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TWEAK activates the non-canonical NFkappaB pathway in murine renal tubular cells: modulation of CCL21.

Sanz AB, Sanchez-Niño MD, Izquierdo MC, Jakubowski A, Justo P, Blanco-Colio LM, Ruiz-Ortega M, Selgas R, Egido J, Ortiz A - PLoS ONE (2010)

Bottom Line: TWEAK-induced sustained NFkappaB activation was associated with NFkappaB2 p100 processing to p52 via proteasome and nuclear translocation and DNA-binding of p52 and RelB.Moreover, anti-TWEAK treatment prevented the recruitment of T cells to the kidney in this model (4.1+/-1.4 vs 1.8+/-1-fold over healthy control).Our results thus identify TWEAK as a regulator of non-canonical NFkappaB activation and CCL21 expression in tubular cells thus promoting lymphocyte recruitment to the kidney during acute injury.

View Article: PubMed Central - PubMed

Affiliation: Servicio de Nefrologia, Fundación para la Investigación Biomédica del Hospital Universitario La Paz, Madrid, Spain.

ABSTRACT
TWEAK is a member of the TNF superfamily of cytokines that contribute to kidney tubulointerstitial injury. It has previously been reported that TWEAK induces transient nuclear translocation of RelA and expression of RelA-dependent cytokines in renal tubular cells. Additionally, TWEAK induced long-lasting NFkappaB activation suggestive of engagement of the non-canonical NFkappaB pathway. We now explore TWEAK-induced activation of NFkappaB2 and RelB, as well as expression of CCL21, a T-cell chemotactic factor, in cultured murine tubular epithelial cells and in healthy kidneys in vivo. In cultured tubular cells, TWEAK and TNFalpha activated different DNA-binding NFkappaB complexes. TWEAK-induced sustained NFkappaB activation was associated with NFkappaB2 p100 processing to p52 via proteasome and nuclear translocation and DNA-binding of p52 and RelB. TWEAK, but not TNFalpha used as control), induced a delayed increase in CCL21a mRNA (3.5+/-1.22-fold over control) and CCL21 protein (2.5+/-0.8-fold over control), which was prevented by inhibition of the proteasome, or siRNA targeting of NIK or RelB, but not by RelA inhibition with parthenolide. A second NFkappaB2-dependent chemokine, CCL19, was upregulates by TWEAK, but not by TNFalpha. However, both cytokines promoted chemokine RANTES expression (3-fold mRNA at 24 h). In vivo, TWEAK induced nuclear NFkappaB2 and RelB translocation and CCL21a mRNA (1.5+/-0.3-fold over control) and CCL21 protein (1.6+/-0.5-fold over control) expression in normal kidney. Increased tubular nuclear RelB and tubular CCL21 expression in acute kidney injury were decreased by neutralization (2+/-0.9 vs 1.3+/-0.6-fold over healthy control) or deficiency of TWEAK (2+/-0.9 vs 0.8+/-0.6-fold over healthy control). Moreover, anti-TWEAK treatment prevented the recruitment of T cells to the kidney in this model (4.1+/-1.4 vs 1.8+/-1-fold over healthy control). Our results thus identify TWEAK as a regulator of non-canonical NFkappaB activation and CCL21 expression in tubular cells thus promoting lymphocyte recruitment to the kidney during acute injury.

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TWEAK induces the expression of NFκB2 targets, CCL21 and CCL19, in tubular cells.A) Time-dependent increase in CCL21a, CCL19 and RANTES mRNA expression in tubular cells treated with 100 ng/mL TWEAK or 30 ng/ml TNFα. Real time RT-PCR. Mean ±SEM of four independent experiments. *p<0.04 vs control. B) Time-dependent increase in CCL21 protein expression in tubular cells treated with 100 ng/mL TWEAK or 30 ng/mL TNFα. Mean ±SEM of four independent experiments. *p<0.03 vs control. C) Parthenolide (PTN) does not prevent TWEAK-induced CCL21a mRNA up-regulation at 24 h. Real time RT-PCR. Mean ±SEM of four experiments. *p<0.04 vs control; #p<0.04 vs TWEAK alone. D) Lactacystin prevents TWEAK-induced CCL21a mRNA up-regulation at 24 h. Real time RT-PCR. Mean ±SEM of four experiments. *p<0.04 vs control; #p<0.03 vs TWEAK alone.
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pone-0008955-g004: TWEAK induces the expression of NFκB2 targets, CCL21 and CCL19, in tubular cells.A) Time-dependent increase in CCL21a, CCL19 and RANTES mRNA expression in tubular cells treated with 100 ng/mL TWEAK or 30 ng/ml TNFα. Real time RT-PCR. Mean ±SEM of four independent experiments. *p<0.04 vs control. B) Time-dependent increase in CCL21 protein expression in tubular cells treated with 100 ng/mL TWEAK or 30 ng/mL TNFα. Mean ±SEM of four independent experiments. *p<0.03 vs control. C) Parthenolide (PTN) does not prevent TWEAK-induced CCL21a mRNA up-regulation at 24 h. Real time RT-PCR. Mean ±SEM of four experiments. *p<0.04 vs control; #p<0.04 vs TWEAK alone. D) Lactacystin prevents TWEAK-induced CCL21a mRNA up-regulation at 24 h. Real time RT-PCR. Mean ±SEM of four experiments. *p<0.04 vs control; #p<0.03 vs TWEAK alone.

Mentions: CCL21 was previously reported to be a gene target of the non-canonical NFκB2 pathway in splenocytes [19], [33]. We selected CCL21 as a potential target of NFκB2 activation in renal cells because it modulates T cell recruitment and CCL21 has recently been reported to play a role in tubulointerstitial injury [26]. Additionally, the pathways that regulate its expression in the kidney are unknown. As previously reported basal CCL21a mRNA levels were 40-fold higher in control spleen than in control kidney (not shown) [34]. TWEAK induced the expression of CCL21a mRNA in cultured tubular cells. The time-course of CCL21a stimulation paralleled that of RelB/p52 accumulation in nuclei, increasing up to 24 h (Figure 4A). We used RANTES as a TNF-inducible control. At the protein level, TWEAK, but not TNFα, induced the expression of the CCL21 cytokine (Figure 4B). Unsupervised hierarchical cluster analysis of an array of 94 immune response-related genes did not disclose differences between TNF and TWEAK treated cells at either 4 or 24 h: samples stimulated with either cytokine clustered together and could not be separated. However, induction of CCL19 mRNA, a known target of NFκB2 [19], [35], was only observed in TWEAK stimulated cells. Induction of CCL19 mRNA with a time-course consistent with the RelB/p52 accumulation in nuclei by TWEAK, but not by TNF, was then confirmed by qRT-PCR (Figure 4A).


TWEAK activates the non-canonical NFkappaB pathway in murine renal tubular cells: modulation of CCL21.

Sanz AB, Sanchez-Niño MD, Izquierdo MC, Jakubowski A, Justo P, Blanco-Colio LM, Ruiz-Ortega M, Selgas R, Egido J, Ortiz A - PLoS ONE (2010)

TWEAK induces the expression of NFκB2 targets, CCL21 and CCL19, in tubular cells.A) Time-dependent increase in CCL21a, CCL19 and RANTES mRNA expression in tubular cells treated with 100 ng/mL TWEAK or 30 ng/ml TNFα. Real time RT-PCR. Mean ±SEM of four independent experiments. *p<0.04 vs control. B) Time-dependent increase in CCL21 protein expression in tubular cells treated with 100 ng/mL TWEAK or 30 ng/mL TNFα. Mean ±SEM of four independent experiments. *p<0.03 vs control. C) Parthenolide (PTN) does not prevent TWEAK-induced CCL21a mRNA up-regulation at 24 h. Real time RT-PCR. Mean ±SEM of four experiments. *p<0.04 vs control; #p<0.04 vs TWEAK alone. D) Lactacystin prevents TWEAK-induced CCL21a mRNA up-regulation at 24 h. Real time RT-PCR. Mean ±SEM of four experiments. *p<0.04 vs control; #p<0.03 vs TWEAK alone.
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pone-0008955-g004: TWEAK induces the expression of NFκB2 targets, CCL21 and CCL19, in tubular cells.A) Time-dependent increase in CCL21a, CCL19 and RANTES mRNA expression in tubular cells treated with 100 ng/mL TWEAK or 30 ng/ml TNFα. Real time RT-PCR. Mean ±SEM of four independent experiments. *p<0.04 vs control. B) Time-dependent increase in CCL21 protein expression in tubular cells treated with 100 ng/mL TWEAK or 30 ng/mL TNFα. Mean ±SEM of four independent experiments. *p<0.03 vs control. C) Parthenolide (PTN) does not prevent TWEAK-induced CCL21a mRNA up-regulation at 24 h. Real time RT-PCR. Mean ±SEM of four experiments. *p<0.04 vs control; #p<0.04 vs TWEAK alone. D) Lactacystin prevents TWEAK-induced CCL21a mRNA up-regulation at 24 h. Real time RT-PCR. Mean ±SEM of four experiments. *p<0.04 vs control; #p<0.03 vs TWEAK alone.
Mentions: CCL21 was previously reported to be a gene target of the non-canonical NFκB2 pathway in splenocytes [19], [33]. We selected CCL21 as a potential target of NFκB2 activation in renal cells because it modulates T cell recruitment and CCL21 has recently been reported to play a role in tubulointerstitial injury [26]. Additionally, the pathways that regulate its expression in the kidney are unknown. As previously reported basal CCL21a mRNA levels were 40-fold higher in control spleen than in control kidney (not shown) [34]. TWEAK induced the expression of CCL21a mRNA in cultured tubular cells. The time-course of CCL21a stimulation paralleled that of RelB/p52 accumulation in nuclei, increasing up to 24 h (Figure 4A). We used RANTES as a TNF-inducible control. At the protein level, TWEAK, but not TNFα, induced the expression of the CCL21 cytokine (Figure 4B). Unsupervised hierarchical cluster analysis of an array of 94 immune response-related genes did not disclose differences between TNF and TWEAK treated cells at either 4 or 24 h: samples stimulated with either cytokine clustered together and could not be separated. However, induction of CCL19 mRNA, a known target of NFκB2 [19], [35], was only observed in TWEAK stimulated cells. Induction of CCL19 mRNA with a time-course consistent with the RelB/p52 accumulation in nuclei by TWEAK, but not by TNF, was then confirmed by qRT-PCR (Figure 4A).

Bottom Line: TWEAK-induced sustained NFkappaB activation was associated with NFkappaB2 p100 processing to p52 via proteasome and nuclear translocation and DNA-binding of p52 and RelB.Moreover, anti-TWEAK treatment prevented the recruitment of T cells to the kidney in this model (4.1+/-1.4 vs 1.8+/-1-fold over healthy control).Our results thus identify TWEAK as a regulator of non-canonical NFkappaB activation and CCL21 expression in tubular cells thus promoting lymphocyte recruitment to the kidney during acute injury.

View Article: PubMed Central - PubMed

Affiliation: Servicio de Nefrologia, Fundación para la Investigación Biomédica del Hospital Universitario La Paz, Madrid, Spain.

ABSTRACT
TWEAK is a member of the TNF superfamily of cytokines that contribute to kidney tubulointerstitial injury. It has previously been reported that TWEAK induces transient nuclear translocation of RelA and expression of RelA-dependent cytokines in renal tubular cells. Additionally, TWEAK induced long-lasting NFkappaB activation suggestive of engagement of the non-canonical NFkappaB pathway. We now explore TWEAK-induced activation of NFkappaB2 and RelB, as well as expression of CCL21, a T-cell chemotactic factor, in cultured murine tubular epithelial cells and in healthy kidneys in vivo. In cultured tubular cells, TWEAK and TNFalpha activated different DNA-binding NFkappaB complexes. TWEAK-induced sustained NFkappaB activation was associated with NFkappaB2 p100 processing to p52 via proteasome and nuclear translocation and DNA-binding of p52 and RelB. TWEAK, but not TNFalpha used as control), induced a delayed increase in CCL21a mRNA (3.5+/-1.22-fold over control) and CCL21 protein (2.5+/-0.8-fold over control), which was prevented by inhibition of the proteasome, or siRNA targeting of NIK or RelB, but not by RelA inhibition with parthenolide. A second NFkappaB2-dependent chemokine, CCL19, was upregulates by TWEAK, but not by TNFalpha. However, both cytokines promoted chemokine RANTES expression (3-fold mRNA at 24 h). In vivo, TWEAK induced nuclear NFkappaB2 and RelB translocation and CCL21a mRNA (1.5+/-0.3-fold over control) and CCL21 protein (1.6+/-0.5-fold over control) expression in normal kidney. Increased tubular nuclear RelB and tubular CCL21 expression in acute kidney injury were decreased by neutralization (2+/-0.9 vs 1.3+/-0.6-fold over healthy control) or deficiency of TWEAK (2+/-0.9 vs 0.8+/-0.6-fold over healthy control). Moreover, anti-TWEAK treatment prevented the recruitment of T cells to the kidney in this model (4.1+/-1.4 vs 1.8+/-1-fold over healthy control). Our results thus identify TWEAK as a regulator of non-canonical NFkappaB activation and CCL21 expression in tubular cells thus promoting lymphocyte recruitment to the kidney during acute injury.

Show MeSH
Related in: MedlinePlus