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

Linda Demer,Rita B. Effros,Aneela Reddy,Wendy Tseng,Jinxiu Lu,Yin Tintut,Yifan Geng,Lucia S. Graham

doi:10.1074/jbc.m110.117366

Linda Demer, Rita B. Effros + Show 6 more

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https://doi.org/10.1074/jbc.m110.117366

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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 receptor-activator of nuclear κB 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.

Highlights

Effects of Forskolin on smooth muscle cells (SMC) Expression of Osteoclast Regulatory Cytokines—To examine the effects of forskolin on expression of osteoclast regulatory cytokines, aortic SMC were treated with forskolin for the indicated times, and RANKL, OPG, and IL-6 mRNA expression was assessed by real-time RT-qPCR
This study demonstrates that protein kinase A (PKA) activation in murine vascular SMC induces expression of osteoclastogenic cytokines, including RANKL, and that PKA-treated vascular cells upregulate osteoclastogenesis in adjacent preosteoclasts (Fig. 6A)
Because monocyte/macrophages are known precursors of osteoclasts, and they are present in large numbers in atherosclerotic plaque [19], the results suggest that PKA activation may promote remodeling of ectopic bone in the vasculature, as it does in skeletal bone

Summary

Introduction

2 The abbreviations used are: RANKL, receptor-activator of nuclear ␬B ligand; SMC, smooth muscle cell; OPG, osteoprotegerin; PAPC, 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine; TRAP, tartrate-resistant acid phosphatase. PKA Induction of RANKL in Vascular Cells and matrix calcification. Consistent with our previous findings [26], the control vascular SMC in co-culture significantly reduced the number of TRAP-positive osteoclasts compared with RAW cell monoculture (Fig. 3, C and D).

Results

Conclusion