Sung-Young Kim

Autophagy Impairment Induces Premature Senescence in Primary Human Fibroblasts

Background Recent studies have demonstrated that activation of autophagy increases the lifespan of organisms from yeast to flies. In contrast to the lifespan extension effect in lower organisms, it has been reported that overexpression of unc-51-like kinase 3 (ULK3), the mammalian homolog of autophagy-specific gene 1 (ATG1), induces premature senescence in human fibroblasts. Therefore, we assessed whether the activation of autophagy would genuinely induce premature senescence in human cells. Methodology/Principal Findings Depletion of ATG7, ATG12, or lysosomal-associated membrane protein 2 (Lamp2) by transfecting siRNA or infecting cells with a virus containing gene-specific shRNA resulted in a senescence-like state in two strains of primary human fibroblasts. Prematurely senescent cells induced by autophagy impairment exhibited the senescent phenotypes, similar to the replicatively senescent cells, such as increased senescence associated β-galactosidase (SA-β-gal) activity, reactive oxygen species (ROS) generation, and accumulation of lipofuscin. In addition, expression levels of ribosomal protein S6 kinase1 (S6K1), p-S6K1, p-S6, and eukaryotic translation initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) in the mammalian target of rapamycin (mTOR) pathway and beclin-1, ATG7, ATG12-ATG5 conjugate, and the sequestosome 1 (SQSTM1/p62) monomer in the autophagy pathway were decreased in both the replicatively and the autophagy impairment-induced prematurely senescent cells. Furthermore, it was found that ROS scavenging by N-acetylcysteine (NAC) and inhibition of p53 activation by pifithrin-α or knockdown of p53 using siRNA, respectively, delayed autophagy impairment-induced premature senescence and restored the expression levels of components in the mTOR and autophagy pathways. Conclusion Taken together, we concluded that autophagy impairment induces premature senescence through a ROS- and p53-dependent manner in primary human fibroblasts.

The role of transient receptor potential channel blockers in human gastric cancer cell viability

Transient receptor potential cation channel, subfamily M, receptor 7 (TRPM7) is a ubiquitous divalent-selective ion channel with its own kinase domain. Human gastric cancer cells express the TRPM7 channel, and the presence of this channel is essential for cell survival. Recent studies have suggested that 5-lipoxygenase (5-LOX) inhibitors are potent blockers of the TRPM7 channels. The aim of this study was to show the effects of 5-LOX inhibitors on the growth and survival of gastric cancer cells. Among 5-LOX inhibitors, nordihydroguaiaretic acid (NDGA), 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), and 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2,2-dimethylpropanoic acid (MK886) were potent blockers of TRPM7-like currents in gastric cancer cells and also induced cell death. However, zileuton was ineffective in suppressing TRPM7-like current activity and inducing cell death. Moreover, a specific transient receptor potential cation channel, subfamily C, member 3 (TRPC3) inhibitor, a pyrazole compound (Pyr3), and a specific melastatin TRP (TRPM4) inhibitor, 9-phenanthrol, did not affect TRPM7-like currents or induce cell death. We conclude that TRPM7 has an important role in the growth and survival of gastric cancer cells and a likely potential target for the pharmacological treatment of gastric cancer.

Quercetin induces apoptosis by inhibiting MAPKs and TRPM7 channels in AGS cells.

The worldwide incidence and mortality rate of gastric cancer remain high, and thus, novel treatment concepts are required. Quercetin, a bioflavonoid, has been proposed to have anti-cancer properties. The aim of this study was to determine the nature of the apoptotic mechanisms responsible for the effects of quercetin on AGS cells (a commonly used human gastric adenocarcinoma cell line). AGS cell viability was assessed by MTT assay and flow cytometric analysis, mitochondrial membrane depolarization was assessed, and caspase-3 was used to determine the involvement of apoptosis. Whole-cell configuration patch-clamp experiments were used to regulate the transient receptor potential melastatin (TRPM)7 channels. To investigate the signaling pathway of quercetin-induced apoptosis in the AGS cells, western blot analysis and MTT assay were performed. Quercetin was found to induce the apoptosis of these cells, and this apoptosis was inhibited by SB203580 (a p38 kinase inhibitor), SP600125 (a JNK inhibitor) and PD98059 (an ERK inhibitor). In addition, quercetin inhibited TRPM7 currents in the AGS cells and in human embryo kidney (HEK)293 cells which overexpress TRPM7 channels. Furthermore, treatment with quercetin increased the apoptosis of HEK293 cells, which overexpress TRPM7, indicating that the upregulation of TRPM7 channels underlies quercetin-induced cell death. These results suggest that quercetin plays an important pathophysiological role in AGS cells through mitogen‑activated protein kinase (MAPK) signaling pathways and TRPM7 channels, and that quercetin has potential as a pharmacological agent for the treatment of gastric cancer.

Biliverdin reductase A in the prevention of cellular senescence against oxidative stress

Biliverdin reductase A (BLVRA), an enzyme that converts biliverdin to bilirubin, has recently emerged as a key regulator of the cellular redox cycle. However, the role of BLVRA in the aging process remains unclear. To study the role of BLVRA in the aging process, we compared the stress responses of young and senescent human diploid fibroblasts (HDFs) to the reactive oxygen species (ROS) inducer, hydrogen peroxide (H2O2). H2O2 markedly induced BLVRA activity in young HDFs, but not in senescent HDFs. Additionally, depletion of BLVRA reduced the H2O2-dependent induction of heme oxygenase-1 (HO-1) in young HDFs, but not in senescent cells, suggesting an aging-dependent differential modulation of responses to oxidative stress. The role of BLVRA in the regulation of cellular senescence was confirmed when lentiviral RNAitransfected stable primary HDFs with reduced BLVRA expression showed upregulation of the CDK inhibitor family members p16, p53, and p21, followed by cell cycle arrest in G0-G1 phase with high expression of senescence-associated β-galactosidase. Taken together, these data support the notion that BLVRA contributes significantly to modulation of the aging process by adjusting the cellular oxidative status.

Senescence-related functional nuclear barrier by down-regulation of nucleo-cytoplasmic trafficking gene expression

One of the characteristic natures of senescent cells is the hypo- or irresponsiveness not only to growth factors but also to apoptotic stress. In the present study, we confirmed the inhibition of nuclear translocation of activated p-ERK1/2 and NF-kB p50 in response to growth stimuli or LPS in the senescent human diploid fibroblasts. In order to elucidate the underlying mechanism for the senescence-associated hypo-responsiveness, we carried out the comparison study for gene expression profiles through microarray analysis. In consequence, we observed the vast reduction in expression of nucleo-cytoplasmic trafficking genes in senescent cells, when compared with those in young cells. Expression levels of several nucleoporins, karyopherin alpha, karyopherin beta, Ran, and Ran-regulating factors were confirmed to be down-regulated in senescent HDFs by using RT-PCR and Western blot methods. Taken together, these data suggest the operation of certain senescence-associated functional nuclear barriers by down-regulation of the nucleo-cytoplasmic trafficking genes in the senescent cells.

Molecular determinants of PKA-dependent inhibition of TRPC5 channel

Canonical transient receptor potential (TRPC) channels are Ca(2+)-permeable, nonselective cation channels that are widely expressed in numerous cell types. Here, we demonstrate a new mechanism of TPRC isofom 5 (TRPC5) regulation, via cAMP signaling via Gα(s). Monovalent cation currents in human embryonic kidney-293 cells transfected with TRPC5 were induced by G protein activation with intracellular perfusion of GTPγS or by muscarinic stimulation. This current could be inhibited by a membrane-permeable analog of cAMP, 8-bromo-cAMP, by isoproterenol, by a constitutively active form of Gα(s) [Gα(s) (Q227L)], and by forskolin. These inhibitory effects were blocked by the protein kinase A (PKA) inhibitors, KT-5720 and H-89, as well as by two point mutations at consensus PKA phosphorylation sites on TRPC5 (S794A and S796A). Surface expression of several mutated versions of TRPC5, quantified using surface biotinylation, were not affected by Gα(s) (Q227L), suggesting that trafficking of this channel does not underlie the regulation we report. This mechanism of inhibition was also found to be important for the closely related channel, TRPC4, in particular for TRPC4α, although TRPC4β was also affected. However, this form of regulation was not found to be involved in TRPC6 and transient receptor potential vanilloid 6 function. In murine intestinal smooth muscle cells, muscarinic stimulation-induced cation currents were mediated by TRPC4 (>80%) and TRPC6. In murine intestinal smooth muscle cells, 8-bromo-cAMP, adrenaline, and isoproterenol decreased nonselective cation currents activated by muscarinic stimulation or GTPγS. Together, these results suggest that TRPC5 is directly phosphorylated by G(s)/cAMP/PKA at positions S794 and S796. This mechanism may be physiologically important in visceral tissues, where muscarinic receptor and β(2)-adrenergic receptor are involved in the relaxation and contraction of smooth muscles.

Isoform- and receptor-specific channel property of canonical transient receptor potential (TRPC)1/4 channels.

Transient receptor potential canonical (TRPC) 1, the first mammalian homologue of Drosophila trp gene, is distributed widely in mammalian cells and is involved in many physiological functions. TRPC1 is reported to be functional following heteromeric formation with other TRPC channels such as TRPC4 or TRPC5. It is known that the composition of this widely distributed TRPC1 is far from simple; functionality of such channels has been highly controversial. Furthermore, TRPC1 gene is known to have two splicing variants; one encodes long (TRPC1α) and the other encodes short (TRPC1β) TRPC1 isoforms, respectively. In this study, we examined the functionality of TRPC1/4 channels using various activation systems. Gq/11-coupled receptor (e.g., M1 or M3 receptors) stimulation significantly increased TRPC1α/4 currents but induced mild activation of TRPC1β/4. In addition, when expressed with TRPC4, TRPC1α acted as a pore-constituting subunit and not a β ancillary subunit. Multimerized with TRPC4, TRPC1α also generated strong pore field strength. We also found that Gi/o-coupled receptor (e.g., M2 receptor) stimulation was insufficient to activate TRPC1α/4 and TRPC1β/4 channels but selectively activated TRPC4 homomeric channels. These findings demonstrate that TRPC1/4 channel shows dynamic gating property depending on TRPC1 isoform subtypes and receptor stimulation system. Therefore, careful discrimination of the specificity of TRPC1 isoforms and upstream activation system is important in thorough understanding of TRPC1 and TRPC1/4 channels.Transient receptor potential canonical (TRPC) 1, the first mammalian homologue of Drosophila trp gene, is distributed widely in mammalian cells and is involved in many physiological functions. TRPC1 is reported to be functional following heteromeric formation with other TRPC channels such as TRPC4 or TRPC5. It is known that the composition of this widely distributed TRPC1 is far from simple; functionality of such channels has been highly controversial. Furthermore, TRPC1 gene is known to have two splicing variants; one encodes long (TRPC1α) and the other encodes short (TRPC1β) TRPC1 isoforms, respectively. In this study, we examined the functionality of TRPC1/4 channels using various activation systems. Gq/11-coupled receptor (e.g., M1 or M3 receptors) stimulation significantly increased TRPC1α/4 currents but induced mild activation of TRPC1β/4. In addition, when expressed with TRPC4, TRPC1α acted as a pore-constituting subunit and not a β ancillary subunit. Multimerized with TRPC4, TRPC1α also generated strong pore field strength. We also found that Gi/o-coupled receptor (e.g., M2 receptor) stimulation was insufficient to activate TRPC1α/4 and TRPC1β/4 channels but selectively activated TRPC4 homomeric channels. These findings demonstrate that TRPC1/4 channel shows dynamic gating property depending on TRPC1 isoform subtypes and receptor stimulation system. Therefore, careful discrimination of the specificity of TRPC1 isoforms and upstream activation system is important in thorough understanding of TRPC1 and TRPC1/4 channels.

Diet control to achieve euglycemia induces significant loss of heart and liver weight via increased autophagy compared with ad libitum diet in diabetic rats

Intensive glucose control increases the all-cause mortality in type 2 diabetes mellitus (T2DM); however, the underlying mechanisms remain unclear. We hypothesized that strict diet control to achieve euglycemia in diabetes damages major organs, increasing the mortality risk. To evaluate effects on major organs when euglycemia is obtained by diet control, we generated a model of end-stage T2DM in 13-week-old Sprague-Dawley rats by subtotal pancreatectomy, followed by ad libitum feeding for 5 weeks. We divided these rats into two groups and for the subsequent 6 weeks provided ad libitum feeding to half (AL, n=12) and a calorie-controlled diet to the other half (R, n=12). To avoid hypoglycemia, the degree of calorie restriction in the R group was isocaloric (g per kg body weight per day) compared with a sham-operated control group (C, n=12). During the 6-week diet control period, AL rats ate three times more than rats in the C or R groups, developing hyperglycemia with renal hyperplasia. R group achieved euglycemia but lost overall body weight significantly compared with the C or AL group (49 or 22%, respectively), heart weight (39 or 23%, respectively) and liver weight (50 or 46%, respectively). Autophagy levels in the heart and liver were the highest in the R group (P<0.01), which also had the lowest pAkt/Akt levels among the groups (P<0.05 in the heart; P<0.01 in the liver). In conclusion, glycemic control achieved by diet control can prevent hyperglycemia-induced renal hyperplasia in diabetes but may be deleterious even at isocaloric rate when insulin is deficient because of significant loss of heart and liver mass via increased autophagy.

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.

Characteristics of the Cholecystokinin-Induced Depolarization of Pacemaking Activity in Cultured Interstitial Cells of Cajal from Murine Small Intestine

Background/Aims: In this study, we studied the effects of cholecystokinin (CCK) on pacemaker potentials in cultured interstitial cells of Cajal (ICCs) from mouse small intestine using the whole cell patch clamp technique. Methods: ICCs are pacemaker cells that exhibit periodic spontaneous depolarization, which is responsible for the production of slow waves in gastrointestinal smooth muscle, and generate periodic pacemaker potentials in current-clamp mode. Results: Exposure to CCK (100 nM-5 µM) decreased the amplitudes of pacemaker potentials and depolarized resting membrane potentials. To identify the type of CCK receptors involved in ICCs, we examined the effects of CCK agonists and found that the addition of CCK1 agonist (A-71323, 1 µM) depolarized resting membrane potentials, whereas exposure to CCK2 agonist (gastrin , 1 µM) had no effect on pacemaker potentials. To confirm these results, we examined the effects of CCK antagonists and found that pretreatment with CCK1 antagonist (SR 27897, 1 µM) blocked CCK-induced effects. However, pretreatment with CCK2 antagonist (LY 225910, 1 µM) did not. Furthermore, intracellular GDPβS suppressed CCK-induced effects. To investigate the involvements of phospholipase C (PLC), protein kinase C (PKC), and protein kinase A (PKA) in the effects of CCK in cultured ICCs, we used U-73122 (an active PLC inhibitor), chelerythrine (a PKC inhibitor), SQ-22536 (an inhibitor of adenylate cyclase), or mPKAI (an inhibitor of myristoylated PKA). All inhibitors blocked the CCK-mediated effects on pacemaker potentials. In addition, we found that transient receptor potential classical 5 (TRPC5) channel was involved in CCK-activated currents in cultured ICCs. Conclusion: These results suggest that the CCK induced depolarization of pacemaking activity occurs in a G-protein-, PLC-, PKC-, and PKA-dependent manner via CCK1 receptor and TRPC5 channel is a candidate for CCK-activated currents in cultured ICCs in murine small intestine. Therefore, the ICCs are targets for CCK and their interaction can affect intestinal motility.