Fang Zhang

Glycogen synthase kinase-3 (GSK-3) is a critical, negative regulator of diverse signaling pathways. Lithium is a direct inhibitor of GSK-3 and has been widely used to test the putative role of GSK-3 in multiple settings. However, lithium also inhibits other targets, including inositol monophosphatase and structurally related phosphomonoesterases, and thus additional approaches are needed to attribute a given biological effect of lithium to a specific target. For example, lithium is known to increase the inhibitory N-terminal phosphorylation of GSK-3, but the target of lithium responsible for this indirect regulation has not been identified. We have characterized a short peptide derived from the GSK-3 interaction domain of Axin that potently inhibits GSK-3 activity in vitro and in mammalian cells and robustly activates Wnt-dependent transcription, mimicking lithium action. We show here, using the GSK-3 interaction domain peptide, as well as small molecule inhibitors of GSK-3, that lithium induces GSK-3 N-terminal phosphorylation through direct inhibition of GSK-3 itself. Reduction of GSK-3 protein levels, either by RNA interference or by disruption of the mouse GSK-3beta gene, causes increased N-terminal phosphorylation of GSK-3, confirming that GSK-3 regulates its own phosphorylation status. Finally, evidence is presented that N-terminal phosphorylation of GSK-3 can be regulated by the GSK-3-dependent protein phosphatase-1.inhibitor-2 complex.

Genetic defects in the purine salvage enzyme adenosine deaminase (ADA) lead to severe combined immunodeficiency (SCID) with profound depletion of T, B, and natural killer cell lineages. Human leukocyte antigen-matched allogeneic hematopoietic stem cell transplantation (HSCT) offers a successful treatment option. However, individuals who lack a matched donor must receive mismatched transplants, which are associated with considerable morbidity and mortality. Enzyme replacement therapy (ERT) for ADA-SCID is available, but the associated suboptimal correction of immunological defects leaves patients susceptible to infection. Here, six children were treated with autologous CD34-positive hematopoietic bone marrow stem and progenitor cells transduced with a conventional gammaretroviral vector encoding the human ADA gene. All patients stopped ERT and received mild chemotherapy before infusion of gene-modified cells. All patients survived, with a median follow-up of 43 months (range, 24 to 84 months). Four of the six patients recovered immune function as a result of engraftment of gene-corrected cells. In two patients, treatment failed because of disease-specific and technical reasons: Both restarted ERT and remain well. Of the four reconstituted patients, three remained off enzyme replacement. Moreover, three of these four patients discontinued immunoglobulin replacement, and all showed effective metabolic detoxification. All patients remained free of infection, and two cleared problematic persistent cytomegalovirus infection. There were no adverse leukemic side effects. Thus, gene therapy for ADA-SCID is safe, with effective immunological and metabolic correction, and may offer a viable alternative to conventional unrelated donor HSCT.

The aim of this study was to explore the intracellular mechanisms underlying the cardiovascular toxicity of air particulate matter (PM) with an aerodynamic diameter of less than 2.5 µm (PM2.5) in a human umbilical vein cell line, EA.hy926. We found that PM2.5 exposure triggered reactive oxygen species (ROS) generation, resulting in a significant decrease in cell viability. Data from Western blots showed that PM2.5 induced phosphorylation of Jun N-terminal kinase (JNK), extracellular signal regulatory kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and protein kinase B (AKT), and activation of nuclear factor kappa B (NF-κB). We further observed a significant increase in expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) in a time- and dose-dependent manner. Moreover, the adhesion of monocytic THP-1 cells to EA.hy926 cells was greatly enhanced in the presence of PM2.5 . However, N-acetylcysteine (NAC), a scavenger of ROS, prevented the increase of ROS generation, attenuated the phosphorylation of the above kinases, and decreased the NF-κB activation as well as the expression of ICAM-1 and VCAM-1. Furthermore, ERK inhibitor (U0126), AKT inhibitor (LY294002) and NF-κB inhibitor (BAY11-7082) significantly down-regulated PM2.5 -induced ICAM-1 and VCAM-1 expression as well as adhesion of THP-1 cells, but not JNK inhibitor (SP600125) and p38 MAPK inhibitor (SB203580), indicating that ERK/AKT/NF-κB is involved in the signaling pathway that leads to PM2.5 -induced ICAM-1 and VCAM-1 expression. These findings suggest PM2.5 -induced ROS may function as signaling molecules triggering ICAM-1 and VCAM-1 expressions through activating the ERK/AKT/NF-κB-dependent pathway, and further promoting monocyte adhesion to endothelial cells.