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AhR and Arnt differentially regulate NF-κB signaling and chemokine responses in human bronchial epithelial cells.

Øvrevik J, Låg M, Lecureur V, Gilot D, Lagadic-Gossmann D, Refsnes M, Schwarze PE, Skuland T, Becher R, Holme JA - Cell Commun. Signal (2014)

Bottom Line: In contrast, Arnt suppressed only CXCL8, but did not prevent the p65-activation directly.Moreover, ligand-activated AhR suppressed CXCL8 and CCL5 responses by other agents, but AhR ligands alone induced CXCL8 responses when given at sufficiently high concentrations, thus underscoring the duality of AhR in regulation of inflammation.We propose that AhR-signaling may be a weak activator of p65-signaling that suppresses p65-activity induced by strong activators of NF-κB, but that its anti-inflammatory properties also are due to interference with additional pathways.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: The aryl hydrocarbon receptor (AhR) has gradually emerged as a regulator of inflammation in the lung and other tissues. AhR may interact with the p65-subunit of the nuclear factor (NF)-κB transcription factors, but reported outcomes of AhR/NF-κB-interactions are conflicting. Some studies suggest that AhR possess pro-inflammatory activities while others suggest that AhR may be anti-inflammatory. The present study explored the impact of AhR and its binding partner AhR nuclear translocator (Arnt) on p65-activation and two differentially regulated chemokines, CXCL8 (IL-8) and CCL5 (RANTES), in human bronchial epithelial cells (BEAS-2B).

Results: Cells were exposed to CXCL8- and CCL5-inducing chemicals, 1-nitropyrene (1-NP) and 1-aminopyrene (1-AP) respectively, or the synthetic double-stranded RNA analogue, polyinosinic-polycytidylic acid (Poly I:C) which induced both chemokines. Only CXCL8, and not CCL5, appeared to be p65-dependent. Yet, constitutively active unligated AhR suppressed both CXCL8 and CCL5, as shown by siRNA knock-down and the AhR antagonist α-naphthoflavone. Moreover, AhR suppressed activation of p65 by TNF-α and Poly I:C as assessed by luciferase-assay and p65-phosphorylation at serine 536, without affecting basal p65-activity. In contrast, Arnt suppressed only CXCL8, but did not prevent the p65-activation directly. However, Arnt suppressed expression of the NF-κB-subunit RelB which is under transcriptional regulation by p65. Furthermore, AhR-ligands alone at high concentrations induced a moderate CXCL8-response, without affecting CCL5, but suppressed both CXCL8 and CCL5-responses by Poly I:C.

Conclusion: AhR and Arnt may differentially and independently regulate chemokine-responses induced by both inhaled pollutants and pulmonary infections. Constitutively active, unligated AhR suppressed the activation of p65, while Arnt may possibly interfere with the action of activated p65. Moreover, ligand-activated AhR suppressed CXCL8 and CCL5 responses by other agents, but AhR ligands alone induced CXCL8 responses when given at sufficiently high concentrations, thus underscoring the duality of AhR in regulation of inflammation. We propose that AhR-signaling may be a weak activator of p65-signaling that suppresses p65-activity induced by strong activators of NF-κB, but that its anti-inflammatory properties also are due to interference with additional pathways.

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The constitutive activity of unligated AhR and Arnt differentially regulate CXCL8 and CCL5 in BEAS-2B cells. The model summarizes the possible pathways discussed for AhR- and Arnt-mediated regulation of CXCL8 and CCL5 in BEAS-2B cells exposed to 1-NP and 1-AP (A) or Poly I:C (B). “x/X” and “y/Y” represents hitherto unidentified signaling pathways/response elements involved in regulation of CXCL8 and CCL5, respectively. The positioning of the response elements in the CXCL8 or CCL5 promoters is not meant to be representative. Moreover, “y/Y” induced by 1-AP (A) and Poly I:C (B) are not necessarily identical and AhR may also affect other pathways involved in Poly I:C-induced CXCL8 in addition to NF-κB (B). The suggested position and association of Arnt and RelB is partly based on findings reported by Wright and Duckett [49]. The role of AhR and Arnt in chemokine regulation may differ in other cell types.
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Figure 10: The constitutive activity of unligated AhR and Arnt differentially regulate CXCL8 and CCL5 in BEAS-2B cells. The model summarizes the possible pathways discussed for AhR- and Arnt-mediated regulation of CXCL8 and CCL5 in BEAS-2B cells exposed to 1-NP and 1-AP (A) or Poly I:C (B). “x/X” and “y/Y” represents hitherto unidentified signaling pathways/response elements involved in regulation of CXCL8 and CCL5, respectively. The positioning of the response elements in the CXCL8 or CCL5 promoters is not meant to be representative. Moreover, “y/Y” induced by 1-AP (A) and Poly I:C (B) are not necessarily identical and AhR may also affect other pathways involved in Poly I:C-induced CXCL8 in addition to NF-κB (B). The suggested position and association of Arnt and RelB is partly based on findings reported by Wright and Duckett [49]. The role of AhR and Arnt in chemokine regulation may differ in other cell types.

Mentions: In the present study we have compared the effects of AhR and Arnt depletion on the classical NF-κB pathway and expression of two differentially regulated chemokines, CXCL8 and CCL5, in bronchial epithelial BEAS-2B cells exposed to PAHs or the TLR3 agonist Poly I:C. The results showed that regulation of CXCL8, but not CCL5, was dependent on basal and/or induced p65 activity. AhR suppressed both CXCL8 and CCL5 responses in BEAS-2B cells by widely differing stimuli, while Arnt only suppressed CXCL8. Moreover, the AhR-antagonist ANF failed to reproduce the effects of AhR knock-down on CXCL8/CCL5 regulation. This suggests that AhR suppressed chemokine responses independently of Arnt, and the effects were most likely due to the constitutive activity of unligated AhR. AhR suppressed p65 activation induced by strong activators of the classical NF-κB pathway, such as TNF-α and Poly I:C, but did not affect basal p65 activity. In addition, since AhR also suppressed CCL5, as well as the p65-independent increase in CXCL8 by 1-NP (which were unable to enhance p65 activity), the anti-inflammatory effects of AhR were necessarily not restricted to interference with NF-κB-signaling. In contrast to AhR, Arnt seemed unable to suppress the onset of p65 activation. Nevertheless, Arnt may still interfere with p65 signaling through other mechanisms, possibly in collaboration with RelB, which is a well-known suppressor of p65 activity. A schematic presentation of the possible roles of AhR and Arnt in suppression of CXCL8 and CCL5 in BEAS-2B cells, as discussed below, is presented in Figure 10. In addition to the above, we also observed that potent-AhR agonists could suppress both CXCL8 and CCL5-responses by TLR-ligands, but given alone at sufficiently high concentrations, they elicited a moderate activation of CXCL8. Thus ligand-activated AhR appear to possess both pro- and anti-inflammatory effects in BEAS-2B cells.


AhR and Arnt differentially regulate NF-κB signaling and chemokine responses in human bronchial epithelial cells.

Øvrevik J, Låg M, Lecureur V, Gilot D, Lagadic-Gossmann D, Refsnes M, Schwarze PE, Skuland T, Becher R, Holme JA - Cell Commun. Signal (2014)

The constitutive activity of unligated AhR and Arnt differentially regulate CXCL8 and CCL5 in BEAS-2B cells. The model summarizes the possible pathways discussed for AhR- and Arnt-mediated regulation of CXCL8 and CCL5 in BEAS-2B cells exposed to 1-NP and 1-AP (A) or Poly I:C (B). “x/X” and “y/Y” represents hitherto unidentified signaling pathways/response elements involved in regulation of CXCL8 and CCL5, respectively. The positioning of the response elements in the CXCL8 or CCL5 promoters is not meant to be representative. Moreover, “y/Y” induced by 1-AP (A) and Poly I:C (B) are not necessarily identical and AhR may also affect other pathways involved in Poly I:C-induced CXCL8 in addition to NF-κB (B). The suggested position and association of Arnt and RelB is partly based on findings reported by Wright and Duckett [49]. The role of AhR and Arnt in chemokine regulation may differ in other cell types.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4222560&req=5

Figure 10: The constitutive activity of unligated AhR and Arnt differentially regulate CXCL8 and CCL5 in BEAS-2B cells. The model summarizes the possible pathways discussed for AhR- and Arnt-mediated regulation of CXCL8 and CCL5 in BEAS-2B cells exposed to 1-NP and 1-AP (A) or Poly I:C (B). “x/X” and “y/Y” represents hitherto unidentified signaling pathways/response elements involved in regulation of CXCL8 and CCL5, respectively. The positioning of the response elements in the CXCL8 or CCL5 promoters is not meant to be representative. Moreover, “y/Y” induced by 1-AP (A) and Poly I:C (B) are not necessarily identical and AhR may also affect other pathways involved in Poly I:C-induced CXCL8 in addition to NF-κB (B). The suggested position and association of Arnt and RelB is partly based on findings reported by Wright and Duckett [49]. The role of AhR and Arnt in chemokine regulation may differ in other cell types.
Mentions: In the present study we have compared the effects of AhR and Arnt depletion on the classical NF-κB pathway and expression of two differentially regulated chemokines, CXCL8 and CCL5, in bronchial epithelial BEAS-2B cells exposed to PAHs or the TLR3 agonist Poly I:C. The results showed that regulation of CXCL8, but not CCL5, was dependent on basal and/or induced p65 activity. AhR suppressed both CXCL8 and CCL5 responses in BEAS-2B cells by widely differing stimuli, while Arnt only suppressed CXCL8. Moreover, the AhR-antagonist ANF failed to reproduce the effects of AhR knock-down on CXCL8/CCL5 regulation. This suggests that AhR suppressed chemokine responses independently of Arnt, and the effects were most likely due to the constitutive activity of unligated AhR. AhR suppressed p65 activation induced by strong activators of the classical NF-κB pathway, such as TNF-α and Poly I:C, but did not affect basal p65 activity. In addition, since AhR also suppressed CCL5, as well as the p65-independent increase in CXCL8 by 1-NP (which were unable to enhance p65 activity), the anti-inflammatory effects of AhR were necessarily not restricted to interference with NF-κB-signaling. In contrast to AhR, Arnt seemed unable to suppress the onset of p65 activation. Nevertheless, Arnt may still interfere with p65 signaling through other mechanisms, possibly in collaboration with RelB, which is a well-known suppressor of p65 activity. A schematic presentation of the possible roles of AhR and Arnt in suppression of CXCL8 and CCL5 in BEAS-2B cells, as discussed below, is presented in Figure 10. In addition to the above, we also observed that potent-AhR agonists could suppress both CXCL8 and CCL5-responses by TLR-ligands, but given alone at sufficiently high concentrations, they elicited a moderate activation of CXCL8. Thus ligand-activated AhR appear to possess both pro- and anti-inflammatory effects in BEAS-2B cells.

Bottom Line: In contrast, Arnt suppressed only CXCL8, but did not prevent the p65-activation directly.Moreover, ligand-activated AhR suppressed CXCL8 and CCL5 responses by other agents, but AhR ligands alone induced CXCL8 responses when given at sufficiently high concentrations, thus underscoring the duality of AhR in regulation of inflammation.We propose that AhR-signaling may be a weak activator of p65-signaling that suppresses p65-activity induced by strong activators of NF-κB, but that its anti-inflammatory properties also are due to interference with additional pathways.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: The aryl hydrocarbon receptor (AhR) has gradually emerged as a regulator of inflammation in the lung and other tissues. AhR may interact with the p65-subunit of the nuclear factor (NF)-κB transcription factors, but reported outcomes of AhR/NF-κB-interactions are conflicting. Some studies suggest that AhR possess pro-inflammatory activities while others suggest that AhR may be anti-inflammatory. The present study explored the impact of AhR and its binding partner AhR nuclear translocator (Arnt) on p65-activation and two differentially regulated chemokines, CXCL8 (IL-8) and CCL5 (RANTES), in human bronchial epithelial cells (BEAS-2B).

Results: Cells were exposed to CXCL8- and CCL5-inducing chemicals, 1-nitropyrene (1-NP) and 1-aminopyrene (1-AP) respectively, or the synthetic double-stranded RNA analogue, polyinosinic-polycytidylic acid (Poly I:C) which induced both chemokines. Only CXCL8, and not CCL5, appeared to be p65-dependent. Yet, constitutively active unligated AhR suppressed both CXCL8 and CCL5, as shown by siRNA knock-down and the AhR antagonist α-naphthoflavone. Moreover, AhR suppressed activation of p65 by TNF-α and Poly I:C as assessed by luciferase-assay and p65-phosphorylation at serine 536, without affecting basal p65-activity. In contrast, Arnt suppressed only CXCL8, but did not prevent the p65-activation directly. However, Arnt suppressed expression of the NF-κB-subunit RelB which is under transcriptional regulation by p65. Furthermore, AhR-ligands alone at high concentrations induced a moderate CXCL8-response, without affecting CCL5, but suppressed both CXCL8 and CCL5-responses by Poly I:C.

Conclusion: AhR and Arnt may differentially and independently regulate chemokine-responses induced by both inhaled pollutants and pulmonary infections. Constitutively active, unligated AhR suppressed the activation of p65, while Arnt may possibly interfere with the action of activated p65. Moreover, ligand-activated AhR suppressed CXCL8 and CCL5 responses by other agents, but AhR ligands alone induced CXCL8 responses when given at sufficiently high concentrations, thus underscoring the duality of AhR in regulation of inflammation. We propose that AhR-signaling may be a weak activator of p65-signaling that suppresses p65-activity induced by strong activators of NF-κB, but that its anti-inflammatory properties also are due to interference with additional pathways.

Show MeSH
Related in: MedlinePlus