Deciphering biased-agonism complexity reveals a new active AT1 receptor entity.

Functional selectivity of G protein-coupled receptor (GPCR) ligands toward different downstream signals has recently emerged as a general hallmark of this receptor class. However, pleiotropic and crosstalk signaling of GPCRs makes functional selectivity difficult to decode. To look from the initial active receptor point of view, we developed new, highly sensitive and direct bioluminescence resonance energy transfer-based G protein activation probes specific for all G protein isoforms, and we used them to evaluate the G protein-coupling activity of [(1)Sar(4)Ile(8)Ile]-angiotensin II (SII), previously described as an angiotensin II type 1 (AT(1)) receptor-biased agonist that is G protein independent but ß-arrestin selective. By multiplexing assays sensing sequential signaling events, from receptor conformations to downstream signaling, we decoded SII as an agonist stabilizing a G protein-dependent AT(1A) receptor signaling module different from that of the physiological agonist angiotensin II, both in recombinant and primary cells. Thus, a biased agonist does not necessarily select effects from the physiological agonist but may instead stabilize and create a new distinct active pharmacological receptor entity.

Ephrin-B1 is a novel specific component of the lateral membrane of the cardiomyocyte and is essential for the stability of cardiac tissue architecture cohesion.

RATIONALE: Cardiac tissue cohesion relying on highly ordered cardiomyocytes (CM) interactions is critical because most cardiomyopathies are associated with tissue remodeling and architecture alterations. OBJECTIVE: Eph/ephrin system constitutes a ubiquitous system coordinating cellular communications which recently emerged as a major regulator in adult organs. We examined if eph/ephrin could participate in cardiac tissue cyto-organization. METHODS AND RESULTS: We reported the expression of cardiac ephrin-B1 in both endothelial cells and for the first time in CMs where ephrin-B1 localized specifically at the lateral membrane. Ephrin-B1 knock-out (KO) mice progressively developed cardiac tissue disorganization with loss of adult CM rod-shape and sarcomeric and intercalated disk structural disorganization confirmed in CM-specific ephrin-B1 KO mice. CMs lateral membrane exhibited abnormal structure by electron microscopy and notably increased stiffness by atomic force microscopy. In wild-type CMs, ephrin-B1 interacted with claudin-5/ZO-1 complex at the lateral membrane, whereas the complex disappeared in KO/CM-specific ephrin-B1 KO mice. Ephrin-B1 deficiency resulted in decreased mRNA expression of CM basement membrane components and disorganized fibrillar collagen matrix, independently of classical integrin/dystroglycan system. KO/CM-specific ephrin-B1 KO mice exhibited increased left ventricle diameter and delayed atrioventricular conduction. Under pressure overload stress, KO mice were prone to death and exhibited striking tissue disorganization. Finally, failing CMs displayed downregulated ephrin-B1/claudin-5 gene expression linearly related to the ejection fraction. CONCLUSIONS: Ephrin-B1 is necessary for cardiac tissue architecture cohesion by stabilizing the adult CM morphology through regulation of its lateral membrane. Because decreased ephrin-B1 is associated with molecular/functional cardiac defects, it could represent a new actor in the transition toward heart failure.

Lysosomal serine protease CLN2 regulates tumor necrosis factor-alpha-mediated apoptosis in a Bid-dependent manner.

Apoptosis is a highly organized, energy-dependent program by which multicellular organisms eliminate damaged, superfluous, and potentially harmful cells. Although caspases are the most prominent group of proteases involved in the apoptotic process, the role of lysosomes has only recently been unmasked. This study investigated the role of the lysosomal serine protease CLN2 in apoptosis. We report that cells isolated from patients affected with late infantile neuronal ceroid lipofuscinosis (LINCL) having a deficient activity of CLN2 are resistant to the toxic effect of death ligands such as tumor necrosis factor (TNF), CD95 ligand, or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not to receptor-independent stress agents. CLN2-deficient cells exhibited a defect in TNF-induced Bid cleavage, release of cytochrome c, and caspase-9 and -3 activation. Moreover, extracts from CLN2-overexpressing cells or a CLN2 recombinant protein were able to catalyze the in vitro cleavage of Bid. Noteworthy, correction of the lysosomal enzyme defect of LINCL fibroblasts using a medium enriched in CLN2 protein enabled restoration of TNF-induced Bid and caspase-3 processing and toxicity. Conversely, transfection of CLN2-corrected cells with small interfering RNA targeting Bid abrogated TNF-induced cell death. Altogether, our study demonstrates that genetic deletion of the lysosomal serine protease CLN2 and the subsequent loss of its catalytic function confer resistance to TNF in non-neuronal somatic cells, indicating that CLN2 plays a yet unsuspected role in TNF-induced cell death.

Critical role for sphingosine kinase-1 in regulating survival of neuroblastoma cells exposed to amyloid-beta peptide.

We examined the role of sphingosine kinase-1 (SphK1), a critical regulator of the ceramide/sphingosine 1-phosphate (S1P) biostat, in the regulation of death and survival of SH-SY5Y neuroblastoma cells in response to amyloid beta (Abeta) peptide (25-35). Upon incubation with Abeta, SH-SY5Y cells displayed a marked down-regulation of SphK1 activity coupled with an increase in the ceramide/S1P ratio followed by cell death. This mechanism was redox-sensitive; N-acetylcysteine totally abrogated the down-regulation of SphK1 activity and strongly inhibited Abeta-induced cell death. SphK1 overexpression impaired the cytotoxicity of Abeta, whereas SphK1 silencing by RNA interference mimicked Abeta-induced cell death, thereby establishing a critical role for SphK1. We further demonstrated that SphK1 could mediate the well established cytoprotective action of insulin-like growth factor (IGF-I) against Abeta toxicity. A dominant-negative form of SphK1 or its pharmacological inhibition not only abrogated IGF-I-triggered stimulation of SphK1 but also hampered IGF-I protective effect. Similarly to IGF-I, the neuroprotective action of TGF-beta1 was also dependent on SphK1 activity; activation of SphK1 as well as cell survival were impeded by a dominant-negative form of SphK1. Taken together, these results provide the first illustration of SphK1 role as a critical regulator of death and survival of Abeta-treated cells.

Role of FAN in tumor necrosis factor-alpha and lipopolysaccharide-induced interleukin-6 secretion and lethality in D-galactosamine-sensitized mice.

Tumor necrosis factor (TNF) alpha-induced neutral sphingomyelinase-mediated generation of ceramide, a bioactive lipid molecule, is transduced by the adaptor protein FAN, which binds to the intracellular region of the CD120a TNFalpha receptor. FAN-deficient mice do not exhibit any gross abnormality. To further explore the functions of FAN in vivo and because CD120a-deficient mice are resistant to endotoxin-induced liver failure and lethality, we investigated the susceptibility of FAN-deficient animals to lipopolysaccharide (LPS). We show that after d-galactosamine sensitization, FAN-deficient mice were partially resistant to LPS- and TNFalpha-induced lethality. Although LPS challenge resulted in a hepatic ceramide content lower in mutant mice than in control animals, it triggered similar histological alterations, caspase activation, and DNA fragmentation in the liver. Interestingly, LPS-induced elevation of IL-6 (but not TNFalpha) serum concentrations was attenuated in FAN-deficient mice. A less pronounced secretion of IL-6 was also observed after LPS or TNFalpha treatment of cultured peritoneal macrophages and embryonic fibroblasts isolated from FAN-deficient mice, as well as in human fibroblasts expressing a mutated FAN. Finally, we show that d-galactosamine-sensitized IL-6-deficient mice were partially resistant to endotoxin-induced liver apoptosis and lethality. These findings highlight the role of FAN and IL-6 in the inflammatory response initiated by endotoxin, implicating TNFalpha.