Genotoxic stresses activate intracellular signaling molecules, which lead to growth arrest, DNA repair, and͞or apoptosis. Among these molecules are the growth arrest and DNA damage protein 34 (GADD34) and the Src-related protein tyrosine kinase Lyn. Here, we report that these two proteins physically and functionally interact to regulate DNA damage-induced apoptosis. Multiple isolates of GADD34 and the related murine protein MyD116 were identified as binding partners of Lyn in a yeast two-hybrid screen. The specific interaction was confirmed by in vitro association of GADD34 with glutathione S-transferase fusion proteins containing the Src Homology 3 (SH3) domain of Lyn, as well as coimmunoprecipitation of GADD34 and Lyn from mammalian cells. GADD34 was tyrosinephosphorylated in vivo in a Lyn-dependent manner. Lyn efficiently phosphorylated affinity-purified GADD34 in vitro. Lyn negatively regulated the proapoptotic function of GADD34 in a kinasedependent manner. Expression of wild-type, but not kinase-inactive, Lyn weakened promotion of apoptosis by GADD34 following treatment with methyl-methanesulfonate or ionizing radiation in HEK293 and HeLa cells. In contrast, pretreatment of cells with the Src-specific tyrosine kinase inhibitor PP1 strengthened promotion of apoptosis by GADD34. We propose that Lyn regulates the proapoptotic function of GADD34 by binding and phosphorylating it.

Chronic allograft nephropathy (CAN) is the primary cause for late kidney allograft loss. Carbon monoxide (CO), a product of heme metabolism by heme oxygenases, is known to impart protection against various stresses. We hypothesized that CO could minimize the chronic fibroinflammatory process and protect kidney allografts from CAN. Lewis kidney grafts were orthotopically transplanted into binephrectomized Brown-Norway rats under short-course tacrolimus. Recipients were maintained in room air or exposed to CO at 20 parts/million for 30 days after transplant. Efficacy of inhaled CO was studied at day 30 and day 80. Isografts maintained normal kidney function throughout the experiment with creatinine clearance of approximately 1.5 ml/min. Renal allograft function in air controls progressively deteriorated, and creatinine clearance declined to 0.2 +/- 0.1 ml/min by day 80 with substantial proteinuria. CO-treated animals had significantly better creatinine clearance (1.3 +/- 0.2 ml/min) with minimal proteinuria. Histological examination revealed the development of progressive CAN in air-exposed grafts, whereas CO-treated grafts had minimal tubular atrophy and interstitial fibrosis, with negligible collagen IV deposition. In vitro analyses revealed that CO-treated recipients had significantly less T cell proliferation against donor peptides via the indirect allorecognition pathway and less anti-donor IgG antibodies compared with air controls. Intragraft mRNA levels for chemokines (regulated on activation normal T cell expressed and secreted, macrophage inflammatory protein-1alpha, chemokine receptors (CCR1, CXCR3, CXCR5), IL-2, and intercellular adhesion molecule-1 were significantly decreased in CO-treated than in air-treated allografts. Furthermore, reduction of blood flow in air-treated allografts was prevented with CO. In conclusion, inhaled CO at a low concentration efficiently abrogates chronic fibroinflammatory changes associated with CAN and improves long-term renal allograft function.

Incubation of mouse T cells expressing the cell surface enzyme ADP ribosyltransferase with nicotinamide adenine dinucleotide (NAD) had been reported to cause ADP ribosylation of cell surface molecules, inhibition of transmembrane signaling, and suppression of immune responses. In this study, we analyze the reasons for these effects and report that contact of T cells with NAD causes cell death. Naive T cells when incubated with NAD and adoptively transferred into semiallogeneic mice fail to cause graft-vs-host disease, and when injected into syngeneic, T cell-deficient recipients do not reconstitute these mice. Rather, they accumulate in the liver, leading to an increase of apoptotic lymphocytes in this organ. Similar effects are induced by injection of NAD, shown to cause a dramatic increase of apoptotic CD3+, CD4+, and CD8+ cells in the liver. Consistent with this, in vitro incubation of naive T cells with NAD is shown to induce apoptosis. In contrast, no cell death is demonstrable when T cells are activated before incubation with NAD. It is concluded that ecto-NAD, as substrate of ADP ribosyltransferase, acts on naive, but not on activated CD69+ T cells.

No abstract