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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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Nuclear translocation of p52 and RelB in TWEAK-stimulated tubular cells.A, B) Temporal pattern of nuclear translocation of RelA, RelB and p52 in tubular cells stimulated with TWEAK, (A), or TNFα (B). ELISA of nuclear proteins binding to an oligonucleotide containing the NFκB consensus site. C) Tubular cells were stimulated with 100 ng/mL TWEAK and nuclear cell lysates obtained at various time points were analyzed by Western blot for p52 and RelB. Representative Western blot and densitometric quantification. Mean ± SEM of four independent experiments. *p<0.04 vs control; #p<0.009 vs control. No significant differences were observed between controls at different time points and they were grouped. D) TWEAK induces degradation of the inhibitory subunits IkBα and IkBβ, in a time-course consistent with canonical NFκB pathway activation. Representative western blot of three independent experiments. E) TWEAK induces phosporylation of IKKα subunit of IKK complex. Representative Western blot and quantification of three independent experiments. Mean ± SEM of three experiments. *p<0.05 vs control.
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pone-0008955-g002: Nuclear translocation of p52 and RelB in TWEAK-stimulated tubular cells.A, B) Temporal pattern of nuclear translocation of RelA, RelB and p52 in tubular cells stimulated with TWEAK, (A), or TNFα (B). ELISA of nuclear proteins binding to an oligonucleotide containing the NFκB consensus site. C) Tubular cells were stimulated with 100 ng/mL TWEAK and nuclear cell lysates obtained at various time points were analyzed by Western blot for p52 and RelB. Representative Western blot and densitometric quantification. Mean ± SEM of four independent experiments. *p<0.04 vs control; #p<0.009 vs control. No significant differences were observed between controls at different time points and they were grouped. D) TWEAK induces degradation of the inhibitory subunits IkBα and IkBβ, in a time-course consistent with canonical NFκB pathway activation. Representative western blot of three independent experiments. E) TWEAK induces phosporylation of IKKα subunit of IKK complex. Representative Western blot and quantification of three independent experiments. Mean ± SEM of three experiments. *p<0.05 vs control.

Mentions: TWEAK reportedly induced early and transient nuclear translocation of RelA in tubular cells [9]. We now show that TWEAK induces a progressive time-dependent increment in nuclear p52 and RelB, as assessed by ELISA of nuclear extracts (Figure 2A) Western blot (Figure 2C), and confocal microscopy (Figure 3C). An ELISA of nuclear NFκB subunits confirmed the early and transient nature of the increase in nuclear RelA and the progressive increase in nuclear RelB and p52 in MCT cells treated with TWEAK (Figure 2A), whereas TNFα only induces nuclear RelA translocation (Figure 2B). In MCT cells TWEAK induces degradation of IκBα and IκBβ (Figure 2D), and this was temporarily associated with translocation of the RelA subunit of NFκB from the cytoplasm to the nucleus [9]. The activation of the IKKα subunit of the IKK complex is necessary for NFκB2 activation via the non-canonical pathway [18]. In this regard, we observed by western blot that TWEAK induces IKKα phosphorylation (Figure 2E).


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)

Nuclear translocation of p52 and RelB in TWEAK-stimulated tubular cells.A, B) Temporal pattern of nuclear translocation of RelA, RelB and p52 in tubular cells stimulated with TWEAK, (A), or TNFα (B). ELISA of nuclear proteins binding to an oligonucleotide containing the NFκB consensus site. C) Tubular cells were stimulated with 100 ng/mL TWEAK and nuclear cell lysates obtained at various time points were analyzed by Western blot for p52 and RelB. Representative Western blot and densitometric quantification. Mean ± SEM of four independent experiments. *p<0.04 vs control; #p<0.009 vs control. No significant differences were observed between controls at different time points and they were grouped. D) TWEAK induces degradation of the inhibitory subunits IkBα and IkBβ, in a time-course consistent with canonical NFκB pathway activation. Representative western blot of three independent experiments. E) TWEAK induces phosporylation of IKKα subunit of IKK complex. Representative Western blot and quantification of three independent experiments. Mean ± SEM of three experiments. *p<0.05 vs control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2813291&req=5

pone-0008955-g002: Nuclear translocation of p52 and RelB in TWEAK-stimulated tubular cells.A, B) Temporal pattern of nuclear translocation of RelA, RelB and p52 in tubular cells stimulated with TWEAK, (A), or TNFα (B). ELISA of nuclear proteins binding to an oligonucleotide containing the NFκB consensus site. C) Tubular cells were stimulated with 100 ng/mL TWEAK and nuclear cell lysates obtained at various time points were analyzed by Western blot for p52 and RelB. Representative Western blot and densitometric quantification. Mean ± SEM of four independent experiments. *p<0.04 vs control; #p<0.009 vs control. No significant differences were observed between controls at different time points and they were grouped. D) TWEAK induces degradation of the inhibitory subunits IkBα and IkBβ, in a time-course consistent with canonical NFκB pathway activation. Representative western blot of three independent experiments. E) TWEAK induces phosporylation of IKKα subunit of IKK complex. Representative Western blot and quantification of three independent experiments. Mean ± SEM of three experiments. *p<0.05 vs control.
Mentions: TWEAK reportedly induced early and transient nuclear translocation of RelA in tubular cells [9]. We now show that TWEAK induces a progressive time-dependent increment in nuclear p52 and RelB, as assessed by ELISA of nuclear extracts (Figure 2A) Western blot (Figure 2C), and confocal microscopy (Figure 3C). An ELISA of nuclear NFκB subunits confirmed the early and transient nature of the increase in nuclear RelA and the progressive increase in nuclear RelB and p52 in MCT cells treated with TWEAK (Figure 2A), whereas TNFα only induces nuclear RelA translocation (Figure 2B). In MCT cells TWEAK induces degradation of IκBα and IκBβ (Figure 2D), and this was temporarily associated with translocation of the RelA subunit of NFκB from the cytoplasm to the nucleus [9]. The activation of the IKKα subunit of the IKK complex is necessary for NFκB2 activation via the non-canonical pathway [18]. In this regard, we observed by western blot that TWEAK induces IKKα phosphorylation (Figure 2E).

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