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

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.


Introduction
Medial vascular calcification is frequently observed in aging, diabetes mellitus, atherosclerosis, and most extensively in chronic kidney disease (CKD) [32,44]. In large arteries, vascular calcification may lead to increased stiffness, elevated pulse pressure and, thus, to cardiac hypertrophy and impaired coronary perfusion [18,20]. Accordingly, vascular calcification is associated with an increased risk for cardiovascular events and cardiovascular and all-cause morbidity and mortality in CKD [35,41,44].
Therefore, the present study explored the effects of IL-18 on SGK1 expression in VSMCs as well as the possible involvement of SGK1 in IL-18-induced osteo-/chondrogenic transdifferentiation and calcification of VSMCs in vitro.

Methods
Cell culture of primary human aortic smooth muscle cells Primary human aortic smooth muscle cells (HAoSMCs, Thermo Fisher Scientific) [2,5,34] were routinely cultured in medium containing Waymouth's MB 752/1 medium and Ham's F-12 nutrient mixture (Thermo Fisher Scientific) in a 1:1 ratio, supplemented with 10% FBS (Thermo Fisher Scientific), 100 U/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific). HAoSMCs were grown to confluence and used in all experiments from passages 4 to 10.
Where indicated, HAoSMCs were transfected with 10 nM SGK1 siRNA (ID no. s740, Thermo Fisher Scientific) or with 10 nM negative control siRNA (ID no. 4390843, Thermo Fisher Scientific) using siPORT amine transfection agent (Thermo Fisher Scientific) according to the manufacturer's protocol [58]. The cells were used 48 h after transfection and silencing efficiency were analyzed by quantitative RT-PCR.

Quantification of calcium deposition
HAoSMCs were decalcified in 0.6 M HCl for 24 h at 4°C. Calcium content in the supernatant was determined by using QuantiChrom Calcium assay kit (BioAssay Systems) according to the manufacturer's protocol. HAoSMCs were lysed with 0.1 M NaOH/0.1% SDS and protein concentration was measured by the Bradford assay (Bio-Rad Laboratories). The results are shown normalized to total protein concentration [4].

Alizarin red staining
To visualize calcification, HAoSMCs were fixed with 4% paraformaldehyde and stained with 2% Alizarin red (pH 4.5) [3]. The calcified areas are shown as red staining.
ALPL activity assay ALPL activity in HAoSMCs was determined by using the ALP colorimetric assay kit (Abcam) according to the manufacturer's protocol. The results are shown normalized to total protein concentration measured by the Bradford assay (Bio-Rad Laboratories) [5,54].

Western blotting
HAoSMCs were lysed with ice-cold IP lysis buffer (Thermo Fisher Scientific) containing complete protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) [45,58]. After centrifugation at 10000 rpm for 5 min, protein concentrations were measured by the Bradford assay (Bio-Rad Laboratories). Equal amounts of proteins were boiled in Roti-Load1 Buffer (Carl Roth GmbH) at 100°C for 10 min, separated on SDSpolyacrylamide gels and transferred to PVDF membranes. The membranes were incubated overnight at 4°C with primary rabbit anti-SGK1 antibody (1:1000 dilution, cell signaling) or rabbit anti-GAPDH antibody (1:1000 dilution, cell signaling) and then with secondary anti-rabbit HRP-conjugated antibody (1:1000 dilution, cell signaling) for 1 h at room temperature. For loading controls, the membranes were stripped in stripping buffer (Thermo Fisher Scientific) at room temperature for 10 min.

Results
To investigate the effects of IL-18 on SGK1 expression in VSMCs, primary human aortic smooth muscle cells (HAoSMCs) were treated with recombinant human IL-18 protein. As shown in Fig. 1a (Fig. 1b). These effects reached statistical significance at 10 ng/ml IL-18 concentration. Similarly, IL-18 treatment dose-dependently increased the mRNA expression of the osteogenic transcription factors MSX2 and CBFA1 (Fig. 1c, d) and of the osteogenic enzyme ALPL (Fig. 1e)  Next, we explored the effects of IL-18 on SGK1 expression and osteogenic signaling in HAoSMCs during high phosphate conditions. As shown in Fig. 2a, phosphate treatment upregulated SGK1 mRNA expression in HAoSMCs, an effect significantly augmented by additional treatment with IL-18. Moreover, the phosphate-induced osteogenic markers MSX2, CBFA1, and ALPL mRNA expression (Fig. 2b-d) as well as ALPL activity (Fig. 2e) in HAoSMCs were significantly enhanced by IL-18 treatment. Alizarin red staining (Fig. 3a) and quantification of calcium deposition (Fig. 3b) in HAoSMCs revealed extensive calcification following treatment with calcification medium, effects again significantly aggravated by additional treatment with IL-18. Taken together, IL-18 augmented phosphate-induced SGK1 expression, osteogenic signaling, and calcification of HAoSMCs.
To investigate the possible involvement of SGK1 in IL-18induced osteo-/chondrogenic transdifferentiation of VSMCs, the endogenous expression of SGK1 was suppressed by silencing of the SGK1 gene in HAoSMCs followed by additional treatment without or with IL-18. As a result, SGK1 mRNA expression was significantly lower in SGK1 siRNA transfected HAoSMCs as compared to negative control siRNA silenced HAoSMCs (Fig. 4a). IL-18 treatment upregulated SGK1 mRNA expression in negative control silenced HAoSMCs. The IL-18-induced mRNA expression of MSX2, CBFA1, and ALPL in negative control silenced HAoSMCs was significantly blunted in SGK1 silenced HAoSMCs (Fig. 4b-d). Furthermore, the augmentation of HAoSMCs calcification by IL-18 in the presence of calcification medium was reversed by SGK1 knockdown (Fig. 4e). In accordance with the previous observations showing protective effects of SGK1 inhibition during high phosphate conditions, silencing of SGK1 significantly inhibited calcium deposition in HAoSMCs beyond counteracting the procalcific effects of IL-18. Taken together, the procalcific effects of IL-18 in HAoSMCs were, at least in part, dependent on SGK1.
The involvement of SGK1 in IL-18-induced osteo-/ chondrogenic transdifferentiation of VSMCs was confirmed by treatment of HAoSMCs with IL-18 in the presence or absence of the SGK1 specific inhibitor EMD638683. As shown in Fig. 5a-c, SGK1 inhibitor EMD638683 similarly suppressed the IL-18-induced upregulation of MSX2, CBFA1, and ALPL mRNA expression in HAoSMCs.
Additional experiments were performed to elucidate the regulation of SGK1-dependent osteogenic signaling in IL-18 treated HAoSMCs. To this end, the roles of phosphatidylinositol-4,5-bisphosphate 3 kinase (PI3K) and 3phosphoinositide-dependent protein kinase 1 (PDK1), critical upstream kinases in SGK1 regulation were investigated. As shown in Fig. 6a-c, the upregulation of MSX2, CBFA1, and ALPL mRNA expression following treatment with IL-18 was significantly inhibited in the presence of either PI3K inhibitors LY294002 or wortmannin. In addition, IL-18-induced osteogenic markers mRNA expression was suppressed by additional treatment with the PDK1 inhibitor GSK2334470 (Fig. 6d-f). Thus, IL-18induced osteo-/chondrogenic signaling in VSMCs involved PI3K pathway activation. Accordingly, pharmacological inhibition of SGK1, PI3K, or PDK1, all suppressed calcium deposition in HAoSMCs triggered by calcification medium supplemented with IL-18 (Fig. 7).

Discussion
The present study discloses a novel key role of SGK1 in the signaling of IL-18-induced osteo-/chondrogenic transdifferentiation of VSMCs in-vitro. IL-18 upregulates SGK1 mRNA expression in VSMCs while silencing or pharmacological inhibition of SGK1 suppresses IL-18-induced osteogenic markers expression, indicative of increased osteo-/chondrogenic transdifferentiation (Fig. 8).
IL-18, a pro-inflammatory cytokine from the IL-1 cytokine family [17], is produced by various cells including osteoblasts [47], chondrocytes [38], and VSMCs [17]. IL-18 induces cellular inflammatory responses by binding to the IL-18 receptor [37]. VSMCs express a functional IL-18 receptor [17,48]. Conversely, inflammatory processes upregulate IL-18 expression [14]. Increased plasma IL-18 levels are frequently observed in CKD patients [12,16,62]. IL-18 is involved in the The procalcific effects of IL-18 are mediated, at least in part, by SGK1. IL-18 upregulates SGK1 expression and augments phosphate-induced SGK1 expression in VSMCs. Excessive SGK1 is sufficient to promote osteo-/chondrogenic transdifferentiation of VSMCs via NF-kB activation [58] and NF-kB activity can be induced by IL-18 [24,42,50,60]. SGK1 similarly plays a key role in vascular inflammation during atherogenesis via activation of NF-kB [7]. We could show here that silencing or pharmacological inhibition of SGK1 in VSMCs is able to suppress IL-18-induced osteogenic markers expression and calcium deposition, suggesting that SGK1 is a key factor in the signaling leading to osteo-/ chondrogenic transdifferentiation and calcification of VSMCs during conditions of high IL-18 levels.
The present observations identify SGK1 regulation as the key mechanism of IL-18-induced osteo-/chondrogenic transdifferentiation of VSMCs. However, other effects may contribute to the procalcific effects of IL-18 in VSMCs. TRPM7 is a ubiquitously expressed Mg 2+ -permeable ion channel [49] with a complex role during vascular calcification [36], which has been suggested to contribute to the effects of IL-18 [62]. SGK1 is able to enhance TRPM7 expression [49]. Thus, TRPM7 expression may also be a downstream product in the SGK1-dependent signaling during calcifying conditions. Taken together, SGK1 appears critically important in the progression of vascular calcification during inflammatory conditions with high IL-18 levels such as CKD [12,16,62]. In addition, SGK1 mediates the induction of osteo-/chondrogenic transdifferentiation and, thus, vascular calcification by other pathological factors such as hyperphosphatemia or mineralocorticoid excess [58]. Thus, SGK1 may be a central key regulator in the signaling pathways mediating vascular calcification during various pathological conditions, including inflammation.  [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, serumand glucocorticoid-inducible kinase 1; NF-kB, nuclear factor Bkappalight-chain-enhancer^of activated B-cells; IkBα, nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha) Therefore, SGK1 inhibition may be a feasible therapeutic target to reduce the progression of vascular calcification triggered by complex conditions such as CKD [28,29,35,44]. Moreover, SGK1 inhibition may potentially have an overall protective effect during cardiovascular or renal disease progression [10,25,27,30,55].

IL-18
In conclusion, IL-18 upregulates SGK1 expression in VSMCs and SGK1 participates in the intracellular signaling mediating the IL-18-induced osteo-/chondrogenic transdifferentiation of VSMCs. Thus, SGK1 inhibition may be beneficial in reducing the progression of medial vascular calcification during vascular inflammatory conditions such as CKD.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
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