Abstract
The molecular pathways by which long chain polyunsaturated fatty acids (LCPUFA) influence skeletal health remain elusive. Both LCPUFA and parathyroid hormone type 1 receptor (PTH1R) are known to be involved in bone metabolism while any direct link between the two is yet to be established. Here we report that LCPUFA are capable of direct, PTH1R dependent activation of extracellular ligand-regulated kinases (ERK). From a wide range of fatty acids studied, varying in chain length, saturation, and position of double bonds, eicosapentaenoic (EPA) and docosahexaenoic fatty acids (DHA) caused the highest ERK phosphorylation. Moreover, EPA potentiated the effect of parathyroid hormone (PTH(1–34)) in a superagonistic manner. EPA or DHA dependent ERK phosphorylation was inhibited by the PTH1R antagonist and by knockdown of PTH1R. Inhibition of PTH1R downstream signaling molecules, protein kinases A (PKA) and C (PKC), reduced EPA and DHA dependent ERK phosphorylation indicating that fatty acids predominantly activate G-protein pathway and not the β-arrestin pathway. Using picosecond time-resolved fluorescence microscopy and a genetically engineered PTH1R sensor (PTH-CC), we detected conformational responses to EPA similar to those caused by PTH(1–34). PTH1R antagonist blocked the EPA induced conformational response of the PTH-CC. Competitive binding studies using fluorescence anisotropy technique showed that EPA and DHA competitively bind to and alter the affinity of PTH1 receptor to PTH(1–34) leading to a superagonistic response. Finally, we showed that EPA stimulates protein kinase B (Akt) phosphorylation in a PTH1R-dependent manner and affects the osteoblast survival pathway, by inhibiting glucocorticoid-induced cell death. Our findings demonstrate for the first time that LCPUFAs, EPA and DHA, can activate PTH1R receptor at nanomolar concentrations and consequently provide a putative molecular mechanism for the action of fatty acids in bone.
Highlights
As age increases, the risk of bone loss and fracture increases [1]
In untransfected Human embryonic kidney 293 (HEK293) cells, no significant increase in ERK1/2 phosphorylation was found compared to nontreated cells (Fig. 1A; lower panel), indicating that ERK1/2 activation was mediated through the PTH1 receptor
The majority of fatty acids have been reported to remain in the plasma membrane after 10 minutes [46] while the long chain polyunsaturated fatty acids (LCPUFA) metabolizing enzyme, cyclooxygenase is localized in the endoplasmic reticulum [47] making it unlikely that the LCPUFAs metabolites are activating parathyroid hormone type 1 receptor (PTH1R) in our experiments
Summary
The risk of bone loss and fracture increases [1]. It is believed that dietary modifications and physical activity may be considered the primary targets to minimize bone loss and fragility [3]. Parathyroid hormone (PTH) has been shown to play an important role in bone homeostasis [4]. Parathyroid hormone related protein (PTH-rP) is a vital developmental morphogen [5,6]. PTH and PTH-rP both bind to and activate PTH/PTH-rP Receptor (PTH1R), a G-protein coupled receptor with seven transmembrane domains which is highly expressed in bone and kidney [7]. PTH1R stimulates multiple signaling cascades including the Gs-cAMP-PKA [8], Gq/11-PLC-PKC [9], and mitogen-activated protein kinases (MAPKs) leading to various biological effects including anabolic and catabolic actions in bone [10]. PTH has been reported to stimulate phosphorylation of Akt [11], a critical regulator of osteoblast differentiation [12] and survival [13]
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