Investigation on potential roles of necroptosis effectors and Serum amyloid A3 in the mouse uterine epithelial cells = 생쥐 자궁 상피세포에서 necroptosis의 인자와 Serum amyloid A3의 역할 탐색

다국어 초록 (Multilingual Abstract)

Investigation on potential roles of necroptosis effectors and Serum amyloid A3 in the mouse uterine epithelial cells Son, Sujin Department of Veterinary Medicine Graduate School of Konkuk University Necroptosis is a form of programmed cell death that is characterized as a regulate d cellular self-destruction mechanism. Once activated by certain external stimuli such as Tumor necrosis factor alpha (TNFα), the process is mediated by sequential actions of Receptor- interacting protein 1 (RIPK1), Receptor-interacting protein 3 (RIPK3), and Mixed lineage kinase domain-like protein (MLKL). Our laboratory previously reported that the cellular integrity of the uterine epithelial cells (UECs) depends on the presence of Tumor susceptibility gene 101 (Tsg101) in mice. One of the roles of TSG101 is known as a counteraction against necroptos is. Thus, I examined if UECs express necroptos is effectors and respond to certain stimuli in the mouse uterus. First, I examined the expression of Ripk1, Ripk3, and Mlkl in UECs and stromal cells (USCs) in mice by RT-PCR and Western blotting. UECs were stimulated with a mixture of TNFα (T), Smac mimetic LCL-161 (S), and zVAD-FMK (Z), a widely used combination to induce necroptosis. Cells were pre-stained with SYTOX Green® and observed under a live imaging apparatus to monitor changes in cell permeability. Peri- implantation uteri from pregnant mice on days 1, 4, and 8 were collected to examine physiological changes related to necroptosis. To examine steroid hormonal regulation of necroptosis-associated factors, uteri were collected from ovariectomized (OVX) mice injected with steroid hormones. I first confirmed that Ripk1, Ripk3, and Mlkl are expressed in UECs. The expression of Ripk1 and Ripk3 were upregulated in uteri from estrogen-injected OVX mice. TSZ- treated UECs showed increases in SYTOX Green staining by 24 h after TSZ treatment than DMSO-treated UECs. Immunofluorescence staining of pMLKL localized in TSZ- treated UECs at 1 h, suggesting activation of necroptotic machinery. UECs with or without TSZ treatment at 1 h were subjected to RNA sequencing analysis. In TSZ- treated UECs, several inflammation-associated genes were identified as differentially regulated genes (DEGs), Serum amyloid A3 (SAA3) is an acute-phase inflammatory protein that increases during inflammatory response in several tissues. I found that Saa3 is upregulated in necroptosis-induced UECs. TSZ-treated UECs have higher Saa3 mRNA levels than T, S, or Z-treated UECs. TSZ-treated L929 cells, the mouse fibroblast cell line which is used as a necroptosis-positive cell line, exhibited an increase in Saa3 expression. Additionally, inducing necroptotic cell death in Ripk3 knockout UECs resulted in fewer cell deaths compared to Ripk3+/- UECs. Notably, TSZ-treated Ripk3-/- UECs showed lower Saa3 expression levels compared to the control group. In conclusion, this study shows that the necroptosis machinery is present in the mouse uterus and UECs and the increased Saa3 expression by TSZ treatment implies its potential role as a necroptosis-target gene. Keywords: Mouse, Uterus, Uterine epithelial cells (UECs), Necroptosis, Serum amyloid A3 (SAA3)

국문 초록 (Abstract)

세포 괴사(Necroptosis)는 조절된 세포 자체 파괴 메커니즘을 특징으로 한다. 특정 외부 자극, 예를 들어 종양 괴사 인자 알파 (TNFα)에 의해 활성화되면 이 과정은 수용체 상호작용 단백질 카이네이즈 1 (RIPK1), 수용체 상호작용 단백질 카이네이즈 3 (RIPK3), 그리고 혼합 계통 카이네이즈 도메인과 유사한 단백질 (MLKL)의 연속적인 작용에 의해 매개된다. 우리 연구실은 이전에 마우스의 자궁 상피 세포 (UECs)의 세포 무결성이 Tumor susceptibility gene 101 (Tsg101)의 존재에 의존한다고 보고한 논문이 있다. TSG101 의 역할 중 하나는 세포 괴사에 대항하는 것으로
알려져 있다. 따라서 나는 마우스 자궁에서 자궁 상피세포가 세포 괴사인자들을 발현하고 특정 자극에 어떻게 반응하는지 조사했다. 먼저 RIPK1, RIPK3 및 MLKL 이 자궁 상피세포에서 발현되는 것을 확인했다. 난소절제술 (OVX) 된 estrogen 이 주입된 자궁에서는 RIPK1 과 RIPK3의 발현이 증가하였다. 세포 괴사를 유발하는 것으로 잘 알려진 TSZ 결합체 (TNFα (T), Smac mimetic LCL-161 (S), and zVAD-FMK (Z))로 처리된 자궁 상피세포는 TSZ 처리 후 24 시간 동안 DMSO 처리된 자궁 상피세포보다 SYTOX Green 염색이 증가되었다. pMLKL 의 면역 형광
염색은 TSZ 처리 1 시간 후에 UECs 에 반응되어 나타났으며, 이는 세포 괴사 기작의 활성화를 시사했다. 1 시간 후의 TSZ 를 처리한 자궁 상피세포는 RNA seq 분석을 진행하였으며 TSZ 처리된 자궁 상피세포에서 여러 염증 관련 유전자가 차등 조절된 유전자로 식별되었다. 그 중, Serum amyloid A3 (SAA3)는 여러 조직에서 염증 반응 중에 증가하는 급성 상피 염증 단백질이며, Saa3 는 세포 괴사가 유도된 자궁 상피세포에서 발현량이 증가되는 것을 확인하였다. TSZ 처리된 자궁 상피세포는 각각 T, S, 또는 Z 처리된 자궁 상피세포보다 더 높은 Saa3 mRNA level 을 보였다. 세포 괴사 positive cell line 으로 사용된 마우스 섬유아 세포주인 L929 cells 에서도 TSZ 를 처리한 그룹에서 Saa3 발현이 증가되었다. 또한 Ripk3-/- 자궁 상피세포에서 세포 괴사를 유도하였더니 Ripk3+/- 자궁 상피세포에 비해 더 적은 세포 사멸이 나타났으며 이중, TSZ 처리된 Ripk3- /- 자궁 상피세포는 대조군에 비해 낮은 Saa3 발현 수준을 보였다. 요약하자면, 마우스 자궁 및 자궁 상피세포에 세포 괴사 기작이 존재하며, TSZ 처리에 의한 Saa3 발현 증가는 세포 괴사 타겟 유전자 로서의 잠재적인 역할을 시사한다는 것을 보여준다

목차 (Table of Contents)

• I. Introduction . 1
• II. Materials and Methods . 4
• 2.1. Animals 4
• 2.2. Preparation of uterine s amples on days 1, 4, and 8 of pregnancy 6
• 2.3. Ovariectomy and injection of s teroid hormones 6
• 2.4. Isolation of mouse uterine epithelial cells 7
• 2.5. Cell culture and induction of necroptos is 8
• 2.6. RNA isolation and RT-PCR. 9
• 2.7. Western blotting. 11
• 2.8. Immunofluorescence s taining and confocal microscopy. . 13
• 2.9. JuLIF MFL live imaging 15
• 2.10. RNA seq 15
• 2.11. Statistics analys is. 17
• III. Results 18
• 3.1. Expression of necroptosis effectors in mouse uterus during the peri-implantat i on
• period 18
• 3.2. Steroid hormonal regulation of necroptosis effectors in the OV X mouse uterus . 21
• 3.3. The presence of necroptosis effectors in uterine epithelial cells (UECs) 25
• 3.4. Induction of necroptosis by TSZ increases cell death i n UECs . 27
• 3.5. RNA seq analysis of UECs treated with TSZ 33
• 3.6. Express ion of Saa3 is highly induced in TSZ-treated UECs 42
• 3.7. Saa3 as a novel necroptos is target gene in L929 cells 49
• 3.8. Cell death in UECs from Ripk3 +/ - and Ripk3 -/ - mice . 51
• 3.9. The role of SAA3 in the RIPK3-dependent necroptosis pathway. 55
• IV. Discussion . 57
• Reference . 60
• Abstract (in Korean) . 67