These results demonstrate that: (i) VSMCs express a functional CaR; (ii) a reduction in CaR expression is associated with increased mineralization in vivo and in vitro; (iii) calcimimetics decrease mineral deposition by VSMC. These data suggest that calcimimetics may inhibit the development of VC in CKD patients.

The extracellular calcium-sensing receptor (CaSR) monitors the systemic extracellular free ionized calcium level ([Ca 2+ ] o ) in organs involved in systemic [Ca 2+ ] o homeostasis. However, the CaSR is also expressed in the nervous system where its role is unknown. Here we find high levels of the CaSR in perinatal mouse sympathetic neurons when their axons are innervating and branching extensively in their targets. Manipulating CaSR function in these neurons by varying [Ca 2+ ] o , using CaSR agonists and antagonists or expressing a dominant-negative CaSR markedly affects neurite growth in vitro Sympathetic neurons lacking the CaSR have smaller neurite arbors in vitro, and sympathetic innervation density is reduced in CaSR-deficient mice in vivo.Hippocampal pyramidal neurons, which also express the CaSR, have smaller dendrites when transfected with dominant-negative CaSR in postnatal organotypic cultures. Our findings reveal a crucial role for the CaSR in regulating the growth of neural processes in the peripheral and central nervous systems.The growth, guidance and branching of neural processes in the developing nervous system is controlled by numerous locally acting and diffusible signalling proteins that bind specific receptors on the growing tips of these processes 1,2. While changes in cytoplasmic Ca 2+ participate in transducing many of these growth and guidance signals, changes within the narrow physiological range of extracellular Ca 2+ have not been thought to play a direct role in regulating growth cone motility 3. The level of extracellular Ca 2+ is monitored by the CaSR, and in accordance with its crucial regulatory function in maintaining [Ca 2+ ] o within very narrow physiological limits 4, it is conspicuously expressed in all structures and organs involved in systemic calcium homeostasis, namely, the parathyroid glands, kidneys, bone and gut. It is also expressed in several other tissues and in multiple sites within the adult brain, including the subfornical organ, olfactory bulbs, striatum, cerebellum, basal ganglia and hippocampus, where its functions are unclear 5,6.To investigate whether CaSR has a role in neuronal development, we screened for the expression of CaSR transcripts in several experimentally tractable populations of neurons in the peripheral nervous system of fetal mice. We found significant CaSR expression in the superior cervical ganglion (SCG), a population of sympathetic neurons that is extensively

Reverse signaling via members of the tumor necrosis factor (TNF) superfamily is increasingly recognized among cells of the immune system where it controls multiple aspects of immune function. Here we document TNFα reverse signaling in the nervous system for the first time and show that it plays a crucial role in establishing sympathetic innervation. During postnatal development, sympathetic axons express TNFα as they grow and branch in their target tissues which in turn express TNFR1. In culture, soluble forms of TNFR1 act directly on postnatal sympathetic axons to promote growth and branching by a mechanism that depends on membrane integrated TNFα and downstream MEK/ERK activation. Sympathetic innervation density is significantly reduced in several tissues in postnatal and adult mice lacking either TNFα or TNFR1. These findings reveal that target-derived TNFR1 acts as a reverse signaling ligand for membrane-integrated TNFα to promote sympathetic axon growth and branching.

The Ca2+-sensing receptor (CaR) is a pleiotropic, type III G protein-coupled receptor (GPCR) that associates functionally with the cytoskeletal protein filamin. To investigate the effect of CaR signaling on the cytoskeleton, human embryonic kidney (HEK)-293 cells stably transfected with CaR (CaR-HEK) were incubated with CaR agonists in serum-free medium for up to 3 h. Addition of the calcimimetic NPS R-467 or exposure to high extracellular Ca2+ or Mg2+ levels elicited actin stress fiber assembly and process retraction in otherwise stellate cells. These responses were ablated by cotreatment with the calcilytic NPS 89636 and were absent in vector-transfected HEK-293 cells. Cotreatment with the Rho kinase inhibitors Y-27632 and H1152 attenuated the CaR-induced morphological change but not intracellular Ca2+ (Ca2+(i)) mobilization or ERK activation, although transfection with a dominant-negative RhoA-binding protein also inhibited calcimimetic-induced actin stress fiber assembly. CaR effects on morphology were unaffected by inhibition of G(q/11) or G(i/o) signaling, epidermal growth factor receptor, or the metalloproteinases. In contrast, CaR-induced cytoskeletal changes were not induced by the aromatic amino acids, treatments that also failed to potentiate CaR-induced ERK activation despite inducing Ca2+(i) mobilization. Together, these data establish that CaR can elicit Rho-mediated changes in stress fiber assembly and cell morphology, which could contribute to the receptor's physiological actions. In addition, this study provides further evidence that aromatic amino acids elicit differential signaling from other CaR agonists.