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Home医源资料库在线期刊循环研究杂志2005年第95卷第7期

Notch-Dependent Cell Cycle Arrest Is Associated With Downregulation of Minichromosome Maintenance Proteins

来源:循环研究杂志
摘要:MaterialsandMethodsCellCultureThehumanmicrovascularendothelialcellline,HMEC-1(HMEC)andhumanumbilicalveinendothelialcells(HUVEC)wereculturedaspreviouslydescribed。SeminCellDevBiol。MolCellBiol。MolCellBiol。...

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    the Department of Medical Biophysics (M.N., K.N., G.M., L.C., A.K.), Department of Pathology and Laboratory Medicine (A.K.), British Columbia Cancer Agency, Vancouver, Canada
    the Department of Pathology and Laboratory Medicine (M.N., A.K.), Experimental Medicine Program (K.N., G.M., L.C., A.K.), University of British Columbia, Vancouver, Canada.

    Abstract

    Perturbation of the Notch signaling pathway has been implicated in the pathogenesis of human cardiovascular diseases, and animal models have confirmed the requirement of Notch during cardiovascular development. We recently demonstrated that Notch activation delays S-phase entry and contributes to endothelial contact inhibition. Minichromosome maintenance (MCM) proteins, components of the prereplicative complex (pre-RC), are essential for DNA replication. Here, we report that Notch-mediated cell cycle arrest is associated with downregulation of MCM2 and MCM6 in endothelial cells and human fibroblasts. Downregulation of MCM proteins is also observed on activation of C promoter binding factor (CBF1) and is mediated by inhibition of Rb phosphorylation, as demonstrated using a constitutively active Rb mutant. Although the effects of the Notch pathway are cell-type specific and context-dependent, in cell types where Notch has an antiproliferative effect, downregulation of MCM proteins may be a common mechanism to inhibit DNA replication.

    Key Words: minichromosome maintenance proteins  cell cycle  Notch  endothelial cells  retinoblastoma protein

    Introduction

    Intercellular signaling mediated by Notch receptors is essential for proper development and homeostasis of the cardiovascular system; indeed, perturbations of the Notch pathway have been implicated in the pathogenesis of several cardiovascular diseases.1eC3 We have recently shown that activation of Notch in endothelial cells has an antiproliferative effect which may explain the defects of vascular remodeling consequent to dysregulation of Notch activation.3,4 In endothelial cells, Notch delays progression toward S-phase through a mechanism that depends, at least in part, on improper subcellular localization of the cyclin D-cdk4 complex secondary to downregulation of p21Cip1.4 Given that this mechanism seems to be endothelial-specific and targeted downregulation of p21Cip1 by short interfering RNA induces a less efficient block of S-phase entry compared with Notch activation, we postulated that additional mechanisms may contribute to Notch-mediated cell cycle arrest.4

    Minichromosome maintenance (MCM) proteins 2 to 7 form a complex with helicase activity and participate in the formation of prereplicative complexes (pre-RCs) that allow chromatin licensing to ensure that DNA replication initiates at specific sites.5 Thus, MCM proteins are essential for DNA replication and cell cycle progression.5. Indeed, inactivation of MCM2 in Drosophila reduces proliferation in the developing central nervous system and microinjection of antibodies targeting MCM3 and MCM2 inhibits DNA replication.5 Here, we identify repression of MCM2 and MCM6 as a mechanism of Notch-mediated cell cycle arrest.

    Materials and Methods

    Cell Culture

    The human microvascular endothelial cell line, HMEC-1 (HMEC) and human umbilical vein endothelial cells (HUVEC) were cultured as previously described.4,6 Primary human foreskin fibroblasts (HFF) were cultured in Dulbecco’s modified Eagle’s medium with 10% heat inactivated fetal bovine serum (HyClone).

    Plasmids and Gene Transfer

    HUVEC and HFF were transduced using Ampho-Phoenix packaging cells.4 Expression of Notch4IC, Notch1IC, CBF1-VP16, and RbK11 proteins were confirmed by immunoblotting (data not shown). For a description of plasmids, see Material and Methods in the online data supplement at http://circres.ahajournals.org.

    Immunoblotting and Immunofluorescence

    Immunostaining was performed as previously described.4 For list of antibodies used see Material and Methods in the online data supplement.

    Statistical Analysis

    To determine statistical significance, a Student t test for comparison between 2 groups was used, whereas a 1-way ANOVA with a Tukey test was used for multiple comparisons. Statistical significance was taken at P=0.05.

    Results and Discussion

    To identify new pathways mediating the Notch antiproliferative effect, HMEC transduced with vector alone (HMEC-pLNCX) or vector encoding the active intracellular portion of the endothelial-specific Notch4 (HMEC-pLNC-Notch4IC) were analyzed using high-throughput immunoblotting (Power Blot, BD Pharmingen).7 MCM2 and MCM6 were consistently downregulated in the HMEC-pLNC-Notch4IC compared with HMEC-pLNCX (online Table I). As we have previously shown, fibronectin was found to be highly upregulated in HMEC-pLNC-Notch4IC (online Table I).8 These findings were validated in HUVEC transduced with empty vector, vector encoding activated Notch4 (Notch4IC) or Notch1 (Notch1IC), because both Notch family members block S-phase entry.4 Both active Notch4 and Notch1 downregulated MCM2 and MCM6 (Figure 1A and online Figure SIA). Together with previous studies, these data suggest that Notch-mediated endothelial cell cycle arrest is effected by multiple pathways that impinge on DNA replication via downregulation of members of the pre-RC.

    Binding of NotchIC to CBF1, the best described effector of the Notch pathway, induces derepression/activation of CBF1 target gene transcription.9 Notch activation in endothelial cells triggers CBF1 activity, and the Notch pathway induces endothelial quiescence partly by a mechanism that impedes phosphorylation of Rb.4,10 We used a constitutively-active form of CBF1, obtained by fusing the CBF1 cDNA with the transcriptional activation domain of herpes virus protein, VP16 (CBF1-VP16), to determine whether Notch-mediated cell cycle arrest could be mimicked by CBF1 activation alone.10 HUVEC-CBF1-VP16 showed reduction of the proportion of cells entering S-phase and of cells expressing phosphorylated Rb, compared with the HUVEC-vector, suggesting that CBF1-VP16 impedes cell cycle progression at least in part via inhibition of Rb phosphorylation (Figure 1B). As well, CBF1-VP16 markedly repressed MCM2 and MCM6 expression (Figure 1C and online Figure SIB). These data demonstrate that activation of CBF1 is sufficient to inhibit Rb phosphorylation and repress MCM2 and MCM6.

    MCM proteins are essential for progression of the cell cycle, but the expression of MCM2 and MCM6 in proliferating and arrested endothelial cells has not been examined. Rb phosphorylation can be used as an indicator of actively proliferating cells, because it is required for progression toward S-phase. Rb phosphorylation was abolished in contact-inhibited compared with logarithmically growing HUVEC. Rb hypophosphorylation directly correlated with the downregulation of MCM2 and MCM6 (Figure 2A). Thus, expression of MCM2 and MCM6 is regulated in concert with Rb phosphorylation and the proliferative status of endothelial cells.

    Overexpression of E2F transcription factors, which are released on Rb phosphorylation, induce MCM expression.11,12 Because both Notch activation and activated CBF1 inhibit Rb phosphorylation, we tested whether persistently hypophosphorylated Rb, which does not release E2F, is sufficient to repress MCM2 and MCM6 expression. Expression of a phosphorylation-resistant Rb mutant with 11 serine/threonine to alanine substitutions (RbK11) inhibits cell proliferation and E2F transcriptional activity similar to the constitutively-active and hypophosphorylated native Rb (data not shown).13 Attenuation of phosphorylation of endogenous Rb in endothelial cells expressing RbK11, using an antibody specific for 2 phospho-acceptor sites that have been mutated in RbK11, confirmed that this construct maintains Rb in a hypophosphorylated state (Figure 2B). HUVEC transduced with RbK11 (HUVEC-RbK11) show downregulation of MCM2 and MCM6 compared with cells transduced with vector alone (HUVEC-vector) (Figure 2C). Hence, Notch-mediated CBF1-dependent inhibition of Rb phosphorylation appears sufficient to mediate downregulation of MCM2 and MCM6.

    The effects of Notch activation on proliferation can be stimulatory or inhibitory depending on the cell type, and the mechanisms mediating cell cycle inhibition can be cell-type specific.4,14 Thus, we sought to determine whether the Notch/CBF1/Rb-dependent mechanism impeding cell cycle progression is conserved in fibroblasts. Active Notch1 reduced Rb phosphorylation, reduced the proportion of HFF entering S-phase, and downregulated MCM2 and MCM6 (online Figure SIC, SIIA, and SIIB). Similarly, constitutively active CBF1-VP16 inhibited S-phase entry and Rb phosphorylation, as well as downregulated MCM2 and MCM6 expression (online Figure SID, SIIC, and SIID). Finally, we confirmed that RbK11 also downregulates MCM2 and MCM6 in HFF (online Figure SID and SIIE). Together these results suggest that inhibition of Rb phosphorylation and downregulation of MCM proteins via a CBF1-dependent mechanism are conserved elements of Notch-mediated cell cycle arrest in at least 2 different cell types.

    Taken together our results suggest that Notch activation triggers an axis of events that, through the activation of CBF1, interferes with Rb phosphorylation and results in downregulation of MCM2 and MCM6 in endothelial cells and fibroblasts. However, fibroblasts do not show suppression of p21Cip1 after Notch activation (data not shown). Hence, repression of MCM proteins may represent a common downstream mechanism for Notch-mediated cell cycle arrest in some cell types. It remains to be established what the effect on MCM expression is in cell types that are stimulated to grow after Notch activation. However, Notch- and CBF1-dependent hypophosphorylation of Rb with consequent downregulation of MCM proteins could be a downstream effect of the arrest in G0/G1 caused by mechanisms that remain to be elucidated. Nevertheless, these results show that there is a functional correlation between 2 highly conserved cellular pathways: the Notch pathway that regulates cell fate through an intercellular signaling mechanism and the ancestral MCM proteins that are essential for initiation of DNA replication.

    Acknowledgments

    This research was supported by grants to A.K. from the National Cancer Institute of Canada with funds from the Canadian Cancer Society and the Heart and Stroke Foundation of British Columbia and the Yukon. M.N. was supported by a fellowship from the Canadian Institutes of Health Research and a Research Trainee Award from the Michael Smith Foundation for Health Research and G.M. by a Research Trainee Award from the Michael Smith Foundation for Health Research. A.K. is a Scholar of the Michael Smith Foundation for Health Research. We thank Denise McDougal for assistance with cell sorting and Dr B. Larriveee for providing HFF.

    References

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    Krebs LT, Xue Y, Norton CR, Shutter JR, Maguire M, Sundberg JP, Gallahan D, Closson V, Kitajewski J, Callahan R, Smith GH, Stark KL, Gridley T. Notch signaling is essential for vascular morphogenesis in mice. Genes Dev. 2000; 14: 1343eC1352.

    Uyttendaele H, Ho J, Rossant J, Kitajewski J. Vascular patterning defects associated with expression of activated Notch4 in embryonic endothelium. Proc Natl Acad Sci U S A. 2001; 98: 5643eC5648.

    Noseda M, Chang L, McLean G, Grim JE, Clurman BE, Smith LL, Karsan A. Notch activation induces endothelial cell cycle arrest and participates in contact inhibition: role of p21Cip1 repression. Mol Cell Biol. 2004; 24: 8813eC8822.

    Tye BK. MCM proteins in DNA replication. Annu Rev Biochem. 1999; 68: 649eC686.

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    Krummrei U, Baulieu EE, Chambraud B. The FKBP-associated protein FAP48 is an antiproliferative molecule and a player in T cell activation that increases IL2 synthesis. Proc Natl Acad Sci U S A. 2003; 100: 2444eC2449.

    Noseda M, McLean G, Niessen K, Chang L, Pollet I, Montpetit R, Shahidi R, Dorovini-Zis K, Li L, Beckstead B, Durand RE, Hoodless PA, Karsan A. Notch activation results in phenotypic and functional changes consistent with endothelial-to-mesenchymal transformation. Circ Res. 2004; 94: 910eC917.

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    MacKenzie F, Duriez P, Wong F, Noseda M, Karsan A. Notch4 inhibits endothelial apoptosis via RBP-Jkappa-dependent and -independent pathways. J Biol Chem. 2004; 279: 11657eC11663.

    Leone G, DeGregori J, Yan Z, Jakoi L, Ishida S, Williams RS, Nevins JR. E2F3 activity is regulated during the cell cycle and is required for the induction of S phase. Genes Dev. 1998; 12: 2120eC2130.

    Ohtani K, Iwanaga R, Nakamura M, Ikeda M, Yabuta N, Tsuruga H, Nojima H. Cell growth-regulated expression of mammalian MCM5 and MCM6 genes mediated by the transcription factor E2F. Oncogene. 1999; 18: 2299eC2309.

    Jiang Z, Zacksenhaus E. Activation of retinoblastoma protein in mammary gland leads to ductal growth suppression, precocious differentiation, and adenocarcinoma. J Cell Biol. 2002; 156: 185eC198.

    Rangarajan A, Talora C, Okuyama R, Nicolas M, Mammucari C, Oh H, Aster JC, Krishna S, Metzger D, Chambon P, Miele L, Aguet M, Radtke F, Dotto GP. Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation. Embo J. 2001; 20: 3427eC3436.

作者: Michela Noseda, Kyle Niessen, Graeme McLean, Linda 2007-5-18
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