elicited by a large K+ gradient across the apical surface of the syncytium and the syncytium's potential, which is slightly positive relative to perilymph. Although a K+ transport system in fibrocytes seems to contribute to the EP, the mechanism remains uncertain. We examined the electrochemical properties of the lateral wall of guinea pigs with electrodes sensitive to potential and K+ while perfusing into the perilymph of the scala tympani blockers of Na+,K+-ATPase, the K+ pump thought to be essential to the system. Inhibiting Na+,K+-ATPase barely affected [K+] in the IS but greatly decreased [K+] within the syncytium, reducing the K+ gradient across its apical surface. The treatment hyperpolarized the syncytium only moderately. Consequently, both the ISP and the EP declined. Fibrocytes evidently use the Na+,K+-ATPase to achieve local K+ transport, maintaining the syncytium's high [K+] that is crucial for the K+ diffusion underlying the positive ISP.

Autocrine and paracrine loops between cancer cells and macrophages promote lymph node metastasis via CCR4/CCL22 in head and neck squamous cell carcinoma.

Lymph node metastasis is a poor prognostic factor for patients with head and neck squamous cell carcinoma (HNSCC). However, its molecular mechanism has not yet been fully understood. In our study, we investigated the expression of CCR4 and its ligand CCL22 in the HNSCC tumor microenvironment and found that the CCR4/CCL22 axis was involved in lymph node metastasis of HNSCC. CCR4 was expressed in 20 of 31 (64.5%) human tongue cancer tissues, and its expression was significantly correlated with lymph node metastasis (p < 0.01) and lymphatic invasion (p < 0.05). CCR4 was expressed in three of five human HNSCC cell lines tested. CCR4(+) HNSCC cells, but not CCR4(-) cells, showed enhanced migration toward CCL22, indicating that functional CCR4 was expressed in HNSCC cell lines. CCL22 was also expressed in cancer cells (48.4% of tongue cancer tissues) or CD206(+) M2-like macrophages infiltrated in tumors and draining lymph nodes. CCL22 produced by cancer cells or CD206(high) M2-like macrophages increased the cell motility of CCR4(+) HNSCC cells in vitro in an autocrine or paracrine manner. In the mouse SCCVII in vivo model, CCR4(+) cancer cells, but not CCR4(-) cells, metastasized to lymph nodes which contained CCL22 producing M2-like macrophages. These results demonstrate that lymph node metastasis of CCR4(+) HNSCC is promoted by CCL22 in an autocrine or M2-like macrophage-dependent paracrine manner. Therefore, the CCR4/CCL22 axis may be an attractive target for the development of diagnostic and therapeutic strategies for patients with HNSCC.

Does a vascular supercharge improve the clinical outcome for free jejunal transfer?

To clarify whether a supercharged free jejunal transfer would have a different clinical outcome from the usual transfer method, we examined clinical data from cases of esophago-pharyngeal reconstruction. Fifty-three patients in whom the hypopharynx and cervical esophagus was reconstructed with a free jejunal transfer were divided into two groups: 19 normal procedures and 34 supercharged. Clinical outcomes including intraoperative and postoperative events, complications and deglutition were compared statistically. There were no significant differences between the groups in terms of the rates of free flap failure, leakage, stenosis, drinking status, dysphagia, or operating time. There were no significant advantages in clinical outcomes when using a supercharge. However, supercharged flaps with an intraoperative arterial thrombosis were all rescued and survived. Thus, a supercharge in free flap is not necessary for all cases. Its indication should be limited to cases when free flaps are not reliable because of intraoperative thrombosis and arterial insufficiency.

A critical role of IL-33 in experimental allergic rhinitis.

BACKGROUND: We reported previously that serum levels of IL-33 are significantly increased in patients with allergic rhinitis (AR). However, very little is known about the role of IL-33 for the development of AR. OBJECTIVE: We thought to develop a novel murine model of ragweed pollen-specific AR and examined the pathologic role for ragweed-induced IL-33 in the development of AR manifestation using IL-33-deficient (il33(-/-)) mice. METHODS: Ragweed-immunized and ragweed-challenged mice were examined for early- and late-phase nasal responses. IL-33 protein expression in the nasal epithelial cells of the AR murine model and patients with AR were assessed by using confocal microscopy. RESULTS: After nasal challenge with ragweed pollen, ragweed-immunized wild-type mice manifested early-phase (sneezing) and late-phase (eosinophilic and basophilic accumulation) responses. In contrast, il33(-/-) and FcεRI(-/-) mice did not have both early- and late-phase AR responses. IL-33 protein was constitutively expressed in the nucleus of nasal epithelial cells and was promptly released into nasal fluids in response to nasal exposure to ragweed pollen. In human subjects we revealed constitutive expression of IL-33 protein in the nasal epithelial cells of healthy control subjects and downregulated expression of IL-33 protein in inflamed nasal epithelial cells of patients with AR. IL-33-stimulated mast cells and basophils contributed to the early- and late-phase AR manifestation through increasing histamine release and production of chemoattractants for eosinophils/basophils, respectively. CONCLUSIONS: Ragweed pollen-driven endogenous IL-33 contributed to the development of AR responses. IL-33 might present an important therapeutic target for the prevention of AR.

K(+)-Cl(-) cotransporter 1 (KCC1) negatively regulates NGF-induced neurite outgrowth in PC12 cells.

Potassium chloride cotransporters (KCCs) mediate electroneutrally-coupled transport of K(+) and Cl(-), and play crucial roles in various cell functions including regulation of cell volume and homeostasis of cellular Cl(-)content. Four isoforms of KCCs (KCC1, 2, 3, and 4) have been identified. KCC1 is ubiquitously expressed, whereas KCC2 is mainly expressed in neuronal cells of central nervous system. KCC3 is highly expressed in heart, skeletal muscle, kidney, lung and placenta. KCC4 is mainly expressed in epithelial cells. In this study, we investigated roles of KCCs in NGF-induced neurite outgrowth of rat pheochromocytoma PC12 cells. The most abundantly expressed isoform in PC12 cells was KCC1. Inhibition of KCCs using [(dihydronindenyl)oxy] alkanoic acid (DIOA), an inhibitor of KCCs, enhanced the NGF-induced neurite outgrowth of PC12 cells in a dose-dependent manner. Treatment of PC12 cells with NGF significantly decreased mRNA expression of KCC1, whereas other isoforms, KCC2-4, showed no changes in their mRNA expression in response to NGF treatment. Knockdown of KCC1 using small interfering RNA (siRNA) enhanced the NGF-induced neurite outgrowth. These results suggest that KCC1 negatively regulates the NGF-induced neurite outgrowth of PC12 cells.

Successful retrograde arterial inflow through a muscular branch in a free anterolateral thigh chimeric flap transfer.

In this report, we present a case in which a free anterolateral thigh (ALT) flap was transferred for head and neck reconstruction after oropharyngeal cancer ablation, and a retrograde arterial inflow was used to salvage the flap when the main arterial pedicle showed usual repeated spasms. The flap was raised as a chimera flap comprising a fasciocutaneous flap and a vastus lateralis muscle flap. After reperfusion, the pedicle artery exhibited spasms repeatedly and vascular flow was unstable. Therefore, we performed arterial supercharge. In the distal portion of the muscle flap, a small arterial branch was dissected as a reverse-flow arterial pedicle. The recipient artery was also a retrograde limb of the superior thyroid artery. The flap survived; however, postoperative ultrasonographic echo evaluation revealed that the spastic descending branch of the lateral circumflex femoral artery was obstructed and that the reverse-flow muscular perforator alone nourished the whole flap. In free ALT flap transfer, a small perforator level artery was able to nourish a flap, even in a retrograde manner. Moreover, when the vasculature of the free flap is unstable, retrograde arterial supply to a small perforator can be an option to save the flap transfer.

The endocochlear potential depends on two K+ diffusion potentials and an electrical barrier in the stria vascularis of the inner ear.

An endocochlear potential (EP) of +80 mV is essential for audition. Although the regulation of K(+) concentration ([K(+)]) in various compartments of the cochlear stria vascularis seems crucial for the formation of the EP, the mechanism remains uncertain. We have used multibarreled electrodes to measure the potential, [K(+)], and input resistance in each compartment of the stria vascularis. The stria faces two fluids, perilymph and endolymph, and contains an extracelluar compartment, the intrastrial space (IS), surrounded by two epithelial layers, the marginal cell (MC) layer and that composed of intermediate and basal cells. Fluid in the IS exhibits a low [K(+)] and a positive potential, called the intrastrial potential (ISP). We found that the input resistance of the IS was high, indicating this space is electrically isolated from the neighboring extracellular fluids. This arrangement is indispensable for maintaining positive ISP. Inhibiting the K(+) transporters of the stria by anoxia, ouabain, or bumetanide caused the [K(+)] of the IS to increase and the intracellular [K(+)] of MCs to decrease, reducing both the ISP and the EP. Calculations indicate that the ISP represents the K(+) diffusion potential across the apical membranes of intermediate cells through Ba(2+)-sensitive K(+) channels. The K(+) diffusion potential across the apical membranes of MCs also contributes to the EP. Because the EP depends on two K(+) diffusion potentials and an electrical barrier in the stria vascularis, interference with any of these elements can interrupt hearing.

The prevalence and clinical features of spasmodic dysphonia: A review of epidemiological surveys conducted in Japan.

OBJECTIVES: Spasmodic dysphonia (SD) is a rare disease and its epidemiological status is unclear. This review aimed to explore the current prevalence and clinical features of SD in Japan. METHODS: We reviewed Japanese surveys of SD and compared them to surveys reported from other countries. We focused on SD prevalence, clinical features (SD type, sex and age), and treatment modalities. RESULTS: The SD prevalence in Japan was 3.5-7.0/100,000, similar to that in Rochester (NY, USA) and Iceland. Adductor SD predominated (90-95%) and females were four-fold more likely to be affected than males. Mean age at onset was approximately 30 years in Japan. Several years elapsed from onset to diagnosis. The most frequent treatment was botulinum toxin injection, and surgical intervention, particularly type 2 thyroplasty is becoming more popular. CONCLUSIONS: Our review demonstrated some differences of clinical features of SD in Japan compared with other countries, such as a greater female predominance and younger age of onset. Many physicians and patients may be unfamiliar with the clinical features of SD leading to delayed of diagnosis. Therefore, we proposed diagnostic criteria to facilitate early diagnosis and an appropriate choice of treatment modalities.

Synergistic effect between proteasome and autophagosome in the clearance of polyubiquitinated TDP-43.

Cytoplasmic aggregates of ubiquitinated TAR DNA-binding protein 43 (TDP-43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism of TDP-43 polyubiquitination remains elusive. We investigated the effect of nuclear exclusion of TDP-43 on aggregate formation and fragmentation, using TDP-43 expression constructs for WT or mutant TDP-43 with a modified nuclear localizing signal (LQ-NLS). Overexpression of the LQ-NLS mutant alone induced no detectable cytoplasmic aggregates during a 72-hr period. Polyubiquitination of both WT TDP-43 and the LQ-NLS mutant was similar in total cell lysates exposed to the proteasome inhibitor lactacystin. However, analysis of subcellular fractions demonstrated a higher concentration of polyubiquitinated TDP-43 in the nuclear fraction than in the cytosol for WT, and vice versa for the LQ-NLS mutant. Polyubiquitin-charged WT and mutant TDP-43 were highly concentrated in the membrane/microsome fraction, which was also positive for the autophagosome marker LC3. In addition, the autophagy inhibitor 3-methyladenine (3MA) blocked degradation of both TDP-43 types, whereas lactacystin was minimally restorative. Furthermore, lactacystin plus 3MA induced prominent cytoplasmic aggregates. We also demonstrated mediation of TDP-43 polyubiquitination by lysine 48 of ubiquitin, indicating a degradation signal in both TDP-43 types. This is the first report delineating the distribution of polyubiquitinated TDP-43 and the degradation pathway of TDP-43 and clarifying the crucial role of autophagosomes in TDP-43 clearance. We also demonstrate that nuclear exclusion itself is not an immediate trigger for ALS pathology. Further clarification of the mechanism of polyubiquitination of TDP-43 and the role of autophagosomes may help in understanding and treating ALS.

Double arterialized free jejunal flap.

In a standard free jejunal transfer, one artery and one vein are anastomosed. However, when raising the jejunal flap, a one-segment jejunum sometimes has two arteries and one accompanying vein as a vascular pedicle. Free jejunal transfer in which two arteries and one vein are anastomosed was designed. We report on the safety and advantages of using this artery-dominant transfer when performing microvascular anastomosis. This technique was used when a one-segment jejunum had two arteries and an accompanying vein. Eight patients underwent this arterial-supercharged free jejunal transfer. All flaps survived, and no complications developed except for two cases of intraoperative thrombosis before the procedure. It is important to transfer the artery-rich graft into the same physiological environment by reconstructing the similar hemodynamics. The grafts can be transferred without harm. This artery-dominant method can be an option when conditions are unfavorable for safer jejunal transfer.

[Congenital ossicular malformation: a study of 27 ears].

Despite otological surgical progress improving clinical congenital ossicular malformation management, some cases remain inadequately treated. We report 27 cases of congenital ossicular malformation, focusing on reasons for remaining or delayed postoperative hearing loss evaluated in 27 congenital ossicular malformation cases in Kyoto Prefecture from 2002 to 2008. Overall success was 93% (25/27) 6 months postoperatively. Two ears had no hearing improvement and three delayed hearing loss 8 to 48 months postoperatively. The first two ears underwent small fenestration stapedotomy with malleus attachment piston, and the other three tympanoplasty type III using an autologous ossicle or total ossicular replacement prosthesis (TORP) as a columella. We discuss problems and solutions using a malleus attachment piston or prosthesis, preoperative audio-and radiological findings, and operative findings including facial nerve anomaly and congenital cholesteatoma.

The integrity of cochlear hair cells is established and maintained through the localization of Dia1 at apical junctional complexes and stereocilia.

Dia1, which belongs to the diaphanous-related formin family, influences a variety of cellular processes through straight actin elongation activity. Recently, novel DIA1 mutants such as p.R1213X (p.R1204X) and p.A265S, have been reported to cause an autosomal dominant sensorineural hearing loss (DFNA1). Additionally, active DIA1 mutants induce progressive hearing loss in a gain-of-function manner. However, the subcellular localization and pathological function of DIA1(R1213X/R1204X) remains unknown. In the present study, we demonstrated the localization of endogenous Dia1 and the constitutively active DIA1 mutant in the cochlea, using transgenic mice expressing FLAG-tagged DIA1(R1204X) (DIA1-TG). Endogenous Dia1 and the DIA1 mutant were regionally expressed at the organ of Corti and the spiral ganglion from early life; alongside cochlear maturation, they became localized at the apical junctional complexes (AJCs) between hair cells (HCs) and supporting cells (SCs). To investigate HC vulnerability in the DIA1-TG mice, we exposed 4-week-old mice to moderate noise, which induced temporary threshold shifts with cochlear synaptopathy and ultrastructural changes in stereocilia 4 weeks post noise exposure. Furthermore, we established a knock-in (KI) mouse line expressing AcGFP-tagged DIA1(R1213X) (DIA1-KI) and confirmed mutant localization at AJCs and the tips of stereocilia in HCs. In MDCKAcGFP-DIA1(R1213X) cells with stable expression of AcGFP-DIA1(R1213X), AcGFP-DIA1(R1213X) revealed marked localization at microvilli on the apical surface of cells and decreased localization at cell-cell junctions. The DIA1-TG mice demonstrated hazy and ruffled circumferential actin belts at AJCs and abnormal stereocilia accompanied with HC loss at 5 months of age. In conclusion, Dia1 plays a pivotal role in the development and maintenance of AJCs and stereocilia, ensuring cochlear and HC integrity. Subclinical/latent vulnerability of HCs may be the cause of progressive hearing loss in DFNA1 patients, thus suggesting new therapeutic targets for preventing HC degeneration and progressive hearing loss associated with DFNA1.

Quercetin stimulates Na+/K+/2Cl- cotransport via PTK-dependent mechanisms in human airway epithelium.

We investigated regulatory mechanisms of Cl(-) secretion playing an essential role in the maintenance of surface fluid in human airway epithelial Calu-3 cells. The present study reports that quercetin (a flavonoid) stimulated bumetanide-sensitive Cl(-) secretion with reduction of apical Cl(-) conductance, suggesting that quercetin stimulates Cl(-) secretion by activating an entry step of Cl(-) across the basolateral membrane through Na(+)/K(+)/2Cl(-) cotransporter (NKCC1). To clarify the mechanism stimulating NKCC1 by quercetin, we verified involvement of protein kinase (PK)A, PKC, protein tyrosine kinase (PTK), and cytosolic Ca(2+)-dependent pathways. A PKA inhibitor (PKI-14-22 amide), a PKC inhibitor (Gö 6983) or a Ca(2+) chelating agent did not affect the quercetin-stimulated Cl(-) secretion. On the other hand, a PTK inhibitor (AG18) significantly diminished the stimulatory action of quercetin on Cl(-) secretion without inhibitory effects on apical Cl(-) conductance, suggesting that a PTK-mediated pathway is involved in the stimulatory action of quercetin. The quercetin action on Cl(-) secretion was suppressed with brefeldin A (BFA, an inhibitor of vesicular transport from ER to Golgi), and the BFA-sensitive Cl(-) secretion was not observed in the presence of an epidermal growth factor receptor (EGFR) kinase inhibitor (AG1478), suggesting that quercetin stimulates Cl(-) secretion by causing the EGFR kinase-mediated translocation of NKCC1 or an NKC1-activating factor to the basolateral membrane in human airway epithelial Calu-3 cells. However, the surface density of NKCC1 was not increased by quercetin, but quercetin elevated the activity of NKCC1. These observations indicate that quercetin stimulates Cl(-) secretion by activating NKCC1 via translocation of an NKCC1-activating factor through an EGFR kinase-dependent pathway.

RB1CC1 activates the promoter and expression of RB1 in human cancer.

RB1-inducible coiled-coil 1 (RB1CC1, also known as FIP200) is a tumor suppressor implicated in the regulation of RB1 (retinoblastoma 1) expression. However, the molecular mechanism of RB1 regulation by RB1CC1 has not been elucidated. Here, we demonstrate that nuclear RB1CC1 binds to the RB1 promoter using chromatin immunoprecipitation assays with anti-RB1CC1 antibody. Luciferase assays with RB1 promoter reporter plasmids revealed that RB1CC1 activated the RB1 promoter through the 201 bp upstream GC-rich region (from the initiation ATG). Electrophoretic mobility shift assay and Western blot analysis supported RB1CC1 binding to the GC-rich region of the RB1 promoter. In addition, the C-terminus of RB1CC1 was required for nuclear localization and subsequent RB1 promoter activation. Furthermore, the expression levels of RB1CC1 and RB1 significantly correlated with in vivo breast cancer tissues as determined by immunohistochemical analysis. These data indicate that nuclear RB1CC1 directly activates the RB1 promoter to enhance RB1 expression in cancer cells. Evaluation of RB1CC1 in various types of human cancer tissues is expected to provide useful information for clinical practice and future therapeutic strategies.