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Mycoplasma genitalium lipoproteins induce human monocytic cell expression of proinflammatory cytokines and apoptosis by activating nuclear factor kappaB.

Wu Y, Qiu H, Zeng Y, You X, Deng Z, Yu M, Zhu C - Mediators Inflamm. (2008)

Bottom Line: LPs were also found to increase the DNA-binding activity of NF-kappaB, a possible mechanism for the induction of cytokine mRNA expression and the cell apoptosis.These effects were abrogated by PDTC, an inhibitor of NF-kappaB.Our results indicate that M. genitalium-derived LP may be an important etiological factor of certain diseases due to the ability of LP to produce proinflammatory cytokines and induction of apoptosis, which is probably mediated through the activation of NF-kappaB.

View Article: PubMed Central - PubMed

Affiliation: Pathogenic Biology Institute, University of South China, 421001 Hengyang, China. yimouwu@sina.com

ABSTRACT
This study was designed to investigate the molecular mechanisms responsible for the induction of proinflammatory cytokines gene expression and apoptosis in human monocytic cell line THP-1 stimulated by lipoproteins (LPs) prepared from Mycoplasma genitalium. Cultured cells were stimulated with M. genitalium LP to analyze the production of proinflammatory cytokines and expression of their mRNA by ELISA and RT-PCR, respectively. Cell apoptosis was also detected by Annexin V-FITC-propidium iodide (PI) staining and acridine orange (AO)-ethidium bromide (EB) staining. The DNA-binding activity of nuclear factor-kappaB (NF-kappaB) was assessed by electrophoretic mobility shift assay (EMSA). Results showed that LP stimulated THP-1 cells to produce tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in a dose-dependent manner. The mRNA levels were also upregulated in response to LP stimulation. LPs were also found to increase the DNA-binding activity of NF-kappaB, a possible mechanism for the induction of cytokine mRNA expression and the cell apoptosis. These effects were abrogated by PDTC, an inhibitor of NF-kappaB. Our results indicate that M. genitalium-derived LP may be an important etiological factor of certain diseases due to the ability of LP to produce proinflammatory cytokines and induction of apoptosis, which is probably mediated through the activation of NF-kappaB.

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Related in: MedlinePlus

The activity of NF-κB in different groups wasexamined by EMSA. NF-κB activation was measuredby EMSA using a biotin-labeled oligonucleotide encompassing the NF-κB consensusmotif. THP-1 cells were stimulated with M.genitalium LP (3 μg/mL) at different timeintervals (0, 1, 2, and 4 hour(s)). THP-1 cells treated with 100 μL aqueous phase and 0.1 μg/mL LPSwere used as control. The specificity of DNA binding was assessed bypreincubating extracts with unlabeled specific (NF-κB) or unspecific (AP-1)competitor oligonucleotide. The arrow indicates specific NF-κB band.
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fig4: The activity of NF-κB in different groups wasexamined by EMSA. NF-κB activation was measuredby EMSA using a biotin-labeled oligonucleotide encompassing the NF-κB consensusmotif. THP-1 cells were stimulated with M.genitalium LP (3 μg/mL) at different timeintervals (0, 1, 2, and 4 hour(s)). THP-1 cells treated with 100 μL aqueous phase and 0.1 μg/mL LPSwere used as control. The specificity of DNA binding was assessed bypreincubating extracts with unlabeled specific (NF-κB) or unspecific (AP-1)competitor oligonucleotide. The arrow indicates specific NF-κB band.

Mentions: In this study, weinvestigated whether M. genitalium LPwas capable of triggering NF-κB activation.For this purpose, the THP-1 cells were stimulated with LP at different time intervals, NF-κB DNA binding activities in nuclear extracts were assessed by nonradioactive EMSA asdescribed in Section 2. As depicted in Figure 4, M. genitalium LP could activate NF-κB in THP-1 cells: the DNA bindingactivities were maximal by 2 hours of stimulation and then declined. Thespecificity of NF-κB DNA binding was verified by competition analysis with anexcess of unlabeled specific or unspecific oligonucleotides.


Mycoplasma genitalium lipoproteins induce human monocytic cell expression of proinflammatory cytokines and apoptosis by activating nuclear factor kappaB.

Wu Y, Qiu H, Zeng Y, You X, Deng Z, Yu M, Zhu C - Mediators Inflamm. (2008)

The activity of NF-κB in different groups wasexamined by EMSA. NF-κB activation was measuredby EMSA using a biotin-labeled oligonucleotide encompassing the NF-κB consensusmotif. THP-1 cells were stimulated with M.genitalium LP (3 μg/mL) at different timeintervals (0, 1, 2, and 4 hour(s)). THP-1 cells treated with 100 μL aqueous phase and 0.1 μg/mL LPSwere used as control. The specificity of DNA binding was assessed bypreincubating extracts with unlabeled specific (NF-κB) or unspecific (AP-1)competitor oligonucleotide. The arrow indicates specific NF-κB band.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2366083&req=5

fig4: The activity of NF-κB in different groups wasexamined by EMSA. NF-κB activation was measuredby EMSA using a biotin-labeled oligonucleotide encompassing the NF-κB consensusmotif. THP-1 cells were stimulated with M.genitalium LP (3 μg/mL) at different timeintervals (0, 1, 2, and 4 hour(s)). THP-1 cells treated with 100 μL aqueous phase and 0.1 μg/mL LPSwere used as control. The specificity of DNA binding was assessed bypreincubating extracts with unlabeled specific (NF-κB) or unspecific (AP-1)competitor oligonucleotide. The arrow indicates specific NF-κB band.
Mentions: In this study, weinvestigated whether M. genitalium LPwas capable of triggering NF-κB activation.For this purpose, the THP-1 cells were stimulated with LP at different time intervals, NF-κB DNA binding activities in nuclear extracts were assessed by nonradioactive EMSA asdescribed in Section 2. As depicted in Figure 4, M. genitalium LP could activate NF-κB in THP-1 cells: the DNA bindingactivities were maximal by 2 hours of stimulation and then declined. Thespecificity of NF-κB DNA binding was verified by competition analysis with anexcess of unlabeled specific or unspecific oligonucleotides.

Bottom Line: LPs were also found to increase the DNA-binding activity of NF-kappaB, a possible mechanism for the induction of cytokine mRNA expression and the cell apoptosis.These effects were abrogated by PDTC, an inhibitor of NF-kappaB.Our results indicate that M. genitalium-derived LP may be an important etiological factor of certain diseases due to the ability of LP to produce proinflammatory cytokines and induction of apoptosis, which is probably mediated through the activation of NF-kappaB.

View Article: PubMed Central - PubMed

Affiliation: Pathogenic Biology Institute, University of South China, 421001 Hengyang, China. yimouwu@sina.com

ABSTRACT
This study was designed to investigate the molecular mechanisms responsible for the induction of proinflammatory cytokines gene expression and apoptosis in human monocytic cell line THP-1 stimulated by lipoproteins (LPs) prepared from Mycoplasma genitalium. Cultured cells were stimulated with M. genitalium LP to analyze the production of proinflammatory cytokines and expression of their mRNA by ELISA and RT-PCR, respectively. Cell apoptosis was also detected by Annexin V-FITC-propidium iodide (PI) staining and acridine orange (AO)-ethidium bromide (EB) staining. The DNA-binding activity of nuclear factor-kappaB (NF-kappaB) was assessed by electrophoretic mobility shift assay (EMSA). Results showed that LP stimulated THP-1 cells to produce tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in a dose-dependent manner. The mRNA levels were also upregulated in response to LP stimulation. LPs were also found to increase the DNA-binding activity of NF-kappaB, a possible mechanism for the induction of cytokine mRNA expression and the cell apoptosis. These effects were abrogated by PDTC, an inhibitor of NF-kappaB. Our results indicate that M. genitalium-derived LP may be an important etiological factor of certain diseases due to the ability of LP to produce proinflammatory cytokines and induction of apoptosis, which is probably mediated through the activation of NF-kappaB.

Show MeSH
Related in: MedlinePlus