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Study on the role of RIPK3 in lymphomagenesis and hepatic steatosis

  • 하유진 (아주대학교 의생명과학과)

초록/요약

Necroptosis is a programmed form of lytic cell death that culminates in the membrane rupture and release of pro-inflammatory cytokines and damage-associated molecular patterns (DAMPs), triggering an inflammatory immune response. Receptor-interacting protein kinase 3 (RIPK3) is a serine/threonine-protein kinase, which plays an essential role in necroptosis pathway. RIPK3 functions as an important constituent factor in forming the necrosome complex with RIPK1 and activates downstream mixed lineage kinase domain-like pseudokinase (MLKL). RIPK3 is involved in multiple human pathogenesis such as cancers, virus infection and metabolic disorders in a necroptosis-dependent or independent manner, suggesting that there might be the context-dependent roles of RIPK3. Therefore, understanding the mechanisms by which RIPK3 contributes to disease progression could be a promising target for therapy in various pathologies. RIPK3 expression is often silenced in many types of cancer cells, which suggests that it may have tumor suppressor properties. However, the exact mechanism by which RIPK3 negatively regulates cancer development and progression remains unclear. Thus, in the first study, I investigated role of RIPK3 in tumorigenesis. I found that Ripk3 deficiency led to a higher rate of spontaneous tumor formation, mainly in the thymus, in an age-dependent manner. I demonstrated that RIPK3-deficient thymocytes lead to an expanded population of CD4+CD8+ double-positive (DP) T cells in young mice. In addition, mice with RIPK3 deletion led to rapid progression of thymic tumorigenesis in N-ethyl-N-nitrosourea (ENU)-induced and p53-deficient mouse models. Mechanistically, RIPK3 is activated by lymphocyte cell-specific protein-tyrosine kinase (LCK), which in turn to increase phosphatase activity of protein phosphatase 2 (PP2A), suppressing hyper-activation of ERK in DP thymocytes. These findings suggest that a RIPK3-PP2A-ERK signaling axis regulates DP thymocyte homeostasis and may provide a potential therapeutic target to improve clinically relevance for thymic lymphoma therapies. While conducting the first study, I observed an abnormality in the liver of aged RIPK3-deficient mice and found that Ripk3 deficiency exhibited a higher rate of fatty liver incidence than littermate controls in seven-month-old mice. RIPK3-dependent necroptosis in hepatocyte is activated in patients with non-alcoholic fatty liver disease (NAFLD), but specific contribution of RIPK3-dependent signaling in NAFLD pathogenesis remains controversial. In the second study, I demonstrated that liver-specific overexpression of RIPK3 attenuates hepatic steatosis in high fat diet (HFD) model. This observation raised the possibility that RIPK3 was not the causative factor for NAFLD progression. In addition, various lipid metabolism changed in Ripk3-deficient liver, suggesting that RIPK3 acts as a regulator of hepatic lipid metabolism. Collectively, the findings of this study suggest that RIPK3 provides a protective effect during thymic lymphoma and hepatic steatosis development, therefore, detection of RIPK3 may provide prognostic information to physicians. Development of strategies for modulating RIPK3 activity and/or expression may be clinically relevant for disease prognosis and therapy.

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목차

I. INTRODUCTION 1
A. Necroptosis 1
B. Roles of RIPK3 2
C. Necroptosis in human pathogenesis 3
D. T cell homeostasis in thymus 4
E. Non-alcoholic fatty liver disease (NAFLD) 5
F. The aim of this study 6
II. MATERIALS AND METHODS 7
A. Mice 7
B. Adeno associated virus (AAV)-mediated RIPK3 overexpression 8
C. Cell culture 9
D. Flow cytometric analysis 9
E. Serum biochemistry 10
F. Liver triglyceride measurement 11
G. Histological analysis 11
H. Proximal ligation assay (PLA) 13
I. Western Blot 13
J. Immunoprecipitation 14
K. Tandem Affinity Purification and Mass Spectrometry (TAP-MS) analysis 15
L. In vitro kinase assay 15
M. RNA-seq analysis 16
N. RNA extraction, cDNA synthesis and quantitative RT-PCR 16
O. Statistical analysis 17
III. RESULTS 19
Part I . RIPK3 maintains the homeostatic proliferation of CD4+CD8+ double-positive (DP) T cells to restrain early onset of thymic lymphoma 19
A. RIPK3 deficiency leads to hyperproliferation of DP T cells in the thymus. 19
B. Increased DP T cells are independent of RIPK3-MLKL necroptosis pathway. 26
C. RIPK3 deficiency accelerates tumor progression in a carcinogen-induced mouse model. 29
D. RIPK3 deficiency potentiates thymic lymphoma progression in p53-/- mice. 33
E. RIPK3 deficiency enhances thymic lymphoma via TCR-dependent ERK hyperactivation. 38
F. LCK interacts with RIPK3 and phosphorylates its tyrosine residue. 40
G. RIPK3 negatively regulates ERK phosphorylation through potentiation of PP2A activity. 45
H. Pharmacological modulation of PP2A activity regulates DP thymocyte proliferation in a carcinogen-induced mouse model. 48
Part II . RIPK3 protects aberrant hepatic fat accumulation via regulation of hepatic lipid metabolism. 54
A. RIPK3 deficiency leads to age-dependent aberrant hepatic fat accumulation. 54
B. RIPK3 was not the causative factor for hepatic steatosis 61
C. Liver-specific overexpression of RIPK3 attenuates HFD-induced hepatic steatosis. 68
D. RIPK3 gene signatures were correlated with lipid metabolism. 77
IV. DISCUSSION 80
Part I . RIPK3 maintains the homeostatic proliferation of CD4+CD8+ double-positive (DP) T cells to restrain early onset of thymic lymphoma 80
Part II . RIPK3 protects aberrant hepatic fat accumulation via regulation of hepatic lipid metabolism. 83
V. REFERENCES 85

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