(The) role of Hippo signaling core component Mob1a/b in maintenance and differentiation of mouse stem cells
저자
발행사항
서울 : 서울대학교 대학원, 2018
학위논문사항
학위논문(박사)-- 서울대학교 대학원 : 생명과학부 2018. 2
발행연도
2018
작성언어
영어
주제어
DDC
570 판사항(22)
발행국(도시)
서울
기타서명
Hippo 신호전달계의 Mob1a/b에 의한 생쥐 줄기세포 항상성 유지 및 분화에 관한 연구
형태사항
108장 : 삽화, 도표 ; 26 cm
일반주기명
참고문헌 수록
DOI식별코드
소장기관
The Hippo signaling pathway is highly conserved in mammals and exerts a significant impact on cell proliferation, apoptosis, differentiation and cancer development. The Hippo pathway is involved in intestinal epithelial homeostasis with Wnt, BMP, Notch and EGF signaling. I investigated the relationship between Hippo and other signaling pathways and the role of Mob1a/b in intestinal homeostasis and maintenance of intestinal stem cells (ISCs). Mice with intestinal epithelial cell (IEC)-specific depletion of Mob1a/b showed hyperproliferation in IECs, defects in secretory lineage differentiation and loss of ISC markers and eventually died with severe loss of body weight and weakness at 10 to 12 days after tamoxifen treatment. In Mob1a/b-depleted IECs, expression of ISC niche factors and Wnt target genes were down-regulated and Bmp2, TgfβR2, Areg, Ereg and Jag2 were transcriptionally activated with enhanced YAP activity. In in vivo and in vitro experiments with several signaling inhibitors, it has been shown that the BMP inhibitor LDN193189 or Tgfβ inhibitor SB431542 had effects on partial restoration of the intestinal degenerative phenotype. Treatment with these inhibitors restored differentiation of secretory lineage cells in Mob1a/b-deficient mice, but not ISC pools in the crypt region. These studies reveal that IEC-specific depletion of Mob1a/b induced overexpression of Bmp2 and TgfβR2 and inhibited Wnt activity, finally leading to loss of ISCs and functional epithelia in the mouse intestine. These results suggest that Mob1a/b has an essential function for intestinal epithelial homeostasis by regulating YAP, Wnt activity and BMP/Tgfβ signaling.
더보기The Hippo pathway plays a role in cell proliferation, survival, differentiation, and development. Large tumor suppressor 1 and 2 (Lats1/2) kinase is activated by binding MOB kinase activator 1A and 1B (Mob1a/b), and then, it subsequently phosphorylates Yes-associated protein 1 (YAP). Major effectors of the Hippo pathway include two transcriptional co-activators: Yes-associated protein 1 (YAP) and WW domain-containing transcription regulator 1 (TAZ). Hyperactivation of YAP and TAZ or dysregulation of Hippo pathway cause uncontrolled cell proliferation leading to tumor in drosophila, mice, and humans. Hyperactivity of YAP and TAZ has been observed in many cancer types of humans. Moreover, research over the past decade supports that tissue homeostasis, stem cell properties, and embryonic development also can be regulated by the Hippo pathway. Through this study, I demonstrated that Mob1a/b has an essential function in embryonic stem cell differentiation and intestinal epithelial homeostasis beyond as a tumor suppressor.
Mob1 was originally known to regulate mitotic process and modulate cytokinesis in yeast. In vitro, Mob1a and Mob1b are involved in cell proliferation and mitotic exit. In vivo, the human MOB1A gene is infrequently mutated, and MOB1A/B expression is down-regulated in human cancer cell lines. Moreover, it has been reported that Mob1a and Mob1b have a role in skin homeostasis and function as tumor suppressors by regulating Lats1/2 activity. However, in vivo functional studies for Mob1a/b in embryonic stem cells (ESCs) and intestinal epithelial cells (IECs) has not been reported. To verify how Mob1a/b functions in mouse embryonic stem cells and intestinal epithelial cells, I have generated Mob1a/b conditional knockout mice and ES cells (mESCs).
Deletion of both Mob1a and Mob1b genes causes an early embryo lethality in mice, implying that Mob1a/1b is essential for early embryogenesis. Further functional analysis of Mob1a/b in embryonic stem cells, I have generated Mob1af/f, 1bf/f; CreERT2 mESCs in which Mob1a/b could be conditionally deleted by 4-hydroxytamoxifen. In experiments with this cell linelinelineline, I found that depletion of Mob1a/b has little effects on maintenance of stemness/pluripotency and proliferation in mESCs, but is essential for differentiation into three germ layers from mESCs. Mob1a/b depletion caused failure of determining cell fate specification with enhanced YAP and TAZ activity in mESCs. YAP knockdown in Mob1a/b-depleted mESCs resulted in recovery of differentiation defects in vitro and in vivo. These results suggest that function of Mob1a/b in differentiation of mESCs is dependent on YAP activity.
To investigate the roles of Mob1a/b in IECs, a Mob1a/1b conditional knockout mouse was generated and bred with a Villin-creERT2 transgenic mouse. Mice with IEC-specific depletion of Mob1a/b showed hyperproliferation in IECs, defects in secretory lineage differentiation and loss of intestinal stem cell (ISC) markers and eventually died with severe loss of body weight and weakness. In Mob1a/b-depleted IECs, expression of Wnt target genes were significantly down-regulated, but Notch ligand Jag2, BMP ligand Bmp2, and TGF receptor TGFβR2 were transcriptionally increased. In in vivo and in vitro experiments with several signaling inhibitors, the BMP inhibitor LDN193189 or Tgfβ inhibitor SB431542 restored differentiation of secretory lineage cells and Wnt activity in Mob1a/b-depleted IECs. These results indicated that Mob1a/b has an essential function for intestinal epithelial homeostasis by regulating YAP, Wnt activity and BMP/Tgfβ signaling.
Currently, the regulation mechanism of YAP and TAZ is believed to be more complex because of new upstream and downstream regulatory components being identified and novel mechanisms revealed. It is more difficult to understand the Hippo pathway because the regulation of the Hippo pathway differs in each cell or tissue type. Therefore, for therapy of cancer and other human diseases related to the Hippo pathway, further studies are required to understand these complex regulatory mechanisms of the Hippo pathway.
Taken together, I suggest that Mob1a and Mob1b have functional redundancy, and exert their roles as the Hippo core component by inhibiting downstream YAP / TAZ activity. My results clearly demonstrate that the Hippo signaling pathway has an essential function in embryonic stem cell differentiation and intestinal epithelial homeostasis.
The Hippo pathway has a crucial role in cell proliferation and apoptosis. Several studies conducted over the past decade have identified that stem cell maintenance and embryonic development can be regulated by the Hippo pathway.
MOB kinase activator 1A and 1B (Mob1a/b) are the key components of the Hippo pathway, and mice with double homozygous deletion of Mob1a/b show early embryonic lethality at the pre-implantation stage. I observed that Mob1a/b double homozygous knockout mice generated by my gene targeting strategy shows embryonic lethality before embryonic day 8.5. The failure of early embryogenesis implies that Mob1a/b-depleted embryonic stem cells lose their stem cell properties or proper regulation of cell cycle progression. However, Mob1a/b depletion did not affect the stemness and proliferation of mouse embryonic stem cells. To investigate how Mob1a/b causes early embryonic lethality, I performed the embryoid body formation assay for the differentiation of embryonic stem cells. Based on morphological and quantitative assays, I observed that Mob1a/b depletion in embryonic stem cells causes a defect with differentiation into all three germ layers. These defects are dependent on YAP activity. Under differentiation conditions, the combination of Mob1a/b depletion and YAP down-regulation resulted in recovery of differentiation defects in vitro. However, YAP knockdown leads to only partial recovery of the differentiation of Mob1a/b-depleted embryonic stem cells in vivo, because TAZ is up-regulated and have functional redundancy with YAP activity. Taken together, my observations suggest that the Hippo pathway has a critical role in early embryogenesis and embryonic stem cell differentiation.
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