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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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Decreased secretion of pyrophosphate (PPi) by ANKH mediated TNF-augmented calcification. Real-time RT-PCR identified the overexpression of ANKH (A) by retroviral vector in HASMCs; EV, empty vector. (B) Calcium content assay and (C) Alizarin-red staining of protective effect of ANKH overexpression on calcification induced by Pi (3.0 mM) and/or TNF (10 ng/ml) for 7 days. (D) TNF (10 ng/ml) reduced extracellular PPi levels in both control cells and Pi-induced calcified cells. (E) HASMCs were treated with ANKH inhibitor (ANKHi: probenecid, 2 mM) or overexpressed ANKH for 3 days, and PPi levels in the medium were measured. (F) HASMCs were treated with ANKH inhibitor or vehicle, then with Pi and/or TNF (10 ng/ml) for 7 days. Calcium content was assayed. (*P<0.05 n=3)
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Figure 2: Decreased secretion of pyrophosphate (PPi) by ANKH mediated TNF-augmented calcification. Real-time RT-PCR identified the overexpression of ANKH (A) by retroviral vector in HASMCs; EV, empty vector. (B) Calcium content assay and (C) Alizarin-red staining of protective effect of ANKH overexpression on calcification induced by Pi (3.0 mM) and/or TNF (10 ng/ml) for 7 days. (D) TNF (10 ng/ml) reduced extracellular PPi levels in both control cells and Pi-induced calcified cells. (E) HASMCs were treated with ANKH inhibitor (ANKHi: probenecid, 2 mM) or overexpressed ANKH for 3 days, and PPi levels in the medium were measured. (F) HASMCs were treated with ANKH inhibitor or vehicle, then with Pi and/or TNF (10 ng/ml) for 7 days. Calcium content was assayed. (*P<0.05 n=3)

Mentions: ANKH is a multipass transmembrane protein that inhibits mineralization by controlling PPi levels. The deletion of ANKH in mouse results in abnormal mineralization in articular cartilage and soft tissues (15). To determine whether TNF promoted vascular calcification by inhibiting ANKH expression, we examined whether ectopic expression of ANKH could inhibit TNF-enhanced vascular calcification in HASMCs by retroviral transduction (Figure 2A). TNF potently enhanced Pi-induced mineralization in HASMCs expressing empty vector, but mineralization was significantly suppressed in HASMCs expressing ANKH (Figure 2B, C). Consistent with reducing ANKH expression, TNF significantly reduced PPi level secretion from both control and Pi-induced calcified cells (Figure 2D). As well, ANKH inhibitor (probenecid, 2 mM) decreased and ANKH overexpression increased PPi secretion (Figure 2E). The effect of probenecid and TNF on Pi-induced calcification was equivalent, and TNF failed to further increase calcium deposition in the presence of probenecid (Figure 2F).


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)

Decreased secretion of pyrophosphate (PPi) by ANKH mediated TNF-augmented calcification. Real-time RT-PCR identified the overexpression of ANKH (A) by retroviral vector in HASMCs; EV, empty vector. (B) Calcium content assay and (C) Alizarin-red staining of protective effect of ANKH overexpression on calcification induced by Pi (3.0 mM) and/or TNF (10 ng/ml) for 7 days. (D) TNF (10 ng/ml) reduced extracellular PPi levels in both control cells and Pi-induced calcified cells. (E) HASMCs were treated with ANKH inhibitor (ANKHi: probenecid, 2 mM) or overexpressed ANKH for 3 days, and PPi levels in the medium were measured. (F) HASMCs were treated with ANKH inhibitor or vehicle, then with Pi and/or TNF (10 ng/ml) for 7 days. Calcium content was assayed. (*P<0.05 n=3)
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376207&req=5

Figure 2: Decreased secretion of pyrophosphate (PPi) by ANKH mediated TNF-augmented calcification. Real-time RT-PCR identified the overexpression of ANKH (A) by retroviral vector in HASMCs; EV, empty vector. (B) Calcium content assay and (C) Alizarin-red staining of protective effect of ANKH overexpression on calcification induced by Pi (3.0 mM) and/or TNF (10 ng/ml) for 7 days. (D) TNF (10 ng/ml) reduced extracellular PPi levels in both control cells and Pi-induced calcified cells. (E) HASMCs were treated with ANKH inhibitor (ANKHi: probenecid, 2 mM) or overexpressed ANKH for 3 days, and PPi levels in the medium were measured. (F) HASMCs were treated with ANKH inhibitor or vehicle, then with Pi and/or TNF (10 ng/ml) for 7 days. Calcium content was assayed. (*P<0.05 n=3)
Mentions: ANKH is a multipass transmembrane protein that inhibits mineralization by controlling PPi levels. The deletion of ANKH in mouse results in abnormal mineralization in articular cartilage and soft tissues (15). To determine whether TNF promoted vascular calcification by inhibiting ANKH expression, we examined whether ectopic expression of ANKH could inhibit TNF-enhanced vascular calcification in HASMCs by retroviral transduction (Figure 2A). TNF potently enhanced Pi-induced mineralization in HASMCs expressing empty vector, but mineralization was significantly suppressed in HASMCs expressing ANKH (Figure 2B, C). Consistent with reducing ANKH expression, TNF significantly reduced PPi level secretion from both control and Pi-induced calcified cells (Figure 2D). As well, ANKH inhibitor (probenecid, 2 mM) decreased and ANKH overexpression increased PPi secretion (Figure 2E). The effect of probenecid and TNF on Pi-induced calcification was equivalent, and TNF failed to further increase calcium deposition in the presence of probenecid (Figure 2F).

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