RANKL, but Not R-Spondins, Is Involved in Vascular Smooth Muscle Cell Calcification through LGR4 Interaction

Abstract

Vascular calcification has a global health impact that is closely linked to bone loss. The Receptor Activator of Nuclear Factor Kappa B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system, fundamental for bone metabolism, also plays an important role in vascular calcification. The Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4), a novel receptor for RANKL, regulates bone remodeling, and it appears to be involved in vascular calcification. Besides RANKL, LGR4 interacts with R-spondins (RSPOs), which are known for their roles in bone but are less understood in vascular calcification. Studies were conducted in rats with chronic renal failure fed normal or high phosphorus diets for 18 weeks, with and without control of circulating parathormone (PTH) levels, resulting in different degrees of aortic calcification. Additionally, vascular smooth muscle cells (VSMCs) were cultured under non-calcifying (1 mM phosphate) and calcifying (3 mM phosphate) media with different concentrations of PTH. To explore the role of RANKL in VSMC calcification, increasing concentrations of soluble RANKL were added to non-calcifying and calcifying media. The effects mediated by RANKL binding to its receptor LGR4 were investigated by silencing the LGR4 receptor in VSMCs. Furthermore, the gene expression of the RANK/RANKL/OPG system and the ligands of LGR4 was assessed in human epigastric arteries obtained from kidney transplant recipients with calcification scores (Kauppila Index). Increased aortic calcium in rats coincided with elevated systolic blood pressure, upregulated Lgr4 and Rankl gene expression, downregulated Opg gene expression, and higher serum RANKL/OPG ratio without changes in Rspos gene expression. Elevated phosphate in vitro increased calcium content and expression of Rankl and Lgr4 while reducing Opg. Elevated PTH in the presence of high phosphate exacerbated the increase in calcium content. No changes in Rspos were observed under the conditions employed. The addition of soluble RANKL to VSMCs induced genotypic differentiation and calcification, partly prevented by LGR4 silencing. In the epigastric arteries of individuals presenting vascular calcification, the gene expression of RANKL was higher. While RSPOs show minimal impact on VSMC calcification, RANKL, interacting with LGR4, drives osteogenic differentiation in VSMCs, unveiling a novel mechanism beyond RANKL-RANK binding.

Keywords: LGR4; R-spondins; RANKL; phosphorus; vascular calcification.

Figure 1

Aortic Ca content and gene expression from control (SHAM) and nephrectomized (NX) rats with and without parathyroidectomy, fed normal (NP) or high (HP) phosphorus diet for 18 weeks. Aortic Ca content was determined by o-cresolphtalein complexone method (A). Gene expression was evaluated by qRT-PCR of: Lgr4 (B), Rankl (C), Opg (D), Rspo1 (E), Rspo2 (F), Rspo3 (G) and Rspo4 (H). The groups are SHAM NP (Sham-operated rats fed normal phosphorus diet), SHAM HP (Sham-operated rats fed high phosphorus diet), PTX NX NP (parathyroidectomized and nephrectomized rats fed normal phosphorus diet), PTX NX HP (parathyroidectomized and nephrectomized rats fed high phosphorus diet), NX NP (nephrectomized rats fed normal phosphorus diet), and NX HP (nephrectomized rats fed high phosphorus diet). Data are presented as median [interquartile range]. R.U., relative units. ^a p < 0.05, ^aa p < 0.01, ^aaa p < 0.001 versus SHAM NP; ^b p < 0.05, ^bb p < 0.01, versus SHAM HP; ^c p < 0.05 versus PTX NX NP; ^dd p < 0.01 versus PTX NX HP; ^ee p < 0.01 versus NX NP.

Figure 2

Effect of high phosphate (Pi) (3 mM) and different PTH concentrations (10⁻⁹, 10⁻⁸, and 10⁻⁷ M) on A7r5 vascular smooth muscle cells after 4 days of exposure. Ca content was determined by the o-cresolphtalein complexone method (A). Gene expression was evaluated by qRT-PCR for Lgr4 (B), Rankl (C), Opg (D), Rspo1 (E), Rspo2 (F), Rspo3 (G) and Rspo4 (H). Data are presented as median [interquartile range]. R.U., relative units. ^a p < 0.05, ^aa p < 0.01, ^aaa p < 0.001 versus control (non-calcifying medium, 1 mM phosphate); ^b p < 0.05 versus Pi.

Figure 3

Effect of different soluble RANKL concentrations (1, 10, and 100 pM) on A7r5 vascular smooth muscle cells after 4 days of exposure in non-calcifying medium (1 mM phosphate). Ca content determined by o-cresolphtalein complexone method (A). Gene expression was evaluated by qRT-PCR for Alp (B), Runx2 (C), and α-actin (D). Data are presented as median [interquartile range]. R.U., relative units. ^a p < 0.05, ^aa p < 0.01, ^aaa p < 0.001 versus control (non-calcifying medium, 1 mM phosphate); ^b p < 0.05, ^bb p < 0.01, versus non-calcifying medium (1 mM phosphate) + 1 pM RANKL.

Figure 4

Effect of Lgr4 silencing on A7r5 vascular smooth muscle cells exposed to soluble RANKL (10 pM) in non-calcifying medium (1 mM phosphate). Ca content was determined by the o-cresolphtalein complexone method (A). Gene expression was evaluated by qRT-PCR for Alp (B), Runx2 (C), and α-actin (D). White and grey boxes represent the non-addition of RANKL and the addition of 10 pM of RANKL, respectively. Data are presented as median [interquartile range]. R.U., relative units. Mock, scramble transfection control of silencing. ^a p < 0.05, ^aa p < 0.01, ^aaa p < 0.001 versus Mock (0 pM RANKL); ^b p < 0.05, ^bb p < 0.01, ^bbb p < 0.001 versus Mock + 10 pM RANKL.