Some mutations in FOXL2 are responsible for premature ovarian failure accompanied with blepharophimosis-ptosis-epicanthusinversus syndrome (BPES) typeI disease, and FOXL2-null mice exhibit developmental defects of granulosa cells. Recently, a new somatic mutation in FOXL2,c.402C>G, leading top. C134W change, has been identified in a vast majority of adult-type ovarian garnulosa cell tumors (GCTs). In the current study, we investigated possible mechanisms by which the C134W mutation could contribute to GCT development. The wild-type (WT) FOXL2 and its mutant form displayed differential apoptotic activities, in which WT induced a significant granulosa cell death while the mutant exhibited a minimal cell death effect. The FOXL2-induced apoptotic response was greatly dependent on caspase8, BID, or BAK since the depletion of either of them prevented FOXL2 to elicitits full apoptotic responses. Stimulated activation of caspase8, consequently resulting increased production of truncated BID (tBID), up-regulation and oligomerization of BAK, and release of cytochromec were all associated with the apoptosis followed by WT FOXL2 expression. In contrast, the mutant FOXL2 was deficient to elicit the full apoptotic signaling responses. In addition, we found the differential up-regulations of expression of death receptors including Fas and TNF-R1 between the WT and the mutant. Moreover, granulosa cells expressing either the WT FOXL2 or its variant form (C134W) exhibited distinct cell death sensitivities by the activation of death receptors. Thus, these differential activities of FOXL2 and it mutant may partly account for the pathophysiology of GCT development occurred by the somatic mutation (C134W) of FOXL2.

Although fenarimol is a widely used chlorinated fungicide applied to fruits and vegetables and considered as a suspected endocrine disrupter (ED), transgeneration studies of fenarimol at its low doses are not available. Objectives: The aims of this study are to address the effect of perinatal exposure to low doses of fenarimol on reproductive performance and to investigate molecular and cellular mechanisms which are associated with. Methods: The body and organ weights and anogenital distance (AGD) of mice offspring (F1) maternally exposed to fenarimol were determined, and their reproductive performances were assessed by mating and ovarian follicular and sperm analyses. In addition, differentially expressed genes (DEGs) in F1 ovaries were identified by DNA microarray. Up-regulated genes were confirmed by quantitative real-time PCR (qRT-PCR) and immunohistochemical analysis. Results: Fenarimol-exposed F1 mice showed the shortened AGD, increased body weight with altered organ weights, increased number of pub, abundant follicles, and enhanced sperm count and quality. Microarray data showed 82 up-regulated and 742 down-regulated genes on the ovaries of fenarimol-exposed mice, in which Cyp17a1 and Cyp19a1 were up-regulated. Conclusions: Low doses of perinatal fenarimol exposure caused reproductive dysfunction in mice and thus can possibly impose risks on reproductive activities of human and wild-life.

본 논문은 인공적인 단백질인 MCL-1ES BH3M에 관한 것으로 MCL-1ES BH3M를 과발현시 세포사멸을 유도한다. MCL-1L을 주형으로 재조합 PCR을 통해서 MCL-1ES BH3M를 클로닝하였다. 새롭게 클로닝한 단백질인 MCL-1ES BH3M 단백질은 안정성을 유지하기 위해서 PEST 도메인이 제거되어 있으며, 다른 BCL-2 패밀리 단백질과의 결합을 조절하기 위해서 BH3도메인의 Leu-Arg-Arg-Val-Gly-Asp-Gly 서열을 7개의 Ala 잔기로 인위적으로 돌연변이를 유도하였다. MCL-1ES BH3M를 293T 세포에서 과발현할 경우 세포사멸을 유도하였고, 항-세포사멸 단백질인 MCL-1L을 같이 과발현하더라도 세포사멸을 유도하였다. 또한, 과발현시 Caspase 9과 3를 활성화하였으며 면역염색법을 통해서 MCL-1ES BH3M 과발현시 미토콘드리아에 MCL-1ES BH3M 단백질이 부분적으로 위치하는 것을 확인하였다. 이상의 결과로 MCL- 1ES BH3M는 Caspase 9과 3의 활성을 통해서 세포사멸을 유도한다. 결론적으로 본 연구는 세포사멸을 유도하는 새로운 molecule을 클로닝하였고, 이 molecule에 의한 세포사멸 기능을 확인하였다.

BPES(Blepharophimosis/Ptosis/Epicanthus inversus Syndrome)는 FOXL2 유전자의 돌연변이에 의해 유발되는 상염색체 우성질환이다. 눈꺼풀이 갈라지거나 쳐지고 넓은 미간이 나타나는 특징이 있으며, 여성의 조기 난소 부전증(prema-ture ovarian failure, POF)을 일으켜 불임을 유발한다. FOXL2는 forkhead family에 속하는 전사인자로서 FOXL2가 결여된 난소에서는 granulosa cell의 분화가 진행되지 않아 난포 성숙과정의 멈춤과 난자의 폐쇄증을 유발한다. FOXL2를 bait로 하여 rat의 난소 cDNA 라이브러리의 yeast two-hybrid screening을 시행하여 FOXL2 단백질과 상호작용을 하는 small ubiquitin-related modifier(SUMO)-conjugating E2 효소인 UBE2I 단백질을 찾았다. UBC9이라고도 알려진 UBE2I 단백질은 SUMO 변형 과정을 위한 필수적인 단백질이다. Sumoylation은 수 많은 전사인자의 전사능력의 조절을 포함하여 다양한 신호전달체계에 관여하는 번역 후 변형과정이다. 본 연구에서 인간세포인 293T 내에서 면역침전반응 실험을 통해 FOXL2와 UBE2I의 단백질-단백질간의 상호작용을 확인하고, FOXL2의 돌연변이형을 제작하여 yeast two-hybrid system을 이용해 UBE2I와 결합에 필요한 FOXL2의 부분을 규명하였다. 따라서, FOX2에 상호작용하는 UBE2I의 규명은 sumoylation에 의한 FOXL2의 새로운 조절 메커니즘을 시사한다.