http / / cjbmb. bjmu. edu. cn Chinese Journal of Biochemistry and Molecular Biology Cdk6 Cdk6

Transcript

1 ISSN CN / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 965 Cdk6 ES * cyclin-dependent kinase 6 Cdk6 embryonic stem ES RNA Cdk6 Cdk6 ES RA LIF ES Cdk6 RNA Cdk6 ES Cdk6 ES 6 Cdk6 Q71 The Role of Cdk6 in Maintaining the Self-renewal State of ES Cells WANG Juan-Juan LIU Hui-Xian HOU Jie ZHANG Wei-Wei * College of Life Sciences Capital Normal University Beijing China Abstract Cyclin-dependent kinase 6 Cdk6 is an important factor in development however whether it is able to maintain the embryonic stem ES cells in undifferentiated state remained obscure In this study we investigated the function of Cdk6 in mouse ES cells by RNA interference and gene expression analyses Our results showed that the expression level of Cdk6 was correlated with the self-renewal of the ES cells with dramatic decrease upon retinoic acid RA treatment or leukaemia inhibitory factor LIF removal for the induction of ES cell differentiation Cdk6 knockdown by RNA interference caused the ES cells into a undifferentiated state where the expression of self-renewal genes were reduced and the expression of differentiation markers such as Cdx2 were induced Our findings suggested that an important function of Cdk6 was to maintain the mouse ES cells into a undifferentiated and self-renewing state Key words embryonic stem cells cyclin-dependent kinase 6 Cdk6 differentiation embryonic stem ES ES ES No No KM No ES * TRA-1-60 TRA ~ ES 5 Tel ES ES 6 ES SSEA3 SSEA4 SSEA1 ES zhangww@ mail cnu edu cn Received May Accepted August Supported by National Natural Science Foundation of China No Scientific Research Program of Beijing Municipal Commission of Education No KM and Scientific Research Foundation for the Returned Overseas Chinese Scholars from the Education Ministry of China No * Corresponding author Tel ES zhangww@ mail cnu edu cn

2 10 Cdk6 ES 959 ES p27 13 ES 14 Cdk6 D CIP / ES KIP Cdk4 Cdk ~ 9 ES 16 ES G Cdk6 20 Cdk6 ES 10 Oct4 Sox2 Nanog 21 ES Cdk6 ES ES transcriptional regulatory network Cdk6 ES ES RNA Cdk6 ES Cdk6 ES Cdk6 ES RNA ES Cdk6 ES ES ES ES Cdk6 ES ES leukaemia inhibitory factor LIF ES bone morphogenetic protein BMP Wnt wingless int LIF Wnt 1 ES Cartwright 11 LIF Wnt 1 1 ES Myc E14-CRL-1821 ATCC ES LIF DMEM GIBCO STAT-3 Myc mrna 044 GIBCO β- T58 GSK-3 Myc ES LIF Wnt 081 GIBCO LIF Myc ES ESGRO ESG1107 ES 1 2 ES Oct Sox2 Nanog 24 h 2 1 Chambers 12 LIF 3 d 6 d 10 d RNA Nanog ES 2 1 μg / ml RA Nanog Sigma d 10 d RNA Nanog 1 3 Nanog ES GIBCO L- GIBCO lipofeatanine cyclin-dependent 6 24 h kinase 6 Cdk6 D 4 μg 10 μl lipofeatanine2000

3 Invitrogen min 250 μl Opti-MEM GIBCO min 24 h 1 0 μg / ml Puromycin 4 d RNA 1 4 RNA real-time PCR Trizol Invitrogen RNA RNeasy minikit fermentas RNA 500 ng RNA RevertAid Fist strand cdna synthesis kit Invitrogen cdna 2 μl 5 μl SYBR Green Supermix Bio-RAD # AP 50 nmol / L mrna β-actin NP-40 Fluka min r / min 30 min 1 6 BCA BSA X A Y Reagent A Bio-Rad Reagent B Bio-Rad Reagent C Bio-Rad A Y X 50 μg Fig 1 Inducing ES cell differentiation by LIF removal 6 SDS min Supersignal west pico Chemiluminescent Substrate Thermo LJ P PVDF 5 min PVDF ImageQuant TM LIF ES Cdk6 LIF ES RNA real-time RT-PCR Oct4 Sox2 Nanog Fig 1 LIF 5 d ES Fig 1A Real-time RT-PCR Oct4 mrna 25% Sox2 50 nmol / L 35% Nanog 1% ILF ES 20 μl Bio-rad PCR ES Fig 1B Cultured ES cells by using the medium without LIF and changed the medium every twenty-four hours Analyze results 1 7 SDS-PAGE after five days A The phenotype of ES cells after removing LIF for five days Observe the cytomorphology of ES cells 2 through microscope B Reduction of Oct4 Sox2 and 12% 5% SDS-PAGE Nanog expression levels in ES cells cultured in no LIF 1 SDS-PAGE 100V SDSmedium ES cells were cultured in medium removed LIF for PAGE 1 5 h five days The mrna levels of Oct4 Sox2 and Nanog were 1 8 Western SDS-PAGE 100V 1 5 h determined by real-time PCR Data are presented as the mean ± SEM Cdk6 Santa Cruz SC-177 β-tublin Santa Cruz SC retinoic acid RA A ES 22 RA ES RNA real-time RT-PCR LAS4000 Oct4 Sox2 Nanog Fig 2 RA ES 5 d

4 10 Cdk6 ES 961 LIF RNA Western Fig 2A Real- Cdk6 RA LIF time RT-PCR Oct4 mrna 1% Sox2 10% Nanog 1% RA Cdk6 ES ES Fig 2B Fig 3C ES Cdk6 Cdk6 ES 2 3 Cdk6 shrna Cdk6 ES Cdk6 ES Cdk6 2 RNA Cdk6 shrna1 2 Table 1 Table 1 The target sequence of Cdk6 gene Name Cdk6 shrna1 Cdk6 shrna2 Sequence GACCAGCAGTGGACAGATA GAGAAGTTTGTGACAGATA Fig 2 RA-induced ES cell differentiation Treated ES cells with 1μ mol / L RA and changed the medium every twenty-four hours Analyze results after five days A The phenotype of ES cells after RA treatment Observe the cytonorphology of ES cells through microscope B Oct4 Sox2 and Nanog expression levels were reduced in ES cells cultured in RA medium ES cells were cultured in medium with RA for five days The mrna levels of Oct4 Sox2 and Nanog were determined by real-time PCR Data are presented as the mean ± SEM 2 2 ES Cdk6 Cdk6 ES Cdk6 2 4 Cdk6 RA 3 d 10 d Oct4 Sox2 Nanog ES LIF 3 d 6 d 10 d RNA real-time RT-PCR Western Fig 3A mrna RA 3 d 10 d Cdk6 40% 20% Cdk6 RA Real-time RT-PCR RA Cdk6 Cdk6 ES Oct4 Sox2 Nanog Fig 3B mrna LIF 3 d 6 d 10 d Cdk6 2 Cdk6 shrna lipofectamine 2000 Cdk6 shrna ES 1 μg / ml puromycin 4 d RNA real-time RT-PCR Western Fgi 4A Cdk6 shrna ES mrna Cdk6 20% Fgi 4B Cdk6 shrna ES Cdk6 Cdk6 shrna ES Cdk6 ES ES Oct4 Sox2 Nanog Esrrb Tdgf1 Fig 5 Cdk6 ES Oct4 90% 50% 20% 40% Sox2 Nanog Esrrb

5 Fig 3 Cdk6 expression level decreased after ES cell differentiation A Reduction of Cdk6 expression level in ES cells cultured in RA-induced cell differentiation ES cells were cultured in the medium with RA for 3 days and 10 days The mrna expression level of Cdk6 was measured by quantitative real-time PCR analysis Data are presented as the mean ± SEM B Reduction of Cdk6 expression level in ES cells cultured in LIF withdrawn induced cell differentiation ES cells were cultured in medium without LIF for 3 6 and 10 days The mrna expression level of Cdk6 was measured by quantitative real-time PCR analysis Data are presented as the mean ± SEM C Reduction of Cdk6 protein expression level in ES cells cultured in RA-treatment or LIF removal-induced cell differentiation The protein expression levels of Cdk6 were measured by Western blotting with RA or no LIF withdrawn treatment for 3 and 6 days The antibody againstβ-tubulin was used as a loading control Fig 4 The expression levels of Cdk6 upon shrna treatment Transfected the construct of Cdk6 shrna to ES cells and selected cells with 1 μg / ml puromycin for 4 days A The mrna levels of Cdk6 were determined by real-time PCR quantification of reverse transcribed RNAs Data are presented as the mean ± SEM B Reduction of Cdk6 protein expression level in ES cells treatmented with shrna The protein expression levels of Cdk6 were measured by western blotting with shrna treatment for 4 days The antibody againstβ-tubulin was used as a loading control Fig 5 The change of self-renewal-related gene expression in ES cells after shrna-mediated Cdk6 depletion The mrna levels of Oct4 Sox2 Nanog Esrrb and Tdgf5 were determined by real-time PCR quantification of reverse transcribed RNAs Transfected the construct of shrna Cdk6 to 60% ~ 70% Tdgf5 ES cells and selected cells with 1 μg / ml puromycin for 4 50% ~ 60% days Data are presented as the mean ± SEM Cdk6 ES Cdk6 Fig 6 trophectoderm Cdx2 Hand1 Cdk6

6 10 Cdk6 ES 963 ectoderm Fgf5 cyclins mesendoderm Msx1 cyclin-dependent kinase Cdks Cdk6 cyclin-dependent kinase ES inhibitor CKIs Fig 6 The change of differentiation-related gene expression in ES cells after shrna- mediated Cdk6 depletion The mrna levels of differentiation genes were determined by real-time PCR quantification of reverse transcribed RNAs Transfected the construct of shrna Cdk6 to ES cells and selected cells with 1 μg / ml puromycin for four days Data are presented as the mean ± SEM 3 Cdks Cyclins Cdks CKIs ES ES G 1 27 ES 11 h G 1 2 h E2F G 1 / S 30 ES Cdk2-cyclinA / E cyclin E p21cip1 p27kip1 30 Cdk2 Cdk2 30 ES Cdk6 / CyclinD ES Cdk1- Cyclin B Cyclin B Cdk1 Y 15 Cdk1 30 Cdk1 Cdk2 PCNA ES 33 Cdk1 Cdk1 34 Ullah 35 Cdk1 RO3306 Cdk1 Cdk1 36 ES Cdk1 Oct4 Cdk1 Oct4 ES Cdks Cdk1 ES 4 G 1 S G 2 M Cdk6 RA LIF checkpoint ES real-time G 1 / S G 2 / M RT-PCR ES Cdk6 ES Cdk ES Cdk6

7 ES sirna Cdk6 ES 896 Oct4 Sox2 Nanog Cdx2 Hand1 Msx1 ES Cdk Cdk6 14 Grossel MJ Hinds PW Cdk6 ES expanding role for cdk6 J Cell Cycle 2006 Cdk6 ES and INK4 inhibitors J References 1 Thomson JA Kalishman J Golos TG et al Isolation of a primate embryonic stem cell line J Proc Natl Acad Sci U S A Thomson JA Marshall VS Primate embryonic stem cells J Curr Top Dev Biol Gerami-Naini B Dovzhenko OV Durning M et al Trophoblast differentiation in embryoid bodies derived from human embryonic stem cells J Endocrinology Odorico JS Kaufman DS Thomson JA Multilineage differentiation from human embryonic stem cell lines J Stem Cells Xu RH Chen X Li DS et al BMP4 initiates human embryonic stem cell differentiation to trophoblast J Nat Biotechnol Wei CL Miura T Robson P et al Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain the stem cell state J Stem Cells Chinzei R Tanaka Y Shimizu-Saito K et al Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functional hepatocytes J Hepatology Nakano T Kodama H Honjo T Generation of lymphohematopoietic cells from embryonic stem cells in culture J Science as activators of the cell cycle in neuronal death induced by J Shen G Cong XQ Du ZW et al Immortalization of endothelial cells differentiated from mouse embryonic stem cells J Acta Biol Exp Sin Martin G R Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells J Proc Natl Acad Sci U S A Cartwright P McLean C Sheppard A et al LIF / STAT3 controls ES cell self-renewal and pluripotency by a Mycdependent mechanism J Development Chambers I Silva J Colby D et al Nanog safeguards pluripotency and mediates germline development J Nature Sherr CJ Roberts JM CDK inhibitors positive and negative regulators of G1-phase progression J Genes Dev From cell cycle to differentiation an Pavletich NP Mechanisms of cyclin-dependent kinase regulation structures of Cdks their cyclin activators and Cip J Mol Biol Hu Mi G Deshpande A Enos M et al A requirement for cyclin-dependent kinase 6 in thymocyte development and tumorigenesis J Cancer Res Faast R White J Cartwright P et al Cdk6-cyclin D3 activity in murine ES cells is resistant to inhibition by p16 INK4a J Oncogene Savatier P Huang S Szekely L et al Contrasting patterns of retinoblastoma protein expression in mouse embryonic stem cells and embryonic fibroblasts J Oncogene Savatier P Lapillonne H van Grunsven LA et al Withdrawal of differentiation inhibitory activity / leukemia inhibitory factor upregulates D-type cyclins and cyclin- dependent kinase inhibitors in mouse embryonic stem cells J Oncogene Stead E White J Faast R et al Pluripotent cell division cycles are driven by ectopic Cdk2 cyclin A / E and E2F activities J Oncogene Chen X Xu H Yuan P et al Integration of external signaling pathways with the core transcriptional network in embryonic stem cells J Cell Schuldiner M Eiges R Eden A et al Induced neuronal differentiation of human embryonic stem cells J Brain Res Liu P Barkley LR Day T et al The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25A / Cdk2- independent mechanism J J Biol Chem Zhang Y Qu D Morris EJ et al The Chk1 / Cdc25A pathway camptothecin J J Neurosci Li A Blow J J The origin of CDK regulation J Nat Cell Biol E Murray A W Recycling the cell cycle cyclins revisited J Cell Becker KA Ghule PN Therrien JA et al Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase J J Cell Physiol

8 10 Cdk6 ES Fluckiger AC Marcy G Marchand M et al Cell cycle features of primate embryonic stem cells J Stem Cells Savatier P Huang S Szekely L et al Contrasting patterns of retinoblastoma protein expression in mouse embryonic stem cells and embryonic fibroblasts J Oncogene Stead E White J Faast R et al Pluripotent cell division cycles are driven by ectopic Cdk2 cyclin A / E and E2F activities J Oncogene Faast R White J Cartwright P et al Cdk6-cyclin D3 activity in murine ES cells is resistant to inhibition by p16 INK4a J Oncogene Savatier P Lapillonne H van Grunsven LA et al Withdrawal of differentiation inhibitory activity / leukemia inhibitory factor upregulates D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem cells J Oncogene Fujii-Yamamoto H Kim JM Arai K et al Cell cycle and developmental regulations of replication factors in mouse embryonic stem cells J J Biol Chem Santamaría D Barrière C Cerqueira A et al Cdk1 is sufficient to drive the mammalian cell cycle J Nature Ullah Z Kohn MJ Yagi R et al Differentiation of trophoblast stem cells into giant cells is triggered by p57 / Kip2 inhibition of CDK1 activity J Genes Dev Wang J Rao S Chu J et al A protein interaction network for pluripotency of embryonic stem cells J Nature