SGK1-dependent stimulation of vascular smooth muscle cell osteo-/chondrogenic transdifferentiation by interleukin-18

Abstract

The serum- and glucocorticoid-inducible kinase 1 (SGK1) is a key regulator of osteo-/chondrogenic transdifferentiation and subsequent calcification of vascular smooth muscle cells (VSMCs). The phenotypical transdifferentiation of VSMCs is associated with increased interleukin-18 (IL-18) levels and generalized inflammation. Therefore, the present study investigated the possible involvement of SGK1 in IL-18-induced vascular calcification. Experiments were performed in primary human aortic smooth muscle cells (HAoSMCs) treated with recombinant human IL-18 protein in control or high phosphate conditions and following SGK1 knockdown by siRNA or pharmacological inhibition of SGK1, PI3K, and PDK1. As a result, IL-18 treatment increased SGK1 mRNA and protein expression in HAoSMCs. IL-18 upregulated SGK1 mRNA expression in a dose-dependent manner. This effect was paralleled by upregulation of the mRNA expression of MSX2 and CBFA1, osteogenic transcription factors, and of tissue-nonspecific alkaline phosphatase (ALPL), an osteogenic enzyme, as markers of increased osteo-/chondrogenic transdifferentiation. Phosphate treatment increased SGK1 and osteogenic markers mRNA expression as well as ALPL activity and induced calcification of HAoSMCs, all effects significantly augmented by additional treatment with IL-18. Conversely, silencing of SGK1 or cotreatment with the SGK1 inhibitor EMD638683 blunted the effects of IL-18 on osteo-/chondrogenic transdifferentiation and calcification of HAoSMCs. The procalcific effects of IL-18 were similarly suppressed in the presence of PI3K or PDK1 inhibitors. In conclusion, SGK1 expression is upregulated by IL-18 in VSMCs and SGK1 participates in the intracellular signaling of IL-18-induced osteo-/chondrogenic transdifferentiation of VSMCs. Thus, SGK1 may serve as therapeutic target to limit the progression of medial vascular calcification during vascular inflammation.

Keywords: Osteo-/chondrogenic signaling; PI3K; SGK1, interleukin-18; Vascular calcification; Vascular smooth muscle cells.

Fig. 1

Interleukin-18 upregulates SGK1 and osteogenic markers expression in primary human aortic smooth muscle cells in a dose-dependent manner. a Representative original Western blots and scatter dot plots and arithmetic means ± SEM (n = 8; arbitrary units, a.u.) of normalized SGK1/GAPDH protein ratio in HAoSMCs following treatment for the indicated times with 10 ng/ml recombinant human interleukin-18 protein (IL-18). b–e Scatter dot plots and arithmetic means ± SEM (n = 4; a.u.) of SGK1 (b), MSX2 (c), CBFA1 (d), and ALPL (e) relative mRNA expression in HAoSMCs following treatment for 24 h with control (CTR) or with the indicated concentrations of recombinant human interleukin-18 protein (IL-18, 0.1–10 ng/ml). *(p < 0.05), **(p < 0.01) statistically significant vs. control-treated HAoSMCs

Fig. 2

Interleukin-18 augments phosphate-induced SGK1 expression and osteogenic signaling in primary human aortic smooth muscle cells. a–d Scatter dot plots and arithmetic means ± SEM (n = 8, arbitrary units, a.u.) of SGK1 (a), MSX2 (b), CBFA1 (c), and ALPL (d) relative mRNA expression in HAoSMCs following treatment for 24 h with control or with 2 mM β-glycerophosphate (Pi) without or with additional treatment with 10 ng/ml recombinant human interleukin-18 protein (IL-18). e Scatter dot plots and arithmetic means ± SEM (n = 8, U/mg protein) of ALPL activity in HAoSMCs following treatment for 7 days with control or with 2 mM β-glycerophosphate (Pi) without or with additional treatment with 10 ng/ml recombinant human interleukin-18 protein (IL-18). *(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 Pi alone

Fig. 3

Interleukin-18 aggravates phosphate-induced calcification of primary human aortic smooth muscle cells. a Representative original images (n = 4) showing Alizarin red staining in HAoSMCs following treatment for 11 days with control or with calcification medium (calc. medium) without or with additional treatment with 10 ng/ml recombinant human interleukin-18 protein (IL-18). The calcified areas are shown as red staining. b Scatter dot plots and arithmetic means ± SEM (n = 8; μg/mg protein) of calcium content in HAoSMCs following treatment for 11 days with control or with calcification medium (calc.) without or with additional treatment with 10 ng/ml recombinant human interleukin-18 protein (IL-18). ***(p < 0.001) statistically significant vs. control-treated HAoSMCs; †(p < 0.05) statistically significant vs. HAoSMCs treated with calcification medium alone

Fig. 4

Silencing of SGK1 blunts interleukin-18-induced osteogenic markers expression and calcification of primary human aortic smooth muscle cells. a–d Scatter dot plots and arithmetic means ± SEM (n = 6, arbitrary units, a.u.) of SGK1 (a), MSX2 (b), CBFA1 (c), and ALPL (d) relative mRNA expression in HAoSMCs following silencing for 48 h with negative control siRNA (neg.si) or SGK1 siRNA (SGK1si) without or with additional treatment for 24 h with 10 ng/ml recombinant human interleukin-18 protein (IL-18). ***(p < 0.001) statistically significant vs. neg.si-silenced HAoSMCs; †††(p < 0.001) statistically significant vs. neg.si-silenced and IL-18-treated HAoSMCs. e Scatter dot plots and arithmetic means ± SEM (n = 6; μg/mg protein) of calcium content in HAoSMCs following 11 days of silencing with negative control siRNA (neg.si) or SGK1 siRNA (SGK1si) and additional treatment with control or with calcification medium (calc.) without or with additional treatment with 10 ng/ml recombinant human interleukin-18 protein (IL-18). *(p < 0.05), **(p < 0.01) statistically significant vs. neg.si-silenced HAoSMCs; †(p < 0.05), ††(p < 0.01) statistically significant vs. neg.si-silenced and calcification medium alone-treated HAoSMCs; §§(p < 0.01) statistically significant vs. neg.si-silenced and calcification medium with IL-18-treated HAoSMCs

Fig. 5

SGK1 inhibition suppresses interleukin-18-induced osteogenic markers expression in primary human aortic smooth muscle cells. a–c Scatter dot plots and arithmetic means ± SEM (n = 4, a.u.) of MSX2 (a), CBFA1 (b), and ALPL (c) relative mRNA expression in HAoSMCs following treatment for 24 h with control (CTR) or with 10 ng/ml recombinant human interleukin-18 protein (IL-18) without or with additional treatment with 50 μM SGK1 inhibitor EMD638683 (EMD). **(p < 0.01) statistically significant vs. control-treated HAoSMCs; †(p < 0.05), ††(p < 0.01) statistically significant vs. IL-18 alone-treated HAoSMCs

Fig. 6

Interleukin-18-induced osteo-/chondrogenic markers expression is suppressed by inhibition of PI3K pathway in primary human aortic smooth muscle cells. a Scatter dot plots and arithmetic means ± SEM (n = 4; a.u.) of MSX2 (a), CBFA1 (b), and ALPL (c) relative mRNA expression in HAoSMCs following treatment for 24 h with control (CTR) or with 10 ng/ml recombinant human interleukin-18 protein (IL-18) without or with additional treatment with the PI3K inhibitors 1 μM LY294002 (LY) or 100 nM wortmannin (Wo). Scatter dot plots and arithmetic means ± SEM (n = 4; a.u.) of MSX2 (d), CBFA1 (e), and ALPL (f) relative mRNA expression in HAoSMCs following treatment for 24 h with control (CTR) or with 10 ng/ml recombinant human interleukin-18 protein (IL-18) without or with additional treatment with 1 μM PDK1 inhibitor GSK2334470 (GSK). *(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 IL-18 alone

Fig. 7

Inhibition of SGK1 or PI3K pathway blunt the effects of interleukin-18 on calcification of primary human aortic smooth muscle cells. Scatter dot plots and arithmetic means ± SEM (n = 6; μg/mg protein) of calcium content in HAoSMCs following treatment for 11 days with control or with calcification medium (calc.) without or with additional treatment with 10 ng/ml recombinant human interleukin-18 protein (IL-18) alone or together with 50 μM SGK1 inhibitor EMD638683 (EMD), 1 μM PI3K inhibitor LY294002 (LY), or 1 μM PDK1 inhibitor GSK2334470 (GSK). **(p < 0.01), ***(p < 0.001) statistically significant vs. control-treated HAoSMCs; †(p < 0.05) statistically significant vs. calcification medium alone-treated HAoSMCs; §§(p < 0.01) statistically significant vs. calcification medium with IL-18 alone-treated HAoSMCs

Fig. 8

Schematic illustration of SGK1-dependent IL-18-induced VSMC calcification. CKD increases circulating IL-18 levels [12, 16]. In VSMCs, IL-18 may activate PI3K/PDK1 signaling to increase the expression and activity of SGK1. Further, SGK1 may induce transcriptional activation of NF-kB via phosphorylation-dependent ubiquitination and degradation of IkBα [25] and, thus, the expression of NF-kB target genes to promote osteo-/chondrogenic transdifferentiation of VSMCs [58]. The osteo-/chondrogenic transdifferentiation of VSMCs induces a procalcific environment causing vascular calcification (CKD, chronic kidney disease; IL-18, interleukin 18; PI3K, phosphatidylinositol-4,5-bisphosphate 3 kinase; PDK1, 3-phosphoinositide-dependent protein kinase 1; SGK1, serum- and glucocorticoid-inducible kinase 1; NF-kB, nuclear factor “kappa-light-chain-enhancer” of activated B-cells; IkBα, nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha)