The G-protein-coupled lactate receptor, GPR81 (HCA1), is known to promote lipid storage in adipocytes by downregulating cAMP levels. Here, we show that GPR81 is also present in the mammalian brain, including regions of the cerebral neocortex and hippocampus, where it can be activated by physiological concentrations of lactate and by the specific GPR81 agonist 3,5-dihydroxybenzoate to reduce cAMP. Cerebral GPR81 is concentrated on the synaptic membranes of excitatory synapses, with a postsynaptic predominance. GPR81 is also enriched at the blood-brain-barrier: the GPR81 densities at endothelial cell membranes are about twice the GPR81 density at membranes of perivascular astrocytic processes, but about one-seventh of that on synaptic membranes. There is only a slight signal in perisynaptic processes of astrocytes. In synaptic spines, as well as in adipocytes, GPR81 immunoreactivity is located on subplasmalemmal vesicular organelles, suggesting trafficking of the protein to and from the plasma membrane. The results indicate roles of lactate in brain signaling, including a neuronal glucose and glycogen saving response to the supply of lactate. We propose that lactate, through activation of GPR81 receptors, can act as a volume transmitter that links neuronal activity, cerebral energy metabolism and energy substrate availability.
Edited by Alex Toker Connective tissue growth factor (CTGF; now often referred to as CCN2) is a secreted protein predominantly expressed during development, in various pathological conditions that involve enhanced fibrogenesis and tissue fibrosis, and in several cancers and is currently an emerging target in several early-phase clinical trials. Tissues containing high CCN2 activities often display smaller degradation products of full-length CCN2 (FL-CCN2). Interpretation of these observations is complicated by the fact that a uniform protein structure that defines biologically active CCN2 has not yet been resolved. Here, using DG44 CHO cells engineered to produce and secrete FL-CCN2 and cell signaling and cell physiological activity assays, we demonstrate that FL-CCN2 is itself an inactive precursor and that a proteolytic fragment comprising domains III (thrombospondin type 1 repeat) and IV (cystine knot) appears to convey all biologically relevant activities of CCN2. In congruence with these findings, purified FL-CCN2 could be cleaved and activated following incubation with matrix metalloproteinase activities. Furthermore, the C-terminal fragment of CCN2 (domains III and IV) also formed homodimers that were ϳ20-fold more potent than the monomeric form in activating intracellular phosphokinase cascades. The homodimer elicited activation of fibroblast migration, stimulated assembly of focal adhesion complexes, enhanced RANKL-induced osteoclast differentiation of RAW264.7 cells, and promoted mammosphere formation of MCF-7 mammary cancer cells. In conclusion, CCN2 is synthesized and secreted as a preproprotein that is autoinhibited by its two N-terminal domains and requires proteolytic processing and homodimerization to become fully biologically active. This work was supported by the Norwegian Council for Cardiovascular Research (to O. J. K.) and Scientia fellowship program, University of Oslo (to A. K. G.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S7 and Tables S1 and S2.
We recently reported that transgenic mice with cardiac-restricted overexpression of CCN2/CTGF have substantially increased tolerance towards ischemia/reperfusion injury. The purpose of this study was to investigate to what extent fully differentiated cardiac myocytes are direct targets of CCN2, and to resolve the signaling mechanisms that convey the cardioprotective actions of CCN2. Akt and GSK-3β were identified as putative intermediaries of intracellular signaling stimulated by recombinant human CCN2 (rhCCN2). Concentration-effect experiments revealed CCN2-stimulated phosphorylation of Akt (Ser473) and downstream GSK-3β (Ser9) with EC50 ~250 nmol/L. CCN2-stimulated phosphorylation of Akt and GSK-3β was sensitive to inhibition of PI3-kinase (LY294002). Phosphorylation of GSK-3β was also sensitive to Akt-inhibition (API-2), demonstrating CCN2-engendered activation of a PI3-kinase/Akt/GSK-3β-signaling pathway. A C-terminal peptide fragment of CCN2 (11.2 kD) displayed partial agonist activity, while two short peptides derived from the Thrombospondin- and the IGFBP- homology domains of CCN2, respectively, additively inhibited rhCCN2-stimulated Akt-phosphorylation. The viability of cardiac myocytes subjected to hypoxia/reoxygenation injury or doxorubicin-induced oxidative stress was assessed by assays of adenylate kinase and lactate dehydrogenase released from dying cells. Cardiac myocytes exposed to CCN2 displayed increased tolerance towards hypoxia/reoxygenation and doxorubicin-induced oxidative stress, an effect that was abrogated by inhibition of PI3-kinase. The cytoprotective actions of CCN2 reflected in the transcriptome of CCN2-stimulated cardiac myocytes (anti-apoptosis, stress, and wound-response gene programs). In conclusion, this study discloses the novel findings that cardiac myocytes are CCN2 target cells in which CCN2 increases tolerance towards hypoxia and oxidative stress via PI3-kinase-dependent Akt/GSK-3β signaling.
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