1.Involvement of PI3K/AKT and MAPK Pathways for TNF-alpha Production in SiHa Cervical Mucosal Epithelial Cells Infected with Trichomonas vaginalis.
Jung Bo YANG ; Juan Hua QUAN ; Ye Eun KIM ; Yun Ee RHEE ; Byung Hyun KANG ; In Wook CHOI ; Guang Ho CHA ; Jae Min YUK ; Young Ha LEE
The Korean Journal of Parasitology 2015;53(4):371-377
Trichomonas vaginalis induces proinflammation in cervicovaginal mucosal epithelium. To investigate the signaling pathways in TNF-alpha production in cervical mucosal epithelium after T. vaginalis infection, the phosphorylation of PI3K/AKT and MAPK pathways were evaluated in T. vaginalis-infected SiHa cells in the presence and absence of specific inhibitors. T. vaginalis increased TNF-alpha production in SiHa cells, in a parasite burden-dependent and incubation time-dependent manner. In T. vaginalis-infected SiHa cells, AKT, ERK1/2, p38 MAPK, and JNK were phosphorylated from 1 hr after infection; however, the phosphorylation patterns were different from each other. After pretreatment with inhibitors of the PI3K/AKT and MAPK pathways, TNF-alpha production was significantly decreased compared to the control; however, TNF-alpha reduction patterns were different depending on the type of PI3K/MAPK inhibitors. TNF-alpha production was reduced in a dose-dependent manner by treatment with wortmannin and PD98059, whereas it was increased by SP600125. These data suggested that PI3K/AKT and MAPK signaling pathways are important in regulation of TNF-alpha production in cervical mucosal epithelial SiHa cells. However, activation patterns of each pathway were different from the types of PI3K/MAPK pathways.
Cell Line
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Cervix Uteri/enzymology/metabolism/*parasitology
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Epithelial Cells/*enzymology/metabolism/parasitology
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Female
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Humans
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*MAP Kinase Signaling System
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Mucous Membrane/*enzymology/metabolism/parasitology
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Phosphatidylinositol 3-Kinases/genetics/*metabolism
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Proto-Oncogene Proteins c-akt/genetics/*metabolism
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Trichomonas Vaginitis/*enzymology/genetics/metabolism/parasitology
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Trichomonas vaginalis/*physiology
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Tumor Necrosis Factor-alpha/genetics/*metabolism
2.Trichomonas vaginalis Metalloproteinase Induces mTOR Cleavage of SiHa Cells.
Juan Hua QUAN ; In Wook CHOI ; Jung Bo YANG ; Wei ZHOU ; Guang Ho CHA ; Yu ZHOU ; Jae Sook RYU ; Young Ha LEE
The Korean Journal of Parasitology 2014;52(6):595-603
Trichomonas vaginalis secretes a number of proteases which are suspected to be the cause of pathogenesis; however, little is understood how they manipulate host cells. The mammalian target of rapamycin (mTOR) regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. We detected various types of metalloproteinases including GP63 protein from T. vaginalis trophozoites, and T. vaginalis GP63 metalloproteinase was confirmed by sequencing and western blot. When SiHa cells were stimulated with live T. vaginalis, T. vaginalis excretory-secretory products (ESP) or T. vaginalis lysate, live T. vaginalis and T. vaginalis ESP induced the mTOR cleavage in both time- and parasite load-dependent manner, but T. vaginalis lysate did not. Pretreatment of T. vaginalis with a metalloproteinase inhibitor, 1,10-phenanthroline, completely disappeared the mTOR cleavage in SiHa cells. Collectively, T. vaginalis metallopeptidase induces host cell mTOR cleavage, which may be related to survival of the parasite.
Blotting, Western
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Cell Line, Tumor
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Epithelial Cells/metabolism/parasitology
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Humans
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Metalloproteases/genetics/*metabolism
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Proteolysis
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Sequence Analysis, DNA
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TOR Serine-Threonine Kinases/*metabolism
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Trichomonas vaginalis/*enzymology/genetics
3.Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence.
Won Tae KIM ; Hyun Hee KONG ; Young Ran HA ; Yeon Chul HONG ; Hae Jin JEONG ; Hak Sun YU ; Dong Il CHUNG
The Korean Journal of Parasitology 2006;44(4):321-330
The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Protease has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castellanii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba.
Virulence Factors/isolation & purification/*metabolism
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Virulence
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Trophozoites/physiology
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Substrate Specificity
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Soil/parasitology
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Serine Endopeptidases/isolation & purification/*metabolism
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Humans
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Epithelial Cells/parasitology/*pathology
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Encephalitis
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Cornea/cytology/parasitology/*pathology
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Cells, Cultured
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Animals
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Acanthamoeba castellanii/enzymology/growth & development/pathogenicity
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Acanthamoeba Keratitis/parasitology
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Acanthamoeba/classification/*enzymology/growth & development/*pathogenicity