The Roles of T Cell Death-Associated Gene 51 in Stress Response

다국어 초록 (Multilingual Abstract)

T cell death-associated gene 51 (TDAG51), also known as pleckstrin homology-like domain, family A, member 1 (PHLDA1), was identified in T cell hybridoma mutant resistant to the anti-T cell receptor (TCR)-induced apoptosis. TDAG51 has been shown to mediate Fas expression by protein kinase C (PKC) and T cell apoptosis.
Although the role of TDAG51 has been reported mostly as a pro-apoptotic mediator since identification, some reports have demonstrated that TDAG51 has anti-apoptotic function (cell survival). Thus, it remains unclear whether TDAG51 promotes apoptosis or not.
In this study, to determine the role of TDAG51 in stress-induced apoptosis, we used TDAG51-knockout (TDAG51-/-) mouse embryonic fibroblasts (MEFs). Endoplasmic reticulum (ER) stress- and oxidative stress-induced apoptosis were elevated in TDAG51-/- MEFs than in wild-type (TDAG51+/+) MEFs. Cleavage of caspase-3 was more elevated in TDAG51-/- MEFs after ER stress, and level of intracellular reactive oxygen species (ROS) was more elevated in TDAG51-/- MEFs after oxidative stress. These results indicate that TDAG51 is an essential for protection in MEFs during various cellular stress-induced apoptosis, such as ER stress and oxidative stress.
The role of TDAG51 is not limited to apoptosis. TDAG51 is expected to play a regulatory role under diverse cellular stress. Some reports demonstrate relationship between TDAG51 and inflammatory response which is one of the cellular stresses. Microarray analysis of parasite-infected macrophage gene expression shows up-regulation of TDAG51 expression. And, lipopolysaccharide (LPS)-induced global gene expression profiling displays up-regulation of TDAG51 expression in immature rat brain. However, direct involvement of TDAG51 in inflammatory response has not been well studied. To analyze the direct role of TDAG51 in inflammatory response, we used TDAG51-/- bone marrow-derived macrophages (BMMs). LPS-induced pro-inflammatory cytokines expression level was decreased in TDAG51-/- BMMs than in TDAG51+/+ BMMs, and its reduced level was restored by TDAG51 expression. Also, TDAG51 interacted directly with Forkhead box protein O1 (FoxO1) transcription factor, and enhanced FoxO1 activity, which is necessary for inflammatory cytokines expression. These data suggest that TDAG51 mediates LPS/Toll-like receptor 4 (TLR4) inflammatory responses via enhancement of FoxO1 activity.
Meanwhile, role of TDAG51 is implicated in stress-related brain function. The epilepsy patients have elevated expression of TDAG51 in their anterior temporal neocortex. Gene expression profiling in chronic mild stress (CMS)-exposed mice brain shows elevated expression of TDAG51 in hippocampus and cerebral cortex. But, it is not clear whether TDAG51 is directly involved in stress-related brain functions. To investigate TDAG51 affects stress-related brain function, we carried out behavioral test and brain cDNA microarray analysis in TDAG51-/- mice. TDAG51-/- dams had severe maternal defects after parturition, like a postpartum psychiatric disorder (PPD). TDAG51-/- dams did not take care of their offspring and resulting in newborn offspring quickly died. Also, deficiency of TDAG51 caused dysregulation of diverse endocrine factors, including serotonin, dopamine, hypothalamic-pituitary-adrenal (HPA) axis and oxytocin during postpartum period. These results indicate that TDAG51 is an important for maintenance of maternal behavior via regulation of endocrine factors during stress conditions such as pregnancy and parturition.
Taken together, although TDAG51 is still important as mediator in apoptosis, TDAG51 also has crucial role as mediator in cell survival. In addition, TDAG51 shows possible regulatory roles in LPS-TLR4 inflammatory response and PPD pathology. These results suggest that TDAG51 may have various regulatory functions depend on conditions, such as type of cells, tissues and stressors.

목차 (Table of Contents)

• CHAPTER I. GENERAL INTRODUCTION 1
• 1. INTRODUCTION 3
• 2. REFERENCES 9
• CHAPTER II. THE ROLE OF TDAG51 IN ENDOPLASMIC RETICULUM STRESS AND OXIDATIVE STRESS-INDUCED APOPTOSIS 13
• 1. INTRODUCTION 15
• 2. MATERIALS AND METHODS 18
• 2-1. Materials 18
• 2-2. Mice 18
• 2-3. Immunoblotting assay 18
• 2-4. MEF isolation and culture 19
• 2-5. Cell viability assay and DAPI DNA staining 19
• 2-6. PI staining and flow cytometry analysis 19
• 2-7. Determination of generated ROS 20
• 2-8. Statistical analysis 20
• 3. RESULTS 21
• 3-1. Expression of TDAG51 is increased by ER stress and oxidative stress 21
• 3-2. ER stress-induced cell death is elevated in TDAG51-/ MEFs 23
• 3-3. Oxidative stress-induced cell death is elevated in TDAG51-/ MEFs 25
• 3-4. TDAG51 deficiency activates ER stress-induced caspase-3 cleavage, and enhances oxidative stress-induced ROS generation 27
• 4. DISCUSSION 29
• 5. REFERENCES 31
• CHAPTER III. TDAG51 PLAYS A ROLE AS AN ENHANCER IN LPS-TLR4 INFLAMMTORY RESPONSE 35
• 1. INTRODUCTION 37
• 2. MATERIALS AND METHODS 45
• 2-1. Plasmids 45
• 2-2. Reagents, chemicals and antibodies 45
• 2-3. Cell culture 46
• 2-4. Mice 47
• 2-5. RNA isolation, reverse transcription, semi-quantitative RT-PCR, and real-time PCR analysis 47
• 2-6. Transfection, GST pull down assay, and immunoprecipitation 48
• 2-7. Kinase inhibitors test 49
• 2-8. Luciferase reporter assay 49
• 2-9. Retroviral infection of BMMs 49
• 2-10. ELISA assay 50
• 2-11. Determination of intracellular ROS 50
• 2-12. LPS-induced lethality test and histological analysis 51
• 2-13. Cellular fractionation 51
• 2-14. Immunoblotting assay 52
• 2-15. Statistical analysis 52
• 3. RESULTS 54
• 3-1. LPS induces expression of TDAG51 in macrophages 54
• 3-2. TDAG51 deficiency decreases LPS-induced expression of pro-inflammatory cytokines 60
• 3-3. TDAG51 deficiency decreases LPS-induced iNOS and Cox-2 expression, and results in reduced intracellular ROS production 64
• 3-4. TDAG51 deficiency protects mice against LPS-induced lethal shock via the weakening of inflammatory response in vivo 66
• 3-5. Weakening of LPS-induced inflammatory response shown in the TDAG51-/ BMMs is not due to attenuation of signal pathways in initiation phase 68
• 3-6. TDAG51 directly interacts with FoxO1 transcription factor in the nucleus, and enhances FoxO1 activity 70
• 4. DISCUSSION 78
• 5. REFERENCES 82
• CHAPTER IV. DEFICIENCY OF TDAG51 CAUSES POSTPARTUM PSYCHIATRIC DISORDER BY DYSREGULATING DIVERSE ENDOCRINE FACTORS 89
• 1. INTRODUCTION 91
• 2. MATERIALS AND METHODS 99
• 2-1. Mice 99
• 2-2. Chemicals, antibodies and cell culture 99
• 2-3. Offspring survival test, dams pregnancy rate test and confirmation of the number of newborn offspring 99
• 2-4. Tissue histology 100
• 2-5. Behavior test 100
• 2-5-1. Nesting behavior test 100
• 2-5-2. Pup retrieval test 101
• 2-5-3. Tail suspension test 101
• 2-5-4. Forced swim test 101
• 2-5-5. Elevated plus-maze test 102
• 2-5-6. Sucrose water consumption test 102
• 2-5-7. Locomoter activity test 103
• 2-5-8. Chronic mild unpredictable stress (CMUS) procedure 103
• 2-6. Brain tissue lysate preparation, cell lysis, and immunoblotting assay 103
• 2-7. Generation of transgenic mice 104
• 2-8. Comparative gene expression profiling 104
• 2-9. RNA isolation, reverse transcription, semi-quantitative RT-PCR, and real-time PCR analysis 105
• 2-10. Statistical analysis 106
• 3. RESULTS 109
• 3-1. TDAG51-/ dams have severe maternal defects during postpartum period 109
• 3-2. Death of offspring born from TDAG51-/ dams is not due to problem of breast milk formation or breast-feeding 112
• 3-3. TDAG51-/ dams display maternal defects in nesting and nursing behavior 114
• 3-4. TDAG51 abundantly exists in diverse region of mouse brain, and is induced by several stressful conditions 117
• 3-5. TDAG51-/ female mice display depressive-like behavior and anxiety-like behavior because of the hypersensitivity to stress 119
• 3-6. Stress induces severe decrease of sucrose water consumption in TDAG51-/ female mice 122
• 3-7. Severe maternal defects shown in TDAG51-/ dams are restored by brain specific TDAG51 expression 125
• 3-8. TDAG51 deficiency in brain causes the dysregulation of neurotransmitters during postpartum period 127
• 3-9. TDAG51 positively regulates expression of oxytocin and oxytocin receptor during postpartum period 134
• 4. DISCUSSION 136
• 5. REFERENCES 142