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Activation of nuclear factor-kappa B accelerates vascular calcification by inhibiting ankylosis protein homolog expression.

Zhao G, Xu MJ, Zhao MM, Dai XY, Kong W, Wilson GM, Guan Y, Wang CY, Wang X - Kidney Int. (2012)

Bottom Line: Although chronic inflammation is one of the etiologic factors, the underlying mechanism is not fully understood.Furthermore, a rat chronic renal failure model, with increased serum TNF levels, activated NF-κB and decreased ANKH levels.Both human calcified atherosclerotic lesions and arteries from patients with chronic kidney disease had activated NF-κB and decreased ANKH expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pathophysiology, Key Laboratory of Molecular Cardiovascular Science, School of Basic Medical Science, Peking University Health Science Center, Ministry of Education, Beijing, PR China.

ABSTRACT
Vascular calcification is a major risk factor of cardiovascular mortality, particularly for patients with end-stage renal disease and diabetes. Although chronic inflammation is one of the etiologic factors, the underlying mechanism is not fully understood. To clarify this, we studied how nuclear factor-kappa B (NF-κB) induction, a mediator of inflammation, might promote vascular calcification. Activation of NF-κB by tumor necrosis factor (TNF) promoted inorganic phosphate-induced calcification in human aortic smooth muscle cells. Pyrophosphate (an inhibitor of calcification) efflux to the extracellular matrix was suppressed along with the decreased expression of ankylosis protein homolog (ANKH), a transmembrane protein that controls pyrophosphate efflux of cells. The restoration of ANKH expression in these cells overcame the decreased pyrophosphate efflux and calcification. Tristetraprolin, a downstream product of NF-κB activation, may mediate destabilization of ANKH mRNA as its knockdown by shRNA increased ANKH expression and decreased calcification. Furthermore, a rat chronic renal failure model, with increased serum TNF levels, activated NF-κB and decreased ANKH levels. In contrast, the inhibition of NF-κB maintained ANKH expression and attenuated vascular calcification both in vivo and in vitro. Both human calcified atherosclerotic lesions and arteries from patients with chronic kidney disease had activated NF-κB and decreased ANKH expression. Thus, TNF-activated NF-κB promotes inflammation-accelerated vascular calcification by inhibiting ankylosis protein homolog expression and consequent pyrophosphate secretion.

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IKK inhibitor blocked the effects of TNF on ANKH expression and calcification. (A) Real-time PCR analysis of the mRNA levels of interleukin 8 (IL-8), IκBα and monocyte chemoattractant protein 1 (MCP-1). (B) Western blot analysis of TNF (10 ng/ml)-induced translocation of p65, phosphorylation (p) and degradation of IκBα with or without IKK inhibitor (IKKi: IKKβVI, 2 μM) in HASMCs. HASMCs were preincubated with the IKK inhibitor for 30 min and then treated with TNF (10 ng/ml). The internal controls are TFIIB and α-tubulin. (C) Real-time RT-PCR analysis of ANKH mRNA level. HASMCs were preincubated with the IKK inhibitor for 30 min and then treated with Pi and/or TNF (10 ng/ml) for 8 hr, then (D) PPi level and (E) calcium content were measured. (*P<0.05 n=3)
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Figure 3: IKK inhibitor blocked the effects of TNF on ANKH expression and calcification. (A) Real-time PCR analysis of the mRNA levels of interleukin 8 (IL-8), IκBα and monocyte chemoattractant protein 1 (MCP-1). (B) Western blot analysis of TNF (10 ng/ml)-induced translocation of p65, phosphorylation (p) and degradation of IκBα with or without IKK inhibitor (IKKi: IKKβVI, 2 μM) in HASMCs. HASMCs were preincubated with the IKK inhibitor for 30 min and then treated with TNF (10 ng/ml). The internal controls are TFIIB and α-tubulin. (C) Real-time RT-PCR analysis of ANKH mRNA level. HASMCs were preincubated with the IKK inhibitor for 30 min and then treated with Pi and/or TNF (10 ng/ml) for 8 hr, then (D) PPi level and (E) calcium content were measured. (*P<0.05 n=3)

Mentions: Because NF-κB is a major downstream effector, we examined whether TNF activates NF-κB to inhibit ANKH, thereby promoting vascular calcification in HASMCs. TNF strongly induced the expression of NF-κB target genes, including interleukin 8 (IL-8), IκBα, and monocyte chemoattractant protein 1 (MCP-1) (Figure 3A), all associated with vascular inflammation. Furthermore, TNF rapidly induced the phosphorylation and degradation of IκBα in HASMCs; consistently, TNF stimulated the nuclear translocation of p65, which was blocked by IKK inhibition (IKKβVI, 2 μM) (Figure 3B). In accordance, IKK inhibition blocked TNF-induced decrease in ANKH expression (Figure 3C) and PPi secretion (Figure 3D) and pro-calcification effects (Figure 3E).


Activation of nuclear factor-kappa B accelerates vascular calcification by inhibiting ankylosis protein homolog expression.

Zhao G, Xu MJ, Zhao MM, Dai XY, Kong W, Wilson GM, Guan Y, Wang CY, Wang X - Kidney Int. (2012)

IKK inhibitor blocked the effects of TNF on ANKH expression and calcification. (A) Real-time PCR analysis of the mRNA levels of interleukin 8 (IL-8), IκBα and monocyte chemoattractant protein 1 (MCP-1). (B) Western blot analysis of TNF (10 ng/ml)-induced translocation of p65, phosphorylation (p) and degradation of IκBα with or without IKK inhibitor (IKKi: IKKβVI, 2 μM) in HASMCs. HASMCs were preincubated with the IKK inhibitor for 30 min and then treated with TNF (10 ng/ml). The internal controls are TFIIB and α-tubulin. (C) Real-time RT-PCR analysis of ANKH mRNA level. HASMCs were preincubated with the IKK inhibitor for 30 min and then treated with Pi and/or TNF (10 ng/ml) for 8 hr, then (D) PPi level and (E) calcium content were measured. (*P<0.05 n=3)
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Figure 3: IKK inhibitor blocked the effects of TNF on ANKH expression and calcification. (A) Real-time PCR analysis of the mRNA levels of interleukin 8 (IL-8), IκBα and monocyte chemoattractant protein 1 (MCP-1). (B) Western blot analysis of TNF (10 ng/ml)-induced translocation of p65, phosphorylation (p) and degradation of IκBα with or without IKK inhibitor (IKKi: IKKβVI, 2 μM) in HASMCs. HASMCs were preincubated with the IKK inhibitor for 30 min and then treated with TNF (10 ng/ml). The internal controls are TFIIB and α-tubulin. (C) Real-time RT-PCR analysis of ANKH mRNA level. HASMCs were preincubated with the IKK inhibitor for 30 min and then treated with Pi and/or TNF (10 ng/ml) for 8 hr, then (D) PPi level and (E) calcium content were measured. (*P<0.05 n=3)
Mentions: Because NF-κB is a major downstream effector, we examined whether TNF activates NF-κB to inhibit ANKH, thereby promoting vascular calcification in HASMCs. TNF strongly induced the expression of NF-κB target genes, including interleukin 8 (IL-8), IκBα, and monocyte chemoattractant protein 1 (MCP-1) (Figure 3A), all associated with vascular inflammation. Furthermore, TNF rapidly induced the phosphorylation and degradation of IκBα in HASMCs; consistently, TNF stimulated the nuclear translocation of p65, which was blocked by IKK inhibition (IKKβVI, 2 μM) (Figure 3B). In accordance, IKK inhibition blocked TNF-induced decrease in ANKH expression (Figure 3C) and PPi secretion (Figure 3D) and pro-calcification effects (Figure 3E).

Bottom Line: Although chronic inflammation is one of the etiologic factors, the underlying mechanism is not fully understood.Furthermore, a rat chronic renal failure model, with increased serum TNF levels, activated NF-κB and decreased ANKH levels.Both human calcified atherosclerotic lesions and arteries from patients with chronic kidney disease had activated NF-κB and decreased ANKH expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pathophysiology, Key Laboratory of Molecular Cardiovascular Science, School of Basic Medical Science, Peking University Health Science Center, Ministry of Education, Beijing, PR China.

ABSTRACT
Vascular calcification is a major risk factor of cardiovascular mortality, particularly for patients with end-stage renal disease and diabetes. Although chronic inflammation is one of the etiologic factors, the underlying mechanism is not fully understood. To clarify this, we studied how nuclear factor-kappa B (NF-κB) induction, a mediator of inflammation, might promote vascular calcification. Activation of NF-κB by tumor necrosis factor (TNF) promoted inorganic phosphate-induced calcification in human aortic smooth muscle cells. Pyrophosphate (an inhibitor of calcification) efflux to the extracellular matrix was suppressed along with the decreased expression of ankylosis protein homolog (ANKH), a transmembrane protein that controls pyrophosphate efflux of cells. The restoration of ANKH expression in these cells overcame the decreased pyrophosphate efflux and calcification. Tristetraprolin, a downstream product of NF-κB activation, may mediate destabilization of ANKH mRNA as its knockdown by shRNA increased ANKH expression and decreased calcification. Furthermore, a rat chronic renal failure model, with increased serum TNF levels, activated NF-κB and decreased ANKH levels. In contrast, the inhibition of NF-κB maintained ANKH expression and attenuated vascular calcification both in vivo and in vitro. Both human calcified atherosclerotic lesions and arteries from patients with chronic kidney disease had activated NF-κB and decreased ANKH expression. Thus, TNF-activated NF-κB promotes inflammation-accelerated vascular calcification by inhibiting ankylosis protein homolog expression and consequent pyrophosphate secretion.

Show MeSH
Related in: MedlinePlus