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Activation of adenosine A3 receptor alleviates TNF-α-induced inflammation through inhibition of the NF-κB signaling pathway in human colonic epithelial cells.

Ren T, Qiu Y, Wu W, Feng X, Ye S, Wang Z, Tian T, He Y, Yu C, Zhou Y - Mediators Inflamm. (2014)

Bottom Line: Pretreatment with 2-Cl-IB-MECA prior to stimulation with TNF-α attenuated NF-κB p65 nuclear translocation as p65 protein decreased in the nucleus of cells and increased in the cytoplasm, inhibited the degradation of IκB-α, and reduced phosphorylated-IκB-α level significantly, compared to TNF-α-only-treated groups.Furthermore, 2-Cl-IB-MECA significantly decreased TNF-α-stimulated IL-8 and IL-1β mRNA expression and secretion, compared to the TNF-α-only treated group.These results confirm that A3AR is expressed in human colonic epithelial cells and demonstrate that its activation has an anti-inflammatory effect, through the inhibition of NF-κB signaling pathway, which leads to inhibition of downstream IL-8 and IL-1β expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, The Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, China.

ABSTRACT
To investigate the expression of adenosine A3 receptor (A3AR) in human colonic epithelial cells and the effects of A3AR activation on tumor necrosis factor alpha (TNF-α-) induced inflammation in order to determine its mechanism of action in human colonic epithelial cells, human colonic epithelial cells (HT-29 cells) were treated with different concentrations of 2-Cl-IB-MECA prior to TNF-α stimulation, followed by analysis of NF-κB signaling pathway activation and downstream IL-8 and IL-1β production. A3AR mRNA and protein were expressed in HT-29 cells and not altered by changes in TNF-α or 2-Cl-IB-MECA. Pretreatment with 2-Cl-IB-MECA prior to stimulation with TNF-α attenuated NF-κB p65 nuclear translocation as p65 protein decreased in the nucleus of cells and increased in the cytoplasm, inhibited the degradation of IκB-α, and reduced phosphorylated-IκB-α level significantly, compared to TNF-α-only-treated groups. Furthermore, 2-Cl-IB-MECA significantly decreased TNF-α-stimulated IL-8 and IL-1β mRNA expression and secretion, compared to the TNF-α-only treated group. These results confirm that A3AR is expressed in human colonic epithelial cells and demonstrate that its activation has an anti-inflammatory effect, through the inhibition of NF-κB signaling pathway, which leads to inhibition of downstream IL-8 and IL-1β expression. Therefore, A3AR activation may be a potential treatment for gut inflammatory diseases such as inflammatory bowel disease.

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Qualitative expression of A3AR, NF-κB p65, IκB-α, and phosphorylated-IκB-α (p-IκB-α) in HT-29 cells following different treatments. HT-29 cells were treated with 30 nM 2-Cl-IB-MECA (CIM) for 30 min prior to TNF-α (10 ng/mL) stimulation for 30 min for the immunofluorescence (IF) assay. IF was performed by using specific antibodies for target proteins and DAPI for counterstaining of nuclei. ((a)1–3) The strong green fluorescence signal representing A3AR was observed mainly on the HT-29 cell membrane. However, cells stimulated by TNF-α alone or pretreated with 2-Cl-IB-MECA had no obvious change in A3AR expression. ((b)1–3) In untreated cells, NF-κB p65 was limited to the cytoplasm, and nuclear localization was observed in TNF-α-treated HT-29 cells. However, a significant reduction in p65 nuclear translocalization was seen in 2-Cl-IB-MECA + TNF-α cells. ((c)1–3) There was a tendency for IκB-α to decrease in the cytoplasm of TNF-α-treated cells. This effect was inhibited in 2-Cl-IB-MECA + TNF-α cells. ((d)1–3) There was a tendency for p-IκB-α to increase in the cytoplasm of TNF-α-treated cells. This effect was inhibited in 2-Cl-IB-MECA + TNF-α cells. Magnification ×600; scale bars indicated 50 μm.
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fig2: Qualitative expression of A3AR, NF-κB p65, IκB-α, and phosphorylated-IκB-α (p-IκB-α) in HT-29 cells following different treatments. HT-29 cells were treated with 30 nM 2-Cl-IB-MECA (CIM) for 30 min prior to TNF-α (10 ng/mL) stimulation for 30 min for the immunofluorescence (IF) assay. IF was performed by using specific antibodies for target proteins and DAPI for counterstaining of nuclei. ((a)1–3) The strong green fluorescence signal representing A3AR was observed mainly on the HT-29 cell membrane. However, cells stimulated by TNF-α alone or pretreated with 2-Cl-IB-MECA had no obvious change in A3AR expression. ((b)1–3) In untreated cells, NF-κB p65 was limited to the cytoplasm, and nuclear localization was observed in TNF-α-treated HT-29 cells. However, a significant reduction in p65 nuclear translocalization was seen in 2-Cl-IB-MECA + TNF-α cells. ((c)1–3) There was a tendency for IκB-α to decrease in the cytoplasm of TNF-α-treated cells. This effect was inhibited in 2-Cl-IB-MECA + TNF-α cells. ((d)1–3) There was a tendency for p-IκB-α to increase in the cytoplasm of TNF-α-treated cells. This effect was inhibited in 2-Cl-IB-MECA + TNF-α cells. Magnification ×600; scale bars indicated 50 μm.

Mentions: To verify the presence of A3AR, NF-κB p65, IκB-α, and phosphorylated-IκB-α in HT-29 cells, IF was performed. Representative micrographs from these experiments are shown in Figure 2. Firstly, a strong green fluorescence signal representing A3AR was observed mainly on HT-29 cell membranes, consistent with previous reports that A3AR is a transmembrane receptor. However, TNF-α-stimulated cells pretreated with 2-Cl-IB-MECA showed no obvious change in A3AR expression (Figure 2(a)). Secondly, confocal microscopy of NF-κB p65 in HT-29 cells is shown in Figure 2(b). In untreated cells, NF-κB p65 was mainly located in the cytoplasm and was almost absent from the nucleus (Figure 2(b)1). TNF-α stimulation induced NF-κB p65 translocation to the nuclei. Translocation of NF-κB p65 towards the nucleus can be observed in Figure 2(b)2. Meanwhile, impaired IκB-α expression and the increased expression of phosphorylated-IκB-α in the cytoplasm, induced by TNF-α, could be observed in Figures 2(c)2 and 2(d)2. Pretreatment with 2-Cl-IB-MECA and subsequent stimulation with TNF-α attenuated NF-κB p65 nuclear translocation and suppressed the phosphorylation of IκB-α (Figures 2(b)3, 2(c)3, and 2(d)3).


Activation of adenosine A3 receptor alleviates TNF-α-induced inflammation through inhibition of the NF-κB signaling pathway in human colonic epithelial cells.

Ren T, Qiu Y, Wu W, Feng X, Ye S, Wang Z, Tian T, He Y, Yu C, Zhou Y - Mediators Inflamm. (2014)

Qualitative expression of A3AR, NF-κB p65, IκB-α, and phosphorylated-IκB-α (p-IκB-α) in HT-29 cells following different treatments. HT-29 cells were treated with 30 nM 2-Cl-IB-MECA (CIM) for 30 min prior to TNF-α (10 ng/mL) stimulation for 30 min for the immunofluorescence (IF) assay. IF was performed by using specific antibodies for target proteins and DAPI for counterstaining of nuclei. ((a)1–3) The strong green fluorescence signal representing A3AR was observed mainly on the HT-29 cell membrane. However, cells stimulated by TNF-α alone or pretreated with 2-Cl-IB-MECA had no obvious change in A3AR expression. ((b)1–3) In untreated cells, NF-κB p65 was limited to the cytoplasm, and nuclear localization was observed in TNF-α-treated HT-29 cells. However, a significant reduction in p65 nuclear translocalization was seen in 2-Cl-IB-MECA + TNF-α cells. ((c)1–3) There was a tendency for IκB-α to decrease in the cytoplasm of TNF-α-treated cells. This effect was inhibited in 2-Cl-IB-MECA + TNF-α cells. ((d)1–3) There was a tendency for p-IκB-α to increase in the cytoplasm of TNF-α-treated cells. This effect was inhibited in 2-Cl-IB-MECA + TNF-α cells. Magnification ×600; scale bars indicated 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4016939&req=5

fig2: Qualitative expression of A3AR, NF-κB p65, IκB-α, and phosphorylated-IκB-α (p-IκB-α) in HT-29 cells following different treatments. HT-29 cells were treated with 30 nM 2-Cl-IB-MECA (CIM) for 30 min prior to TNF-α (10 ng/mL) stimulation for 30 min for the immunofluorescence (IF) assay. IF was performed by using specific antibodies for target proteins and DAPI for counterstaining of nuclei. ((a)1–3) The strong green fluorescence signal representing A3AR was observed mainly on the HT-29 cell membrane. However, cells stimulated by TNF-α alone or pretreated with 2-Cl-IB-MECA had no obvious change in A3AR expression. ((b)1–3) In untreated cells, NF-κB p65 was limited to the cytoplasm, and nuclear localization was observed in TNF-α-treated HT-29 cells. However, a significant reduction in p65 nuclear translocalization was seen in 2-Cl-IB-MECA + TNF-α cells. ((c)1–3) There was a tendency for IκB-α to decrease in the cytoplasm of TNF-α-treated cells. This effect was inhibited in 2-Cl-IB-MECA + TNF-α cells. ((d)1–3) There was a tendency for p-IκB-α to increase in the cytoplasm of TNF-α-treated cells. This effect was inhibited in 2-Cl-IB-MECA + TNF-α cells. Magnification ×600; scale bars indicated 50 μm.
Mentions: To verify the presence of A3AR, NF-κB p65, IκB-α, and phosphorylated-IκB-α in HT-29 cells, IF was performed. Representative micrographs from these experiments are shown in Figure 2. Firstly, a strong green fluorescence signal representing A3AR was observed mainly on HT-29 cell membranes, consistent with previous reports that A3AR is a transmembrane receptor. However, TNF-α-stimulated cells pretreated with 2-Cl-IB-MECA showed no obvious change in A3AR expression (Figure 2(a)). Secondly, confocal microscopy of NF-κB p65 in HT-29 cells is shown in Figure 2(b). In untreated cells, NF-κB p65 was mainly located in the cytoplasm and was almost absent from the nucleus (Figure 2(b)1). TNF-α stimulation induced NF-κB p65 translocation to the nuclei. Translocation of NF-κB p65 towards the nucleus can be observed in Figure 2(b)2. Meanwhile, impaired IκB-α expression and the increased expression of phosphorylated-IκB-α in the cytoplasm, induced by TNF-α, could be observed in Figures 2(c)2 and 2(d)2. Pretreatment with 2-Cl-IB-MECA and subsequent stimulation with TNF-α attenuated NF-κB p65 nuclear translocation and suppressed the phosphorylation of IκB-α (Figures 2(b)3, 2(c)3, and 2(d)3).

Bottom Line: Pretreatment with 2-Cl-IB-MECA prior to stimulation with TNF-α attenuated NF-κB p65 nuclear translocation as p65 protein decreased in the nucleus of cells and increased in the cytoplasm, inhibited the degradation of IκB-α, and reduced phosphorylated-IκB-α level significantly, compared to TNF-α-only-treated groups.Furthermore, 2-Cl-IB-MECA significantly decreased TNF-α-stimulated IL-8 and IL-1β mRNA expression and secretion, compared to the TNF-α-only treated group.These results confirm that A3AR is expressed in human colonic epithelial cells and demonstrate that its activation has an anti-inflammatory effect, through the inhibition of NF-κB signaling pathway, which leads to inhibition of downstream IL-8 and IL-1β expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, The Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, China.

ABSTRACT
To investigate the expression of adenosine A3 receptor (A3AR) in human colonic epithelial cells and the effects of A3AR activation on tumor necrosis factor alpha (TNF-α-) induced inflammation in order to determine its mechanism of action in human colonic epithelial cells, human colonic epithelial cells (HT-29 cells) were treated with different concentrations of 2-Cl-IB-MECA prior to TNF-α stimulation, followed by analysis of NF-κB signaling pathway activation and downstream IL-8 and IL-1β production. A3AR mRNA and protein were expressed in HT-29 cells and not altered by changes in TNF-α or 2-Cl-IB-MECA. Pretreatment with 2-Cl-IB-MECA prior to stimulation with TNF-α attenuated NF-κB p65 nuclear translocation as p65 protein decreased in the nucleus of cells and increased in the cytoplasm, inhibited the degradation of IκB-α, and reduced phosphorylated-IκB-α level significantly, compared to TNF-α-only-treated groups. Furthermore, 2-Cl-IB-MECA significantly decreased TNF-α-stimulated IL-8 and IL-1β mRNA expression and secretion, compared to the TNF-α-only treated group. These results confirm that A3AR is expressed in human colonic epithelial cells and demonstrate that its activation has an anti-inflammatory effect, through the inhibition of NF-κB signaling pathway, which leads to inhibition of downstream IL-8 and IL-1β expression. Therefore, A3AR activation may be a potential treatment for gut inflammatory diseases such as inflammatory bowel disease.

Show MeSH
Related in: MedlinePlus