Dongki Yang

Convergence of IRBIT, phosphatidylinositol (4,5) bisphosphate, and WNK/SPAK kinases in regulation of the Na+-HCO3− cotransporters family

Fluid and HCO3− secretion is a vital function of secretory epithelia, involving basolateral HCO3− entry through the Na+-HCO3− cotransporter (NBC) NBCe1-B, and luminal HCO3− exit mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and solute carrier family 26 (SLC26) Cl−/HCO3− exchangers. HCO3− secretion is highly regulated, with the WNK/SPAK kinase pathway setting the resting state and the IRBIT/PP1 pathway setting the stimulated state. However, we know little about the relationships between the WNK/SPAK and IRBIT/PP1 sites in the regulation of the transporters. The first 85 N-terminal amino acids of NBCe1-B function as an autoinhibitory domain. Here we have identified a positively charged module within NBCe1-B(37-65) that is conserved in NBCn1-A and all 20 members of the NBC superfamily except NBCe1-A. This module is required for the interaction and activation of NBCe1-B and NBCn1-A by IRBIT and their regulation by phosphatidylinositol 4,5-bisphosphate (PIP2). Activation of the transporters by IRBIT and PIP2 is nonadditive but complementary. Phosphorylation of Ser65 mediates regulation of NBCe1-B by SPAK, and phosphorylation of Thr49 is required for regulation by IRBIT and SPAK. Sequence searches using the NBCe1-B regulatory module as a template identified a homologous sequence in the CFTR R domain and Slc26a6 sulfat transporter and antisigma factor antagonist (STAS) domain. Accordingly, the R and STAS domains bind IRBIT, and the R domain is required for activation of CFTR by IRBIT. These findings reveal convergence of regulatory modalities in a conserved domain of the NBC that may be present in other HCO3− transporters and thus in the regulation of epithelial fluid and HCO3− secretion.

Activation of TRPC4β by Gαi subunit increases Ca2+ selectivity and controls neurite morphogenesis in cultured hippocampal neuron.

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels. TRPC channels are activated by stimulation of Gαq-PLC-coupled receptors. Here, we report that TRPC4/TRPC5 can be activated by Gαi. We studied the essential role of Gαi subunits in TRPC4 activation and investigated changes in ion selectivity and pore dilation of the TRPC4 channel elicited by the Gαi2 subunit. Activation of TRPC4 by Gαi2 increased Ca2+ permeability and Ca2+ influx through TRPC4 channels. Co-expression of the muscarinic receptor (M2) and TRPC4 in HEK293 cells induced TRPC4-mediated Ca2+ influx. Moreover, both TRPC4β and the TRPC4β-Gαi2 signaling complex induced inhibition of neurite growth and arborization in cultured hippocampal neurons. Cells treated with KN-93, a CaMKII inhibitor, prevented TRPC4- and TRPC4-Gαi2(Q205L)-mediated inhibition of neurite branching and growth. These findings indicate an essential role of Gαi proteins in TRPC4 activation and extend our knowledge of the functional role of TRPC4 in hippocampal neurons.

Regulation of calcium influx and signaling pathway in cancer cells via TRPV6-Numb1 interaction.

Ca(2+) is a critical factor in the regulation of signal transduction and Ca(2+) homeostasis is altered in different human diseases. The level of Ca(2+) in cells is highly regulated through a diverse class of regulators. Among them is the transient receptor potential vanilloid 6 (TRPV6), which is a Ca(2+) selective channel that absorbs Ca(2+) in the small intestine. TRPV6 is overexpressed in some cancers and exhibits oncogenic potential, but its exact mechanism is still poorly understood. The Numb protein is a cell fate determinant that functions in endocytosis and as a tumor suppressor via the stabilization of p53. Numb protein consisted of four isoforms. Here, we showed a novel function of Numb1, which negatively regulates TRPV6 activity. The expression of Numb1 decreased cytosolic Ca(2+) concentrations in TRPV6-transfected HEK293 cells. When all the isoforms of Numb were depleted using siRNA in a TRPV6 stable cell line, the levels of cytosolic Ca(2+) increased. We observed an interaction between Numb1 and TRPV6 using co-immunoprecipitation. We confirmed this interaction using Fluorescence Resolution Energy Transfer (FRET). We identified the TRPV6 and Numb1 binding site using TRPV6 C-terminal truncation mutants and Numb1 deletion mutants. The binding site in TRPV6 was an aspartic acid at amino acid residue 716, and that binding site in Numb1 was arginine at amino acid residue 434. A Numb1 mutant, lacking TRPV6 binding activity, failed to inhibit TRPV6 activity. Every isoform of Numb knockdown, using an siRNA-based approach in MCF-7 breast cancer cells, not only showed enhanced TRPV6 expression but also both the cytosolic Ca(2+) concentration and cell proliferation were increased. The down-regulated expression of TRPV6 using siRNA increased Numb protein expression; however, the cytosolic influx of Ca(2+) and proliferation of the cell were decreased. To examine downstream signaling during Ca(2+) influx, we performed Western blotting analysis on TRPV6 upregulated cancer cells (MCF-7, PC-3). Taken together, these results demonstrated that Numb1 interacts with TRPV6 through charged residues and inhibits its activity via the regulation of protein expression. Moreover, we provided evidence for a Ca(2+)-regulated cancer cell signaling pathway and that the Ca(2+) channel is a target of cancer cells.

Nocardia Brain Abscess in an Immunocompetent Patient

Nocardia cerebral abscess is rare, constituting approximately 1-2% of all cerebral abscesses. Mortality for a cerebral abscess of Nocardia is three times higher than that of other bacterial cerebral abscesses, therefore, early diagnosis and therapy is important. Nocardia cerebral abscess is generally occur among immunocompromised patients, and critical infection in immunocompetent patients is extremely rare. We report on a case of a brain abscess by Nocardia farcinica in an immunocompetent patient who received treatment with surgery and antibiotics. This is the second case of a brain abscess caused by N. farcinica in an immunocompetent patient in Korea.

Inhibition of platelet-derived growth factor receptor tyrosine kinase and downstream signaling pathways by Compound C.

AMP-activated protein kinase (AMPK) has been implicated in anti-proliferative actions in a range of cell systems. Recently, it was observed that Compound C, an inhibitor of AMPK, also reduced the cell viability in human diploid fibroblasts (HDFs). Compound C-induced growth arrest was associated with a decrease in the cell cycle regulatory proteins, such as proliferating cell nuclear antigen, phosphorylated pRB, cyclin-dependent protein kinases (Cdk 2 and 4), cyclins (D and E), and the Cdk inhibitors (p21, p16, and p27). Therefore, the present study examined the molecular mechanism of the antiproliferative effects of Compound C. Although Compound C inhibited serum-induced phosphorylation of Akt and its substrate, glycogen synthase kinase-3β, it did not affect the Akt activity in vitro. Compound C significantly inhibited the receptor tyrosine phosphorylation and the activity of downstream signaling molecules, such as p85 phosphoinositide 3-kinase, phospholipase C-γ1, and extracellular signal-regulated kinase 1/2, induced by platelet-derived growth factor (PDGF) but not by epidermal growth factor- and insulin-like growth factor. In vitro growth factor receptor tyrosine kinase activity profiling revealed the IC50 for PDGF receptor-β (PDGFRβ) to be 5.07μM, whereas the IC50 for the epidermal growth factor receptor and insulin-like growth factor receptor was ≥100μM. The inhibitory effect of Compound C on PDGFRβ and Akt was also observed in AMPKα1/α2-knockout mouse embryonic fibroblasts, indicating that its inhibitory effect is independent of the AMPK activity. The inhibitory effect of Compound C on cell proliferation and PDGFRβ tyrosine phosphorylation was also demonstrated in various PDGFR-expressing cells, including MRC-5, BEAS-2B, rat aortic vascular smooth muscle cells, and A172 glioblastoma cells. These results indicate that Compound C can be used as a potential antiproliferative agent for PDGF- or PDGFR-associated diseases, such as cancer, atherosclerosis, and fibrosis.

EX4 stabilizes and activates Nrf2 via PKCδ, contributing to the prevention of oxidative stress-induced pancreatic beta cell damage

ABSTRACT Oxidative stress in pancreatic beta cells can inhibit insulin secretion and promote apoptotic cell death. Exendin‐4 (EX4), a glucagon‐like peptide‐1 receptor agonist, can suppress beta cell apoptosis, improve beta cell function and protect against oxidative damage. In this study, we investigated the molecular mechanisms for antioxidative effects of EX4 in pancreatic beta cells. INS‐1 cells, a rat insulinoma cell line, were pretreated with EX4 and exposed to palmitate or H2O2. Reactive oxygen species (ROS) production, and glutathione and insulin secretion were measured. The mRNA and protein expression levels of antioxidant genes were examined. The level of nuclear factor erythroid 2‐related factor 2 (Nrf2), its binding to antioxidant response element (ARE), and its ubiquination in the presence of EX4 were determined. The Nrf2 signaling pathway was determined using rottlerin (protein kinase [PK]C&dgr; inhibitor), H89 (PKA inhibitor) and LY294002 (phosphatidylinositide 3‐kinase [PI3K] inhibitor). EX4 treatment decreased ROS production, recovered cellular glutathione levels and insulin secretion in the presence of oxidative stress in INS‐1 cells. The expression levels of glutamate‐cysteine ligase catalytic subunit and heme oxygenase‐1 were increased by EX4 treatment. EX4 promoted Nrf2 translocation, ARE binding activity and enhanced stabilization of Nrf2 by inhibition of ubiquitination. Knockdown of Nrf2 abolished the effect of EX4 on increased insulin secretion. Inhibition of PKC&dgr; attenuated Nrf2 translocation and antioxidative gene expression by EX4 treatment. We suggest that EX4 activates and stabilizes Nrf2 through PKC&dgr; activation, contributing to the increase of antioxidant gene expression and consequently improving beta cell function in the presence of oxidative stress. HIGHLIGHTSEX4 protects against oxidative stress‐induced pancreatic beta cell dysfunction.EX4 increases antioxidant gene expression.Antioxidative effect of EX4 is mediated by Nrf2.EX4 increases Nrf2 level by stabilizing Nrf2 protein.EX4 stabilizes Nrf2 by activation of PKC&dgr;.

Baicalein protects rat insulinoma INS-1 cells from palmitate-induced lipotoxicity by inducing HO-1

Objective β-Cell dysfunction plays a central role in the pathogenesis of type 2 diabetes (T2D), and the identification of novel approaches to improve β-cell function is essential to treat this disease. Baicalein, a flavonoid originally isolated from the root of Scutellaria Baicalensis, has been shown to have beneficial effects on β-cell function. Here, the authors investigated the molecular mechanism responsible for the protective effects of baicalein against palmitate (PA)-induced impaired β-cell function, and placed focus on the role of heme oxygenase (HO)-1. Methods Rat pancreatic β-cell line INS-1 cells or mouse pancreatic islets were cultured with PA (500 μM) to induce lipotoxicity in the presence or absence of baicalein (50 μM), and the expressions of the ER stress markers, ATF-3, CHOP and GRP78 were detected by Western blotting and/or qPCR. The involvement of HO-1 was evaluated by HO-1 siRNA transfection and using the HO-1 inhibitor ZnPP. Results Baicalein reduced PA-induced ER stress and inflammation and enhanced insulin secretion, and these effects were associated with the induction of HO-1. Furthermore, these protective effects were attenuated by ZnPP and by HO-1 siRNA. Pretreatment of PD98059 (an ERK inhibitor) significantly inhibited the protective effects of baicalein and blocked HO-1 induction. On the other hand, CO production by RuCO (a CO donor) ameliorated PA-induced ER stress, suggesting that CO production followed by HO-1 induction may contribute to the protective effects of baicalein against PA-induced β-cell dysfunction. Conclusion Baicalein protects pancreatic β-cells from PA-induced ER stress and inflammation via an ERK-HO-1 dependent pathway. The authors suggest HO-1 induction in pancreatic β-cells appears to be a promising therapeutic strategy for T2D.

The interaction domains of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heteromultimeric channels

Transient receptor potential canonical (TRPC) family contains a non-selective cation channel, and four TRPC subunits form a functional tetrameric channel. TRPC4/5 channels form not only the homotetrameric channel but also a heterotetrameric channel with TRPC1. We investigated the interaction domain required for TRPC1/4 or TRPC1/5 heteromultimeric channels using FRET and the patch-clamp technique. TRPC1 only localized at the plasma membrane (PM) when it was coexpressed with TRPC4 or TRPC5. The TRPC1/4 or TRPC1/5 heteromultimeric showed the typical outward rectifying I/V curve. When TRPC1 and TRPC4 form a heteromeric channel, the N-terminal coiled-coil domain (CCD) and C-terminal 725-745 region of TRPC1 interact with the N-terminal CCD and C-terminal 700-728 region of TRPC4. However, when TRPC1 and TRPC5 form a heteromeric channel, the N-terminal CCD and C-terminal 673-725 region of TRPC1 interact with the N-terminal CCD and C-terminal 707-735 region of TRPC5. In conclusion, the N-terminal CCD of TRPC channels is essential for the heteromultimeric structure of TRPC channels, whereas specific C-terminal regions are required for unique heteromerization between subgroups of TRPC channels.

Dexmedetomidine Modulates Histamine-induced Ca(2+) Signaling and Pro-inflammatory Cytokine Expression.

Dexmedetomidine is a sedative and analgesic agent that exerts its effects by selectively agonizing α2 adrenoceptor. Histamine is a pathophysiological amine that activates G protein-coupled receptors, to induce Ca2+ release and subsequent mediate or progress inflammation. Dexmedetomidine has been reported to exert inhibitory effect on inflammation both in vitro and in vivo studies. However, it is unclear that dexmedetomidine modulates histamine-induced signaling and pro-inflammatory cytokine expression. This study was carried out to assess how dexmedetomidine modulates histamine-induced Ca2+ signaling and regulates the expression of pro-inflammatory cytokine genes encoding interleukin (IL)-6 and -8. To elucidate the regulatory role of dexmedetomidine on histamine signaling, HeLa cells and human salivary gland cells which are endogenously expressed histamine 1 receptor were used. Dexmedetomidine itself did not trigger Ca2+ peak or increase in the presence or absence of external Ca2+. When cells were stimulated with histamine after pretreatment with various concentrations of dexmedetomidine, we observed inhibited histamine-induced [Ca2+]i signal in both cell types. Histamine stimulated IL-6 mRNA expression not IL-8 mRNA within 2 hrs, however this effect was attenuated by dexmedetomidine. Collectively, these findings suggest that dexmedetomidine modulates histamine-induced Ca2+ signaling and IL-6 expression and will be useful for understanding the antagonistic properties of dexmedetomidine on histamine-induced signaling beyond its sedative effect.

Calcium permeability of transient receptor potential canonical (TRPC) 4 channels measured by TRPC4-GCaMP6s

Conflicting evidence has been obtained regarding whether transient receptor potential cation channels (TRPC) are store-operated channels (SOCs) or receptor-operated channels (ROCs). Moreover, the Ca/Na permeability ratio differs depending on whether the current-voltage (I-V) curve has a doubly rectifying shape or inward rectifying shape. To investigate the calcium permeability of TRPC4 channels, we attached GCaMP6s to TRPC4 and simultaneously measured the current and calcium signals. A TRPC4 specific activator, (–)-englerin A, induced both current and calcium fluorescence with the similar time course. Muscarinic receptor stimulator, carbachol, also induced both current and calcium fluorescence with the similar time course. By forming heteromers with TRPC4, TRPC1 significantly reduced the inward current with outward rectifying I-V curve, which also caused the decrease of calcium fluorescence intensity. These results suggest that GCaMP6s attached to TRPC4 can detect slight calcium changes near TRPC4 channels. Consequently, TRPC4-GCaMP6s can be a useful tool for testing the calcium permeability of TRPC4 channels.