• Development and Reproduction
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• v.27 no.3
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• pp.149-157
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• 2023

We investigated the involvement of autophagy with steroidogenesis in testicular Leydig cells. Human chorionic gonadotropin (hCG)-stimulated T production in Leydig cells was not remarkably altered in the presence of an autophagy inhibitor 3-methyladenine (3-MA). Although pretreatment with 3-MA demonstrated a tendency to decrease hCG-induced T production, the differences were significant only at a higher time point of 24 h following hCG. Microtubule associated protein light chain 3 (LC3)-II was detectable in the control cells in all the experiments. The hCG-induced increase in steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleave (P450scc) protein levels were not significantly altered by 3-MA. Leydig cells isolated from immature rat testes 12 h following hCG treatment showed relatively increased levels of LC3-II protein compared to the control group. Furthermore, LC3-II levels shown in these cells reached almost the identical to those from normal adult testes. However, LC3-II protein levels were almost comparable or even slightly lower than the controls at 48 h following hCG. Expression of StAR and P450scc was upregulated at both 12 and 48 h after hCG. We also used MA-10 cells, the mouse Leydig cell line, in this experiment. When dibutyryl cyclic-AMP was treated with MA-10 cells, P4 levels were significantly increased in the cell culture medium. However, P4 levels tended to decrease in the presence of 3-MA, but the difference was not statistically significant. This was consistent with the results of the rat Leydig cell experiments. Together, we believe that although autophagy participates in steroidogenesis and enhances steroidogenic efficacy of Leydig cells, it may not be a decisive cellular process for steroidogenesis, specifically in the mature Leydig cells.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.4
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• pp.592-598
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• 2019

Objective: Autophagy is a bulk degradation system for intracellular proteins which contributes to skeletal muscle homeostasis, according to previous studies in humans and rodents. However, there is a lack of information on the physiological role of autophagy in the skeletal muscle of meat animals. This study was planned as a pilot study to investigate changes in expression of two major autophagy-related genes, microtubule-associated protein 1 light chain $3{\beta}$ (MAP1LC3B) and autophagy related 7 (ATG7) in fattening beef cattle, and to compare them with skeletal muscle growth. Methods: Six castrated Japanese Black cattle (initial body weight: $503{\pm}20kg$) were enrolled in this study and fattened for 7 months. Three skeletal muscles, M. longissimus, M. gluteus medius, and M. semimembranosus, were collected by needle biopsy three times during the observation period, and mRNA levels of MAP1LC3B and ATG7 were determined by quantitative reverse-transcription polymerase chain reaction. The expression levels of genes associated with the ubiquitin-proteasome system, another proteolytic mechanism, were also analyzed for comparison with autophagy-related genes. In addition, ultrasonic scanning was repeatedly performed to measure M. longissimus area as an index of muscle growth. Results: Our results showed that both MAP1LC3B and ATG7 expression increased over the observation period in all three skeletal muscles. Interestingly, the increase in expression of these two genes in M. longissimus was highly correlated with ultrasonic M. longissimus area and body weight. On the other hand, the expression of genes associated with the ubiquitin-proteasome system was unchanged during the same period. Conclusion: These findings suggest that autophagy plays an important role in the growth of skeletal muscle of fattening beef cattle and imply that autophagic activity affects meat productivity.

• Journal of Life Science
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• v.23 no.8
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• pp.978-983
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• 2013

Kinesin-II, a molecular motor, consists of two different motor subunits, KIF3A and KIF3B, and one large kinesin superfamily-associated protein 3 (KAP3), forming a heterotrimeric complex. KAP3 is associated with the tail domains of motor subunits. However, its exact role remains unclear. Here, we demonstrated KAP3 binding to the carboxyl (C)-terminal tail region of HS-associated protein X-1 (HAX-1). HAX-1 bound to the C-terminal region of KAP3, but not to KIFs (KIF3A, KIF3B, and KIF5B) and the kinesin light chain (KLC) in the yeast two-hybrid assays. The interaction was further confirmed in the glutathione S-transferase (GST) pull-down assay and by co-immunoprecipitation. Anti- HAX-1 antibody as well as anti-KIF3A antibody co-immunoprecipitated KIF3B and KAP3 from mouse brain extracts. These results suggest that KAP3 could mediate the interaction between Kinesin-II and HAX-1.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.3
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• pp.349-360
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• 2018

Autophagy has been studied as a therapeutic strategy for cardiovascular diseases. However, insufficient studies have been reported concerning the influence of vascular smooth muscle cells (VSMCs) through autophagy regulation. The aim of the present study was to determine the effects of VSMCs on the regulation of autophagy under in vitro conditions similar to vascular status of the equipped micro-tubule target agent-eluting stent and increased release of platelet-derived growth factor-BB (PDGF-BB). Cell viability and proliferation were measured using MTT and cell counting assays. Immunofluorescence using an $anti-{\alpha}-tubulin$ antibody was performed to determine microtubule dynamic formation. Cell apoptosis was measured by cleavage of caspase-3 using western blot analysis, and by nuclear fragmentation using a fluorescence assay. Autophagy activity was assessed by microtubule-associated protein light chain 3-II (LC-II) using western blot analysis. Levels of intracellular reactive oxygen species (ROS) were measured using $H_2DCFDA$. The proliferation and viability of VSMCs were inhibited by microtubule regulation. Additionally, microtubule-regulated and PDGF-BB-stimulated VSMCs increased the cleavage of caspase-3 more than only the microtubule-regulated condition, similar to that of LC3-II, implying autophagy. Inhibitory autophagy of microtubule-regulated and PDGF-BB-stimulated VSMCs resulted in low viability. However, enhancement of autophagy maintained survival through the reduction of ROS. These results suggest that the apoptosis of conditioned VSMCs is decreased by the blocking generation of ROS via the promotion of autophagy, and proliferation is also inhibited. Thus, promoting autophagy as a therapeutic target for vascular restenosis and atherosclerosis may be a good strategy.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.1
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• pp.1-7
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• 2011

Many anticancer agents as well as ionizing radiation have been shown to induce autophagy which is originally described as a protein recycling process and recently reported to play a crucial role in various disorders. In HCT116 human colon cancer cells, we found that curcumin, a polyphenolic phytochemical extracted from the plant Curcuma longa, markedly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II and degradation of sequestome-1 (SQSTM1) which is a marker of autophagosome degradation. Moreover, we found that curcumin caused GFP-LC3 formation puncta, a marker of autophagosome, and decrease of GFP-LC3 and SQSTM1 protein level in GFP-LC3 expressing HCT116 cells. It was further confirmed that treatment of cells with hydrogen peroxide induced increase of LC3 conversion and decrease of GFP-LC3 and SQSTM1 levels, but these changes by curcumin were almost completely blocked in the presence of antioxidant, N-acetylcystein (NAC), indicating that curcumin leads to reactive oxygen species (ROS) production, which results in autophagosome development and autolysosomal degradation. In parallel with NAC, SQSTM1 degradation was also diminished by bafilomycin A, a potent inhibitor of autophagosome-lysosome fusion, and cell viability assay was further confirmed that cucurmin-induced cell death was partially blocked by bafilomycin A as well as NAC. We also observed that NAC abolished curcumin-induced activation of extracelluar signal-regulated kinases (ERK) 112 and p38 mitogen-activated protein kinases (MAPK), but not Jun N-terminal kinase (JNK). However, the activation of ERK1/2 and p38 MAPK seemed to have no effect on the curcumin-induced autophagy, since both the conversion of LC3 protein and SQSTM1 degradation by curcumin was not changed in the presence of NAC. Taken together, our data suggest that curcumin induced ROS production, which resulted in autophagic activation and concomitant cell death in HCT116 human colon cancer cell. However, ROS-dependent activation of ERK1/2 and p38 MAPK, but not JNK, might not be involved in the curcumin-induced autophagy.

• Journal of Life Science
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• v.33 no.7
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• pp.531-537
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• 2023

Intracellular and axonal transport is mediated by microtubule-dependent motor proteins, such as kinesins and cytoplasmic dynein. Kinesin moves along the microtubule to the positive end of the microtubule, while dynein moves to the negative end of the microtubule. Kinesin-1 was first identified as a kinesin superfamily protein (KIF) that functions in the intracellular transport of various cargoes, including organelles, neurotransmitter receptors, and mRNA-protein complexes, through interactions between the carboxyl (C)-terminal domain and the cargo. It interacts with other cargoes, but the adapter/scaffold proteins that mediate between kinesin-1 and the cargo have yet to be fully identified. In this study, a yeast two-hybrid screen was used to identify adapter proteins that interact with the C-terminal region of KIF5A. We found an association between the C-terminal region of KIF5A and the cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), originally identified in malignant hamster oral keratinocytes. CDK2AP1 bound to the C-terminal region of KIF5A and did not interact with KIF3A (the motor of kinesin-2), KIF5B, KIF5C, and kinesin light chain 1 (KLC1). The C-terminal region of CDK2AP1 is essential for its interaction with KIF5A. When co-expressed in HEK-293T cells, CDK2AP1 and kinesin-1 co-immunoprecipitated and co-localized in the cells. These results suggest that the KIF5A-CDK2AP1 interaction serves as an adapter protein connecting kinesin-1 and the cargo when kinesin-1 transports cargo in cells.

• Bulletin of the Korean Chemical Society
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• v.35 no.10
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• pp.2985-2989
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• 2014

Autophagy is a self-digestion process in which intracellular structures are degraded in response to stress. Notably, prolonged autophagy leads to cell death. In this study, we investigated whether CX-4945, an orally available protein kinase 2 (CK2) inhibitor, induces autophagic cell death in human cervical cancer-derived HeLa cells and in human prostate cancer-derived LNCaP cells. CX-4945 treatment of both cell lines resulted in the formation of autophagosomes, in the conversion of microtubule-associated protein 1 light chain 3 (LC3), and in down-regulation of the Akt-mammalian target of rapamycin (mTOR)-p70 ribosomal protein S6 kinase (S6K) signaling cascade. Thus, pharmacologic inhibition of CK2 by CX-4945 induced autophagic cell death in human cancer cells by down-regulating Akt-mTOR-S6K. These results suggest that autophagy-inducing agents have potential as anti-cancer drugs.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.48 no.2
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• pp.105-112
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• 2022

2'-fucosyllactose (2'-FL) is the most abundant human milk oligosaccharide (HMO) present in breast milk, promoting the growth of beneficial microorganisms in the gut and aiding in the relief of allergic and inflammatory reactions. In this study, the anti-melanogenic effects of 2'-FL, and its potential for application in whitening cosmetics, were evaluated. MTT assay was performed on MNT-1 cells, human-derived melanocytes. 2'-FL was treated and replaced at 48 h intervals for 7 days, and it was confirmed that there was no cytotoxicity at 20 g/L or less, while a 40% reduction in melanin production was also observed. Western blot analysis of TYR and TYRP1, factors involved in melanogenesis, revealed that 2'-FL treatment reduced their expression levels. In addition, 2'-FL application and observation of the autophagy marker microtubule-associated protein 1 light chain 3 (LC3) revealed it was converted from LC3-I to LC3-𝚷, indicating increased autophagy. Likewise, confocal microscopy revealed an increase in LC3 puncta after 2'-FL treatment. Therefore, it is suggested that 2'-FL-mediated activation of autophagy reduces melanogenesis by inhibiting the expression levels of TYR and TYRP1 proteins. In conclusion, it has been confirmed that 2'-FL induces autophagy and suppresses melanin production, so its potential as a whitening cosmetic material is expected.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.5
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• pp.449-456
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• 2023

N-methyl-D-aspartate (NMDA) receptors are ionic glutamine receptors involved in brain development and functions such as learning and memory formation. NMDA receptor inhibition is associated with autophagy activation. In this study, we investigated whether the NMDA receptor antagonists, memantine and ifenprodil, induce autophagy in human retinal pigment epithelial cells (ARPE-19) to remove N-retinylidene-N-retinylethanolamine (A2E), an intracellular lipofuscin component. Fluorometric analysis using labeled A2E (A2E-BDP) and confocal microscopic examination revealed that low concentrations of NMDA receptor antagonists, which did not induce cytotoxicity, significantly reduced A2E accumulation in ARPE-19 cells. In addition, memantine and ifenprodil activated autophagy in ARPE-19 cells as measured by microtubule-associated protein 1A/1B-light chain3-II formation and phosphorylated p62 protein levels. Further, to understand the correlation between memantine- and ifenprodil-mediated A2E degradation and autophagy, autophagy-related 5 (ATG5) was depleted using RNA interference. Memantine and ifenprodil failed to degrade A2E in ARPE-19 cells lacking ATG5. Taken together, our study indicates that the NMDA receptor antagonists, memantine and ifenprodil, can remove A2E accumulated in cells via autophagy activation in ARPE-19 cells.

• Biomolecules & Therapeutics
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• v.27 no.1
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• pp.34-40
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• 2019

Transglutaminase 2 (TGase 2) plays a key role in p53 regulation, depleting p53 tumor suppressor through autophagy in renal cell carcinoma. We found that microtubule-associated protein 1A/1B-light chain 3 (LC3), a hallmark of autophagy, were tightly associated with the level of TGase 2 in cancer cells. TGase 2 overexpression increased LC3 levels, and TGase 2 knockdown decreased LC3 levels in cancer cells. Transcript abundance of LC3 was inversely correlated with level of wild type p53. TGase 2 knockdown using siRNA, or TGase 2 inhibition using GK921 significantly reduced autophagy through reduction of LC3 transcription, which was followed by restoration of p53 levels in cancer cells. TGase 2 overexpression promoted the autophagy process by LC3 induction, which was correlated with p53 depletion in cancer cells. Rapamycin-resistant cancer cells also showed higher expression of LC3 compared to the rapamycin-sensitive cancer cells, which was tightly correlated with TGase 2 levels. TGase 2 knockdown or TGase 2 inhibition sensitized rapamycin-resistant cancer cells to drug treatment. In summary, TGase 2 induces drug resistance by potentiating autophagy through LC3 induction via p53 regulation in cancer.