We demonstrated that miR-200c directly targets SIRT1, eNOS, and FOXO1; via this mechanism, miR-200c decreased NO and increased the acetylation of SIRT1 targets, that is, FOXO1 and p53. FOXO1 acetylation inhibited its transcriptional activity on target genes, that is, SIRT1 and the ROS scavengers, catalase and manganese superoxide dismutase. In keeping, miR-200c increased ROS production and induced p66Shc protein phosphorylation in Ser-36; this mechanism upregulated ROS and inhibited FOXO1 transcription, reinforcing this molecular circuitry. These in vitro results were validated in three in vivo models of oxidative stress, that is, human skin fibroblasts from old donors, femoral arteries from old mice, and a murine model of hindlimb ischemia. In all cases, miR-200c was higher versus control and its targets, that is, SIRT1, eNOS, and FOXO1, were downmodulated. In the mouse hindlimb ischemia model, anti-miR-200c treatment rescued these targets and improved limb perfusion. Innovation and Conclusion: miR-200c disrupts SIRT1/FOXO1/eNOS regulatory loop. This event promotes ROS production and decreases NO, contributing to endothelial dysfunction under conditions of increased oxidative stress such as aging and ischemia. Antioxid. Redox Signal. 27, 328-344.

Accumulation of senescent cells contributes to the reduced regenerative capacity in aged tissues. By evaluating the molecular pathways of senescence in relation to proliferative potential of primary keratinocyte cultures from young and old healthy donors, and from young patients with inherited defects leading to premature aging, we demonstrated that p16(INK4a) is a reliable marker of both physiological and premature epidermal aging. Analysis of the expression and activity of p16(INK4a) regulators showed that stem cell depletion, reduced proliferation, and p16(INK4a) upregulation in keratinocytes derived from the chronologically and prematurely aged epidermis strongly correlate with Bmi-1 downregulation. In highly proliferative tissues, replicative and premature senescence participate in determining senescent cell accumulation. Our findings demonstrated that Bmi-1 is downregulated in human keratinocytes during both in vitro processes, in parallel with p16(INK4a) upregulation and accomplishment of clonal conversion. When premature senescence was induced by specific exogenous stimuli, concomitant Ets-1 upregulation was also observed. Moreover, Bmi-1 inhibited Ets-1-mediated p16(INK4a) upregulation. Finally, Bmi-1 overexpression reduced p16(INK4a) promoter activity and decreased protein expression in aged and diseased keratinocytes, inducing a delay of clonal conversion and an increase of cell clonogenic ability. Altogether these findings underline a key role of Bmi-1 downregulation in enforcing aging in primary human keratinocytes.

NMR technology has dramatically contributed to the revolution of image diagnostic. NMR apparatuses use combinations of microwaves over a homogeneous strong (1 Tesla) static magnetic field. We had previously shown that low intensity (0.3-66 mT) static magnetic fields deeply affect apoptosis in a Ca2+ dependent fashion (Fanelli et al., 1999 FASEBJ., 13;95-102). The rationale of the present study is to examine whether exposure to the static magnetic fields of NMR can affect apoptosis induced on reporter tumor cells of haematopoietic origin. The impressive result was the strong increase (1.8-2.5 fold) of damage-induced apoptosis by NMR. This potentiation is due to cytosolic Ca2+ overload consequent to NMR-promoted Ca2+ influx, since it is prevented by intracellular (BAPTA-AM) and extracellular (EGTA) Ca2+ chelation or by inhibition of plasma membrane L-type Ca2+ channels. Three-days follow up of treated cultures shows that NMR decrease long term cell survival, thus increasing the efficiency of cytocidal treatments. Importantly, mononuclear white blood cells are not sensitised to apoptosis by NMR, showing that NMR may increase the differential cytotoxicity of antitumor drugs on tumor vs normal cells. This strong, differential potentiating effect of NMR on tumor cell apoptosis may have important implications, being in fact a possible adjuvant for antitumor therapies.

Hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders, characterized by a progressive sensory neuropathy often complicated by ulcers and amputations, with variable motor and autonomic involvement. Several pathways have been implicated in the pathogenesis of neuronal degeneration in HSAN, while recent observations point to an emerging role of cytoskeleton organization and function. Here, we report novel biallelic mutations in the DST gene encoding dystonin, a large cytolinker protein of the plakin family, in an adult form of HSAN type VI. Affected individuals harbored the premature termination codon variant p.(Lys4330*) in trans with the p.(Ala203Glu) change affecting a highly conserved residue in an isoform-specific N-terminal region of dystonin. Functional studies showed defects in actin cytoskeleton organization and consequent delayed cell adhesion, spreading and migration, while recombinant p.Ala203Glu dystonin loses the ability to bind actin. Our data aid in the clinical and molecular delineation of HSAN-VI and suggest a central role for cell-motility and cytoskeletal defects in its pathogenesis possibly interfering with the neuronal outgrowth and guidance processes. K E Y W O R D S BPAG1, bullous pemphigoid antigen 1, cell adhesion, cell migration, cytoskeleton, dermal fibroblasts, DST, dystonin, hereditary sensory and autonomic neuropathies, HSAN 106