摘要
间充质干细胞（Mesenchymal Stem Cells, MSCs）是一类广泛存在于出生后机体多种组织的多能干细胞，较少涉及到诸如胚胎干细胞的伦理学问题。一般认为在适合条件下，MSCs可以分化为成骨细胞、成脂细胞及成软骨细胞。近些年来又发现了MSCs的一些新功能，例如支持造血、抑制T细胞免疫反应等。不仅如此，一些基于MSCs的细胞疗法已经展开尝试和应用，并取得了良好的进展。
目前肥胖、糖尿病以及其他代谢性疾病已经成为世界范围内的首要健康威胁，这些疾病与脂肪细胞的发育和功能异常有着非常密切的联系。成脂分化是MSCs多向分化的重要组成部分，这一过程受到复杂而又精密的调控。简而言之，MSCs受到外界成脂分化信号刺激后，会激活一系列关键转录因子的表达，而这些转录因子继而转录出大量脂肪细胞特异的基因来执行特化的功能，最终完成MSCs向成熟脂肪细胞的转变。目前认为，C/EBPα和PPARγ是成脂分化中的最为关键的转录因子，对它们的转录调控研究有重要的意义。
CKIP-1是我们实验室开展研究多年的基因，其蛋白产物N端含有PH结构域，C端含有亮氨酸拉链结构域，认为其主要定位在细胞近质膜区。近年来我们实验室建立了ckip-1~（-/-）小鼠模型，并在体内水平证明CKIP-1可以增强泛素连接酶Smurf1的活性，抑制成骨前体细胞的分化和矿化能力。因为MSCs是成骨、成脂前体细胞的共同干祖细胞，所以我们在本论文中利用敲除小鼠模型来探讨CKIP-1缺失对MSCs成脂分化的影响。
我们采用了从密质骨中分离MSCs的方法，得到了表面抗原均一的高纯度ckip-1~(+/+)和ckip-1~（-/-）MSCs，并进行了相关干细胞性能验证。在随后的实验中我们发现：
1. ckip-1~（-/-）小鼠MSCs成脂分化能力增强，关键标志物（marker）基因表达水平显著升高，其中C/EBPα尤为明显，而且这种升高早在转录水平已经发生。我们首先发现CKIP-1蛋白表达在MSCs成脂分化中发生明显变化，随后在比较ckip-1~(+/+)和ckip-1~（-/-）MSCs成脂分化能力时发现，ckip-1~（-/-）MSCs中出现的脂肪细胞显著增多。在将不同培养代数的ckip-1~(+/+)和ckip-1~（-/-）MSCs进行成脂分化诱导时，我们发现ckip-1~（-/-）MSCs显示出更高水平的成脂marker基因表达，其中C/EBPα最为显著。而且通过报告基因实验，我们证明了过表达CKIP-1也可以抑制C/EBPα的下游效应。
2.首次证明生理状态下CKIP-1可以定位在细胞核中，并与C/EBPα的转录抑制因子——组蛋白去乙酰化酶HDAC1存在相互作用。目前对C/EBPα的转录调控机制了解得比较清楚的是：成脂分化启动后，其他两个C/EBP转录因子家族成员C/EBPβ与δ会结合在C/EBPα的启动子区激活其转录，而组蛋白去乙酰化酶HDAC1作为转录共抑制子，也会被招募到该区域。HDAC1会降低组蛋白H3及H4乙酰化水平，延缓迟滞RNA聚合酶II对C/EBPα的转录。我们的数据表明CKIP-1可以和上述蛋白复合体中的HDAC1共定位在MSCs的细胞核，并与HDAC1发生内源、外源相互作用，且这种相互作用不需要借助其他分子。
3. CKIP-1可以增强HDAC1与C/EBPα启动子区的结合，从而抑制C/EBPα的转录。我们发现与脂肪前体细胞系不同，在MSCs中HDAC1组成型结合于C/EBPα的启动子区，而非被招募；在成脂分化初期，HDAC1与该区结合能力减弱，从其上发生解离。与ckip-1~(+/+)MSCs相比，在ckip-1~(-/-)MSCs中，成脂分化启动后结合在该启动子区的HDAC1显著减少，相应地，结合在该区的Ac-H3与RNA聚合酶II显著增多。过表达CKIP-1可以减弱HDAC1与该启动子区的解离，这证明CKIP-1确实可以通过增强HDAC1与该启动子区的结合能力来抑制C/EBPα的转录。
4.在高脂饮食下，ckip-1~(-/-)小鼠体重增长比ckip-1~(+/+)小鼠更为显著。经过脂肪含量60%的饲料喂养后，ckip-1~(-/-)小鼠体内主要的脂肪团质量以及瘦素水平也显著高于ckip-1~(+/+)小鼠，并显示出更为严重的脂肪肝表型。
综上，我们发现了CKIP-1基因的另一新功能——抑制MSCs成脂分化，并首次揭示了其在生理条件下的核定位。近期本实验室与中国航天员训练科研中心的合作研究也显示CKIP-1可以抑制心肌肥大，再联系到前期CKIP-1抑制成骨细胞分化的结果，我们认为CKIP-1是一个广谱系抑制干祖细胞分化的基因。此外，对成脂分化而言，前期的认识大多来自脂肪前体细胞系中（如3T3-L1）。我们通过基因敲除小鼠的模型，揭示了CKIP-1可以增强HDAC1对C/EBPα的转录抑制而负调控MSCs的成脂分化，并发现在MSCs中，HDAC1对C/EBPα的转录调控具有自身的特点，这些工作都丰富了对成脂分化中C/EBPα转录调控的认识。
另外，我们在实验中还得到了一些意外的发现。虽然小鼠作为应用最为广泛的模式生物，但是依照传统方法从骨髓中分离MSCs存在着许多困难。近些年来有研究者指出小鼠长骨密质骨是MSCs的一个重要来源，并发展出全新的分离方法，而且有数据表明这种方法可以分离到比骨髓中更多更纯的MSCs。根据已有认识，密质骨也存在于顶骨。应用相似的方法，我们从小鼠顶骨密质骨中也分离到了与长骨来源的MSCs细胞形态、表面抗原都高度相似的细胞。尽管如此，我们发现顶骨来源的这些细胞内成骨转录因子osterix表达远高于长骨来源的MSCs。随后我们将这两种不同骨来源的细胞进行了诱导分化实验。与长骨来源的MSCs可以轻易分化为脂肪细胞不同，顶骨来源的细胞丧失了成脂分化的能力。与这一表型相一致，成脂分化的marker基因如PPARγ、C/EBPα及aP-2mRNA水平在顶骨来源的细胞中远低于长骨来源的MSCs。而对于成骨分化而言，经过诱导后，顶骨来源的细胞中成骨分化的marker基因如Runx2，osterix及osteocalcin mRNA水平显著高于长骨来源的MSCs，并展示出显著增强的分化活性及矿化细胞外基质的能力。这些数据说明了这群顶骨来源的细胞更像是分化命运已经决定的祖细胞(committed progenitor cells)而非MSCs。这也提示我们驻留在小鼠机体不同部位密质骨内的间充质干/祖细胞的分化命运已经决定，并不是所有部位的密质骨都可以作为MSCs的来源。
Mesoderm-derived mesenchymal stem cells (MSCs) are present in many tissuespostnatally and can differentiate into osteocytes, adipocytes and chondrocytes underappropriate conditions. Aside from their multiple differentiation capacity, MSCs alsoplay important roles in supporting hematopoiesis and in suppressing theimmunoresponse of T lymphocytes. Because of these characteristics, the applicationof MSCs in cytotherapy is promising, such as their use in tissue repair and in reducingimmunological rejection.
In present world, improved knowledge of all aspects of adipose biology will berequired to counter the burgeoning epidemic of obesity and relevant metabolicdiseases, which has closed association with abnormal development and function ofadipocytes. The adipocytes constituting white fat tissue are originated from MSCs,which differentiate in response to a series of cues. The extracellular signals aretransduced into nuclear transcription factors via cascades reactions to transcribe manyhundreds of genes, which responsible for establishing the mature fat cell shape andfunction. Transcriptional regulation of adipogenic differentiation is a tightlycontrolled process that is regulated by an elaborate network of transcription factors,cofactors and signalling intermediates from numerous pathways.
Among these transcription factors, the most notable are CCAAT/enhancer bindingproteins(C/EBP) and the peroxisome proliferator-activated receptors gamma(PPAR). Actually, it is well accepted that both C/EBPα and PPARγ act as criticalregulators of adipogenesis, since deficiency of either of them shows thedevelopmental defects of white adipose in mouse model. Consequently, in our effortsto gain a complete understanding of the processes regulating the function ofadipocytes, it is important to identify the mechanisms regulating transcription of theseadipogenic transcription factors.
CKIP-1is a PH domain containing protein, involving in apoptosis, cell skeletonmaintaining and so on. In recent years, it was found CKIP-1promoted E3ligaseSmurf1's activities and negatively regulated osteogenesis in mouse model. It is wellknown that osteoblasts and adipoblasts are descended from common progenitors-- MSCs. Yet, the function of CKIP-1in the adipogenic differentiation of MSCs is notknown. In the present study, we isolated and richened MSCs from murine compactbone of CKIP-1wild-type (WT) and knockout (KO) mice, and further identified theirpurity and CFU-F capacity. In the subsequent multiple differentiation assays, wefound that MSCs lacking CKIP-1display enhanced adipogenesis after exposed tostimuli than their wild-type counterparts, and increased transcriptional levels of thecritical adipogenic marker genes, especially for C/EBPα. Due to the HDAC1represscomplex is curial for the transcription of C/EBPα, we investigated the association ofCKIP-1and HDAC1in adipogenesis of MSCs. It is found that CKIP-1can localize inthe nucleus and interact with HDAC1directly. This is the frist time that CKIP-1hasbeen observed in the nucleus under physiological conditions. Our date also show thatHDAC1constitutively binds the promoter of C/EBPα in MSCs, but dislodges duringthe early phase of adipogenesis. In the presence of CKIP-1, the dislodgment ofHDAC1from the promoter of C/EBPα is weakened, which suppresses thetranscription of C/EBPα and adipogenesis. Furthermore, CKIP-1deficiency leadsmore Ac-histone3and RNA pol II in the promoter of C/EBPα during adipogenesis ofMSCs, but less HDAC1. Moreover, on high fat diet, CKIP-1KO mice showaccelerated body weight gain, increased total fat mass and severe fatty liverphenotype than their WT counterparts. Taken together, we conclude that CKIP-1is anovel negative regulator of adipogenesis of MSCs.
In addition, we observed some interesting phenomenons by chance. MSCs culturewas first developed by Friedenstein et al. from bone marrow, and since then, bonemarrow has become the common source for isolating MSCs. Thus far, MSCs havebeen isolated from the bone marrow of many species, including humans, rats and pigs.In contrast to other species, murine MSCs cannot be easily harvested from the bonemarrow due to contamination by hematopoietic cells, and this contamination isdifficult to eliminate using the characteristic plastic adherence of MSCs. Recentstudies showed that the compact bone tissue of long bones is a novel source of MSCsfor both humans and mice. These studies developed a novel method to obtainhigh-purity murine MSCs by culturing collagenase-digested compact bone fragments.Some procedures in this method help avoid hematopoietic cell contamination, such asby removing bone marrow before collagenase digestion and discarding the releasedcells after collagenase digestion. Furthermore, subsequent experiments showed thatcompact bone is a richer source of MSCs than the marrow plug within it. Based on these findings, we postulated that murine MSCs could be isolated from the compactbone of the calvaria, which has a relatively low bone marrow content. In this study,we found that cells migrating from the calvaria possess morphological characteristicsand surface antigen profiles similar to those of MSCs derived from long bones.However, these calvaria-derived cells highly expressed the osteogenic transcriptionfactor osterix. The calvaria-derived cells lost their adipogenic capacity but gained ahigher osteogenic capacity. These results suggest that not all types of murine compactbones in the body are sources of MSCs and that the differentiation fate ofmesenchymal stem/progenitor cells in different types of compact bones is alreadycommitted. The cells that migrate from the calvaria should be considered progenitorcells rather than MSCs.
引文
1. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues.Science1997;276:71-74.
2. Friedenstein AJ, Chailakhyan RK, Latsinik NV, Panasyuk AF, Keiliss-Borok IV.Stromal cells responsible for transferring the microenvironment of thehemopoietic tissues. Cloning in vitro and retransplantation in vivo.Transplantation1974;17:331-34.
3. Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblastcol-onies in monolayer cultures of guinea-pig bone marrow and spleen cells.Cell Tissue Kinet1970;4:393-403.
4. Friedenstein AJ, Gorskaja JF, Kulagina, NN. Fibroblast precursors in normaland irradiated mouse hematopoietic organs. Exp. Hematol1976;4:267-274.
5. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD,Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential ofadult human mesenchymal stem cells. Science1999;284:143-147.
6. Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease.Nat Rev Immunol2008;9:726-36.
7. Harada S, Rodan GA. Control of osteoblast function and regulation of bone mass.Nature2003;423:326-31.
8. Chamberlain G, Fox J, Ashton B, Middleton J. Mesenchymal Stem Cells: TheirPhenotype, Differentiation Capacity, Immunological Features, and Potential forHoming. Stem cells2007;25:2739-2749.
9. Kolf CM, Cho E, Tuan RS. Biology of adult mesenchymal stem cells: regulationof niche, self-renewal and differentiation. Arthritis Res Ther2007;9:204.
10. Muguruma Y, Yahata T, Miyatake H, Sato T, Uno T, Itoh J, Kato S, Ito M, HottaT, Ando K. Reconstitution of the functional human hematopoietic microenviro-nment derived from human mesenchymal stem cells in the murine bone marrowcompartment. Blood2006;107:1878-1887.
11. Schofield R. The relationship between the spleen colony-forming cell and thehaemopoietic stem cell. Blood Cells1978;4:7-25.
12. Wilson A, Trumpp A. Bone-marrow haematopoietic-stem-cell niches. Nature RevImmunol2006;6:93-106.
13. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneicimmune cell responses. Blood2005;105:1815-1822.
14. Beyth S, Borovsky Z, Mevorach D, Liebergall M, Gazit Z, Aslan H, Galun E,Rachmilewitz J. Human mesenchymal stem cells alter antigen-presenting cellmaturation and induce T-cell unresponsiveness. Blood2005;105:2214-2219.
15. Maccario R, Podestà M, Moretta A, Cometa A, Comoli P, Montagna D, Daudt L,Ibatici A, Piaggio G, Pozzi S, Frassoni F, Locatelli F. Interaction of humanmesenchymal stem cells with cells involved in alloantigen-specific immuneresponse favors the differentiation of CD4+T-cell subsets expressing aregulatory/suppressive phenotype. Haematologica2005;90:516-525.
16. Spaggiari GM, Capobianco A, Becchetti S, Mingari MC, Moretta L. Mesenchy-mal stem cell-natural killer cell interactions: evidence that activated NK cells arecapable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell prolif-eration. Blood2006;107:1484-1490.
17. Spaggiari GM, Capobianco A, Abdelrazik H, Becchetti F, Mingari MC, MorettaL. Mesenchymal stem cells inhibit natural killer-cell proliferation, cytotoxicity,and cytokine production: role of indoleamine2,3-dioxygenase and prostaglandinE2. Blood2008;111:1327-1333.
18. Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P,Grisanti S, Gianni AM. Human bone marrow stromal cells suppressT-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood2002;99:3838-3843.
19. Bartholomew A, Sturgeon C, Siatskas M, Ferrer K, McIntosh K, Patil S, HardyW, Devine S, Ucker D, Deans R, Moseley A, Hoffman R. Mesenchymal stemcells suppress lymphocyte proliferation in vitro and prolong skin graft survival invivo. Exp. Hematol2002;30:42-48.
20. Ren G, Zhang L, Zhao X, Xu G, Zhang Y, Roberts AI, Zhao RC, Shi Y.Mesenchymal stem cell-mediated immunosuppression occurs via concertedaction of chemokinesand nitric oxide. Cell Stem Cell2008;2:141-50.
21. Pereira RF, O'Hara MD, Laptev AV, Halford KW, Pollard MD, Class R, Simon D,Livezey K, Prockop DJ. Marrow stromal cells as a source of progenitor cells fornonhematopoietic tissues in transgenic mice with a phenotype of osteogenesisimperfecta. Proc. Natl. Acad. Sci. USA1998;95:1142-1147.
22. Lee RH, Seo MJ, Reger RL, Spees JL, Pulin AA, Olson SD, Prockop DJ.Multipotent stromal cells from human marrow home to and promote repair ofpancreatic islets and renal glomeruli in diabetic NOD/scid mice. Proc. Natl.Acad. Sci. USA2006;103:17438-17443.
23. Le Blanc K, Rasmusson I, Sundberg B, G therstr m C, Hassan M, Uzunel M,Ringdén O. Treatment of severe acute graft-versus-host disease with third partyhaploidentical mesenchymal stem cells. Lancet2004;363:1439-1441.
24. Li H, Guo Z, Jiang X, Zhu H, Li X, Mao N. Mesenchymal stem cells altermigratory property of T and dendritic cells to delay the development of murinelethal acute graftversus-host disease. Stem Cells2008;26:2531-2541.
25. Djouad F, Fritz V, Apparailly F, Louis-Plence P, Bony C, Sany J, Jorgensen C,No l D. Reversal of the immunosuppressive properties of mesenchymal stemcells by tumor necrosis factor alpha in collageninduced arthritis. Arthritis Rheum2005;52:1595-1603.
26. Augello A, Tasso R, Negrini SM, Cancedda R, Pennesi G. Cell therapy usingallogeneic bone marrow mesenchymal stem cells prevents tissue damage incollagen-induced arthritis. Arthritis Rheum2007;56:1175-1186.
27. da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stem cells residein virtually all post-natal organs and tissues. J Cell Sci2006;119:2204-2213.
28. Short B, Brouard N, R. Driessen R, et al. Prospective isolation of stromalprogenitor cells from mouse BM. Cytotherapy2001;3:407-408.
29. Short B, Brouard N, Simmons PJ. Purification of MSC from mouse compactbone. Blood2002;100:62a.
30. Sakaguchi Y, Sekiya I, Yagishita K. Ichinose S, Shinomiya K, Muneta T. Suspen-ded cells from trabecular bone by collagenase digestion become virtuallyidentical to mesenchymal stem cells obtained from marrow aspirates. Blood2004;104:2728-2735.
31. Short BJ, Brouard N, Simmons PJ.Prospective isolation of mesenchymal stemcells from mouse compact bone. Methods Mol Biol2009;482:259-268.
32. Nuttall ME, Patton AJ, Olivera DL, Nadeau DP, Gowen M. Human trabecularbone cells are able to express both osteoblastic and adipocytic phenotype:implications for osteopenic disorders. J Bone Miner Res1998;13:371-382.
33. Tuli R, Seghatoleslami MR, Tuli S, Wang ML, Hozack WJ, Manner PA,Danielson KG, Tuan RS. A simple, high-yield method for obtaining multi-potential mesenchymal progenitor cells from trabecular bone. Mol Biotechnol2003;23:37-49.
34. N th U, Osyczka AM, Tuli R, Hickok NJ, Danielson KG, Tuan RS. Multilineagemesenchymal differentiation potential of human trabecular bone-derived cells. JOrthop Res2002;20:1060-1069.
35. Sottile V, Halleux C, Bassilana F, Keller H, Seuwen K. Stem cell characteristicsof human trabecular bone-derived cells. Bone2002;30:699-704.
36. Tuli R, Tuli S, Nandi S, Wang ML, Alexander PG, Haleem-Smith H, Hozack WJ,Manner PA, Danielson KG, Tuan RS. Characterization of multipotentialmesenchymal progenitor cells derived from human trabecular bone. Stem Cells2003;21:681-693.
37. Guo Z, Li H, Li X, Yu X, Wang H, Tang P, Mao N. In vitro characteristics andin vivo immunosuppressive activity of compact bone-derived murinemesenchymal progenitor cells. Stem Cells2006;24:992-1000.
38. Zhu H, Guo ZK, Jiang XX, Li H, Wang XY, Yao HY, Zhang Y, Mao N. Aprotocol for isolation and culture of mesenchymal stem cells from mousecompact bone. Nat Protoc2010;5:550-560.
39. Cristancho AG, Lazar MA. Forming functional fat: a growing understanding ofadipocyte differentiation. Nat Rev Mol Cell Biol2011;12:722-734.
40. Rosen ED, MacDougald OA. Adipocyte differentiation from the inside out. NatRev Mol Cell Biol2006;7:885-896.
41. Guilherme A, Virbasius JV, Puri V, Czech MP. Adipocyte dysfunctions linkingobesity to insulin resistance and type2diabetes. Nat Rev Mol Cell Biol2008;9:367-377.
42. Rosen ED, Walkey CJ, Puigserver P, Spiegelman BM. Transcriptional regulationof adipogenesis. Genes Dev2000;14:1293-1307.
43. Mueller E, Drori S, Aiyer A, Yie J, Sarraf P, Chen H, Hauser S, Rosen ED, Ge K,Roeder RG, Spiegelman BM. Genetic analysis of adipogenesis throughperoxisome proliferator-activated receptor γ isoforms. J Biol Chem2002;277:41925-41930.
44. Zhang J, Fu M, Cui T, Xiong C, Xu K, Zhong W, Xiao Y, Floyd D, Liang J, Li E,Song Q, Chen YE. Selective disruption of PPARγ2impairs the development ofadipose tissue and insulin sensitivity. Proc Natl Acad Sci USA2004;101:10703-10708.
45. Medina-Gomez G, Virtue S, Lelliott C, Boiani R, Campbell M, ChristodoulidesC, Perrin C, Jimenez-Linan M, Blount M, Dixon J, Zahn D, Thresher RR,Aparicio S, Carlton M, Colledge WH, Kettunen MI, Sepp nen-Laakso T, SethiJK, O'Rahilly S, Brindle K, Cinti S, Oresic M, Burcelin R, Vidal-Puig A. Thelink between nutritional status and insulin sensitivity is dependent on theadipocyte-specific peroxisome proliferator-activated receptor-γ2isoform.Diabetes2005;54:1706-1716.
46. Imai T, Takakuwa R, Marchand S, Dentz E, Bornert JM, Messaddeq N,Wendling O, Mark M, Desvergne B, Wahli W, Chambon P, Metzger D.Peroxisome proliferator-activated receptor γ is required in mature white andbrown adipocytes for their survival in the mouse. Proc Natl Acad Sci USA2004;101:4543-4547.
47. Wang ND, Finegold MJ, Bradley A, Ou CN, Abdelsayed SV, Wilde MD, TaylorLR, Wilson DR, Darlington GJ. Impaired energy homeostasis in C/EBPalphaknockout mice. Science1995;269:1108-1112.
48. Linhart HG, Ishimura-Oka K, DeMayo F, Kibe T, Repka D, Poindexter B, BickRJ, Darlington GJ. C/EBPα is required for differentiation of white, but notbrown, adipose tissue. Proc Natl Acad Sci USA2001;98:12532-12537.
49. Tang QQ, Otto TC, Lane MD. CCAAT/enhancer-binding protein β is requiredfor mitotic clonal expansion during adipogenesis.Proc Natl Acad Sci USA2003;100:850-855.
50. Tanaka T, Yoshida N, Kishimoto T, Akira S. Defective adipocyte differentiationin mice lacking the C/EBPδ and/or C/EBPδ gene. EMBO J1997;16:7432-7443.
51. Wiper-Bergeron N, Wu D, Pope L, Schild-Poulter C, Haché RJ. Stimulation ofpreadipocyte differentiation by steroid through targeting of an HDAC1complex.EMBO J2003;22:2135-2145.
52. Yoo EJ, Chung JJ, Choe SS, Kim KH, Kim JB. Down-regulation of histonedeacetylases stimulates adipocyte differentiation. J Biol Chem2006;281:6608-
6615.
53. Haberland M, Montgomery RL, Olson EN. The many roles of histonedeacetylases in development and physiology: implications for disease andtherapy. Nat Rev Genet2009;10:32-42.
54. Zuo Y, Qiang L, Farmer SR. Activation of CCAAT/enhancer-binding protein(C/EBP) alpha expression by C/EBP beta during adipogenesis requires aperoxisome proliferator-activated receptor-gamma-associated repression ofHDAC1at the C/EBP alpha gene promoter. J Biol Chem2006;281:7960-7967.
55. Lu K, Yin X, Weng T, et al. Targeting WW domains linker of HECT-typeubiquitin ligase Smurf1for activation by CKIP-1. Nature Cell Biol2008;10:994-1002.
56. Olsten ME, Canton DA, Zhang C, et al. The Pleckstrin homology domain ofCK2interacting protein-1is required for interactions and recruitment of proteinkinase CK2to the plasma membrane. J Biol Chem2004;279:42114-42127.
57. Safi A, Vandromme M, Caussanel S, Valdacci L, Baas D, Vidal M, Brun G,Schaeffer L, Goillot E. Role for the pleckstrin homology domain-containingprotein CKIP-1in phosphatidylinositol3-kinase-regulated muscle differentiation.Mol Cell Biol2004;24:1245-1255.
58. Canton DA, Olsten ME, Kim K, Doherty-Kirby A, Lajoie G, Cooper JA,Litchfield DW. The pleckstrin homology domain-containing protein CKIP-1isinvolved in regulation of cell morphology and the actin cytoskeleton andinteraction with actin capping protein. Mol Cell Biol2005;25:3519-3534.
59. Canton DA, Olsten ME, Niederstrasser H, Cooper JA, Litchfield DW. The roleof CKIP-1in cell morphology depends on its interaction with actin-cappingprotein. J Biol Chem2006;281:36347-36359.
60. Tokuda E, Fujita N, Oh-hara T, Sato S, Kurata A, Katayama R, Itoh T, TakenawaT, Miyazono K, Tsuruo T. Casein kinase2-interacting protein-1, a novel Aktpleckstrin homology domain-interacting protein,down-regulates PI3K/Aktsignaling and suppresses tumor growth in vivo. Cancer Res2007;67:9666-9676.
61. Yu Y, Zhang C, Zhou G, Wu S, Qu X, Wei H, Xing G, Dong C, Zhai Y, Wan J,Ouyang S, Li L, Zhang S, Zhou K, Zhang Y, Wu C, He F. Gene expressionprofiling in human fetal liver and identification of tissue-and developmental-stage-specific genes through compiled expression profiles and efficient cloningof full-length cDNAs. Genome Res2001;11:1392-1403.
62. Zhang L, Xing G, Tie Y, Tang Y, Tian C, Li L, Sun L, Wei H, Zhu Y, He F. Rolefor the pleckstrin homology domain-containing protein CKIP-1in AP-1regulation and apoptosis. EMBO J2005;24:766-778.
63. Zhang L, Tie Y, Tian C, Xing G, Song Y, Zhu Y, Sun Z, He F. CKIP-1recruitsnuclear ATM partially to the plasma membrane through interaction with ATM.Cell Signal2006;18:1386-1395.
64. Xi S, Tie Y, Lu K, Zhang M, Yin X, Chen J, Xing G, Tian C, Zheng X, He F,Zhang L. N-terminal PH domain and C-terminal auto-inhibitory region ofCKIP-1coordinate to determine its nucleus-plasma membrane shuttling. FEBSLett2010;584:1223-30.
65. Elks ML, Manganiello VC. A role for soluble cAMP phosphodiesterases indifferentiation of3T3-L1adipocytes. J Cell Physiol1985;124:191-198.
66. Nakae J, Kitamura T, Kitamura Y, Biggs WH3rd, Arden KC, Accili D. Theforkhead transcription factor Foxo1regulates adipocyte differentiation. Dev Cell2003;4:119-129.
67. Hotamisligil G. S., In Diabetes Mellitus,(Lippincott Williams&Wilkins,Philadelphia,2003), pp.953–962.
68. Hirosumi J, Tuncman G, Chang L, G rgün CZ, Uysal KT, Maeda K, Karin M,Hotamisligil GS. A central role for JNK in obesity and insulin resistance. Nature2002;420:333-336.
69. Ozcan U, Cao Q, Yilmaz E, Lee AH, Iwakoshi NN, Ozdelen E, Tuncman G,G rgün C, Glimcher LH, Hotamisligil GS. Endoplasmic reticulum stress linksobesity, insulin action, and type2diabetes. Science2004;306:457-461.
70. Simons A, Melamed-Bessudo C, Wolkowicz R, Sperling J, Sperling R,Eisenbach L, Rotter V. PACT: cloning and characterization of a cellular p53binding protein that interacts with Rb. Oncogene1997;14:145-155.
71. Witte MM, Scott RE. The proliferation potential protein-related (P2P-R) genewith domains encoding heterogeneous nuclear ribonucleoprotein association andRb1binding shows repressed expression during terminal differentiation. ProcNatl Acad Sci USA1997;94:1212-1217.
72. Pugh DJ, Ab E, Faro A, Lutya PT, Hoffmann E, Rees DJ. DWNN, a novelubiquitin-like domain, implicates RBBP6in mRNA processing and ubiquitin-like pathways. BMC Struct Biol2006;6:1.
73. Gao S, Witte MM, Scott RE. P2P-R protein localizes to the nucleolus ofinterphase cells and the periphery of chromosomes in mitotic cells which showmaximum P2P-R immunoreactivity. J Cell Physiol2002;191:145-154.
74. Gao S, Scott RE. P2P-R protein overexpression restricts mitotic progression atprometaphase and promotes mitotic apoptosis. J Cell Physiol2002,193:199-207.
75. Gao S, Scott RE. Stable overexpression of specific segments of the P2P-Rprotein in human MCF-7cells promotes camptothecin-induced apoptosis. J CellPhysiol2003;197:445-452.
76. Scott RE, Gao S. P2P-R deficiency modifies nocodazole-induced mitotic arrestand UV-induced apoptosis. Anticancer Res2002;22:3837-3842.
77. Li L, Deng B, Xing G, Teng Y, Tian C, Cheng X, Yin X, Yang J, Gao X, Zhu Y,Sun Q, Zhang L, Yang X, He F. PACT is a negative regulator of p53andessential for cell growth and embryonic development. Proc Natl Acad Sci U S A2007;104:7951-7956.
78. Rosen ED, Hsu CH, Wang X, Sakai S, Freeman MW, Gonzalez FJ, SpiegelmanBM. C/EBPalpha induces adipogenesis through PPARgamma: a unified pathway.Genes Dev2002;16:22–26.
79. Salma N, Xiao H, Mueller E, Imbalzano AN. Temporal recruitment oftranscription factors and SWI/SNF chromatin-remodeling enzymes duringadipogenic induction of the peroxisome proliferator-activated receptor gammanuclear hormone receptor. Mol Cell Biol2004;24:4651–4663.
80. Hollenberg AN, Susulic VS, Madura JP, Zhang B, Moller DE, Tontonoz P, SarrafP, Spiegelman BM, Lowell BB. Functional antagonism between C/EBPalpha andperoxisome proliferator-activated receptor-gamma on the leptin promoter. J BiolChem1997;272:5283–5290.
81. Kronenberg HM. Developmental regulation of the growth plate. Nature2003;423:332-336.
82. Helms JA, Schneider RA.Cranial skeletal biology. Nature2003;423:326-331.
83. Zelzer E, Olsen BR.The genetic basis for skeletal diseases. Nature2003;423:343-348.
84. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y,Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S,Kishimoto T.Targeted disruption of Cbfa1results in a complete lack of boneformation owing to maturational arrest of osteoblasts. Cell1997;89:755-764.
85. Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, deCrombrugghe B.The novel zinc finger-containing transcription factor osterix isrequired for osteoblast differentiation and bone formation. Cell2002;12:17-29.
86. Soussi T, Béroud C. Assessing TP53status in human tumours to evaluateclinical outcome. Nat Rev Cancer2001;1:233-240.
87. Muller P, Hrstka R, Coomber D, Lane DP, Vojtesek B. Chaperone-dependentstabilization and degradation of p53mutants. Oncogene2008,27:3371-3383.