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p35 Is Required for CDK5 Activation in Cellular Senescence*

  • Daqin Mao
    Affiliations
    Molecular Oncology Research Institute, Tufts Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111

    Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111
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  • Philip W. Hinds
    Correspondence
    To whom correspondence should be addressed: Tufts Medical Center, 800 Washington St. no. 5609, Boston, MA 02111. Tel.: 617-636-7947; Fax: 617-636-7813
    Affiliations
    Molecular Oncology Research Institute, Tufts Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants CA104322 and AG020208 (to P. W. H.).
Open AccessPublished:February 24, 2010DOI:https://doi.org/10.1074/jbc.M109.066118
      The retinoblastoma tumor suppressor gene (RB-1) is a key regulator of cellular senescence. Expression of the retinoblastoma protein (pRB) in human tumor cells that lack it results in senescence-like changes. The induction of the senescent phenotype by pRB requires the postmitotic kinase CDK5, the best known function of which is in neuronal development and postmitotic neuronal activities. Activation of CDK5 in neurons depends on its activators p35 and p39; however, little is known about how CDK5 is activated in non-neuronal senescent cells. Here we report that p35 is required for the activation of CDK5 in the process of cellular senescence. We demonstrate that: (i) p35 is expressed in osteosarcoma cells, (ii) p35 is required for CDK5 activation induced by pRB during senescence, (iii) p35 is required for the senescent morphological changes in which CDK5 is known to be involved as well as for expression of the senescence secretome, and (iv) p35 is up-regulated in senescing cells. Taken together, these results suggest that p35 is at least one of the activators of CDK5 that is mobilized in the process of cellular senescence, which may provide insight into cancer cell proliferation and future cancer therapeutics.

      Introduction

      Cellular senescence was originally described as the process of cell cycle arrest that accompanies the exhaustion of replicative potential in cultured somatic cells (
      • Hayflick L.
      ). Senescent cells display characteristic changes in cell morphology, physiology, gene expression, and typically express a senescent-associated β-galactosidase (SA-β-gal)
      The abbreviations used are: SA-β-gal
      senescence-associated β-galactosidase
      RB-1
      retinoblastoma gene
      pRB
      retinoblastoma protein
      CDK5
      cyclin-dependent kinase 5
      HDF
      human diploid fibroblasts
      MMP
      matrix metalloproteinase.
      activity (
      • Dimri G.P.
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      • Basile G.
      • Acosta M.
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      ). Although the term replicative senescence indicates the widely accepted model of a terminal growth arrest because of telomere attrition, an apparently identical process called premature senescence can be acutely produced in response to activated oncogenes, DNA damage, oxidative stress, and suboptimal cell culture conditions (
      • Campisi J.
      • d'Adda di Fagagna F.
      ). These observations imply that senescence is a cellular response to stress that limits the proliferation of damaged cells. Based on such antiproliferative effects, cellular senescence was proposed to be a tumor-suppressive, fail-safe mechanism that shares conceptual and possibly therapeutic similarities with the apoptosis machinery (
      • Campisi J.
      ,
      • Mathon N.F.
      • Lloyd A.C.
      ,
      • Braig M.
      • Schmitt C.A.
      ). There is now substantial evidence that cellular senescence is a bona fide barrier to tumorigenesis and cells must overcome it to progress to full-blown malignancy. For example, recent studies suggest that oncogene-induced senescence occurs and suppresses tumorigenesis in vivo. Together, the findings identify senescent cells in premalignant hyperplastic lesions but not in malignant ones, and show that oncogene-induced senescence potently restricts tumor progression at an early stage. Mutations in certain tumor suppressor genes compromise senescence, thereby contributing to cell immortalization and cancer (
      • Braig M.
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      • Kuilman T.
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      • Bartkova J.
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      • Takaoka M.
      • Nakagawa H.
      • Tort F.
      • Fugger K.
      • Johansson F.
      • Sehested M.
      • Andersen C.L.
      • Dyrskjot L.
      • Ørntoft T.
      • Lukas J.
      • Kittas C.
      • Helleday T.
      • Halazonetis T.D.
      • Bartek J.
      • Gorgoulis V.G.
      ). Furthermore, cytotoxic agents used in cancer chemotherapy can induce cellular senescence, and defects in this process contribute to drug resistance in vivo (
      • Chang B.D.
      • Broude E.V.
      • Dokmanovic M.
      • Zhu H.
      • Ruth A.
      • Xuan Y.
      • Kandel E.S.
      • Lausch E.
      • Christov K.
      • Roninson I.B.
      ,
      • Schmitt C.A.
      • Fridman J.S.
      • Yang M.
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      ,
      • te Poele R.H.
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      ,
      • Roberson R.S.
      • Kussick S.J.
      • Vallieres E.
      • Chen S.Y.
      • Wu D.Y.
      ).
      The RB-1 and p53 tumor suppressors are important senescence regulators. p16INK4a/pRB and p14ARF/p53 pathways are typically activated during senescence, and enforced expression of components of either signaling pathway induces senescence in some cell types (
      • Stein G.H.
      • Beeson M.
      • Gordon L.
      ,
      • Lin A.W.
      • Barradas M.
      • Stone J.C.
      • van Aelst L.
      • Serrano M.
      • Lowe S.W.
      ,
      • Stein G.H.
      • Drullinger L.F.
      • Soulard A.
      • Dulić V.
      ,
      • Lin A.W.
      • Lowe S.W.
      ,
      • Kelly-Spratt K.S.
      • Gurley K.E.
      • Yasui Y.
      • Kemp C.J.
      ,
      • Dimri G.P.
      • Itahana K.
      • Acosta M.
      • Campisi J.
      ,
      • Ferbeyre G.
      • de Stanchina E.
      • Lin A.W.
      • Querido E.
      • McCurrach M.E.
      • Hannon G.J.
      • Lowe S.W.
      ). Oncogenic lesions that disable these tumor suppressor systems bypass senescence (
      • Shay J.W.
      • Pereira-Smith O.M.
      • Wright W.E.
      ,
      • Brown J.P.
      • Wei W.
      • Sedivy J.M.
      ,
      • Serrano M.
      • Lin A.W.
      • McCurrach M.E.
      • Beach D.
      • Lowe S.W.
      ,
      • Brookes S.
      • Rowe J.
      • Ruas M.
      • Llanos S.
      • Clark P.A.
      • Lomax M.
      • James M.C.
      • Vatcheva R.
      • Bates S.
      • Vousden K.H.
      • Parry D.
      • Gruis N.
      • Smit N.
      • Bergman W.
      • Peters G.
      ,
      • Rheinwald J.G.
      • Hahn W.C.
      • Ramsey M.R.
      • Wu J.Y.
      • Guo Z.
      • Tsao H.
      • De Luca M.
      • Catricalà C.
      • O'Toole K.M.
      ,
      • Beauséjour C.M.
      • Krtolica A.
      • Galimi F.
      • Narita M.
      • Lowe S.W.
      • Yaswen P.
      • Campisi J.
      ). Significantly, the role of p16INK4a/pRB in the senescence of primary cells can be recapitulated in tumor cells. The reintroduction of pRB or p16INK4a into tumor cells that lack either protein induces a premature senescence requiring p21CIP1 or, in the absence of an intact p53 pathway, p27KIP1 (
      • Xu H.J.
      • Zhou Y.
      • Ji W.
      • Perng G.S.
      • Kruzelock R.
      • Kong C.T.
      • Bast R.C.
      • Mills G.B.
      • Li J.
      • Hu S.X.
      ,
      • Dai C.Y.
      • Enders G.H.
      ,
      • Alexander K.
      • Hinds P.W.
      ). Intriguingly, cyclin-dependent kinase inhibitors like p14ARF, p21CIP1, and p27KIP1, which are required for senescence, can induce markers of senescence on their own. However, they cannot mediate the senescent shape change, demonstrating that these two processes in senescence are separable (
      • Alexander K.
      • Hinds P.W.
      ,
      • Dulić V.
      • Beney G.E.
      • Frebourg G.
      • Drullinger L.F.
      • Stein G.H.
      ,
      • Collado M.
      • Medema R.H.
      • Garcia-Cao I.
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      • Barradas M.
      • Glassford J.
      • Rivas C.
      • Burgering B.M.
      • Serrano M.
      • Lam E.W.
      ).
      Using several model systems of senescence, including long-term passage and acute expression of Ras or pRB, work in our laboratory has shown that cyclin-dependent kinase 5 (CDK5), a serine/threonine kinase that displays kinase activity predominantly in postmitotic neurons, plays a central role in the morphology change of senescent cells (
      • Alexander K.
      • Yang H.S.
      • Hinds P.W.
      ,
      • Yang H.S.
      • Hinds P.W.
      ,
      • Yang H.S.
      • Hinds P.W.
      ). Expression of pRB in pRB-deficient SAOS-2 cells activates CDK5 during the course of senescence. Induction of CDK5 activity leads to the phosphorylation and activation of the ERM family member, Ezrin, as well as the repression of Rac GTPase activation, which are coincident with acquisition of the pRB-induced senescent phenotypes. However, little is known about how CDK5 is activated in senescent cells induced by pRB.
      In this study, we show that p35, one of the known activators of CDK5 in neurons, is required for CDK5 activation and the cell morphology change in pRB-induced SAOS-2 senescence. An increase of p35 at the mRNA level was also detected upon pRB expression in SAOS-2 cells, as well as in senescing IMR90 human diploid fibroblasts after long-term passage. These results further support a role for the CDK5/p35 pathway in regulating cellular senescence, which may provide insight into the regulatory mechanism underlying the induction of the senescent phenotype and its impact on cell proliferation and tumorigenesis.

      DISCUSSION

      We have previously reported the activation of CDK5 in human primary and tumor cells induced to senesce by a variety of stimuli, and the activity of CDK5 is necessary for proper acquisition of the cytoskeletal changes accompanying senescence. Discovery of this role for CDK5 was unexpected, because its activity has primarily been associated with post-mitotic neurons. However, given that CDK5 is ubiquitously expressed in mammalian tissues, an increasing body of evidence has established CDK5 kinase activity and functions in non-neuronal cells. In this report we show the presence of the CDK5 activators, p35 and p39, in our model systems of senescence. Up-regulation of the expression of p35 is concomitant with CDK5 activation in senescing SAOS2 and IMR90 cells. Knockdown of p35 by shRNA markedly suppresses the morphologic changes of senescent SAOS-2 cells induced by pRB, which coincides with a decrease in CDK5 activity. The polymerization of actin filaments is inhibited in the p35 knockdown cells, and the levels of actin and the F-actin-associated Ezrin are reduced as well. These findings underscore a role for CDK5/p35 activity in mediating the cytoskeletal reorganization in the non-neuronal senescent cells. Furthermore, the use of model systems in which senescence is induced by pRB reintroduction in p16Ink4a/pRB-deficient tumor cells strongly implicates the pRB pathway in CDK5/p35 up-regulation at least in part through transcriptional up-regulation of p35. Although the pRB pathway is essential for the transcriptional repression of loci in senescent cells (
      • Narita M.
      • Nũnez S.
      • Heard E.
      • Lin A.W.
      • Hearn S.A.
      • Spector D.L.
      • Hannon G.J.
      • Lowe S.W.
      ), its role in the induction of gene expression in senescence is poorly understood. Because pRB/E2F complexes are usually repressive, they most likely do not directly regulate the genes that are highly expressed by senescent cells, although they could indirectly control such genes, for example, by silencing a repressor. At present, we have been unable to test for a direct role for pRB in regulating p35 mRNA production, as reporter constructs containing the p35 promoter do not respond to pRB (data not shown). It is possible that pRB effects on the p35 promoter, whether direct or indirect, require an appropriate chromatin context as observed for differentiation-specific promoters (
      • Thomas D.M.
      • Carty S.A.
      • Piscopo D.M.
      • Lee J.S.
      • Wang W.F.
      • Forrester W.C.
      • Hinds P.W.
      ).
      Senescent cells show striking changes in gene expression. Interestingly, many changes in gene expression appear not to be directly related to growth arrest. Despite the universality of morphological changes observed in a wide variety of senescent cells, little is known about the potential contribution of this phenotype to the establishment or maintenance of the irreversible growth arrest that accompanies senescence. Significant additional studies of the role of cytoskeletal rearrangement in the biochemical and proliferative aspects typical of senescence are needed to fully appreciate the role of CDK5/p35 in this process. The role of CDK5/p35 activity in regulating the differentiation of monocytes might provide some clues. In promyelocytic HL60 cells, treatment with 1,25-dihydroxyvitamin D3 (1,25D3) results in the up-regulation of the Egr1 gene, which in turn activates p35 transcription and expression, and subsequently enhances CDK5 activity. p35-associated CDK5 phosphorylates MEK1 on Thr-286, preventing MAPK/ERK phosphorylation and cell proliferation. However, MEK1 phosphorylation at Thr-286 requires prior Ser-218 and Ser-222 phosphorylation and activation by Raf1 induced by growth factors or cytokines. These events are suggested to result in the up-regulation of p27 and eventually, monocytic differentiation (
      • Chen F.
      • Wang Q.
      • Wang X.
      • Studzinski G.P.
      ,
      • Rosales J.L.
      • Lee K.Y.
      ). Given the established role of pRB in osteoblast differentiation (
      • Thomas D.M.
      • Carty S.A.
      • Piscopo D.M.
      • Lee J.S.
      • Wang W.F.
      • Forrester W.C.
      • Hinds P.W.
      ), it is possible that complex mechanisms such as these are stimulated by pRB in senescing mesenchymal cells that are unable to properly differentiate.
      There is now substantial evidence that induction of senescence constitutes an important block to tumor progression. It has also become clear that senescent cells have characteristic alterations in secreted growth factors, inflammatory cytokines, extracellular-matrix components, and matrix-degrading enzymes, which could influence the growth of adjacent neighboring tumor cells by altering the tissue microenvironment (
      • Campisi J.
      • d'Adda di Fagagna F.
      , ). We find that p35/cdk5 can influence this senescence secretome, implicating cdk5 in the physiological response to this aspect of senescence. Further, recent studies suggest that, as with CDK5 in neurons, the CDK5 activity in non-neuronal cells may influence phenotypic changes mostly through its direct or indirect effect on the organization of cytoskeletal structures. CDK5 regulates cellular processes such as cell-cell and cell-matrix adhesion, cell migration and wound healing in a variety of tissues (
      • Rosales J.L.
      • Lee K.Y.
      ). Thus the induction of senescent shape change may also impact the neighboring tumor cells and their microenvironment.
      Tumor cells are exposed to many sources of stress, especially those derived from the aberrant proliferative signals of oncogenes. As one of the cellular responses to these stresses, senescence has emerged as a compelling target for future cancer therapeutics and may determine the response of tumor cells to chemotherapeutic drugs. Because senescence effector mechanisms are still largely unknown, understanding the molecular mechanisms through which CDK5 affects cellular senescence may give clues to potential therapeutic approaches for cancer and age-related diseases. Finally, the intriguing relationship of this aspect of senescence to neuronal and non-neuronal differentiation (
      • Chen F.
      • Wang Q.
      • Wang X.
      • Studzinski G.P.
      ,
      • Lazaro J.B.
      • Kitzmann M.
      • Poul M.A.
      • Vandromme M.
      • Lamb N.J.
      • Fernandez A.
      ,
      • Sarker K.P.
      • Lee K.Y.
      ) may help to unravel the role of senescence effectors in the physiology of tissues that accumulate a senescent cell burden following acute or chronic stress.

      Acknowledgments

      We thank Li-Huei Tsai for critical CDK5-related reagents.

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