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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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NF-κB dependent expression of tristetraprolin (TTP) mediated the effects of TNF on ANKH expression and calcification. ANKH-promoter activity assay with pGL3 luciferase reporter in (A) HASMCs and (B) HEK293T cells. (C) IKK inhibitor was added to block NF-κB signal; RT-PCR analysis of TTP mRNA level. After transfection of scramble or TTP shRNA (pSingle-tTS-TTP-shRNA) for 24 hr, RT-PCR analysis of mRNA level of (D) TTP and (E) ANKH with or without TNF (10 ng/ml). (F) Calcium content assay: after transfection of scramble or TTP shRNA for 24 hr, cells were treated with Pi or Pi+TNF (10 ng/ml) for 7 days. (G) Calcium content assay: after transfection of empty or TTP vectors for 24 h, cells were treated with Pi or Pi+TNF (10 ng/ml) for 7 days. (*P<0.05, n=3)
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Figure 5: NF-κB dependent expression of tristetraprolin (TTP) mediated the effects of TNF on ANKH expression and calcification. ANKH-promoter activity assay with pGL3 luciferase reporter in (A) HASMCs and (B) HEK293T cells. (C) IKK inhibitor was added to block NF-κB signal; RT-PCR analysis of TTP mRNA level. After transfection of scramble or TTP shRNA (pSingle-tTS-TTP-shRNA) for 24 hr, RT-PCR analysis of mRNA level of (D) TTP and (E) ANKH with or without TNF (10 ng/ml). (F) Calcium content assay: after transfection of scramble or TTP shRNA for 24 hr, cells were treated with Pi or Pi+TNF (10 ng/ml) for 7 days. (G) Calcium content assay: after transfection of empty or TTP vectors for 24 h, cells were treated with Pi or Pi+TNF (10 ng/ml) for 7 days. (*P<0.05, n=3)

Mentions: Genome-wide analysis of the ANKH promoter predicted the existence of a transcription-factor NF-κB binding site (−194~−203 bp), which suggests that TNF might inhibit ANKH transcription through NF-κB. We subcloned the 0.3-kb upstream sequence of the ANKH promoter from the transcription start site (pGL3-ANKH-prom) from human genomic DNA into the pGL3 luciferase reporter. The basal luciferase activity was higher for pGL3-ANKH-prom than the pGL3 control vector in both HASMCs and HEK293T cells (Figure 5A, B). However, TNF was unable to modulate the basal luciferase activity of pGL3-ANKH-prom. As a control, TNF potently stimulated NF-κB luciferase reporter activities in both HASMCs and HEK293T cells. Therefore, NF-κB may not regulate ANKH expression at the transcriptional level.


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 dependent expression of tristetraprolin (TTP) mediated the effects of TNF on ANKH expression and calcification. ANKH-promoter activity assay with pGL3 luciferase reporter in (A) HASMCs and (B) HEK293T cells. (C) IKK inhibitor was added to block NF-κB signal; RT-PCR analysis of TTP mRNA level. After transfection of scramble or TTP shRNA (pSingle-tTS-TTP-shRNA) for 24 hr, RT-PCR analysis of mRNA level of (D) TTP and (E) ANKH with or without TNF (10 ng/ml). (F) Calcium content assay: after transfection of scramble or TTP shRNA for 24 hr, cells were treated with Pi or Pi+TNF (10 ng/ml) for 7 days. (G) Calcium content assay: after transfection of empty or TTP vectors for 24 h, cells were treated with Pi or Pi+TNF (10 ng/ml) for 7 days. (*P<0.05, n=3)
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Related In: Results  -  Collection

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Figure 5: NF-κB dependent expression of tristetraprolin (TTP) mediated the effects of TNF on ANKH expression and calcification. ANKH-promoter activity assay with pGL3 luciferase reporter in (A) HASMCs and (B) HEK293T cells. (C) IKK inhibitor was added to block NF-κB signal; RT-PCR analysis of TTP mRNA level. After transfection of scramble or TTP shRNA (pSingle-tTS-TTP-shRNA) for 24 hr, RT-PCR analysis of mRNA level of (D) TTP and (E) ANKH with or without TNF (10 ng/ml). (F) Calcium content assay: after transfection of scramble or TTP shRNA for 24 hr, cells were treated with Pi or Pi+TNF (10 ng/ml) for 7 days. (G) Calcium content assay: after transfection of empty or TTP vectors for 24 h, cells were treated with Pi or Pi+TNF (10 ng/ml) for 7 days. (*P<0.05, n=3)
Mentions: Genome-wide analysis of the ANKH promoter predicted the existence of a transcription-factor NF-κB binding site (−194~−203 bp), which suggests that TNF might inhibit ANKH transcription through NF-κB. We subcloned the 0.3-kb upstream sequence of the ANKH promoter from the transcription start site (pGL3-ANKH-prom) from human genomic DNA into the pGL3 luciferase reporter. The basal luciferase activity was higher for pGL3-ANKH-prom than the pGL3 control vector in both HASMCs and HEK293T cells (Figure 5A, B). However, TNF was unable to modulate the basal luciferase activity of pGL3-ANKH-prom. As a control, TNF potently stimulated NF-κB luciferase reporter activities in both HASMCs and HEK293T cells. Therefore, NF-κB may not regulate ANKH expression at the transcriptional level.

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