Hyun Jun Jung

Aquaporin-5: A Marker Protein for Proliferation and Migration of Human Breast Cancer Cells

Aquaporin (AQP) is a family of transmembrane proteins for water transport. Recent studies revealed that AQPs are likely to play a role in tumor progression and invasion. We aimed to examine the potential role of AQP5 in the progression of human breast cancer cells. Expression of AQP5 mRNA and protein was seen in human breast cancer cell line (both MCF7 and MDA-MB-231) by RT-PCR and immunoblotting analysis. Immunoperoxidase labeling of AQP5 was observed at ductal epithelial cells of human breast tissues. In benign tumor, AQP5 labeling was mainly seen at the apical domains of ductal epithelial cells. In contrast, in invasive ductal carcinoma, prominent AQP5 labeling was associated with cancer cells, whereas some ducts were unlabeled and apical polarity of AQP5 in ducts was lost. Cell proliferation (BrdU incorporation assay) and migration of MCF7 cells were significantly attenuated by lentivirus-mediated AQP5-shRNA transduction. Hyperosmotic stress induced by sorbitol treatment (100 mM, 24 h) reduced AQP5 expression in MCF7 cells, which was also associated with a significant reduction in cell proliferation and migration. Taken together, prominent AQP5 expression in breast cancer cells with the loss of polarity of ductal epithelial cells was seen during the progression of breast carcinoma. shRNA- or hyperosmotic stress-induced reduction in AQP5 expression of MCF7 cells was associated with significantly reduced cell proliferation and migration. In conclusion, AQP5 overexpression is likely to play a role in cell growth and metastasis of human breast cancer and could be a novel target for anti-breast cancer treatment.

Fungicidal effect of resveratrol on human infectious fungi.

Resveratrol, a phenolic antioxidant found in grapes, has been known to mediate various biological activities on the human body. In the present study, we tested the antifungal activity of resveratrol against human pathogenic fungi before carrying out further studies to elucidate the antifungal mechanism(s) of resveratrol. Resveratrol displayed potent antifungal activity against human pathogenic fungi at concentration levels of 10–20 μg/mL Furthermore, time-kill curve exhibited fungicidal effect of resveratrol on C. albicans, but the compound had no hemolytic activity against human erythrocytes. The destruction of C. albicans cells by resveratrol was confirmed by scanning electron microscopy. These results suggest that resveratrol could be employed as a therapeutic agent to treat fungal infections of humans.

Antimicrobial property of (+)-lyoniresinol-3α-O-β-d-Glucopyranoside isolated from the root bark ofLycium chinense Miller against human pathogenic microorganisms

Abstract(+)-Lyoniresinol-3α-O-β-d-glucopyranoside (1) was isolated from an ethyl acetate extract of the root bark fromLycium chinense Miller, and its structure was determined using 1D and 2D NMR spectroscopy including DEPT, HMQC, and HMBC. (+)-Lyoniresinol-3α-O-β-d-glucopyranoside exhibited potent antimicrobial activity against antibiotic-resistant bacterial strains, methicillinresistantStaphylococcus aureus (MRSA) isolated from patients, and human pathogenic fungi without having any hemolytic effect on human erythrocytes. In particular, compound1 induced the accumulation of intracellular trehalose onC. albicans as stress response to the drug, and disrupted the dimorphic transition that forms pseudo-hyphae caused by the pathogenesis. This indicates that (+)-lyoniresinol-3α-O-β-d-glucopyranoside has excellent potential as a lead compound for the development of antibiotic agents.

E3 ubiquitin-protein ligases in rat kidney collecting duct: response to vasopressin stimulation and withdrawal

The E3 ubiquitin (Ub)-protein ligases (E3s) play a role as regulators of protein trafficking and degradation. We aimed to integrate the profile of E3s in rat kidney and examine the changes in protein abundance of the selected E3s in response to 1-deamino-8-D-arginine vasopressin (dDAVP) stimulation/withdrawal. Sprague-Dawley rats were infused with vehicle (n = 13), dDAVP for 5 days (n = 13), or dDAVP was withdrawn for periods (15 min, 30 min, 1, 3, 6, 12, or 24 h) after 5-day infusion (n = 46). Total RNA was isolated from the inner medulla (IM) for transcriptome analysis. Plasma membrane (PM)- or intracellular vesicle (ICV)-enriched fractions of whole kidney were immunoisolated for liquid chromatography-tandem mass spectrometry analysis. dDAVP infusion for 5 days (D5d) significantly increased urine osmolality, which was maintained during 3-h withdrawal of dDAVP after 5-day infusion (D5d-3h). Consistent with this, aquaporin-2 (AQP2) expression in the PM fractions of D5d and D5d-3h increased, whereas AQP2 expression in the ICV fractions of D5d-3h was further increased, indicating internalization of AQP2. Transcriptome analysis revealed 86 genes of E3s and LC-MS/MS analysis demonstrated 16 proteins of E3s. Among these, seven E3s (BRCA1, UBR4, BRE1B, UHRF1, NEDD4, CUL5, and FBX6) were shared. RT-PCR demonstrated mRNA expressions of the seven identified E3s in the kidney, and immunoblotting demonstrated changes in protein abundance of the selected E3s (BRE1B, NEDD4, and CUL5) in response to dDAVP stimulation/withdrawal or lithium-induced nephrogenic diabetes insipidus. The rate of AQP2 degradation was retarded in mpkCCDc14 cells with small interfering RNA-mediated knockdown of NEDD4 or CUL5. Taken together, identified E3s could be involved in the degradation of proteins associated with vasopressin-induced urine concentration.

Emerging role of Akt substrate protein AS160 in the regulation of AQP2 translocation

AS160, a novel Akt substrate of 160 kDa, contains a Rab GTPase-activating protein (GAP) domain. The present study examined the role of Akt and AS160 in aquaporin-2 (AQP2) trafficking. The main strategy was to examine the changes in AQP2 translocation in response to small interfering RNA (siRNA)-mediated AS160 knockdown in mouse cortical collecting duct cells (M-1 cells and mpkCCDc14 cells). Short-term dDAVP treatment in M-1 cells stimulated phosphorylation of Akt (S473) and AS160, which was also seen in mpkCCDc14 cells. Conversely, the phosphoinositide 3-kinase (PI3K) inhibitor LY 294002 diminished phosphorylation of Akt (S473) and AS160. Moreover, siRNA-mediated Akt1 knockdown was associated with unchanged total AS160 but decreased phospho-AS160 expression, indicating that phosphorylation of AS160 is dependent on PI3K/Akt pathways. siRNA-mediated AS160 knockdown significantly decreased total AS160 and phospho-AS160 expression. Immunocytochemistry revealed that AS160 knockdown in mpkCCDc14 cells was associated with increased AQP2 density in the plasma membrane [135 ± 3% of control mpkCCDc14 cells (n = 65), P < 0.05, n = 64] despite the absence of dDAVP stimulation. Moreover, cell surface biotinylation assays of mpkCCDc14 cells with AS160 knockdown exhibited significantly higher AQP2 expression [150 ± 15% of control mpkCCDc14 cells (n = 3), P < 0.05, n = 3]. Taken together, PI3K/Akt pathways mediate the dDAVP-induced AS160 phosphorylation, and AS160 knockdown is associated with higher AQP2 expression in the plasma membrane. Since AS160 contains a GAP domain leading to a decrease in the active GTP-bound form of AS160 target Rab proteins for vesicle trafficking, decreased expression of AS160 is likely to play a role in the translocation of AQP2 to the plasma membrane.

Tankyrase-mediated β-catenin activity regulates vasopressin-induced AQP2 expression in kidney collecting duct mpkCCDc14 cells

Aquaporin-2 (AQP2) mediates arginine vasopressin (AVP)-induced water reabsorption in the kidney collecting duct. AVP regulates AQP2 expression primarily via Gsα/cAMP/PKA signaling. Tankyrase, a member of the poly(ADP-ribose) polymerase family, is known to mediate Wnt/β-catenin signaling-induced gene expression. We examined whether tankyrase plays a role in AVP-induced AQP2 regulation via ADP-ribosylation of G protein-α (Gα) and/or β-catenin-mediated transcription of AQP2. RT-PCR and immunoblotting analysis revealed the mRNA and protein expression of tankyrase in mouse kidney and mouse collecting duct mpkCCDc14 cells. dDAVP-induced AQP2 upregulation was attenuated in mpkCCDc14 cells under the tankyrase inhibition by XAV939 treatment or small interfering (si) RNA knockdown. Fluorescence resonance energy transfer image analysis, however, revealed that XAV939 treatment did not affect dDAVP- or forskolin-induced PKA activation. Inhibition of tankyrase decreased dDAVP-induced phosphorylation of β-catenin (S552) and nuclear translocation of phospho-β-catenin. siRNA-mediated knockdown of β-catenin decreased forskolin-induced AQP2 transcription and dDAVP-induced AQP2 expression. Moreover, inhibition of phosphoinositide 3-kinase/Akt, which was associated with decreased nuclear translocation of β-catenin, diminished dDAVP-induced AQP2 upregulation, further indicating that β-catenin mediates AQP2 expression. Taken together, tankyrase plays a role in AVP-induced AQP2 regulation, which is likely via β-catenin-mediated transcription of AQP2, but not ADP-ribosylation of Gα. The results provide novel insights into vasopressin-mediated urine concentration and homeostasis of body water metabolism.

Synergistic antibacterial effect between silybin and N,N'-dicyclohexylcarbodiimide in clinical Pseudomonas aeruginosa isolates.

Silybin is a composition of the silymarin group as a hepatoprotective agent, and it exhibits various biological activities, including an antibacterial activity. In this study, the effects of a combination of silybin with N,N′-dicyclohexylcarbodiimide (DCCD) against clinical isolates of Pseudomonas aeruginosa were investigated. In the results of susceptibility assay, silybin showed more potent antibacterial activity in methicilin-resistant Staphylococcus aureus (MRSA) than in P. aeruginosa, but DCCD significantly increased the antibacterial activity of silybin in P. aeruginosa. The antibacterial activity of silybin was affected by the strong action of multidrug-resistant pumps rather than by a permeable disruption of lipopolysaccharide and silybin showed a remarkable synergistic activity in combination with some antibiotic agents against drug-resistant bacteria. Therefore, silybin has a potential as a combination therapeutic agent for treatment of infectious diseases by multidrug-resistant bacteria.

Extracellular pH affects phosphorylation and intracellular trafficking of AQP2 in inner medullary collecting duct cells

Kidney collecting duct cells are continuously exposed to the changes of extracellular pH (pHe). We aimed to study the effects of altered pHe on desmopressin (dDAVP)-induced phosphorylation (Ser(256), Ser(261), Ser(264), and Ser(269)) and apical targeting of aquaporin-2 (AQP2) in rat kidney inner medullary collecting duct (IMCD) cells. When freshly prepared IMCD tubule suspensions exposed to HEPES buffer with pH 5.4, 6.4, 7.4, or 8.4 for 1 h were stimulated with dDAVP (10(-10) M, 3 min), AQP2 phosphorylation at Ser(256), Ser(264), and Ser(269) was significantly attenuated under acidic conditions. Next, IMCD cells primary cultured in transwell chambers were exposed to a transepithelial pH gradient for 1 h (apical pH 6.4, 7.4, or 8.4 vs. basolateral pH 7.4 and vice versa). Immunocytochemistry and cell surface biotinylation assay revealed that exposure to either apical pH 6.4 or basolateral pH 6.4 for 1 h was associated with decreased dDAVP (10(-9) M, 15 min, basolateral)-induced apical targeting of AQP2 and surface expression of AQP2. Fluorescence resonance energy transfer analysis revealed that the dDAVP (10(-9) M)-induced increase of PKA activity was significantly attenuated when LLC-PK1 cells were exposed to pHe 6.4 compared with pHe 7.4 and 8.4. In contrast, forskolin (10(-7) M)-induced PKA activation and dDAVP (10(-9) M)-induced increases of intracellular Ca(2+) were not affected. Taken together, dDAVP-induced phosphorylation and apical targeting of AQP2 are attenuated in IMCD cells under acidic pHe, likely via an inhibition of vasopressin V2 receptor-G protein-cAMP-PKA actions.

Membrane Trafficking of Collecting Duct Water Channel Protein AQP2 Regulated by Akt/AS160

Akt (protein kinase B (PKB)) is a serine/threonine kinase that acts in the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The PI3K/Akt signaling pathway, triggered by growth factors and hormones including vasopressin, is an important pathway that is widely involved in cellular mechanisms regulating transcription, translation, cell growth and death, cell proliferation, migration, and cell cycles. In particular, Akt and Akt substrate protein of 160 kDa (AS160) are likely to participate in the trafficking of aquaporin-2 (AQP2) in the kidney collecting duct. In this study, we demonstrated that 1) small interfering RNA (siRNA)-mediated gene silencing of Akt1 significantly decreased Akt1 and phospho-AS160 protein expression; and 2) confocal laser scanning microscopy of AQP2 in mouse cortical collecting duct cells (M-1 cells) revealed AS160 knockdown by siRNA increased AQP2 expression in the plasma membrane compared with controls, despite the absence of dDAVP stimulation. Thus, the results suggest that PI3K/Akt pathways could play a role in AQP2 trafficking via the AS160 protein.

Vasopressin V2R-Targeting Peptide Carrier Mediates siRNA Delivery into Collecting Duct Cells

Internalization of receptor proteins after interacting with specific ligands has been proposed to facilitate siRNA delivery into the target cells via receptor-mediated siRNA transduction. In this study, we demonstrated a novel method of vasopressin V2 receptor (V2R)-mediated siRNA delivery against AQP2 in primary cultured inner medullary collecting duct (IMCD) cells of rat kidney. We synthesized the dDAVP conjugated with nine D-arginines (dDAVP-9r) as a peptide carrier for siRNA delivery. The structure of synthetic peptide carrier showed two regions (i.e., ligand domain to V2R (dDAVP) and siRNA carrying domain (nine D-arginine)) bisected with a spacer of four glycines. The results revealed that 1) synthesized dDAVP-9r peptides formed a stable polyplex with siRNA; 2) siRNA/dDAVP-9r polyplex could bind to the V2R of IMCD cells and induced AQP2 phosphorylation (Ser 256); 3) siRNA/dDAVP-9r polyplex was stable in response to the wide range of different osmolalities, pH levels, or to the RNases; 4) fluorescein-labeled siRNA was delivered into V2R-expressing MDCK and LLC-PK1 cells by siRNA/dDAVP-9r polyplex, but not into the V2R-negative Cos-7 cells; and 5) AQP2-siRNA/dDAVP-9r polyplex effectively delivered siRNA into the IMCD cells, resulting in the significant decrease of protein abundance of AQP2, but not AQP4. Therefore, for the first time to our knowledge, we demonstrated that V2R-mediated siRNA delivery could be exploited to deliver specific siRNA to regulate abnormal expression of target proteins in V2R-expressing kidney cells. The methods could be potentially used in vivo to regulate abnormal expression of proteins associated with disease conditions in the V2R-expressing kidney cells.