SGK1-dependent stimulation of vascular smooth muscle cell osteo-/chondrogenic transdifferentiation by interleukin-18
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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.
KeywordsSGK1, interleukin-18 PI3K Vascular calcification Osteo-/chondrogenic signaling Vascular smooth muscle cells
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].
Vascular calcification is an active pathological process, promoted mainly by osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs) [18, 28]. In CKD, various pathological factors, most importantly phosphate, induce the osteo-/chondrogenic transdifferentiation of VSMCs via complex intracellular signaling pathways [3, 18, 29, 34, 54]. These cells are characterized by increased expression and activity of osteogenic transcription factors such as msh homeobox 2 (MSX2)  and core-binding factor α-1 (CBFA1)  as well as osteogenic enzymes such as tissue-nonspecific alkaline phosphatase (ALPL) [31, 43]. The osteoblast- and chondroblast-like VSMCs promote mineralization of vascular tissue via mechanisms similar to physiological bone calcification . Inflammatory processes are involved in osteo-/chondrogenic transdifferentiation of VSMCs and vascular calcification in CKD [18, 46].
Increased plasma levels of interleukin-18 (IL-18), a pro-inflammatory cytokine , are frequently observed in CKD patients [12, 16] and associated with medial vascular calcification [23, 62]. Recent reports indicate that IL-18 enhances osteo-/chondrogenic transdifferentiation of VSMCs , but the intracellular signaling pathways contributing to these procalcific effects are still ill-defined.
The serum- and glucocorticoid-inducible kinase 1 (SGK1) is upregulated at transcriptional level by various pathologic factors known to trigger osteo-/chondrogenic transdifferentiation and calcification of VSMCs, such as hyperphosphatemia, mineralocorticoid or glucocorticoid excess, hyperglycemia, or inflammatory cytokines [25, 56, 57, 58]. SGK1 has been shown to contribute to the pathophysiology of several disorders [25, 30], including cardiac remodeling [53, 56, 57], hypertension , stroke , diabetes mellitus , kidney disease [10, 55], or inflammation . SGK1 promotes osteo-/chondrogenic transdifferentiation of VSMCs . Overexpression of constitutively active SGK1, but not inactive SGK1, is sufficient to induce osteo-/chondrogenic transdifferentiation of VSMCs . Conversely, inhibition, deficiency, or knockdown of SGK1 are able to inhibit osteo-/chondrogenic transdifferentiation and calcification of VSMCs during phosphate exposure .
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.
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 . The cells were used 48 h after transfection and silencing efficiency were analyzed by quantitative RT-PCR.
HAoSMCs were treated for 24 h (qRT-PCR), 7 days (ALPL activity), or 11 days (calcification) with 2 mM β-glycerophosphate (Sigma-Aldrich), the indicated concentrations of recombinant human interleukin-18 protein (R&D Systems), 50 μM SGK1 inhibitor EMD638683 (stock in DMSO) [57, 58], 1 μM LY294002 (Enzo Life Sciences, stock in DMSO), 100 nM wortmannin (Enzo Life Sciences, stock in DMSO) , or 1 μM PDK1 inhibitor GSK2334470 (Cayman Chemical, stock in DMSO). Equal amounts of vehicle were used as control. Treatment with calcification medium containing 10 mM β-glycerophosphate and 1.5 mM CaCl2 (Sigma-Aldrich) for 11 days was used for quantification of mineralization and Alizarin red staining [52, 59]. Fresh media with agents were added every 2–3 days.
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 .
Alizarin red staining
To visualize calcification, HAoSMCs were fixed with 4% paraformaldehyde and stained with 2% Alizarin red (pH 4.5) . 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].
Total RNA was isolated from HAoSMCs by using Trizol Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions [1, 33, 55]. Reverse transcription of 2 μg total RNA was performed using oligo(dT)12–18 primers (Thermo Fisher Scientific) and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). Quantitative RT-PCR was performed with the iCycler iQ™ Real-Time PCR Detection System (Bio-Rad Laboratories) and iQ™ Sybr Green Supermix (Bio-Rad Laboratories) according to the manufacturer’s instructions. The specificity of the PCR products was confirmed by analysis of the melting curves. All PCRs were performed in duplicate and relative mRNA expression was calculated by the 2-ΔΔCt method using GAPDH as housekeeping gene, normalized to the control group. The following human primers were used (Thermo Fisher Scientific; 5′ → 3′ orientation):
ALPL fw: GGGACTGGTACTCAGACAACG;
ALPL rev: GTAGGCGATGTCCTTACAGCC;
CBFA1 fw: GCCTTCCACTCTCAGTAAGAAGA;
CBFA1 rev: GCCTGGGGTCTGAAAAAGGG;
GAPDH fw: GAGTCAACGGATTTGGTCGT;
GAPDH rev: GACAAGCTTCCCGTTCTCAG;
MSX2 fw: TGCAGAGCGTGCAGAGTTC;
MSX2 rev: GGCAGCATAGGTTTTGCAGC;
SGK1 fw: GCAGAAGAAGTGTTCTATGCAGT;
SGK1 rev: CCGCTCCGACATAATATGCTT.
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 SDS-polyacrylamide 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. Antibody binding was detected with ECL detection reagent (Thermo Fisher Scientific), and bands were quantified by using ImageJ software. Results are shown as the ratio of total protein to GAPDH normalized to the control group.
Data are shown as scatter dot plots and arithmetic means ± SEM. N indicates the number of independent experiments performed at different passages of the cells. Normality was tested with Shapiro-Wilk test. Non-normal datasets were transformed (log) prior to statistical testing to provide normality according to Shapiro-Wilk test. Statistical testing was performed by one-way ANOVA followed by Tukey test for homoscedastic data or Games-Howell test for heteroscedastic data. Non-normal data were tested by the Steel-Dwass method. P < 0.05 was considered statistically significant.
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) in HAoSMCs, as markers of osteo-/chondrogenic transdifferentiation. Thus, the increased SGK1 expression in IL-18 treated HAoSMCs was paralleled by increased osteo-/chondrogenic transdifferentiation.
IL-18, a pro-inflammatory cytokine from the IL-1 cytokine family , is produced by various cells including osteoblasts , chondrocytes , and VSMCs . IL-18 induces cellular inflammatory responses by binding to the IL-18 receptor . VSMCs express a functional IL-18 receptor [17, 48]. Conversely, inflammatory processes upregulate IL-18 expression . Increased plasma IL-18 levels are frequently observed in CKD patients [12, 16, 62]. IL-18 is involved in the progression of CKD [46, 51] and its related complications including vascular calcification [46, 62]. Similar to previous reports , our observations show that IL-18 treatment enhances phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of VSMCs. In addition, IL-18 treatment alone is able to induce the expression of osteogenic markers in primary human aortic VSMCs.
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  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 . 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.
Moreover, IL-18 is able to activate PI3K-dependent signaling in various cell types [8, 61, 63] including VSMCs , while PI3K activation leads to increased expression and activity of SGK1 [6, 13, 19, 25, 30]. Accordingly, PI3K and PDK1 inhibition suppresses IL-18-dependent osteoinduction in VSMCs.
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 Mg2+-permeable ion channel  with a complex role during vascular calcification , which has been suggested to contribute to the effects of IL-18 . SGK1 is able to enhance TRPM7 expression . 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 . Thus, SGK1 may be a central key regulator in the signaling pathways mediating vascular calcification during various pathological conditions, including inflammation. 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].
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.
J.V. and I.A. designed research; N.S., T.T.D.L., and I.A. performed experiments; F.L., B.P., J.V., and I.A. analyzed and interpreted data; J.V. and I.A. wrote the manuscript with comments and edits from all authors.
Open access funding provided by Johannes Kepler University Linz. This work was supported by the Berlin Institute of Health (BIH) Translational Postdoc Grant, the Deutsche Forschungsgemeinschaft (AL2054/1-1; VO2259/2-1), the European Union Seventh Framework Programme (FP7/2007-2013–603288-SysVasc), and the Else Kröner-Fresenius-Stiftung and the DZHK (German Centre for Cardiovascular Research).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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