Homozygous male and female mice were analyzed, and wild-type littermates (mice were immunolabeled. Introduction Chronic kidney disease (CKD) is usually a global public health problem that shortens lifespan, primarily by increasing risk of cardiovascular disease (Eckardt et al., 2013). Novel therapeutic targets are Mouse monoclonal to ApoE urgently needed to reduce the burden of cardiovascular disease in CKD. Left ventricular hypertrophy (LVH) is usually a common pattern of cardiovascular injury in CKD that affects up to 75% of individuals by the time they reach end-stage renal disease (Faul et al., 2011). By promoting heart failure and atrial and ventricular arrhythmias, LVH is usually a powerful risk factor for cardiovascular events and death (de Simone et al., 2008). The complex pathogenesis of LVH entails ventricular pressure and volume overload, but emerging data also implicate a novel role for the bone-derived, phosphate-regulating hormone, fibroblast NVP-AAM077 Tetrasodium Hydrate (PEAQX) growth factor (FGF) 23 (Gutierrez et al., 2009). The primary physiological effects of FGF23 to stimulate urinary phosphate excretion and reduce circulating calcitriol concentrations are mediated by FGF23 binding to FGF receptors (FGFR) in the kidney, with -klotho providing as the co-receptor that enhances binding affinity (Urakawa et al., 2006). Serum levels of FGF23 rise progressively as kidney function declines, presumably as a compensation to maintain neutral phosphate balance in the setting of reduced glomerular filtration of phosphate (Wolf, 2012). However, chronically elevated FGF23 levels may be ultimately maladaptive in patients with CKD, given the powerful dose-dependent associations of higher FGF23 with increased risks of NVP-AAM077 Tetrasodium Hydrate (PEAQX) LVH, congestive heart failure and death (Gutierrez et al., 2009; Gutierrez et al., 2008; Isakova et al., 2011). FGF23 induces hypertrophic growth of cardiac myocytes in vitro and LVH in rodents through a direct FGFR-dependent mechanism, but independently of -klotho, which is not expressed in cardiac myocytes (Faul et al., 2011). Whereas -klotho-expressing cells in the kidney respond to FGF23 by activating the Ras/mitogen-activated protein kinase (MAPK) cascade (Urakawa et NVP-AAM077 Tetrasodium Hydrate (PEAQX) al., 2006), the pro-hypertrophic effects of FGF23 on cardiac myocytes were blocked by pharmacologic inhibition of phospholipase C (PLC) and calcineurin (Faul et al., 2011). In contrast, the pro-hypertrophic effects of the prototypical paracrine FGF family member, FGF2, were blocked by inhibitors of the Ras/MAPK cascade (Faul et al., 2011). These findings suggest NVP-AAM077 Tetrasodium Hydrate (PEAQX) that different FGF ligands can activate unique downstream signaling pathways in cardiac myocytes, and that in the absence of -klotho, FGF23 activates the PLC/calcineurin/nuclear factor of activated T cells (NFAT) signaling axis, which is a potent inducer of pathological LVH (Molkentin et al., 1998). However, the identity of the specific FGFR that mediates the cardiac effects of FGF23 is usually unknown. The mammalian genome encodes four FGFR isoforms, FGFR1C4, that are receptor tyrosine kinases (Ornitz and Itoh, 2001). Following ligand-induced auto-phosphorylation of FGFR, FGF receptor substrate (FRS) 2 undergoes tyrosine phosphorylation by FGFR and stimulates Ras/MAPK and PI3K/Akt signaling (Eswarakumar et al., 2005). In contrast to FRS2, which is usually constitutively bound to FGFR independent of the receptors activation state, PLC can also be recruited to bind directly to one specific phosphorylated tyrosine residue (pY751 in mouse FGFR4) within a consensus sequence (YLDL) in the FGFR cytoplasmic tail (Mohammadi et al., 1991; Vainikka et al., 1994). Subsequent phosphorylation of PLC by FGFR activates PLC (Burgess et al., 1990), which induces generation of diacylglycerol and inositol 1,4,5-triphosphate, and increases cytoplasmic Ca2+ that activates calcineurin and its substrate, NFAT (Eswarakumar et al., 2005). Here, we statement our investigation into the specific FGFR isoform that mediates PLC signaling and the pro-hypertrophic effects of FGF23 in cardiac myocytes. Results FGF23 activates FGFR4 and PLC signaling in the absence of -klotho To study FGF-FGFR-dependent signaling, we used HEK293 cells that express all FGFR isoforms but lack -klotho (data not shown), much like cardiac myocytes (Faul et al., 2011). As a read-out of calcineurin/NFAT versus Ras/MAPK activation, we analyzed phosphorylation of PLC and FRS2. In response to 30 minutes of treatment, FGF23 increased phosphorylated PLC levels without changing overall PLC expression, but did not induce phosphorylation of FRS2 (Physique 1A). In contrast, FGF2 experienced no effect on phospho-PLC levels but increased phosphorylation of FRS2 and ERK1/2. Thus, in HEK293 cells, FGF23 and FGF2 activate unique FGFR adaptor proteins, which could explain their differential downstream signaling in cardiac myocytes (Faul et al., 2011). Open in a separate window.
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