Limits...
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.

Show MeSH

Related in: MedlinePlus

NF-κB signalling mediated the effects of TNF on ANKH expression and calcification in HASMCs. (A) Western blot analysis confirmed the overexpression of flag-tagged p65 via retrovirus vector in HASMCs; EV, empty vector. (B) HASMCs were stably transfected with EV or p65, then treated with Pi (3.0 mM) for 7 days; Alizarin-red staining and calcium content assay of calcium deposition. (C) HASMCs transfected with EV or p65 were treated with vehicle or Pi for 3 days; RT-PCR analysis of IL-8 and ANKH mRNA levels. (D) Western blot analysis confirmed p65 knockdown via lentivirus vector in HASMCs. (E) HASMCs stably expressing scramble or p65-shRNA were treated with Pi (3.0 mM) and/or TNF (10 ng/ml) for 7 days; Alizarin-red staining and calcium content assay of calcium deposition. (F) HASMCs expressing scramble or p65-shRNA were treated with Pi and/or TNF (10 ng/ml) for 3 days; RT-PCR analysis of IL-8 and ANKH mRNA levels. (G) HASMCs stably expressing p65 and/or ANKH via retrovirus vector were treated with Pi for 7 days; calcium content assay of calcium deposition. (*P<0.05 vs EV or scramble, n=3)
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3376207&req=5

Figure 4: NF-κB signalling mediated the effects of TNF on ANKH expression and calcification in HASMCs. (A) Western blot analysis confirmed the overexpression of flag-tagged p65 via retrovirus vector in HASMCs; EV, empty vector. (B) HASMCs were stably transfected with EV or p65, then treated with Pi (3.0 mM) for 7 days; Alizarin-red staining and calcium content assay of calcium deposition. (C) HASMCs transfected with EV or p65 were treated with vehicle or Pi for 3 days; RT-PCR analysis of IL-8 and ANKH mRNA levels. (D) Western blot analysis confirmed p65 knockdown via lentivirus vector in HASMCs. (E) HASMCs stably expressing scramble or p65-shRNA were treated with Pi (3.0 mM) and/or TNF (10 ng/ml) for 7 days; Alizarin-red staining and calcium content assay of calcium deposition. (F) HASMCs expressing scramble or p65-shRNA were treated with Pi and/or TNF (10 ng/ml) for 3 days; RT-PCR analysis of IL-8 and ANKH mRNA levels. (G) HASMCs stably expressing p65 and/or ANKH via retrovirus vector were treated with Pi for 7 days; calcium content assay of calcium deposition. (*P<0.05 vs EV or scramble, n=3)

Mentions: To determine whether TNF-activated NF-κB was sufficient to promote calcification, we overexpressed Flag-tagged p65 in HASMCs by retroviral transduction (Figure 4A). Overexpression of p65 significantly enhanced Pi-induced mineralization (Figure 4B) and induced IL-8 mRNA level but repressed ANKH level (Figure 4C). To determine whether NF-κB was essential for TNF-enhanced mineralization, we depleted p65 in HASMCs using lentivirus-mediated expression of short hairpin RNA (shRNA) and confirmed the knockdown by western blot analysis (Figure 4D). In contrast to p65 overexpression, p65 knockdown significantly reduced TNF-augmented mineralization in Pi-treated HASMCs (Figure 4E). In accordance, p65 knockdown inhibited TNF-induced IL-8 expression but rescued TNF-decreased ANKH level (Figure 4F). ANKH overexpression in HASMCs prevented p65-augmented calcium deposition (Figure 4G). Therefore, TNF may activate NF-κB to promote calcification by downregulating ANKH expression and PPi secretion.


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)

NF-κB signalling mediated the effects of TNF on ANKH expression and calcification in HASMCs. (A) Western blot analysis confirmed the overexpression of flag-tagged p65 via retrovirus vector in HASMCs; EV, empty vector. (B) HASMCs were stably transfected with EV or p65, then treated with Pi (3.0 mM) for 7 days; Alizarin-red staining and calcium content assay of calcium deposition. (C) HASMCs transfected with EV or p65 were treated with vehicle or Pi for 3 days; RT-PCR analysis of IL-8 and ANKH mRNA levels. (D) Western blot analysis confirmed p65 knockdown via lentivirus vector in HASMCs. (E) HASMCs stably expressing scramble or p65-shRNA were treated with Pi (3.0 mM) and/or TNF (10 ng/ml) for 7 days; Alizarin-red staining and calcium content assay of calcium deposition. (F) HASMCs expressing scramble or p65-shRNA were treated with Pi and/or TNF (10 ng/ml) for 3 days; RT-PCR analysis of IL-8 and ANKH mRNA levels. (G) HASMCs stably expressing p65 and/or ANKH via retrovirus vector were treated with Pi for 7 days; calcium content assay of calcium deposition. (*P<0.05 vs EV or scramble, n=3)
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: NF-κB signalling mediated the effects of TNF on ANKH expression and calcification in HASMCs. (A) Western blot analysis confirmed the overexpression of flag-tagged p65 via retrovirus vector in HASMCs; EV, empty vector. (B) HASMCs were stably transfected with EV or p65, then treated with Pi (3.0 mM) for 7 days; Alizarin-red staining and calcium content assay of calcium deposition. (C) HASMCs transfected with EV or p65 were treated with vehicle or Pi for 3 days; RT-PCR analysis of IL-8 and ANKH mRNA levels. (D) Western blot analysis confirmed p65 knockdown via lentivirus vector in HASMCs. (E) HASMCs stably expressing scramble or p65-shRNA were treated with Pi (3.0 mM) and/or TNF (10 ng/ml) for 7 days; Alizarin-red staining and calcium content assay of calcium deposition. (F) HASMCs expressing scramble or p65-shRNA were treated with Pi and/or TNF (10 ng/ml) for 3 days; RT-PCR analysis of IL-8 and ANKH mRNA levels. (G) HASMCs stably expressing p65 and/or ANKH via retrovirus vector were treated with Pi for 7 days; calcium content assay of calcium deposition. (*P<0.05 vs EV or scramble, n=3)
Mentions: To determine whether TNF-activated NF-κB was sufficient to promote calcification, we overexpressed Flag-tagged p65 in HASMCs by retroviral transduction (Figure 4A). Overexpression of p65 significantly enhanced Pi-induced mineralization (Figure 4B) and induced IL-8 mRNA level but repressed ANKH level (Figure 4C). To determine whether NF-κB was essential for TNF-enhanced mineralization, we depleted p65 in HASMCs using lentivirus-mediated expression of short hairpin RNA (shRNA) and confirmed the knockdown by western blot analysis (Figure 4D). In contrast to p65 overexpression, p65 knockdown significantly reduced TNF-augmented mineralization in Pi-treated HASMCs (Figure 4E). In accordance, p65 knockdown inhibited TNF-induced IL-8 expression but rescued TNF-decreased ANKH level (Figure 4F). ANKH overexpression in HASMCs prevented p65-augmented calcium deposition (Figure 4G). Therefore, TNF may activate NF-κB to promote calcification by downregulating ANKH expression and PPi secretion.

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