Bài 2: Cơ sở phân tử tính đa tiềm năng

CƠ SỞ PHÂN TỬ TÍNH ĐA TIỀM NĂNG TS. Trần Hồng Diễm PTN Nghiên cứu và Ứng dụng Tế bào gốc Trường Đại học KHTN - Đại học Quốc Gia Tp. HCM 03/10/2015 •  Tế bào gốc (stem cell) là tế bào có khả năng tự làm mới (self-renewal) và biệt hoá (differentiate) thành những tế bào chức năng khác TẾ BÀO GỐC (STEM CELLS) •  Tiềm năng biệt hoá (differentated potential) của tế bào gốc: -Toàn năng (totipotent) -Vạn năng (pluripotent) - Đa năng (multipotent) -  - Đơn năng (unipotent) TẾ BÀO GỐC VẠN NĂNG (PLURIPOTENT STEM CELLS) •  Pluri bắt nguồn từ tiếng la tinh “plures” nghĩa là một vài (several) hoặc nhiều (many). •  Thuật ngữ vạn năng (Pluripotent) được dùng để mô tả tế bào gốc có khả năng biệt hoá thành các tế bào xuất sứ từ ba lớp phôi: trung bì, nội bì, ngoại bì. •  Tế bào vạn năng (pluripotent cell) có khả năng tạo nên bất cứ loại tế bào nào, một đặc tính được thấy trong sự phát triển tự nhiên của phôi và trong một số điều kiện xác định của PTN Totipotent Pluripotent Multipotent Progenitor Organs Physiol Rev 85: 635–678, 2005; •  Tế bào gốc phôi (Embryonic stem cells) •  Tế bào ung thư biểu mô phôi (Embryonic carcinoma cells) •  Tế bào gốc sinh dục- Tế bào mầm (Embryonic germ cellS) •  Tế bào đa tiềm năng cảm ứng (Induced pluripotent stem cells) CÁC LOẠI TẾ BÀO GỐC VẠN NĂNG MOUSE Physiol Rev 85: 635–678, 2005; HUMAN Physiol Rev 85: 635–678, 2005; •  Unlimited self renewal •  Stable karyotype •  Immortal •  Highly efficient and reproducible differentiation potential •  Germ line colonization •  Clonogenicity •  High versatility to genetic manipulation without loss of pluripotency ĐẶC TÍNH TẾ BÀO GỐC VẠN NĂNG CÁC YẾU TỐ DUY TRÌ TRẠNG THÁI KHÔNG BIỆT HOÁ CỦA TẾ BÀO GỐC PHÔI •  Các yếu tố bên ngoài: -  LIF and Cytokines -  BMP4 -  bFGF •  Các yếu tố bên trong: -  Oct4 -  Nanog -  Foxd3 -  Sox2 -  miRNA LIF và Cytokine REVIEW: THỤ THỂ CYTOKINE Phân loại thụ thể cytokine (dựa trên cấu trúc) REVIEW: THỤ THỂ CYTOKINE Phân loại thụ thể cytokine (dựa trên cấu trúc) REVIEW: CYTOKINE RECEPTORS •  LIF: nhân tố ức chế bạch cầu •  LIF cũng được biết đến như 1 nhân tố có hoạt tính ức chế biệt hoá •  LIF thuộc họ cytokine kiểu IL-6 •  LIF hoạt hoá chuỗi tín hiệu hoạt động -  tăng điều hoà các gen biểu hiện trong tế bào đa tiềm năng -  giảm điều hoà các gen biểu hiện trong tế bào biệt hoá LEUKEMIA INHIBITORY FACTOR- LIF •  Họ IL-6 kích thích tế bào thông qua receptor gp130; gp130 làm việc ở dạng heteromer cùng với một receptor chuyên biệt ligand (như IL-6R,IL-11R,LIFR,..) •  gp130 được biểu hiện ở tất cả các tế bào •  receptor chuyên biệt ligand biểu hiện giới hạn ở tế bào chuyên biệt CYTOKINE HỌ IL-6 VÀ THỤ THỂ Signal Transduction: Cascades to the Stem Cell Nucleus 31 between trophoblast and ICM whereby production of LIF by trophoblast could sustain the pluripotent ICM. Expression of cytokine receptors and ligands: Although gp130 is widely expressed in various tissues, ligand-specific receptor components display a more restricted expression. LIFR, OSMR, and CTNFR are expressed in ES cells; conse- quently, CT-1, OSM, and CNTF and LIF are interchangeable in preventing ES cell differentiation and supporting ES cell derivation and maintenance of ES cells in culture. IL-6 and IL-11 cannot substitute for CT-1; OSM and CNTF as the IL- 6 and IL-11 receptors are not expressed in ES cells. However, IL-6 can prevent ES differentiation if delivered in conjunction with a soluble form of the IL-6 receptor, which retains ligand binding activity and capability to induce gp130 homodimer- ization (see Figure 4-1). Genetic studies: LIFR-null embryos die shortly after birth, and exhibit reduced bone mass and profound loss of moto- neurons. Embryos homozygous for the gp130 mutation die between 12 and 18 days postcoitum (dpc) because of placen- tal, myocardial, hematological, and neurological disorders. CTFR-deficient mice exhibit perinatal death and display pro- found motor neuron deficits. Receptor gene function in ES cells and diapause: The late embryonic lethality of the gp130 -/- fetuses is in conflict with the gp130 requirement for ES cell self-renewal in vitro. Delayed or quiescent blastocysts were used for the initial experiments of ES cell derivation. Lactating females can con- ceive while still nursing their pups but cannot support blasto- cyst implantation because they do not produce estrogen at the fourth day of gestation. Consequently, embryonic develop- ment is arrested and resumes under favorable conditions for optimal development. This phenomenon, termed diapause, can be artificially induced by ovariectomy after fertilization. The embryos reach the blastocyst stage, hatch from the zona pellucida, and float in uterus in a quiescent status for up to 4 weeks. In this scenario, the epiblast, which normally preserves its pluripotent status for about three days (from 3.5 dpc, when it forms, up to 6.5 dpc, when gastrulation starts), can be main- tained for longer time periods and resumes development when an estrogen-rich environment is established. The possibility that cytokine receptors may then have an embryonic function in the quiescence embryo state was inves- tigated. LIFR-/- and gp130 -/- delayed embryos are unable to resume embryogenesis after 12 and 6 days of diapause, respectively. The number of cells that constitute the ICM of delayed gp130 -/- blastocysts is gradually reduced by apop- tosis during the 6-day period of diapause. Moreover, ICMs isolated from delayed gp130 null blastocysts cannot form a pluripotent outgrowth in vitro, as they differentiate exclu- sively into parietal endoderm. Thus, it appears that maintenance of the epiblast during diapause is temporally dependent on different cytokines and that Gp130 plays a more critical role than LIFR in this process. Two models may explain why epiblast cells enter apoptosis in the absence of gp130 signaling: 1. gp130 relays a cell survival signal from the extracellular compartment to the nucleus. This model is supported by the anti-apoptotic activity of the transcription factor STAT3 in a variety of cells. 2. gp130 suppresses epiblast differentiation. In the absence of gp130 signal, epiblast cells may differentiate inappropri- ately (as shown by the endoderm formation solely) and consequently die. Signal Transduction: Cascades to the Stem Cell Nucleus Homo- or heterodimerization of gp130 results in the activa- tion of receptor-associated kinases of the Janus family Jak1, Jak2 and Tyk2. These tyrosine kinases constitutively interact with the conserved regions box 1 and box 2 of gp130. The receptor complex is inactive until ligand-induced receptor dimerization Figure 4-1. Schematic structure of the cytokine receptor complexes sharing gp130 as a common subunit. IL-6/IL6R and IL-11/IL-11-R complexes induce gp130 homodimerization. Both LIF and CT-1 bind to LIFR and induce LIFR/gp130 heterodimerization. CTNF engages LIFR/gp130 as a signaling compe- tent complex through an association with CTNF/CTNFR, whereas OSM engages the LIFR/gp130 or OSMR/gp130 by binding to the gp130 portion of the heterodimers. •  LIF và LIFR có sự biểu hiện qua lại trong phôi nang (blastocyst) chuột: - LIF biểu hiện trong trophectoderm - LIFR biểu hiện trong ICM -> nhờ tương tác cận tiết giữa lớp dưỡng bào (trophoblast) và khối tế bào bên trong (ICM), sự sinh tạo LIF bởi trophoblast có thể duy trì ICM đa tiềm năng. •  LIFR, OSMR, và CNTFR biểu hiện ở ES cellS -> CT-1, OSM, CNTF và LIF ngăn sự biệt hoá của ES và củng cố nguồn gốc tế bào và duy trì tế bào ES trong nuôi cấy. •  IL- 6R và IL-11R không biểu hiện ở ES cellS -> Tuy nhiên, IL-6 ngăn sự biệt hoá ES khi gắn với soluble form của IL-6R -> duy trì hoạt tính gắn cơ chất và có khả năng cảm ứng gp130 homodimerization Maintaining the undifferentiated state in ESC BioDiscovery | www.biodiscoveryjournal.co.uk 4 September 2012 | Issue 3 | 1 constitutively bound to the cytoplasmic part of the complex Lifr/gp130. JAK is activated by binding of LIF to the receptor complex, which results in phosphorylation of specific tyrosine residues of gp130 and Lifr. The latter recruits the transcription factors STAT1 (Signal transducer and activator of transcription) and STAT3 [34, 35]. STAT proteins, in their bound state, are in turn phosphorylated by JAK forming activated homo- or heterodimers. The latter travel from the cytoplasm to the nucleus, where they act to transactivate the expression of other target proteins [36]. STAT3 has numerous target genes. Using chromatin immunoprecipitation (ChIP) in 2008 Chen et al. identified 2546 genomic sites for binding of STAT3, approximately one-third of which (718 sites) were target sites for binding of Oct-4, Sox2 and Nanog [37]. Many of the STAT3 binding sites are in genes directly responsible for the maintenance of the undifferentiated state, including Oct4 and Nanog [38]. Among the target genes of STAT3 in ESC there are transcriptionally active as well as transcriptionally inactive genes. As could only be expected, the transcriptionally active genes targeted by STAT3 generally code for products responsible for the maintenance of the pluripotent state, while the transcriptionally inactive genes are characterized by tissue-specific pattern of expression. Among the latter are gata (specific for the ectodermal lineage), gata4 (endodermal lineage), lhx1 (LIM homeobox protein 1, mesodermal lineage), eomes (trophectoderm), etc. [39]. Among the crucially important target genes activated by STAT3 is the cellular proto-oncogene c-Myc [40]. Its protein product functions in the positive regulation of the cell cycle. The levels of mRNA of c-Myc are regulated via the LIF signalling pathway by transactivation of c- Myc transcription by means of binding of phosphorylated STAT dimers to the c-Myc promoter region. Forced expression of stabilized c-Myc can sustain the pluripotent state of mESC in the absence of LIF [40]. Stabilization of c-Myc is most often achieved by targeted mutagenesis affecting the T58 codon in c-Myc, which is the target for GSK3β-dependent phosphorylation of c- Myc, resulting in its subsequent ubiquitination and degradation [41]. mESC expressing T58A mutant c-Myc sustain their stemness characteristics in LIF-free growth medium and in presence of mutated STAT3, which is devoid of transactivation properties. GSK3β is the beta isoform of the glycogen synthase kinase 3, a serine/threonin kinase phosphorylating target proteins such as p53, Axin, Notch, and SMAD3 (see below). Inhibition of GSK3β in mESC results in growth acceleration, producing tumour-like structures [42]. Inhibition of GSK3 together with inhibition of ERK in very early murine embryos supports derivation of ‘naïve’ mESC [43, 44]. The role c-Myc plays in the maintenance of the pluripotent state and the capability for self-renewal of mESC is likely to be implemented via more than one mechanism. Among these, prominent is the ability of c- Myc to inhibit endodermal differentiation by suppressing its crucial regulator, Gata6 [45], by stimulating the expression of the catalytic subunit of the telomerase complex (TERT) [46] and of microRNAs characteristic of the undifferentiated state [47]. Figure 1. LIF signalling in mESC. Curved arrows indicate phosphorylation, straight arrows indicate activation LIF/gp130/STAT3 4. Molecular Bases of Pluripotency 32 brings the associated Jak kinases within sufficient proximity to allow transphosphorylation and activation of the kinase catalytic domain. Activated Jaks phosphorylate specific tyrosines on the intracellular domain of gp130, creating docking sites for the recruitment of SH2 proteins to the activated receptor complex. When gp130 is phoshorylated, several signaling pathways are activated, involving STAT1 and STAT3, the SH2-domain containing tyrosine phosphatase (SHP2), ERK1 and ERK2 (extracellular signal receptor kinases or mitogen-activated kinases (MAPK), growth-factor receptor-bound protein (Grb) 2, Grb2-associated binder protein (Gab) 1, and phosphatidylinositol-3 kinase (PI3K) (see Figure 4-2). STAT: Latent Transcription Factors Transmitting Signals The STAT family: STAT proteins belong to a group of latent cytoplasmic transcription factors that play a central role in transmitting signals from the membrane to the nucleus, hence their name (signal transducers and activator of transcription). Seven major STAT proteins have thus far been identified in mouse (STAT1 to STAT6, including STAT5a and STAT5b). With the exception of STAT4, which is restricted to myeloid cells and testis, STAT factors are ubiquitously expressed. They are activated in many cell types by a broad range of cytokines, growth factors, and interferons (IFNs), and they are substrates for tyrosine kinases of the Src and Jak families. Structure: STAT proteins share several conserved struc- tural and functional domains. A tetramerization and a leucine zipper-like domain are located at the amino terminus, fol- lowed by a DNA binding domain, a Src homology domain 3- like region (SH3, proline rich motif binding domain), a Src homology domain 2 (SH2), a critical site of tyrosine phos- phorylation (Y705 in STAT3), and a carboxy-terminal trans- activation domain. No evidence has emerged so far to suggest an SH3 function. The SH2 domain plays three important roles: • Recruitment to activated receptor complexes. • Interaction with Jaks. • STAT dimerization and DNA binding. Regulation: The regulation of STAT signaling is mostly post-translational and involves both tyrosine and serine phoshorylation. Figure 4-2. Schematic description of the signaling pathways induced by IL-6 family cytokine. Following gp130 hetero- or homodimerization, activated JAKs phosphorylate the intracellular domain of gp130 on Y126,173, 265, 275, and 118. STAT3 association with phosphorylated Y126–275 leads to STAT3 phosphorylation, dimerization, and translocation to the stem cell nucleus. Association of SHP-2 with phosphorylated Y118 leads via adaptor proteins to activation of the Ras pathway and translocation of ERK1/2 to nucleus. STAT3 activation induces ES self-renewal, whereas ERK activation causes cell differentiation. mES Hoạt hoá STAT3 Hoạt hoá ERK -> Sự cân bằng trạng thái hoạt hoá STAT3 và ERK sẽ quyết định số phận ESC hoặc phân chia bình hườ g hoặc biệt hoá -> Con đường tín hiệu PI3K cũng được cảm ứng bởi LIF -> ức chế PI3K -> gia tăng các ERK hoạt hoá -> cảm ứng biệt hoá Darr H, Benvenisty N.(2006) Factors involved in self- renewal and pluripotency of embryonic stem cells. Handb Exp Pharmacol. (174):1-19. been treated with reverse transcriptase were run in parallel to verify that the obtained PCR products were amplified cDNA with no conta- mination of genomic DNA (results not shown). Densitometric scannings of the PCR bands were performed using the object image 2.08 ƒ software. Real-time quantitative PCR was performed on 0.1 !g of cDNA using the QuantiTect SYBR green PCR kit (Qiagen) and DNA Engine Opticon (MJ Research) according to the manufacturers’ instructions. RESULTS Expression Levels and Kinase Activity of cYes in Mouse and Human ES Cells—To determine whether cYes protein expres- sion or activity was regulated in ES cells under different con- ditions, Western blotting, and in vitro kinase assays were per- formed on undifferentiated mouse and human ES cells as well as on 10-day-old mouse and human EBs. As shown in Fig. 1A, cYes is highly active in both undifferentiated human and mouse ES cells, as assessed by autophosphorylation of cYes. cYes activity was significantly down-regulated when mouse ES cells differentiated and the decreased cYes activity could be detected as early as 4 days after LIF withdrawal. By contrast, the level of cYes protein was unchanged or only marginally reduced in mouse EBs cultured for up to 21 days in the absence of LIF (data not shown). To evaluate whether cYes is part of an LIF signal transduction pathway, as shown for Hck (20, 22), mES cells were serum-starved overnight and stimulated with different concentrations of LIF for various periods of time. Cells were lysed, immunoprecipitated for cYes, and subjected to an in vitro kinase assay. cYes activity was stimulated by LIF in a dose- (Fig. 1B) and time-dependent (Fig. 1C) manner. The Western blots of immunoprecipitated cYes were probed with anti-cYes antibody and a phospho-specific antibody that binds the autophosphorylation site in cYes. These data confirm that LIF treatment increase autophosphorylation of cYes in vivo (Fig. 1E). Serum-starved mES cells treated with 15% FBS for 20 min also exhibited increased cYes kinase activity compared with untreated cells. The degree of cYes-activation in response to serum was similar to that following LIF activation (Fig. 1E). SU6656, at concentrations above 0.5 !M, inhibited the LIF-de- pendent activation of cYes in mES cells (Fig. 1D). The SU6656 inhibitor potently and selectively inhibits cYes, cSrc, Fyn, and Lyn of the Src family. In contrast to other frequently used Src family inhibitors, SU6656 does not inhibit non-Src family ty- rosine kinases such as PDGFR", FGFR1, IGF1R, Met, Csk, Kit Bcr-Abl, or Cdk2, nor does it inhibit the Src family members Lck or Frk/Rak (23, 26). It is unclear whether SU6656 inhibits Hck kinase activity. In summary, cYes protein is easily detected in both mouse and human ES cells, and its kinase activity is activated by LIF and it is serum- and down-regulated when cells differentiate. The JAK1/STAT3 and Ras/MAPK Pathways Are Not Dis- rupted by Src Family Suppression in Mouse ES Cells—To as- sess if the JAK/STAT3 or MAPK pathways are activated down- FIG. 1. cYes is activated by LIF and serum. A, undifferentiated human and mouse ES cells and 10-day-old EBs were lysed, immunopre- cipitated with anti-Yes antibody, and subjected to an in vitro kinase assay. cYes phosphorylation was assessed by autoradiography. The amount of cYes protein in each sample was determined by Western blotting for cYes. The anti-cYes antibody used is highly specific for cYes and does not recognize any other Src family members, thus confirming that the #[32P]ATP-incorporated protein is indeed cYes. B and C, mES cells were serum-starved overnight and stimulated with different concentrations of LIF (B) for various periods of time (C). Cells cultured in serum with 500 pM LIF were run in parallel as a control (B, 500 on). cYes autophosphorylation was assessed as above. D, the selective Src family inhibitor SU6656 inhibits LIF-induced activation of cYes in mES cells. mES cells were serum-starved overnight and stimulated with 32 nM LIF for 20 min with or without the presence of 0.5, 2, or 5 !M SU6656, which was added 30 min prior to LIF; cYes kinase activity was then assessed. E, mouse ES cells were serum-starved overnight and treated with serum (15% FBS) or 32 nM LIF for 20 min; cYes kinase activity was then assessed. Western blots were probed with anti-cYes and anti-phospho 416 Src antibody. The Src Family Is Important for ES Cell Self-renewal31592 by guest on September 29, 2015 Downloaded from -> cYes được hoạt hoá bởi LIF và huyết thanh been treated with reverse transcriptase were run in parallel to verify that the obtained PCR products were amplified cDNA with no conta- mination of genomic DNA (results not shown). Densitometric scannings of the PCR bands were performed using the object image 2.08 ƒ software. Real-time quantitative PCR was performed on 0.1 !g of cDNA u