SGK1 induces vascular smooth muscle cell calcification through NF-κB signaling
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
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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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 5

SGK1 inhibition ameliorates phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of primary HAoSMCs.

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SGK1 inhibition ameliorates phosphate-induced osteo-/chondrogenic transd...
(A) Scatter dot plots and arithmetic means ± SEM (n = 7 per group; AU) of SGK1 relative mRNA expression in HAoSMCs following treatment with control or β-glycerophosphate (Pi) without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). (B) Representative confocal microscopy images (n = 3 per group) showing NF-κB p65 protein expression and localization in HAoSMCs following treatment with control or β-glycerophosphate (Pi) without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). Green labeling, NF-κB p65 expression; magenta labeling, nuclei. Scale bars: 20 μm. (C) Representative original images (n = 4 per group) showing alizarin red staining in HAoSMCs following treatment with control or with calcification medium without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). Calcified areas are shown as red staining. (D–H) Scatter dot plots and arithmetic means ± SEM of calcium content (D, n = 6 per group, μg/mg protein), alkaline phosphatase activity (E, n = 4 per group, U/mg protein), and MSX2 (F), CBFA1 (G), and ALPL (H) relative mRNA expression (n = 7 per group; AU) in HAoSMCs following treatment with control or phosphate (Pi) without or with additional treatment with SGK1 inhibitor EMD638683 (EMD). *P < 0.05, **P < 0.01, ***P < 0.001 statistically significant vs. control-treated HAoSMCs; †P < 0.05, ††P < 0.01, †††P < 0.001 statistically significant vs. HAoSMCs treated with calcification medium/Pi alone (Steel-Dwass method for A and 1-way ANOVA with Tukey-HSD post hoc test for D–H).