Abstract-Several studies underline the role of the transcription factor NF-B in the development of left cardiac hypertrophy (LVH). We have demonstrated recently that the RGS homology domain within the amino terminus of GRK5 (GRK5-NT) is able to inhibit NF-B transcription activity and its associated phenotypes. The aim of this study was to evaluate the ability of GRK5-NT to regulate LVH through the inhibition of NF-B both in vitro and in vivo. In cardiomyoblasts, GRK5-NT inhibits phenylephrine-induced transcription of both NF-B and atrial natriuretic factor promoters, assessed by luciferase assay, thus confirming a role for this protein in the regulation of cardiomyocyte hypertrophy. In vivo, we explored 2 rat models of LVH, the spontaneously hypertensive rat and the normotensive Wistar Kyoto rat exposed to chronic administration of phenylephrine. Intracardiac injection of an adenovirus encoding for GRK5-NT reduces cardiac mass in spontaneously hypertensive rats and prevents the development of phenylephrineinduced LVH in Wistar Kyoto rats. This associates with inhibition of NF-B signaling (assessed by NF-B levels), transcriptional activity and phenotypes (fibrosis and apoptosis). Such phenomenon is independent from hemodynamic changes, because adenovirus encoding for GRK5-NT did not reduce blood pressure levels in spontaneously hypertensive rats or in Wistar Kyoto rats. In conclusion, our study supports the regulation of LVH based on the GRK5-NT inhibition of the NF-B transduction signaling. (Hypertension. 2010;56:696-704.)Key Words: cardiac hypertrophy Ⅲ intracardiac injection Ⅲ spontaneously hypertensive rats Ⅲ NF-B Ⅲ transcription factors N F-B is an ubiquitously expressed transcription factor that modulates the expression of genes involved in the regulation of cell functions, such as survival, apoptosis, growth, division, innate immunity, differentiation, and cellular responses to stress, hypoxia, and ischemia. 1-4 The classic cellular model in which this factor is studied is the immune system for its central role in cytokine production. 4,5 It has been reported recently that NF-B is relevant in the development of left ventricular hypertrophy (LVH) and remodeling through mechanisms independent from inflammation. NF-B mediates hypertrophic growth of cardiomyocytes in response to G protein-coupled receptor agonists, including norepinephrine, endothelin 1, and angiotensin II. 6,7 Also, NF-B inhibition attenuates LVH in different animal models of disease. 8,9 This evidence suggests that NF-B blockade may be an effective strategy to inhibit LVH and remodeling. The family of G protein-coupled receptor kinases (GRKs) and, in particular GRK2 and GRK5, possesses the ability to bind both NF-B and its inhibitor, IB␣. 10,11 In particular, GRK5, by means of its RGS homology (RH) domain within the amino terminus, interacts with IB␣ leading to the stabilization and accumulation of the IB␣/NF-B complex in the nucleus and, consequently, to the inhibition of NF-B transcriptional activity. 11 This feature of the RH domain of GR...

Metabolic stimuli such as insulin and insulin like growth factor cause cellular accumulation of G protein Coupled Receptor Kinase 2 (GRK2), which in turn is able to induce insulin resistance. Here we show that in fibroblasts, GRK2 is able to increase ATP cellular content by enhancing mitochondrial biogenesis; also, it antagonizes ATP loss after hypoxia/reperfusion. Interestingly, GRK2 is able to localize in the mitochondrial outer membrane, possibly through one region within the RGS homology domain and one region within the catalytic domain. In vivo, GRK2 removal from the skeletal muscle results in reduced ATP production and impaired tolerance to ischemia. Our data show a novel sub-cellular localization of GRK2 in the mitochondria and an unexpected role in regulating mitochondrial biogenesis and ATP generation.

T cell ontogeny is a sophisticated process, which takes place within the thymus through a series of well-defined discrete stages. The process requires a proper lympho-stromal interaction. In particular, cortical and medullary thymic epithelial cells (cTECs, mTECs) drive T cell differentiation, education, and selection processes, while the thymocyte-dependent signals allow thymic epithelial cells (TECs) to maturate and provide an appropriate thymic microenvironment. Alterations in genes implicated in thymus organogenesis, including Tbx1, Pax1, Pax3, Pax9, Hoxa3, Eya1, and Six1, affect this well-orchestrated process, leading to disruption of thymic architecture. Of note, in both human and mice, the primordial TECs are yet unable to fully support T cell development and only after the transcriptional activation of the Forkhead-box n1 (FOXN1) gene in the thymic epithelium this essential function is acquired. FOXN1 is a master regulator in the TEC lineage specification in that it down-stream promotes transcription of genes, which, in turn, regulate TECs differentiation. In particular, FOXN1 mainly regulates TEC patterning in the fetal stage and TEC homeostasis in the post-natal thymus. An inborn null mutation in FOXN1 leads to Nude/severe combined immunodeficiency (SCID) phenotype in mouse, rat, and humans. In Foxn1−/− nude animals, initial formation of the primordial organ is arrested and the primordium is not colonized by hematopoietic precursors, causing a severe primary T cell immunodeficiency. In humans, the Nude/SCID phenotype is characterized by congenital alopecia of the scalp, eyebrows, and eyelashes, nail dystrophy, and a severe T cell immunodeficiency, inherited as an autosomal recessive disorder. Aim of this review is to summarize all the scientific information so far available to better characterize the pivotal role of the master regulator FOXN1 transcription factor in the TEC lineage specifications and functionality.

BACKGROUND AND PURPOSE There is much evidence supporting the role of β2‐adrenoceptors (β2AR) in angiogenesis but the mechanisms underlying their effects have not been elucidated. Hence, we studied post‐ischaemic angiogenesis in the hindlimb (HL) of β2AR knock‐out mice (β2AR−/−) in vivo and explored possible molecular mechanisms in vitro. EXPERIMENTAL APPROACH Femoral artery resection (FAR) was performed in wild‐type and β2AR−/− mice and adaptive responses to chronic HL ischaemia were explored; blood flow was measured by ultrasound and perfusion of dyed beads, bone rarefaction, muscle fibrosis and skin thickness were evaluated by immunoflourescence and morphometric analysis. Intrafemoral delivery of an adenovirus encoding the human β2AR (ADβ2AR) was used to reinstate β2ARs in β2AR−/− mice. Molecular mechanisms were investigated in mouse‐derived aortic endothelial cells (EC) in vitro, focusing on NFκB activation and transcriptional activity. RESULTS Angiogenesis was severely impaired in β2AR−/− mice subjected to FAR, but was restored by gene therapy with ADβ2AR. The proangiogenic responses to a variety of stimuli were impaired in β2AR−/− EC in vitro. Moreover, removal of β2ARs impaired the activation of NFκB, a transcription factor that promotes angiogenesis; neither isoprenaline (stimulates βARs) nor TNFα induced NFκB activation in β2AR−/− EC. Interestingly, cAMP response element binding protein (CREB), a transcription factor that counter regulates NFκB, was constitutively increased in β2AR−/− ECs. ADβ2AR administration restored β2AR membrane density, reduced CREB activity and reinstated the NFκB response to isoprenaline and TNFα. CONCLUSIONS AND IMPLICATIONS Our results suggest that β2ARs control angiogenesis through the tight regulation of nuclear transcriptional activity.

We have analyzed the human mineralocorticoid receptor (hMR) gene in 14 families with autosomal dominant or sporadic pseudohypoaldosteronism (PHA1), a rare form of mineralocorticoid resistance characterized by neonatal renal salt wasting and failure to thrive. Six heterozygous mutations were detected. Two frameshift mutations in exon 2 (insT1354, del8bp537) and one nonsense mutation in exon 4 (C2157A, Cys645stop) generate truncated proteins due to premature stop codons. Three missense mutations (G633R, Q776R, L979P) differently affect hMR function. The DNA binding domain mutant R633 exhibits reduced maximal transactivation, although its binding characteristics and ED(50) of transactivation are comparable with wild-type hMR. Ligand binding domain mutants R776 and P979 present reduced or absent aldosterone binding, respectively, which is associated with reduced or absent ligand-dependent transactivation capacity. Finally, P979 possesses a transdominant negative effect on wild-type hMR activity, whereas mutations G633R and Q776R probably result in haploinsufficiency in PHA1 patients. We conclude that hMR mutations are a common feature of autosomal dominant PHA1, being found in 70% of our familial cases. Their absence in some families underscores the importance of an extensive investigation of the hMR gene and the role of precise diagnostic procedures to allow for identification of other genes potentially involved in the disease.