Neuronal Nitric Oxide Synthase Regulates Cerebellar Parallel Fiber Slow EPSC in Purkinje Neurons by Modulating STIM1-Gated TRPC3-Containing Channels.

Responding to burst stimulation of parallel fibers (PFs), cerebellar Purkinje neurons (PNs) generate a convolved synaptic response displaying a fast excitatory postsynaptic current (EPSCFast) followed by a slow EPSC (EPSCSlow). The latter is companied with a rise of intracellular Ca2+ and critical for motor coordination. The genesis of EPSCSlow in PNs results from activation of metabotropic type 1 glutamate receptor (mGluR1), oligomerization of stromal interaction molecule 1 (STIM1) on the membrane of endoplasmic reticulum (ER) and opening of transient receptor potential canonical 3 (TRPC3) channels on the plasma membrane. Neuronal nitric oxide synthase (nNOS) is abundantly expressed in PFs and granule neurons (GNs), catalyzing the production of nitric oxide (NO) hence regulating PF-PN synaptic function. We recently found that nNOS/NO regulates the morphological development of PNs through mGluR1-regulated Ca2+-dependent mechanism. This study investigated the role of nNOS/NO in regulating EPSCSlow. Electrophysiological analyses showed that EPSCSlow in cerebellar slices of nNOS knockout (nNOS-/-) mice was significantly larger than that in wildtype (WT) mice. Activation of mGluR1 in cultured PNs from nNOS-/- mice evoked larger TRPC3-channel mediated currents and intracellular Ca2+ rise than that in PNs from WT mice. In addition, nNOS inhibitor and NO-donor increased and decreased, respectively, the TRPC3-current and Ca2+ rise in PNs. Moreover, the NO-donor effectively decreased TRPC3 currents in HEK293 cells expressing WT STIM1, but not cells expressing a STIM1 with cysteine mutants. These novel findings indicate that nNOS/NO inhibits TRPC3-containig channel mediated cation influx during EPSCSlow, at least in part, by S-nitrosylation of STIM1.

Increased GABA signaling in liver macrophage promotes HBV replication in HBV-carrier mice.

Gamma-aminobutyric acid (GABA) signals in various non-neuronal cells including hepatocytes and some immune cells. Studies, including ours, show that type A GABA receptors (GABAARs)-mediated signaling occurs in macrophages regulating tissue-specific functions. Our recent study reveals that activation of GABAARs in liver macrophages promotes their M2-like polarization and increases HBV replication in mice. This short article briefly summarizes the GABA signaling system in macrophages and discusses potential mechanisms by which GABA signaling promotes HBV replication.

The hepatic GABAergic system promotes liver macrophage M2 polarization and mediates HBV replication in mice.

Macrophages display functional phenotypic plasticity. Hepatitis B virus (HBV) infection induces polarizations of liver macrophages either to M1-like pro-inflammatory phenotype or to M2-like anti-inflammatory phenotype. Gamma-aminobutyric acid (GABA) signaling exists in various non-neuronal cells including hepatocytes and some immune cells. Here we report that macrophages express functional GABAergic signaling components and activation of type A GABA receptors (GABAARs) promotes M2-polarization thus advancing HBV replication. Notably, intraperitoneal injection of GABA or the GABAAR agonist muscimol increased HBV replication in HBV-carrier mice that were generated by hydrodynamical injection of adeno-associated virus/HBV1.2 plasmids (pAAV/HBV1.2). The GABA-augmented HBV replication in HBV-carrier mice was significantly reduced by the GABAAR inhibitor picrotoxin although picrotoxin had no significant effect on serum HBsAg levels in control HBV-carrier mice. Depletion of liver macrophages by liposomal clodronate treatment also significantly reduced the GABA-augmented HBV replication. Yet adoptive transfer of liver macrophages isolated from GABA-treated donor HBV-carrier mice into the liposomal clodronate-pretreated recipient HBV-carrier mice restored HBV replication. Moreover, GABA or muscimol treatment increased the expression of "M2" cytokines in macrophages, but had no direct effect on HBV replication in the HepG2.2.15 cells, HBV1.3-transfected Huh7, HepG2, or HepaRG cells, or HBV-infected Huh7-NTCP cells. Taken together, these results suggest that increasing GABA signaling in the liver promotes HBV replication in HBV-carrier mice by suppressing the immunity of liver macrophages, but not by increasing the susceptibility of hepatocytes to HBV infection. Our study shows that a previously unknown GABAergic system in liver macrophage has an essential role in HBV replication.

Association of the nasopharyngeal carcinoma and the subsequent open glaucoma development: a nationwide cohort study.

This study aimed to investigate the possible association between nasopharyngeal carcinoma (NPC) and following open angle glaucoma (OAG). A retrospective research applying the National Health Insurance Research Database (NHIRD) of Taiwan was conducted with a follow up period from January 1, 2000 to December 31, 2016. There were 4184 and 16736 participants that selected and categorized into the NPC and non-NPC groups after exclusion. The major outcome of our study was the development of OAG according to diagnostic codes, exam and managements. The Cox proportional hazard regression was employed to estimate the adjusted hazard ratio (aHR) and 95% confidence interval (CI) of OAG between the two groups. In this study, a numbers of 151 and 513 OAG episodes occurred in the NPC and non-NPC groups and the NPC population showed a significantly higher incidence of OAG compared to the non-NPC population in multivariable analysis (aHR: 1.293, 95% CI: 1.077-1.551, p = 0.0057). Besides, the cumulative probability of OAG was significantly higher in the NPC group than that in the non-NPC population (p = 0.0041). About other risk factor for OAG, age older than 40 years old, diabetes mellitus and persistent steroid usage were related to OAG occurrence (all p < 0.05). In conclusion, the NPC may be an independent risk factor of following OAG development.

Diabetic macular edema and proliferative diabetic retinopathy treated with anti-vascular endothelial growth factor under the reimbursement policy in Taiwan.

The purpose of this retrospective interventional case series is to compare the functional and anatomical outcomes in eyes with diabetic macular edema (DME) and proliferative diabetic retinopathy (PDR) treated intravitreally with aflibercept or ranibizumab under the Taiwan National Insurance Bureau reimbursement policy. 84 eyes were collected and all eyes were imaged with spectral-domain optical coherence tomography (SD-OCT), color fundus photographs (CFPs), and fluorescein angiography (FA). At 24 months after therapy initiation, the logMAR BCVA improved from 0.58 ± 0.33 to 0.47 ± 0.38 (p < 0.01), the CRT decreased from 423.92 ± 135.84 to 316.36 ± 90.02 (p < 0.01), and the number of microaneurysms decreased from 142.14 ± 57.23 to 75.32 ± 43.86 (p < 0.01). The mean injection count was 11.74 ± 5.44. There was no intergroup difference in logMAR BCVA (p = 0.96), CRT (p = 0.69), or injection count (p = 0.81). However, the mean number of microaneurysms was marginally reduced (p = 0.06) in eyes treated with aflibercept at the end of the follow-up, and the incidence rates of supplementary panretinal photocoagulation (PRP) (p = 0.04) and subthreshold micropulse laser (SMPL) therapy sessions (p = 0.01) were also reduced. Multivariate analysis revealed that only initial logMAR BCVA influenced the final VA improvements (odds ratio (OR) 0.49, 95% confidence interval (CI) 0.21 ~ 0.93, p < 0.01); in contrast, age (OR - 0.38, 95% CI - 6.97 ~ - 1.85, p < 0.01) and initial CRT (OR 0.56, 95% CI 0.34 ~ 0.84, p < 0.01) both influenced the final CRT reduction at 24 months. To sum up, both aflibercept and ranibizumab are effective in managing DME with PDR in terms of VA, CRT and MA count. Eyes receiving aflibercept required less supplementary PRP and SMPL treatment than those receiving ranibizumab. The initial VA influenced the final VA improvements at 24 months, while age and initial CRT were prognostic predictors of 24-month CRT reduction.

XB130 Deficiency Causes Congenital Hypothyroidism in Mice due to Disorganized Apical Membrane Structure and Function of Thyrocytes.

Background: Congenital hypothyroidism is often caused by genetic mutations that impair thyroid hormone (TH) production, resulting in growth and development defects. XB130 (actin filament associated protein 1 like 2) is an adaptor/scaffold protein that plays important roles in cell proliferation, migration, intracellular signal transduction, and tumorigenesis. It is highly expressed in thyrocytes, however, its function in the thyroid remains largely unexplored. Methods:Xb130-/- mice and their littermates were studied. Postnatal growth and growth hormone levels were measured, and responses to low or high-iodine diet, and levothyroxine treatment were examined. TH and thyrotropin in the serum and TH in the thyroid glands were quantified. Structure and function of thyrocytes in embryos and postnatal life were studied with histology, immunohistochemistry, immunofluorescence staining, Western blotting, and quantitative reverse transcription polymerase chain reaction. Results:Xb130-/- mice exhibited transient growth retardation postnatally, due to congenital hypothyroidism with reduced TH synthesis and secretion, which could be rescued by exogenous thyroxine supplementation. The thyroid glands of Xb130-/- mice displayed diminished thyroglobulin iodination and release at both embryonic and early postnatal stages. XB130 was found mainly on the apical membrane of thyroid follicles. Thyroid glands of embryonic and postnatal Xb130-/- mice exhibited disorganized apical membrane structure, delayed folliculogenesis, and abnormal formation of thyroid follicle lumina. Conclusion: XB130 critically regulates folliculogenesis by maintaining apical membrane structure and function of thyrocytes, and its deficiency leads to congenital hypothyroidism.

Nitric oxide attenuates microglia proliferation by sequentially facilitating calcium influx through TRPV2 channels, activating NFATC2, and increasing p21 transcription.

Microglia proliferation is critical for proper development and function of the central nervous system (CNS), while dysregulation of proliferation contributes to pathology. We recently reported that male inducible nitric oxide synthase knockout (iNOS-/-) mice displayed significantly more proliferating microglia in their postnatal cortex than age-matched wildtype (WT) male mice. Moreover, nitric oxide (NO) signaling in mouse microglia greatly upregulates calcium entry through transient receptor potential vanilloid type 2 (TRPV2) channels. Considering that TRPV2 activity restricts astrocytic proliferation within glioma tissues, we investigated the roles of iNOS/NO signaling and TRPV2 expression in the regulation of microglial proliferation in vitro using assays of calcium imaging, immunocytochemistry, western blot, and polymerase chain reaction. Results showed that non-dividing microglia exhibited substantially higher expression of TRPV2 on the plasma membrane and significantly larger calcium influx through TRPV2 channels in comparison to dividing microglia. Additionally, non-dividing WT microglia exhibited significantly more NO production than dividing WT microglia. Furthermore, the NO-donor NOC18 increased the nuclear translocation of nuclear factor of activated T-cells cytoplasmic 2 (NFATC2) and the mRNA of the cyclin-dependent kinase inhibitor p21 and decreased the percentage of dividing WT and iNOS-/- microglia in culture. Importantly, the presence of the TRPV2 inhibitor tranilast abolished these effects of NOC18. Together, results from this study indicated that iNOS/NO signaling inhibits microglial proliferation through TRPV2-mediated calcium influx, nuclear translocation of the transcription factor NFATC2, and p21 expression. [Figure: see text].

Autocrine GABA signaling distinctively regulates phenotypic activation of mouse pulmonary macrophages.

In response to micro-environmental cues such as microbial infections or T-helper 1 and 2 (TH1 and TH2) cytokines, macrophages (Mϕs) develop into M1- or M2-like phenotypes. Phenotypic polarization/activation of Mϕs are also essentially regulated by autocrine signals. Type-A γ-aminobutyric acid receptor (GABAAR)-mediated autocrine signaling is critical for phenotypic differentiation and transformation of various cell types. The present study explored whether GABAAR signaling regulates lung Mϕ (LMϕ) phenotypic activation under M1/TH1 and M2/TH2 environments. Results showed that GABAAR subunits were expressed by primary LMϕ of mice and the mouse Mϕ cell line RAW264.7. The expression levels of GABAAR subunits in mouse LMϕs and RAW264.7 cells decreased or increased concurrently with classical (M1) or alternative (M2) activation, respectively. Moreover, activation or blockade of GABAARs distinctively influenced the phenotypic characteristics of Mϕ. These results suggested that microenvironments leading to LMϕ phenotypic polarization concurrently modulates autocrine GABA signaling and its role in Mϕ activation.

S-Nitrosylation of STIM1 by Neuronal Nitric Oxide Synthase Inhibits Store-Operated Ca2+ Entry.

Store-operated Ca2+ entry (SOCE) mediated by stromal interacting molecule-1 (STIM1) and Orai1 represents a major route of Ca2+ entry in mammalian cells and is initiated by STIM1 oligomerization in the endoplasmic or sarcoplasmic reticulum. However, the effects of nitric oxide (NO) on STIM1 function are unknown. Neuronal NO synthase is located in the sarcoplasmic reticulum of cardiomyocytes. Here, we show that STIM1 is susceptible to S-nitrosylation. Neuronal NO synthase deficiency or inhibition enhanced Ca2+ release-activated Ca2+ channel current (ICRAC) and SOCE in cardiomyocytes. Consistently, NO donor S-nitrosoglutathione inhibited STIM1 puncta formation and ICRAC in HEK293 cells, but this effect was absent in cells expressing the Cys49Ser/Cys56Ser STIM1 double mutant. Furthermore, NO donors caused Cys49- and Cys56-specific structural changes associated with reduced protein backbone mobility, increased thermal stability and suppressed Ca2+ depletion-dependent oligomerization of the luminal Ca2+-sensing region of STIM1. Collectively, our data show that S-nitrosylation of STIM1 suppresses oligomerization via enhanced luminal domain stability and rigidity and inhibits SOCE in cardiomyocytes.

Protective roles of hepatic GABA signaling in acute liver injury of rats.

γ-Aminobutyric acid (GABA) is produced by various cells through the catalytic activity of glutamic acid decarboxylase (GAD). Activation of type-A GABA receptor (GABAAR) inhibits stem cell proliferation but protects differentiated cells from injures. The present study investigated hepatic GABA signaling system and the role of this system in liver physiology and pathophysiology. RT-PCR and immunoblot assays identified GAD and GABAAR subunits in rat livers and in HepG2 and Clone 9 hepatocytes. Patch-clamp recording detected GABA-induced currents in Clone 9 hepatocytes and depolarization in WITT cholangiocytes. The function of hepatic GABA signaling system in rats was examined using models of d-galactosamine (GalN)-induced acute hepatocytic injury in vivo and in vitro. The expression of GAD increased whereas GABAAR subunits decreased in the liver of GalN-treated rats. Remarkably, treating rats with GABA or the GABAAR agonist muscimol, but not the GABABR agonist baclofen, protected hepatocytes against GalN toxicity and improved liver function. In addition, muscimol treatment decreased the formation of pseudobile ductules and the enlargement of hepatocytic canaliculi in GalN-treated rats. Our results revealed that a complex GABA signaling system exists in the rat liver. Activation of this intrahepatic GABAergic system protected the liver against toxic injury.NEW & NOTEWORTHY Auto- and paracrine GABAergic signaling systems exist in the rat hepatocytes and cholangiocytes. Activation of GABA signaling protects liver function from d-galactosamine injury by reducing toxic impairment of hepatocytes and by decreasing cholangiocyte proliferation.

Isoflurane regulates atypical type-A γ-aminobutyric acid receptors in alveolar type II epithelial cells.

BACKGROUND: Volatile anesthetics act primarily through upregulating the activity of γ-aminobutyric acid type A (GABAA) receptors. They also exhibit antiinflammatory actions in the lung. Rodent alveolar type II (ATII) epithelial cells express GABAA receptors and the inflammatory factor cyclooxygenase-2 (COX-2). The goal of this study was to determine whether human ATII cells also express GABAA receptors and whether volatile anesthetics upregulate GABAA receptor activity, thereby reducing the expression of COX-2 in ATII cells. METHODS: The expression of GABAA receptor subunits and COX-2 in ATII cells of human lung tissue and in the human ATII cell line A549 was studied with immunostaining and immunoblot analyses. Patch clamp recordings were used to study the functional and pharmacological properties of GABAA receptors in cultured A549 cells. RESULTS: ATII cells in human lungs and cultured A549 cells expressed GABAA receptor subunits and COX-2. GABA induced currents in A549 cells, with half-maximal effective concentration of 2.5 µM. Isoflurane (0.1-250 µM) enhanced the GABA currents, which were partially inhibited by bicuculline. Treating A549 cells with muscimol or with isoflurane (250 µM) reduced the expression of COX-2, an effect that was attenuated by cotreatment with bicuculline. CONCLUSIONS: GABAA receptors expressed by human ATII cells differ pharmacologically from those in neurons, exhibiting a higher affinity for GABA and lower sensitivity to bicuculline. Clinically relevant concentrations of isoflurane increased the activity of GABAA receptors and reduced the expression of COX-2 in ATII cells. These findings reveal a novel mechanism that could contribute to the antiinflammatory effect of isoflurane in the human lung.

Mature miR-17-5p and passenger miR-17-3p induce hepatocellular carcinoma by targeting PTEN, GalNT7 and vimentin in different signal pathways.

To study the physiological role of a single microRNA (miRNA), we generated transgenic mice expressing the miRNA precursor miR-17 and found that the mature miR-17-5p and the passenger strand miR-17-3p were abundantly expressed. We showed that mature miR-17-5p and passenger strand miR-17-3p could synergistically induce the development of hepatocellular carcinoma. The mature miR-17-5p exerted this function by repressing the expression of PTEN. In contrast, the passenger strand miR-17-3p repressed expression of vimentin, an intermediate filament with the ability to modulate metabolism, and GalNT7, an enzyme that regulates metabolism of liver toxin galactosamine. Hepatocellular carcinoma cells, HepG2, transfected with miR-17 formed larger tumors with more blood vessels and less tumor cell death than mock-treated cells. Expression of miR-17 precursor modulated HepG2 proliferation, migration, survival, morphogenesis and colony formation and inhibited endothelial tube formation. Silencing of PTEN, vimentin or GalNT7 with their respective siRNAs enhanced proliferation and migration. Re-expressing these molecules reversed their roles in proliferation, migration and tumorigenesis. Further experiments indicated that these three molecules do not interact with each other, but appear to function in different signaling pathways. Our results demonstrated that a mature miRNA can function synergistically with its passenger strand leading to the same phenotype but by regulating different targets located in different signaling pathways. We anticipate that our assay will serve as a helpful model for studying miRNA regulation.

Versican 3'-untranslated region (3'-UTR) functions as a ceRNA in inducing the development of hepatocellular carcinoma by regulating miRNA activity.

This study was designed to explore the role of versican in the development of hepatocellular carcinoma (HCC). Ectopic expression of the versican 3'-untranslated region (3'-UTR) was studied as a competitive endogenous RNA for regulating miRNA functions. We used this approach to modulate the expression of versican and its related proteins in 3'-UTR transgenic mice and in the liver cancer cell line HepG2, stably transfected with the 3'-UTR or a control vector. We demonstrated that transgenic mice expressing the versican 3'-UTR developed HCC and increased expression of versican isoforms V0 and V1. HepG2 cells transfected with versican 3'-UTR displayed increased proliferation, survival, migration, invasion, colony formation, and enhanced endothelial cell growth, but decreased apoptosis. We found that versican 3'-UTR could bind to miRNAs miR-133a, miR-199a*, miR-144, and miR-431 and also interacted with CD34 and fibronectin. As a consequence, expression of versican, CD34, and fibronectin was up-regulated by ectopic transfection of the versican 3'-UTR, which was confirmed in HepG2 cells and in transgenic mice as compared with wild-type controls. Transfection with siRNAs targeting the versican 3'-UTR abolished the effects of the 3'-UTR. Taken together, these results demonstrate that versican V0 and V1 isoforms play important roles in HCC development and that versican mRNAs compete with endogenous RNAs in regulating miRNA functions.

TRIM59, a novel multiple cancer biomarker for immunohistochemical detection of tumorigenesis.

OBJECTIVES AND DESIGN: We identified a novel TRIM59 gene, as an early signal transducer in two (SV40Tag and Ras) oncogene pathways in murine prostate cancer (CaP) models. We explore its clinical applications as a multitumour marker detecting early tumorigenesis by immunohistochemistry (IHC). SETTING AND PARTICIPANTS: 88 CaP patients were from a tissue microarray (TMA) of radical prostatectomy specimen, 42 patients from a 35 multiple tumour TMA, 75 patients with renal cell carcinoma (RCC) and 92 patients from eight different tumour groups (breast, lung, parotid, gastrointestinal, female genital tract, bladder, kidney and prostate cancer). RESULTS: TRIM59 upregulation specifically in tumour area was determined by IHC in 291 cases of 37 tumour types. To demonstrate that TRIM59 upregulation is 'tumour-specific', we characterised a significant correlation of TRIM59 IHC signals with tumorigenesis and progression, while in control and normal area, TRIM59 IHC signal was all negative or significantly low. TRIM59 protein upregulation in prostate and kidney cancers was detectable in both intensity and extent in early tumorigenesis of prostate intraepithelial neoplasia (p<0.05) and grade 1 of RCC (p<0.05), and stopped until high grades cancer. The results of the correlation in these two large cohorts of tumour types confirmed and repeated murine CaP model studies. Enhanced TRIM59 expression was identified in most of the 37 different tumours, while the highest intensities were in lung, breast, liver, skin, tongue and mouth (squamous cell cancer) and endometrial cancers. Multiple tumour upregulation was further confirmed by comparing relative scores of TRIM59 IHC signals in eight tumours with a larger patient population; and by a mouse whole-mount embryo (14.5 days post conception) test on the origin of TRIM59 upregulation in epithelial cells. CONCLUSIONS: TRIM59 may be used a novel multiple tumour marker for immunohistochemical detecting early tumorigenesis and could direct a novel strategy for molecular-targeted diagnosis and therapy of cancer.

Differential detection and distribution of microglial and hematogenous macrophage populations in the injured spinal cord of lys-EGFP-ki transgenic mice.

The acute inflammatory response that follows spinal cord injury (SCI) contributes to secondary injury that results in the expansion of the lesion and further loss of neurologic function. A cascade of receptor-mediated signaling events after SCI leads to activation of innate immune responses including the migration of microglia and active recruitment of circulating leukocytes. Because conventional techniques do not always distinguish macrophages derived from CNS-resident microglia from blood-derived monocytes, the role that each macrophage type performs cannot be assessed unambiguously in these processes. We demonstrate that, in the normal and spinal cord-injured lys-EGFP-ki transgenic mouse, enhanced green fluorescent protein (EGFP) is expressed only in mature hematopoietic granulomyelomonocytic cells and not in microglia. This allowed us to assess the temporal and spatial relationships between microglia-derived and hematogenous macrophages as well as neutrophils during a period of 6 weeks after clip compression SCI. Within the lesion, EGFP-positive monocyte-derived macrophages were found at the epicenter surrounded by EGFP-negative-activated microglia and microglia-derived macrophages. Neutrophils were not present when EGFP-positive monocyte-derived macrophages were depleted, indicating that neutrophil persistence in the lesion depended on the presence of these monocytes. Thus, these 2 distinct macrophage populations can be independently identified and tracked, thereby allowing their roles in acute and chronic stages of SCI-associated inflammation to be defined.

GABA coordinates with insulin in regulating secretory function in pancreatic INS-1 ß-cells.

Pancreatic islet ß-cells produce large amounts of γ-aminobutyric acid (GABA), which is co-released with insulin. GABA inhibits glucagon secretion by hyperpolarizing α-cells via type-A GABA receptors (GABA(A)Rs). We and others recently reported that islet ß-cells also express GABA(A)Rs and that activation of GABA(A)Rs increases insulin release. Here we investigate the effects of insulin on the GABA-GABA(A)R system in the pancreatic INS-1 cells using perforated-patch recording. The results showed that GABA produces a rapid inward current and depolarizes INS-1 cells. However, pre-treatment of the cell with regular insulin (1 µM) suppressed the GABA-induced current (I(GABA)) by 43%. Zinc-free insulin also suppressed I(GABA) to the same extent of inhibition by regular insulin. The inhibition of I(GABA) occurs within 30 seconds after application of insulin. The insulin-induced inhibition of I(GABA) persisted in the presence of PI3-kinase inhibitor, but was abolished upon inhibition of ERK, indicating that insulin suppresses GABA(A)Rs through a mechanism that involves ERK activation. Radioimmunoassay revealed that the secretion of C-peptide was enhanced by GABA, which was blocked by pre-incubating the cells with picrotoxin (50 µM, p<0.01) and insulin (1 µM, p<0.01), respectively. Together, these data suggest that autocrine GABA, via activation of GABA(A)Rs, depolarizes the pancreatic ß-cells and enhances insulin secretion. On the other hand, insulin down-regulates GABA-GABA(A)R signaling presenting a feedback mechanism for fine-tuning ß-cell secretion.

GABA exerts protective and regenerative effects on islet beta cells and reverses diabetes.

Type 1 diabetes (T1D) is an autoimmune disease characterized by insulitis and islet ß-cell loss. Thus, an effective therapy may require ß-cell restoration and immune suppression. Currently, there is no treatment that can achieve both goals efficiently. We report here that GABA exerts antidiabetic effects by acting on both the islet ß-cells and immune system. Unlike in adult brain or islet α-cells in which GABA exerts hyperpolarizing effects, in islet ß-cells, GABA produces membrane depolarization and Ca(2+) influx, leading to the activation of PI3-K/Akt-dependent growth and survival pathways. This provides a potential mechanism underlying our in vivo findings that GABA therapy preserves ß-cell mass and prevents the development of T1D. Remarkably, in severely diabetic mice, GABA restores ß-cell mass and reverses the disease. Furthermore, GABA suppresses insulitis and systemic inflammatory cytokine production. The ß-cell regenerative and immunoinhibitory effects of GABA provide insights into the role of GABA in regulating islet cell function and glucose homeostasis, which may find clinical application.

Deletion of the ubiquitin ligase Nedd4L in lung epithelia causes cystic fibrosis-like disease.

Cystic fibrosis is caused by impaired ion transport due to mutated cystic fibrosis transmembrane conductance regulator, accompanied by elevated activity of the amiloride-sensitive epithelial Na(+) channel (ENaC). Here we show that knockout of the ubiquitin ligase Nedd4L (Nedd4-2) specifically in lung epithelia (surfactant protein C-expressing type II and Clara cells) causes cystic fibrosis-like lung disease, with airway mucus obstruction, goblet cell hyperplasia, massive inflammation, fibrosis, and death by three weeks of age. These effects of Nedd4L loss are likely caused by enhanced ENaC function, as reflected by increased ENaC protein levels, increased lung dryness at birth, amiloride-sensitive dehydration of lung explants, and elevated ENaC currents in primary alveolar type II cells analyzed by patch clamp recordings. Moreover, the lung defects were rescued with administration of amiloride into the lungs of young knockout pups via nasal instillation. Our results therefore suggest that the ubiquitin ligase Nedd4L can suppress the onset of cystic fibrosis symptoms by inhibiting ENaC in lung epithelia.

The protein kinase C cascade regulates recruitment of matrix metalloprotease 9 to podosomes and its release and activation.

Podosomes are transient cell surface structures essential for degradation of extracellular matrix during cell invasion. Protein kinase C (PKC) is involved in the regulation of podosome formation; however, the roles of individual PKC isoforms in podosome formation and proteolytic function are largely unknown. Recently, we reported that PDBu, a PKC activator, induced podosome formation in normal human bronchial epithelial cells. Here, we demonstrate that phorbol-12,13-dibutyrate (PDBu)-induced podosome formation is mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes. Interestingly, although blocking atypical PKCζ did not affect PDBu-induced podosome formation, it significantly reduced matrix degradation at podosomes. Inhibition of PKCζ reduced recruitment of matrix metalloprotease 9 (MMP-9) to podosomes and its release and activation. Downregulation of MMP-9 by small interfering RNA (siRNA) or neutralization antibody also significantly reduced matrix degradation. The regulatory effects of PKCζ on matrix degradation and recruitment of MMP-9 to podosomes were PKCζ kinase activity dependent. PDBu-induced recruitment of PKCζ and MMP-9 to podosomes was blocked by inhibition of novel PKC with rottlerin or PKCδ siRNA. Our data suggest that multiple PKC isozymes form a signaling cascade that controls podosome formation and dynamics and MMP-9 recruitment, release, and activation in a coordinated fashion.