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G-protein-coupled receptor (GPCR) kinases, GRKs, are known as serine/threonine kinases that regulate GPCR signaling, but recent findings propose functions for these kinases besides receptor desensitization. Indeed, GRK5 can translocate to the nucleus by means of a nuclear localization sequence, suggesting that this kinase regulates transcription events in the nucleus. To evaluate the effect of GRK5-IkappaB alpha interaction on NFkappaB signaling, we induced the overexpression and the knockdown of GRK5 in cell cultures. GRK5 overexpression causes nuclear accumulation of IkappaB alpha, leading to the inhibition of NFkappaB transcriptional activity. Opposite results are achieved by GRK5 knockdown through siRNA. A physical interaction between GRK5 and IkappaB alpha, rather than phosphorylative events, appears as the underlying mechanism. We identify the regulator of gene protein signaling homology domain of GRK5 (RH) and the N-terminal domain of IkappaB alpha as the regions involved in such interaction. To confirm the biological relevance of this mechanism of regulation for NFkappaB, we evaluated the effects of GRK5-RH on NFkappaB-dependent phenotypes. In particular, GRK5-RH overexpression impairs apoptosis protection and cytokine production in vitro and inflammation and tissue regeneration in vivo. Our results reveal an unexpected role for GRK5 in the regulation of NFkappaB transcription activity. Placing these findings in perspective, this mechanism may represent a therapeutic target for all those conditions involving excessive NFkappaB activity.

Helper-dependent adenoviral (HD-Ad) vectors have great potential for gene therapy applications; however, their administration induces acute toxicity that impairs safe clinical applications. We previously observed that PEGylation of HD-Ad vectors strongly reduces the acute response in murine and primate models. To evaluate whether PEGylated HD-Ad vectors combine reduced toxicity with the correction of pathological phenotypes, we administered an HD-Ad vector expressing the human apolipoprotein A-I (hApoA-I) to low-density lipoprotein (LDL)-receptor-deficient mice (a model for familial hypercholesterolemia) fed a high-cholesterol diet. Mice were treated with high doses of HD-Ad-expressing apo A-I or its PEGylated version. Twelve weeks later, LDL levels were lower and high-density lipoprotein (HDL) levels higher in mice treated with either of the vectors than in untreated mice. After terminal killing, the areas of atherosclerotic plaques were much smaller in the vector-treated mice than in the control animals. Moreover, the increase in pro-inflammatory cytokines was lower and consequently the toxicity profile better in mice treated with PEGylated vector than in mice treated with the unmodified vector. This finding indicates that the reduction in toxicity resulting from PEGylation of HD-Ad vectors does not impair the correction of pathological phenotypes. It also supports the clinical potential of these vectors for the correction of genetic diseases.

"Until recently, free d-amino acids were thought to be involved only in bacterial physiology. Nevertheless, today there is evidence that D-serine, by acting as co-agonist at NMDARs, plays a role in controlling neuronal functions in mammals. Besides D-serine, another D-amino acid, D-aspartate (D-Asp), is found in the mammalian brain with a temporal gradient of occurrence: high in embryo and low in adult. In this study, we demonstrate that D-Asp acts as an endogenous NMDAR agonist, since it triggers currents via interaction with each of NR2A-D receptor subunits. According to its pharmacological features, we showed that oral administration of D-Asp strongly enhances NMDAR-dependent LTP in adulthood and, in turn, completely rescues the synaptic plasticity decay observed in the hippocampus of aged animals. Therefore, our findings suggest a tantalizing hypothesis for which this in-embryo-occurring D-amino acid, when "forced" over its physiological content, may disclose plasticity windows inside which it counteracts the age-related reduction of NMDAR signaling."

Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4), regulate the lifespan of dendritic cells (DCs) by largely undefined mechanisms. Here, we identify a role for calcium-calmodulin–dependent kinase IV (CaMKIV) in this survival program. The pharmacologic inhibition of CaMKs as well as ectopic expression of kinase-inactive CaMKIV decrease the viability of monocyte-derived DCs exposed to bacterial LPS. The defect in TLR4 signaling includes a failure to accumulate the phosphorylated form of the cAMP response element-binding protein (pCREB), Bcl-2, and Bcl-xL. CaMKIV null mice have a decreased number of DCs in lymphoid tissues and fail to accumulate mature DCs in spleen on in vivo exposure to LPS. Although isolated Camk4−/− DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive. The transgenic expression of Bcl-2 in CaMKIV null mice results in full recovery of DC survival in response to LPS. These results reveal a novel link between TLR4 and a calcium-dependent signaling cascade comprising CaMKIV-CREB-Bcl-2 that is essential for DC survival.

We have recently demonstrated that endothelial beta(2) adrenergic receptors beta(2)AR regulate eNOS activity and consequently vascular tone, through means of PKB/AKT. In this work we explored the signal transduction pathway leading to AKT/eNOS activation in endothelial cells (EC). Using pharmacological and molecular inhibitors both in cultured EC cells and in ex vivo rat carotid preparations, we found that G(i) coupling of the beta 2AR is needed for AKT activation and vasorelaxation. Since endothelial activation is sensitive to pertussis toxin but not to G(i beta gamma), inhibition by beta ARKct, we conclude that G(alpha i) mediates beta AR induced AKT activation. Downstream, beta AR signalling requires the soluble tyrosine kinase SRC, as both in cultured EC and rat carotid, the mutant dominant negative of SRC prevent beta(2)AR induced endothelial activation and vasodilation. In EC, G(alpha i) directly interacts with SRC and this interaction leads to SRC activation and phosphorylation in a manner that is regulated by Balk stimulation. We propose a novel signal transduction pathway for beta(2)AR stimulation trough G(alpha i) and SRC, leading to activation of AKT. (c) 2007 Elsevier Inc. All rights reserved.