• BMB Reports
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• 제44권8호
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• pp.541-546
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• 2011

It is generally accepted that spermatozoa capacitation is associated with protein kinase A-mediated tyrosine phosphorylation. In our previous study, we identified the fibrous sheath CABYR binding protein (FSCB), which was phosphorylated by PKA. However, the phosphorylation status of FSCB protein during spermatozoa capacitation should be further investigated. To this aim, in this study, we found that phosphorylation of this 270-kDa protein occurred as early as 1 min after mouse spermatozoa capacitation, which increased over time and remained stable after 60 min. Immunoprecipitation assays demonstrated that the tyrosine and Ser/Thr phosphorylation of FSCB occurred during spermatozoa capacitation. The extent of phosphorylation and was closely associated with the PKA activity and spermatozoa motility characteristics. FSCB phosphorylation could be induced by PKA agonist DB-cAMP, but was blocked by PKA antagonist H-89.Therefore, FSCB contributes to spermatozoa capacitation in a tyrosine-phosphorylated format, which may help in further elucidating the molecular mechanism of spermatozoa capacitation.

• Asian Pacific Journal of Cancer Prevention
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• 제16권4호
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• pp.1343-1348
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• 2015

Human UHRF1 (ubiquitin-like PHD and RING finger domain-containing 1) has been reported to be over-expressed in many cancers, but its role in ovarian cancer remains elusive. Here, we determined whether knockdown of UHRF1 by lentivirus-mediated shRNA could inhibit ovarian cancer cell growth. Lentivirus-mediated short hairpin RNAs (lv-shRNAs-UHRF1) were designed to trigger the gene silencing RNA interference (RNAi) pathway. The efficiency of lentivirus-mediated shRNA infection into HO-8910 and HO-8910 PM cells was determined using fluorescence microscopy to observe lentivirus-mediated GFP expression and was confirmed to be over 80 percent. UHRF1 expression in infected HO-8910 and HO-8910 PM was evaluated by real-time PCR and Western blot analysis. The Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability; flow cytometry and Hoechst 33342 assay was applied to measure cell cycle arrest and apoptosis. Cell invasion was assessed using transwell chambers. Our results demonstrated that the loss of UHRF1 promoted HO-8910 and HO-8910 PM cell apoptosis, while inhibiting cell proliferation. In addition, UHRF1 knockdown significantly inhibited the invasion of human ovarian cancer cells. In the present study, we also showed that depleting HO-8910 cells of UHRF1 caused activation of the DNA damage response pathway, with the cell cycle arrested in G2/M-phase. The DNA damage response in cells depleted of UHRF1 was illustrated by phosphorylation of CHK (checkpoint kinase) 2 on Thr68, phosphorylation of CDC25 (cell division control 25) on Ser 216 and phosphorylation of CDK1 (cyclin-dependent kinase 1) on Tyr 15.

• BMB Reports
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• 제55권5호
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• pp.244-249
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• 2022

Characterized by abnormal proliferation and migration of vascular smooth muscle cells (VSMCs), neointima hyperplasia is a hallmark of vascular restenosis after percutaneous vascular interventions. Vaccinia-related kinase 1 (VRK1) is a stress adaption-associated ser/thr protein kinase that can induce the proliferation of various types of cells. However, the role of VRK1 in the proliferation and migration of VSMCs and neointima hyperplasia after vascular injury remains unknown. We observed increased expression of VRK1 in VSMCs subjected to platelet-derived growth factor (PDGF)-BB by western blotting. Silencing VRK1 by shVrk1 reduced the number of Ki-67-positive VSMCs and attenuated the migration of VSMCs. Mechanistically, we found that relative expression levels of β-catenin and effectors of mTOR complex 1 (mTORC1) such as phospho (p)-mammalian target of rapamycin (mTOR), p-S6, and p-4EBP1 were decreased after silencing VRK1. Restoration of β-catenin expression by SKL2001 and re-activation of mTORC1 by Tuberous sclerosis 1 siRNA (siTsc1) both abolished shVrk1-mediated inhibitory effect on VSMC proliferation and migration. siTsc1 also rescued the reduced expression of β-catenin caused by VRK1 inhibition. Furthermore, mTORC1 re-activation failed to recover the attenuated proliferation and migration of VSMC resulting from shVrk1 after silencing β-catenin. We also found that the vascular expression of VRK1 was increased after injury. VRK1 inactivation in vivo inhibited vascular injury-induced neointima hyperplasia in a β-catenin-dependent manner. These results demonstrate that inhibition of VRK1 can suppress the proliferation and migration of VSMC and neointima hyperplasia after vascular injury via mTORC1/β-catenin pathway.

• 한방비만학회지
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• 제22권2호
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• pp.102-114
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• 2022

Objectives: We investigated the effects of Allii Fistulosi Bulbus (AFB) on high fat diet (HFD)-induced obesity in mice and the regulation of energy metabolism in muscle tissues of mice. Methods: The C57BL/6 mice (6 weeks, male) were fed a HFD for 8 weeks and then administrated with AFB extract at 500 mg/kg (p.o.) once daily for 4 weeks. The body weight (BW), muscle weight, calorie intake, fasting blood glucose (FBG) and serum glucose, insulin, and low-density lipoprotein-cholesterol (LDL-C) levels were measured in mice. It was also observed the histological changes of pancreas, liver, and fat tissues with hematoxylin and eosin staining. It was investigated the phosphorylation of insulin receptor substrate 1 (IRS-1), Ser/Thr kinase (AKT), and adenosine monophosphate-activated protein kinase (AMPK), and the expression of phosphoinositide 3-kinase, glucose transporter type 4 (GLUT4), and sirtuin1 (Sirt1) in gastrocnemius tissues by western blot, respectively. Results: The increases of BWs, calorie intakes and FBG levels in obesity mice were decreased significantly by the administration of AFB extract. The AFB extract administration was reduced significantly serum levels of glucose, insulin, and LDL-C in obesity mice. The AFB extract inhibited lipid accumulation in liver tissues, hyperplasia of pancreatic islets, and enlargement of fat tissues in obesity mice. The phosphorylation of IRS-1 and AKT was increased significantly in muscle tissues and AMPK phosphorylation and the GLUT4 and Sirt1 expression were decreased significantly in muscle tissues after the AFB administration. Conclusions: Our study indicates that AFB extract improves symptoms of obesity through regulation of energy regulating proteins in muscle tissues.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2019년도 춘계학술대회
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• pp.33-33
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• 2019

The AKT signaling pathway is a highly regulated cell signaling system that forms a network with other cell signaling pathways. Hence, the AKT signaling pathway mediates several important cellular functions that include cell survival, proliferation, cell migration, and et cetera. Irregularities that led overactive AKT signaling have been linked to many diseases such as cancer and metabolic-associated diseases. Hence, modulating the overactive AKT signaling pathway via inhibitor is a tantalizing prospect for treatment of cancer and metabolic-associated diseases. Two inhibitors of the AKT signaling pathway will be presented in this symposium: 1) Bisleuconothine A (BisA), a bisindole alkaloid that inhibit autophagy and 2) Ceramicine B (CerB), a limonoid that inhibit adipogenesis. The first topic is on a bisindole alkaloid, BisA and its mechanism in inducing autophagosome formation in lung cancer cell line, A549.(1) Since most autophagy inducing agents generally induce apoptosis, we found that BisA does not induce apoptosis even in high dose. BisA up-regulation of LC3 lipidation is achieved through mTOR inactivation. The phosphorylation of PRAS40, a mTOR repressor was suppressed by BisA. This observation suggested that BisA inactivates mTOR via suppression of PRAS40 phosphorylation. Interestingly, the phosphorylation of AKT, an upstream regulator of PRAS40 phosphorylation was also down-regulated by BisA. These findings suggested that Bis-A induces autophagosomes formation by interfering with the AKT-mTOR signaling pathway. The second topic is on CerB and its mechanism in inhibiting adipogenesis in preadipocytes cell line, MC3T3-G2/PA6.(2,3) CerB inhibits the phosphorylation of protein kinase B (AKT) at the Thr308 position but not the Ser473. Consequently, the phosphorylation of FOXO3 which is located downstream of AKT is also inhibited. Considering that FOXO3 is an important regulator of PPARγ which is a key factor in adipogenesis, CerB may inhibit adipogenesis via the AKT-FOXO3 signaling pathway. Taken together, both BisA and CerB highlighted the potential of AKT signaling pathway modulation as an approach to induce autophagy and inhibit the formation of fat cells, respectively.

• Parasites, Hosts and Diseases
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• 제54권6호
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• pp.759-768
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• 2016

Cystic echinococcosis (CE) treatment urgently requires a novel drug. The p38 mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases, but still have to be characterized in Echinococcus granulosus. We identified a 1,107 bp cDNA encoding a 368 amino acid MAPK protein (Egp38) in E. granulosus. Egp38 exhibits 2 distinguishing features of p38-like kinases: a highly conserved T-X-Y motif and an activation loop segment. Structural homology modeling indicated a conserved structure among Egp38, EmMPK2, and H. sapiens $p38{\alpha}$, implying a common binding mechanism for the ligand domain and downstream signal transduction processing similar to that described for $p38{\alpha}$. Egp38 and its phosphorylated form are expressed in the E. granulosus larval stages vesicle and protoscolices during intermediate host infection of an intermediate host. Treatment of in vitro cultivated protoscolices with the p38-MAPK inhibitor ML3403 effectively suppressed Egp38 activity and led to significant protoscolices death within 5 days. Treatment of in vitro-cultivated protoscolices with $TGF-{\beta}1$ effectively induced Egp38 phosphorylation. In summary, the MAPK, Egp38, was identified in E. granulosus, as an anti-CE drug target and participates in the interplay between the host and E. granulosus via human $TGF-{\beta}1$.

• 생명과학회지
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• 제23권5호
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• pp.689-697
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• 2013

오메가-3 지방산은 많은 암에서 세포독성을 나타낸다고 보고 되어 왔으나 구강암에 대한 연구는 전혀 없다. 이에 본 연구에서는 구강암세포에서 오메가-3 지방산 중 DHA의 세포독성 기전을 규명하여 다음과 같은 결과를 얻었다. DHA는 구강암 세포주 SCC-4 및 SCC-9의 증식을 농도 의존적으로 억제하였으며, FACS 분석, TUNEL assay 및 PARP cleavage 등에 의해 자가사멸을 유도함이 확인 되었다. 또한 DHA는 LC-3II 단백증가, GFP-LC-3 dot 형성 및 autophagic flux assay 등에 의해 자가포식도 유도됨이 규명되었다. SCC-9 세포에서 AMPK의 인산화는 DHA 에 의해 증가 하였으나, p-$AKT^{Thr308}$, p-$AKT^{Ser473}$ 및 mTOR단백양은 감소하였다. 이상의 결과로 DHA는 구강암세포에서 AMPK 활성증가 및 AKT 억제에 통한 mTOR 신호경로 차단에 따른 자가사멸 및 자가포식에 의해 세포독성을 나타낼 수 있음을 시사하며, 따라서 DHA는 구강암의 예방 및 치료에 유용하게 사용될 수 있으리라 생각된다.