SGK1 induces vascular smooth muscle cell calcification through NF-κB signaling
Jakob Voelkl, … , Florian Lang, Ioana Alesutan
Jakob Voelkl, … , Florian Lang, Ioana Alesutan
Published June 11, 2018
Citation Information: J Clin Invest. 2018;128(7):3024-3040. https://doi.org/10.1172/JCI96477.
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Research Article Cell biology Vascular biology

SGK1 induces vascular smooth muscle cell calcification through NF-κB signaling

Abstract

Medial vascular calcification, associated with enhanced mortality in chronic kidney disease (CKD), is fostered by osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Here, we describe that serum- and glucocorticoid-inducible kinase 1 (SGK1) was upregulated in VSMCs under calcifying conditions. In primary human aortic VSMCs, overexpression of constitutively active SGK1S422D, but not inactive SGK1K127N, upregulated osteo-/chondrogenic marker expression and activity, effects pointing to increased osteo-/chondrogenic transdifferentiation. SGK1S422D induced nuclear translocation and increased transcriptional activity of NF-κB. Silencing or pharmacological inhibition of IKK abrogated the osteoinductive effects of SGK1S422D. Genetic deficiency, silencing, and pharmacological inhibition of SGK1 dissipated phosphate-induced calcification and osteo-/chondrogenic transdifferentiation of VSMCs. Aortic calcification, stiffness, and osteo-/chondrogenic transdifferentiation in mice following cholecalciferol overload were strongly reduced by genetic knockout or pharmacological inhibition of Sgk1 by EMD638683. Similarly, Sgk1 deficiency blunted vascular calcification in apolipoprotein E–deficient mice after subtotal nephrectomy. Treatment of human aortic smooth muscle cells with serum from uremic patients induced osteo-/chondrogenic transdifferentiation, effects ameliorated by EMD638683. These observations identified SGK1 as a key regulator of vascular calcification. SGK1 promoted vascular calcification, at least partly, via NF-κB activation. Inhibition of SGK1 may, thus, reduce the burden of vascular calcification in CKD.

Authors

Jakob Voelkl, Trang T.D. Luong, Rashad Tuffaha, Katharina Musculus, Tilman Auer, Xiaoming Lian, Christoph Daniel, Daniel Zickler, Beate Boehme, Michael Sacherer, Bernhard Metzler, Dietmar Kuhl, Maik Gollasch, Kerstin Amann, Dominik N. Müller, Burkert Pieske, Florian Lang, Ioana Alesutan

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Figure 3

NF-κB–dependent effects of SGK1 on osteo-/chondrogenic signaling in primary HAoSMCs.

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NF-κB–dependent effects of SGK1 on osteo-/chondrogenic signaling in prim...
(A) Representative confocal microscopy images (n = 3 per group) showing NF-κB p65 protein expression and localization in HAoSMCs following transfection with empty vector (V), constitutively active SGK1S422D (SGK1SD), or inactive SGK1K127N (SGK1KN). Green labeling, NF-κB p65 expression; magenta labeling, nuclei. Scale bars: 20 μm. (B) Scatter dot plots and arithmetic means ± SEM (n = 4 per group; AU) of NF-κB–dependent transcriptional activity measured by luciferase reporter assay in HAoSMCs following transfection with NF-κB–responsive luciferase/Renilla constructs and with empty vector (V), constitutively active SGK1S422D (SGK1SD), or inactive SGK1K127N (SGK1KN). (C) Scatter dot plots and arithmetic means ± SEM (n = 8 per group; AU) of ZFP36 relative mRNA expression in HAoSMCs following transfection with empty vector (V), constitutively active SGK1S422D (SGK1SD), or inactive SGK1K127N (SGK1KN). (D–F) Scatter dot plots and arithmetic means ± SEM (n = 6 per group; AU) of MSX2 (D), CBFA1 (E), and ALPL (F) relative mRNA expression in HAoSMCs following transfection with empty vector (V) or constitutively active SGK1S422D (SGK1SD) and additional treatment with control, BAY11-7082 (BAY), parthenolide (PAR), or BMS-345541 (BMS). (G–I) Scatter dot plots and arithmetic means ± SEM (n = 6 per group; AU) of MSX2 (G), CBFA1 (H), and ALPL (I) relative mRNA expression in HAoSMCs following transfection with empty vector (V) or constitutively active SGK1S422D (SGK1SD) and additional silencing with negative control siRNA (Neg.si), IKKα siRNA (IKKαsi), or IKKβ siRNA (IKKβsi). *P < 0.05, ***P < 0.001 statistically significant vs. V-transfected HAoSMCs; †P < 0.05, ††P < 0.01, †††P < 0.001 statistically significant vs. SGK1SD-transfected HAoSMCs (1-way ANOVA with Tukey-HSD post hoc test for B–E and H or with Games-Howell post hoc test for G and I and Steel-Dwass method for F).