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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.

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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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P65 is required for CXCL8 but not CCL5 responses in 1-NP-or 1-AP-exposed BEAS-2B cells. Cells were transfected with siRNA against p65 (siP65) or non-targeting control siRNA (siNT), and exposed to 20 μM 1-NP, 1-AP or vehicle (DMSO) alone. CXCL8 (A) and CCL5 (B) gene expression were measured after 6 h by real-time PCR. CXCL8 (C) and CCL5 (D) protein levels in the medium were measured by ELISA after 18 h exposure as described under “Materials and methods”. Efficiency of transfection was assessed by Western blotting (E). The results are expressed as mean ± SEM (A/B: n = 1 (triplicate determinations); C/D: n ≥ 3; E: representative blot, n = 3). *Significantly different from unexposed controls; †Significantly different from cells transfected with non-targeting siRNA.
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Figure 1: P65 is required for CXCL8 but not CCL5 responses in 1-NP-or 1-AP-exposed BEAS-2B cells. Cells were transfected with siRNA against p65 (siP65) or non-targeting control siRNA (siNT), and exposed to 20 μM 1-NP, 1-AP or vehicle (DMSO) alone. CXCL8 (A) and CCL5 (B) gene expression were measured after 6 h by real-time PCR. CXCL8 (C) and CCL5 (D) protein levels in the medium were measured by ELISA after 18 h exposure as described under “Materials and methods”. Efficiency of transfection was assessed by Western blotting (E). The results are expressed as mean ± SEM (A/B: n = 1 (triplicate determinations); C/D: n ≥ 3; E: representative blot, n = 3). *Significantly different from unexposed controls; †Significantly different from cells transfected with non-targeting siRNA.

Mentions: We have previously shown that 1-NP induces CXCL8, while 1-AP induces CCL5 in BEAS-2B cells [23],[24]. As a first step, we explored the involvement of the classical NF-κB pathways in the 1-NP- and 1-AP-induced chemokine responses by transfecting the cells with siRNA against p65 or non-targeting control siRNA. The classical NF-κB pathway is generally considered necessary for transcription of CXCL8 [40]. In line with this, we found that p65 silencing completely blocked both basal and induced CXCL8 responses (Figure 1A and C). In contrast, p65 siRNA had little impact on, or rather increased, the 1-AP-induced CCL5 response (Figure 1B and D). The protein level of p65 was markedly down-regulated in cells treated with the p65 siRNAs, confirming the efficiency of the transfection (Figure 1E). The above results suggest that the classical NF-κB pathway is needed for the CXCL8 response, but not for CCL5 in our cell model.


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)

P65 is required for CXCL8 but not CCL5 responses in 1-NP-or 1-AP-exposed BEAS-2B cells. Cells were transfected with siRNA against p65 (siP65) or non-targeting control siRNA (siNT), and exposed to 20 μM 1-NP, 1-AP or vehicle (DMSO) alone. CXCL8 (A) and CCL5 (B) gene expression were measured after 6 h by real-time PCR. CXCL8 (C) and CCL5 (D) protein levels in the medium were measured by ELISA after 18 h exposure as described under “Materials and methods”. Efficiency of transfection was assessed by Western blotting (E). The results are expressed as mean ± SEM (A/B: n = 1 (triplicate determinations); C/D: n ≥ 3; E: representative blot, n = 3). *Significantly different from unexposed controls; †Significantly different from cells transfected with non-targeting siRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: P65 is required for CXCL8 but not CCL5 responses in 1-NP-or 1-AP-exposed BEAS-2B cells. Cells were transfected with siRNA against p65 (siP65) or non-targeting control siRNA (siNT), and exposed to 20 μM 1-NP, 1-AP or vehicle (DMSO) alone. CXCL8 (A) and CCL5 (B) gene expression were measured after 6 h by real-time PCR. CXCL8 (C) and CCL5 (D) protein levels in the medium were measured by ELISA after 18 h exposure as described under “Materials and methods”. Efficiency of transfection was assessed by Western blotting (E). The results are expressed as mean ± SEM (A/B: n = 1 (triplicate determinations); C/D: n ≥ 3; E: representative blot, n = 3). *Significantly different from unexposed controls; †Significantly different from cells transfected with non-targeting siRNA.
Mentions: We have previously shown that 1-NP induces CXCL8, while 1-AP induces CCL5 in BEAS-2B cells [23],[24]. As a first step, we explored the involvement of the classical NF-κB pathways in the 1-NP- and 1-AP-induced chemokine responses by transfecting the cells with siRNA against p65 or non-targeting control siRNA. The classical NF-κB pathway is generally considered necessary for transcription of CXCL8 [40]. In line with this, we found that p65 silencing completely blocked both basal and induced CXCL8 responses (Figure 1A and C). In contrast, p65 siRNA had little impact on, or rather increased, the 1-AP-induced CCL5 response (Figure 1B and D). The protein level of p65 was markedly down-regulated in cells treated with the p65 siRNAs, confirming the efficiency of the transfection (Figure 1E). The above results suggest that the classical NF-κB pathway is needed for the CXCL8 response, but not for CCL5 in our cell model.

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