Regulation of the memory T cell development and islet beta-cell survival by DAPK-related apoptosis-inducing protein kinase 2 (Drak2)

Drak2 is a member of the death-associated protein family, and is a serine threonine kinase. In Drak2 null mutant mice, T cells have no apparent defect in activation-induced apoptosis after stimulation with anti-CD3 and anti-CD28, but have a lowered threshold to stimulation, compared with wild type (WT) T cells. In our study, in situ hybridization analysis has revealed that Drak2 expression is ubiquitous at the mid-gestation stage in embryos, followed by more focal expression in various organs in the perinatal period and adulthood, notably in the thymus, spleen, lymph nodes, cerebellum, suprachiasmatic nuclei, pituitary, olfactory lobes, adrenal medulla, stomach, skin and testes. We generated Drak2 transgenic (Tg) mice using the human beta-actin promoter. These Tg mice showed normal T cell versus B cell and CD4 versus CD8 populations in the spleen, but their spleen weight cellularity was lower in comparison with wild type mice. After TCR activation, the proliferation response in Drak2 Tg T cells was normal, although their interleukin (IL)-2 and IL-4 but not interferon-r production was augmented. Activated Drak2 Tg T cells demonstrated significantly enhanced apoptosis in the presence of exogenous IL-2. At the molecular level, Drak2 Tg T cells manifested a lower increase of anti-apoptotic factors during activation; such a change probably rendered the cells vulnerable to subsequent IL-2 insults. The heightened apoptosis in Drak2 Tg T cells was associated with reduced numbers of T cells with the memory cell phenotype (CD62Llo) and repressed secondary T cell responses in delayed type hypersensitivity. These results demonstrate that Drak2expresses in the T cell compartment but is not T cell-specific; and it plays critical roles in T cell apoptosis and memory T cell development. Further, we investigated the role of Drak2 in beta-cell survival and diabetes. Drak2 mRNA and protein were rapidly induced in islet beta-cells after exogenous inflammatory cytokine or free fatty acid stimulation, which is present endogenously in either type 1 or type 2 diabetes. Drak2 upregulation was accompanied by increased beta-cell apoptosis. The beta-cells apoptosis caused by the said stimuli was inhibited by Drak2 knockdown using siRNA. Conversely, transgenic (Tg) Drak2 overexpression led to aggravated beta-cell apoptosis triggered by the stimuli. Drak2 overexpression in islets compromised the increase of anti-apoptotic factors, such as Bcl-2, Bcl-xL and Flip, upon the cytokine and free fatty acid stimulation. Further in vivo experiments demonstrated that Drak2 Tg mice were prone to type 1 diabetes in a multiple-low-dose streptozotocin-induced diabetes model, and they were also prone to type 2 diabetes in a diet-induced obesity model. Our data show that Drak2 is detrimental to beta-cell survival. We also investigated the signalling pathway of Drak2. We found that purified Drak2 could phosphorylate p70S6 kinase in an in vitro kinase assay. Drak2 overexpression in NIT-1 cells led to enhanced p70S6 kinase phosphorylation, while Drak2 knockdown in these cells reduced the phosphorylation. These mechanistic studies proved that p70S6 kinase was a bona fide Drak2 substrate in vitro and in vivo.This study has discovered the important functions of Drak2 in T cell homeostasis and diabetes. We proved that p70S6 kinase was a substrate of Drak2. Our findings have broadened our knowledge of Drak2 in the immune and endocrine system. Some of our findings, such as the roles of Drak2 in memory T cell development and beta cell survival could be explored for clinical application in the areas of transplantation and diabetes.

Keyphrases: Drak2, transgenic, memory T cell, apoptosis, islet, diabetes, cellule mémoire T