PKA-induced Receptor Activator of NF-κB Ligand (RANKL) Expression in Vascular Cells Mediates Osteoclastogenesis but Not Matrix Calcification

Abstract

Vascular calcification is a predictor of cardiovascular mortality and is prevalent in patients with atherosclerosis and chronic renal disease. It resembles skeletal osteogenesis, and many bone cells as well as bone-related factors involved in both formation and resorption have been localized in calcified arteries. Previously, we showed that aortic medial cells undergo osteoblastic differentiation and matrix calcification both spontaneously and in response to PKA agonists. The PKA signaling pathway is also involved in regulating bone resorption in skeletal tissue by stimulating osteoblast-production of osteoclast regulating cytokines, including receptoractivator of nuclear B ligand (RANKL) and interleukins. Therefore, we investigated whether PKA activators regulate osteoclastogenesis in aortic smooth muscle cells (SMC). Treatment of murine SMC with the PKA agonist forskolin stimulated RANKL expression at both mRNA and protein levels. Forskolin also stimulated expression of interleukin-6 but not osteoprotegerin (OPG), an inhibitor of RANKL. Consistent with these results, osteoclastic differentiation was induced when monocytic preosteoclasts (RAW264.7) were cocultured with forskolin-treated aortic SMC. Oxidized phospholipids also slightly induced RANKL expression in T lymphocytes, another potential source of RANKL in the vasculature. Because previous studies have shown that RANKL treatment alone induces matrix calcification of valvular and vascular cells, we next examined whether RANKL mediates forskolin-induced matrix calcification by aortic SMC. RANKL inhibition with OPG had little or no effect on osteoblastic differentiation and matrix calcification of aortic SMC. These findings suggest that, as in skeletal tissues, PKA activation induces bone resorptive factors in the vasculature and that aortic SMC calcification specifically induced by PKA, is not mediated by RANKL.

Enhanced Mineralization Potential of Vascular Cells from SM22α-Rankl tg Mice

“…Epidemiological studies show vascular calcification is inversely related to serum RANKL and positively related to serum OPG [14–18]. Moreover, some in vitro studies showed that RANKL treatment induced vascular calcification [19, 20], whereas we and others found no effect [21, 22], albeit in different species and culture conditions.…”

“…RANKL has been implicated in vascular calcification in some studies [19, 20, 28] but not others [21, 22, 29]. These differences may relate to different model systems and cell types.…”

RANKL Enhances Macrophage Paracrine Pro-Calcific Activity in High Phosphate-Treated Smooth Muscle Cells: Dependence on IL-6 and TNF-α

“…In vitro , SMCs have been reported to express RANKL in response to oxidative stress. In addition, endothelial cells express RANKL in response to inflammatory cytokines, as do activated T-cells[32–35]. Other in vitro studies have shown that RANK is expressed on endothelial cells, SMCs and monocytes-macrophages[36, 37].…”

Where do we stand on vascular calcification?

“…These cells may be derived from monocytic cells in the circulation that are triggered to undergo osteoclastic differentiation in the calcific milieu of the diseased vascular wall [31]. …”