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Molecular and Cellular Biology, April 2004, p. 2808-2819, Vol. 24, No. 7
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.7.2808-2819.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cellular Senescence Requires CDK5 Repression of Rac1 Activity

Kamilah Alexander,{dagger} Hai-Su Yang,{dagger},{ddagger} and Philip W. Hinds*

Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

Received 1 August 2003/ Returned for modification 5 September 2003/ Accepted 22 December 2003


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ABSTRACT
 
Cellular senescence is a tumor-suppressive process characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated ß-galactosidase (SA-ß-Gal). We report here a role for CDK5 in induction of senescent cytoskeletal changes. CDK5 activation is upregulated in senescing cells. The increased activity of CDK5 further reduces GTPase Rac1 activity and Pak activation. The repression of the activity of the GTPase Rac1 by CDK5 is required for expression of the senescent phenotype. CDK5 regulation of Rac1 activity is necessary for actin polymerization accompanying senescent morphology in response to expression of pRb, activated Ras, or continuous passage. Inhibition of CDK5 attenuates SA-ß-Gal expression and blocks actin polymerization. These results point to a unique, nonneuronal role for CDK5 in regulation of Rac1 activity in senescence, illuminating the mechanisms underlying induction of senescence and the senescent shape change.


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INTRODUCTION
 
Somatic cells undergo a set number of cell divisions and subsequently stop dividing. This proliferative exhaustion of cells, termed cellular replicative senescence, has been observed upon the culture of many cell types from a variety of species (6). Senescent cells can typically be identified by an enlarged, flattened phenotype and the expression of an enzymatic ß-galactosidase activity at pH 6.0 (SA-ß-Gal) of unknown regulation. In addition, senescent cells are characterized by an irreversible G1 growth arrest involving the repression of genes that drive cell cycle progression and the upregulation of cell cycle inhibitors like p16INK4a, p53, and its transcriptional target, p21CIP1 (4). The irreversibility of the senescent cellular state suggests that senescence may be antioncogenic. The adoption of this terminal exit from the cell cycle in response to cellular stress could prevent the accumulation of deleterious mutations that lead to cellular immortality and malignant transformation (22). Consistent with this model, common tumor suppressors have been implicated in senescence.

In mouse cells, the loss of the p19ARF/p53 pathway is critical for cellular immortalization, while in human cells, the bypass of senescence requires inactivation of the p16INK4a/pRb pathway (22). Indeed, these negative regulators of proliferation accumulate with continued cell division in cultured primary cells, suggesting that they are part of a mechanism that counts the number of cell divisions and thus limits proliferative capacity. The initiating signal of this molecular clock has been attributed to the shortening of telomeres, the progressive erosion of which is thought to eventually trigger growth arrest. In support of this hypothesis, hTERT overexpression and the subsequent restoration of telomerase activity block telomere shortening and ultimately immortalize cells (3). In addition, though hTERT activity is undetectable in normal cells, it is upregulated in tumor cells, further suggesting that dysregulation of hTERT activity is involved in the malignant transformation of cells (20, 25). Significantly, it has been found that immortal cells arising out of hTERT-transduced cells require spontaneous or oncogene-induced disruption of the pRb pathway (10, 19, 30, 34). Thus, pRb likely controls a senescence-instigating pathway that works in synergy with, but is distinct from, that engendered by telomere loss.

The strongest evidence for a telomere-independent senescence pathway is provided by the observation that primary cells will senesce prematurely upon oncogenic stimuli. For instance, the oncogenic, persistently-activated form of the small GTPase Ras, which initially causes proliferation, eventually triggers cell cycle arrest and premature senescence in both mouse embryo fibroblasts and human diploid fibroblasts (HDFs) (38). Ras-induced senescence requires an intact mitogen-activated protein kinase pathway. Indeed, the same senescence effect can be mediated by Ras's downstream effectors, Raf and MEK (21, 38, 47). The senescence induced by Ras is accompanied by an increase in p16INK4a and p19ARF levels and pRb and p53 activation. However, while either p53 or pRb is dispensable in Ras-induced senescence in MEFs, only elimination of pRb function in HDFs by E1A results in senescence inhibition (38). In a conceptually similar but biochemically distinct manner, E2F1 overexpression has been observed to induce a p14ARF-dependent cell cycle arrest and senescence in HDFs (11). However, fibroblasts from patients with constitutive inactivating mutations in p16INK4a are resistant to Ras-induced senescence, although hTERT expression is still required for immortalization (5). Because these cells express functional p14ARF, this work underscores the critical role of the p16INK4a/pRb pathway in premature senescence and its obligate inactivation in the immortalization of human cells.

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 have lost expression of either protein induces a premature senescence requiring p21CIP1 or, in the absence of an intact p53 pathway, p27KIP1 (1, 8, 9). 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 (1, 8, 13).

Despite clear evidence for a role for the p16INK4a/pRb pathway in senescence, little is known about the mechanism by which this pathway commits cells to the senescent phenotype characterized by expression of SA-ß-Gal and morphological alteration. Using several model systems of senescence, including long-term passage and acute expression of Ras or pRb, we have found that CDK5, a kinase that hitherto has been considered to be almost solely involved in regulating neuronal activities, plays a central role in the shape change of senescent cells through regulation of Rac1 activity and influences SA-ß-Gal expression in senescent human cells.


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MATERIALS AND METHODS
 
Cell culture and plasmids. The human osteosarcoma cell line SAOS-2 subclone 2.4 (16) was maintained in Dulbeco's modified Eagle's medium (DMEM) (GIBCO/BRL) supplemented with 15% heat-inactivated fetal bovine serum (FBS). The human breast carcinoma cell line MDA-MB-468 (ATCC) was maintained in L15 medium (Leibovitz) supplemented with 10% FBS. IMR90 and WI38 HDFs were maintained in DMEM supplemented with 10% FBS. Cells were cultured in a 5% CO2 incubator at 37°C. The pSVE and pSVE-RB expression plasmids have been previously described (16, 26, 40, 42). pcDNA3-CDK5 and pcDNA3-dnCDK5 were generous gifts of Li-Huei Tsai. pEBG-RacV12 and pEBG-RacN17 were gifts from the laboratory of John Blenis and have been described previously (7). pBabe-puro and pBabe-RasV12 were provided by the laboratory of Robert Weinberg. Adenovirus constructs Ad-GFP and Ad-RB have been previously described (15). SAOS-2 cells were transfected at 80% confluency with the indicated plasmids by using Effectene (QIAGEN) or Fugene6 (Roche). SAOS-2 transfectants and IMR90 cells infected with pBabe-puro or pBabe-RasV12 were selected with puromycin 24-h posttransfection or infection and maintained under selection for the duration of the experiment.

Immunoblotting and immunoprecipitation. Protein expression was detected by immunoblotting. Cells were lysed in 100 to 200 µl of ELB (50 mM HEPES, [pH 7.2], 250 mM NaCl, 2 mM EDTA, 0.1% NP-40, 1 mM dithiothreitol) plus protease and phosphatase inhibitors (1 mg of aprotinin/ml, 1 µg of leupeptin/ml, 100 µg of phenylmethylsulfonyl fluoride, 4 mM sodium orthovanadate, 2 mM sodium PPi) per 10-cm plate. Protein concentrations in cell lysates were determined by Bio-Rad protein assay. For immunoblotting, 30 to 50 µg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose by standard procedures. Antibodies used for immunoblotting include: anti-CDK5 DC17 (Santa Cruz), anti-Rac1 (Transduction Laboratories), anti-RhoA (Santa Cruz), anti-Cdc42 (Santa Cruz), anti-Pak1 (Santa Cruz), anti-phospho-Pak1 (Sigma) and anti-pRb Ab-5 (Calbiochem). Proteins were detected by using horseradish peroxidase-conjugated donkey anti-mouse or donkey anti-rabbit antibodies (Jackson Immunosciences). One hundred micrograms of cell lysates was immunoprecipitated with the indicated antibody overnight at 4°C, 30 µl of protein A/G beads was added to the immunoprecipitation mixture for 1 h, and then the beads were washed four times with lysate buffer and separated by SDS-PAGE.

Kinase assay. To detect CDK5 kinase activity, immunoprecipitation for CDK5 was performed by using 100 µg of cell lysate with anti-CDK5 F8 (Santa Cruz). The immunoprecipitation mixtures were subjected to an in vitro kinase assay essentially as described previously, with slight modifications (1). One microgram of an N-terminal fragment of PSD-95, bacterially expressed, purified His-N-PDZ1-2S (17), was used as a substrate for immunoprecipitated CDK5 (gift of Li-Huei Tsai). For the Pak1 kinase assay, immunoprecipitation with Pak1 antibody was performed as described previously (27). Briefly, the assay was done in the presence of 1 mM ATP, 1 µCi of [32P-{gamma}]ATP, 3 µg of histone H4, and kinase buffer (50 mM HEPES [pH 7.5], 10 mM MgCl2) for 30 min at 30°C.

siRNA synthesis. The CDK5 small interfering RNA (siRNA) construct was made as described previously by using the pBS/U6 plasmid (39). The oligonucleotides used to create pBS/U6-siCDK5 were as follows: 1a-GGGAGCTGAAATTGGCTGATA, 1b-AGCTTATCAGCCAATTTCAGCTCCCC, 2a-AGCTTATCAGCCAATTTCAGCTCCCCTTTTTG, and 2b-AATTCAAAAAGGGGAGCT-GAAATTGGCTGATA.

GTPase activity assays. The activity of GTP-Rac, GTP-Rho, and GTP-Cdc42 was determined by using an assay developed by Ren and Schwartz (33). The effectors rhotekin and Pak were used as glutathione S-transferase (GST) fusions to affinity precipitate endogenous, cellular, active Rho, and Rac and Cdc42, respectively. The pEGB-Pak1 and -rhotekin plasmids, kind gifts of J. Settleman, were bacterially expressed and bound to GST beads. The assay was performed as previously described (33).

Immunofluorescence. Transfected cells fixed in methanol for 5 min and acetone for 2 min at the indicated times were immunostained as described previously (1). Antiactin (Sigma) was detected with Cy3-conjugated donkey anti-rabbit secondary antibody (Jackson Immunosciences). Fluorescent images were acquired by using DeltaVision deconvolution microscopy.

SA-ß-Gal assay. The SA-ß-Gal assay was performed as previously described (12). Briefly, cells were washed in phosphate-buffered saline and fixed in 2% formaldehyde-0.2% glutaraldehyde. Then the cells were washed and incubated at 37°C overnight with fresh senescence-associated ß-Gal stain solution (1 mg of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside [X-Gal] per ml, 40 mM citric acid-sodium phosphate [pH 6.0], 150 mM NaCl, 2 mM MgCl2, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide). All visualization and photography of cells were performed on a Leica microscope with Sony digital imaging.


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RESULTS
 
The kinase inhibitor roscovitine blocks senescence. As cell cycle arrest accompanied by loss of CDK2 activity is invariably observed in senescent cells, we asked if chemical inhibition of CDKs would augment induction of senescence. To do this, we employed roscovitine, a general kinase inhibitor that at low concentrations specifically inhibits cell cycle-regulating kinases CDK2 and CDK1, causing a G1 cell cycle arrest (24). Unexpectedly, we found that roscovitine inhibited senescence induced by a variety of stimuli.

In the osteosarcoma cell line SAOS-2 lacking wild-type pRb and p53 expression, reintroduction of retinoblastoma protein (pRb) results in induction of SA-ß-Gal 7- to 10-days posttransfection (43). Treatment of SAOS-2 cells 48 h after RB transfection with 10 µM roscovitine for the remaining time course of 7 to 10 days completely inhibited SA-ß-Gal induction (Fig. 1A). Similarly, in the pRb/p53 mutant breast carcinoma cell line MDA-MB-468, infection of the cells with adenovirus RB (Ad-RB) can cause senescence after 7 days (43). Treatment of Ad-RB-infected MDA-MB-468 cells with roscovitine 1 through 6 days after infection inhibited SA-ß-Gal induction by approximately 50% (Fig. 1A). The difference in the effect of roscovitine on the two cell lines may be due to our observation that there are higher expression levels of pRb in the MDA-MB-468-infected cells than in SAOS-2-transfected cells (data not shown). Retroviral infection of the normal HDF strain IMR90 with a constitutively active Ras1 mutant, RasV12, causes cell cycle arrest by 5 days after infection and eventual senescence (38). Treatment of RasV12-infected cells with roscovitine 5 days after infection for a subsequent 5 days reduced SA-ß-Gal expression back to the level of the controls (Fig. 1A). Finally, we tested the effects of roscovitine on senescence induced by the continued passage of HDF strains WI38 and IMR90. Treatment of these cells with roscovitine at early passage would cause cell cycle arrest and prevent their continued passage and senescence. Thus, HDFs that had already become senescent were treated with roscovitine for 48 h and showed a reduction in expression of SA-ß-Gal compared to that for dimethyl sulfoxide-treated cells (Fig. 1A). Similarly, we have seen that pRb-expressing SAOS-2 cells treated with roscovitine after the onset of senescence display a diminution of SA-ß-Gal expression (data not shown) but not complete inhibition. Hence, these results suggest that there are differences in the sensitivity of cells to roscovitine's effects on SA-ß-Gal expression dependent upon whether initiation or maintenance of senescence is being assayed. Overall, these results indicate that roscovitine inhibits a phenotypic marker of senescence under a variety of conditions.



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  		FIG. 1. Roscovitine blocks senescence. (A) SAOS-2 cells were transfected with pBabe-puromycin and pSVE or pSVE-RB, and cells were selected with puromycin. Transfected cells were treated with roscovitine 2- to 10-days posttransfection. MDA-MB-468 cells were infected with Ad-GFP or Ad-RB and treated 1- to 7-days postinfection with roscovitine. IMR90 cells were infected with pBabe-puromycin or pBabe-RasV12 and treated 5- to 10-days posttransfection with roscovitine. Passaged (psg) senescent IMR90 and WI38 cells were treated with roscovitine for 48 h. After being treated with roscovitine, all cells were assayed for SA-ß-Gal expression, and the percentage of SA-ß-Gal-positive cells relative to the total number of cells was determined. DMSO, dimethyl sulfoxide. (B) The change in morphology among cycling presenescent, senescent, and roscovitine-treated senescent cells is shown. All images were obtained by using phase contrast microscopy at a magnification of x60.

Strikingly, roscovitine inhibited not only induction of the SA-ß-Gal marker in these assays but also the senescent morphology change. Senescent cells tend to take on a flattened, enlarged morphology. However, roscovitine treatment of SA0S-2 cells for 2- to 10-days post-RB transfection or MDA-MB-468 cells infected with Ad-RB or senescent IMR90 cells resulted in the formation of small shortened cells larger than wild-type cycling cells but clearly smaller than senescent cells (Fig. 1B). This was also the case with roscovitine-treated, RasV12-infected HDFs and senescent WI38 cells (data not shown). Because expression of SA-ß-Gal and growth arrest in senescent cells can be uncoupled from morphological alteration (1, 8, 13), roscovitine may act early to prevent senescence induction.

CDK5 activity is required for senescence. The drug roscovitine inhibits a variety of cellular kinases in a dose-dependent manner by competing for their ATP-binding domain. At the concentration employed, 10 µM, roscovitine is a specific inhibitor of CDK2, CDK5, and CDK1. However, both CDK2 (1, 8, 13) and CDK1 (K. Alexander and H. Yang, unpublished data) activities are absent in senescent cells, and overexpressed dnCDK2 or dnCDK1 has no inhibitory effect on pRb-induced senescence. Thus, we decided to investigate a possible role for CDK5, a postmitotic kinase known to regulate neuronal cytoskeletal organization (23), in cells undergoing the process of senescence.

SAOS-2 cells transfected with RB or MDA-MB-468 cells infected with Ad-RB were harvested for cell lysates 2, 4, and 7 days after RB reintroduction. Immunoblot analysis showed an increase in CDK5 protein levels as early as 2 days after reintroduction of pRb in both cell lines (Fig. 2A). Performance of a CDK5 immunoprecipitation and a subsequent in vitro kinase assay using a CDK5 substrate, PSD-95 (M. Morabito, personal communication), showed a concomitant increase in CDK5 activity (Fig. 2A) that was inhibited by roscovitine (data not shown). Interestingly, MDA-MB-468 cells, which had higher expression levels of pRb than SAOS-2, also had higher expression levels of CDK5 and much more CDK5 activity. Similarly, in senescent RasV12-infected IMR90 cells, there was a small but detectable increase in CDK5 levels and a comparable increase in CDK5 activity (Fig. 2A). In senescence induced by the passage of IMR90 HDFs, we found no significant changes in CDK5 levels between early passage (EP), middle passage (MP), and senescent (S) cells (Fig. 2A). However, CDK5 activity in IMR90 cells increased at middle passage and remained high in senescent fibroblasts (Fig. 2A). In addition, in both passaged and RasV12-infected fibroblasts, reduction of pRb phosphorylation correlated with increased CDK5 activity (Fig. 2A).



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  		FIG.2. A requirement for CDK5 in senescence. (A) Lysates harvested from cells undergoing senescence by a variety of stimuli were immunoblotted for pRb or CDK5 or immunoprecipitated for CDK5 to perform an in vitro kinase assay using PSD-95 as the substrate. SAOS-2 cells were transfected with pBabe-puromycin and pSVE or pSVE-RB; MDA-MB-468 cells were infected with Ad-GFP or Ad-RB and lysates harvested 2, 4, and 7 days after pRb reintroduction. Lysates from IMR90 cells infected with pBabe-puromycin or pBabe-RasV12 were harvested 10 days after infection and puromycin selection. IMR90 cells were passaged in culture, and cells were harvested for lysates at early passage (EP), middle passage (MP), or after undergoing senescence (S). (B) Cell lysates from SAOS-2 cells transfected with pRb, MDA-MB-468 cells infected with Ad-RB, and differently passaged IMR90 cells were immunoprecipitated with Pak1 antibody. Immunocomplexes were detected with anti-phospho-Pak1 antibody. Phosph-Pak, phosphorylated Pak1. (C) SAOS-2 cells were cotransfected with pBabe-puromycin and pSVE-RB and dnCDK5 at increasing concentrations. After 10 days, cells were assayed for the senescent shape change and SA-ß-Gal expression. The fraction of flat or SA-ß-Gal-positive cells is expressed as a percentage of the total number of cells counted. (D) SAOS-2 cells were transfected with pBabe-puromycin, pBS/U6, and pSVE or pSVE-RB or cotransfected with pSVE-RB and a CDK5 siRNA construct, pBS/U6-siCDK5, for 10-days posttransfection. Cell lysates were immunoblotted with CDK5 or CDK2 antibody. An in vitro CDK5 kinase assay as described for panel A was performed to determine CDK5 kinase activity. (E) SAOS-2 cells transfected as described for panel C were assayed for shape change (flat) or SA-ß-Gal expression 10-days posttransfection, and the percentage of flat or SA-ß-Gal-positive cells was determined.

Previous studies found that CDK5 directly phosphorylates the Rac1 effector Pak1 at Thr-212 (2, 27, 32, 46). We therefore examined whether Pak1 is phosphorylated at this residue in senescent cells. We utilized anti-phospho-Pak1 antibody, which specifically recognizes the phosphorylation of Pak1 at Thr-212. SAOS-2 cells transfected with RB or MDA-MB-468 cells infected with Ad-RB for 2, 5, and 10 days after RB reintroduction or differently passaged IMR90 HDFs were immunoprecipitated with Pak antibody followed by immunoblotting with anti-phospho-Pak antibody (Fig. 2B). The phosphorylation of Pak1 was indeed increased in senescent cells, although total Pak expression levels changed little, consistent with a potential role for pRb in regulating CDK5 expression and/or activity in senescent cells.

To further determine if the ability of roscovitine to block senescence was due to its inhibition of CDK5 function, SAOS-2 cells were cotransfected with pRb and increasing concentrations of a dominant-negative form of CDK5, CDK5N144 (dnCDK5). dnCDK5 was able to block the senescent morphology change in a dose-dependent manner, demonstrating that CDK5 activity is required to attain the senescent phenotype. However, dnCDK5 only partially blocked induction of the senescence marker SA-ß-Gal (Fig. 2C), which might suggest the existence of a CDK5-independent mechanism that participates in SA-ß-Gal induction or, alternatively, that dnCDK5 is not completely effective at blocking CDK5 activity even at higher concentrations.

As dnCDK5 seemed an imperfect mechanism to inhibit CDK5 function in these cells, we made a plasmid-based siRNA construct of CDK5 (siCDK5) to more specifically and effectively inhibit CDK5. Ten days after SAOS-2 cells were transfected with pRb and siCDK5, immunoblot analysis showed that siCDK5 specifically inhibited CDK5 expression levels but had no impact on the homologous kinase CDK2. In addition, in vitro kinase analysis indicated that the pRb-induced elevation of CDK5 activity was inhibited by cointroduction of siCDK5 (Fig. 2D). Most strikingly, siCDK5 inhibited both the senescent morphology change and, to a lesser degree, expression of SA-ß-Gal (Fig. 2E), supporting a role for CDK5 as a regulator of the senescent phenotype.

CDK5 regulates SA-ß-Gal induction through inhibition of Rac1 activity. The data shown above link CDK5 activation to SA-ß-Gal expression and cytoskeletal changes in senescent cells. Because CDK5 has been demonstrated to influence actin polymerization in neurons through interaction with Rac1 (27) and Rac1 has been implicated in controlling cell shape (28, 35), we investigated the role of Rac1 in the senescence phenotype. SAOS-2 cells were cotransfected with pRb and either an activated (RacV12) or dominant-negative (RacN17) form of Rac1. Cells cotransfected with pRb and RacV12 displayed little SA-ß-Gal expression and did not display a flat phenotype, while those cotransfected with pRb and RacN17 had SA-ß-Gal expression levels and morphology comparable to those of senescent cells expressing only pRb (Fig. 3A).



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  		FIG. 3. CDK5 represses Rac1 activity to regulate SA-ß-Gal expression. (A) SAOS-2 cells were transfected with pBabe-puromycin and pSVE-RB plus pCMV or activated RacV12 or dominant-negative RacN17. After 10 days of selection, the cells were stained for SA-ß-Gal expression and photographed. (B) SAOS-2 cells were transfected with pSVE-RB or pSVE-RB and pEBG-RacV12 or pEBG-RacN17 together with either pBS/U6 CDK5 siRNA, pcDNA3-dnCDK5, or pcDNA3-CDK5. One set of transfectants was treated with roscovitine. After 10 days, the number of SA-ß-Gal-positive cells was assayed and expressed as a percentage of the number of total cells.

To determine if CDK5 might act through Rac1 in its ability to regulate senescence, SAOS-2 cells were transfected with pRb alone, pRb and RacV12, or RacN17 together with either dnCDK5 or CDK5 or, alternatively, were treated with roscovitine or cotransfected with siCDK5. After 10 days, the number of SA-ß-Gal-positive cells was assayed. As previously observed, roscovitine completely blocked pRb-induced SA-ß-Gal activity. siCDK5 or dnCDK5 partially inhibited this activity, and interestingly, CDK5 overexpression enhanced the number of SA-ß-Gal-positive cells (Fig. 3B). In the presence of activated Rac1, most pRb induction of SA-ß-Gal was compromised, a phenotype that was exacerbated by expression of dnCDK5, roscovitine, or siCDK5 treatment (Fig. 3B). Coexpression of CDK5 with pRb and RacV12 resulted in a partial rescue of SA-ß-Gal expression, suggesting that CDK5 might be antagonizing some aspect of Rac1 function. Alternatively, these results could indicate that CDK5 and Rac1 operate in different pathways that regulate senescence. However, in cells transfected with RB and RacN17, coexpression of dnCDK5 and siCDK5 or roscovitine treatment in no way inhibited pRb-induced SA-ß-Gal expression (Fig. 3B). This finding strongly suggests that CDK5 regulates SA-ß-Gal activity through inhibition of Rac1 activity, a function that is unnecessary when Rac1 activity is inhibited by its dominant-negative form.

Rac1 activity decreases in senescing cells. In an effort to test the hypothesis that CDK5 acts to regulate the senescent phenotype through modulation of the activity of small GTPases of the Rho family, GST-Pak1 and GST-rhotekin fusion proteins were used to measure the GTP loading of endogenous Rac1, Cdc42, and RhoA GTPases in senescent cells with and without CDK5 inhibition. To achieve this, cell lysates were incubated with GST beads bound to the effector Pak1 or rhotekin, and the GST-bound proteins were subjected to SDS-PAGE and immunoblotted with Rac1, Cdc42, or RhoA antibody (33). In SAOS-2 cells, there were not detectable levels of Cdc42 and RhoA, although these cells have high expression levels of Rac1. Therefore, SAOS-2 cells transfected with pRb or pRb and CDK5 siRNA were assayed for Rac1 activity as the cells underwent senescence. Rac1 activity decreased in senescing SAOS-2 cells after pRb transfection. However, active Rac1 was accumulated in the presence of CDK5 siRNA, while total Rac1 levels were constant, as determined by direct immunoblotting (Fig. 4A). Similarly, Rac1 activity was reduced in senescent MDA-MB-468 cells infected with Ad-RB, whereas no change in the level of the RhoA- or Cdc42-GTP was detected in the senescent cells compared with that for the parental cells (Fig. 4B). These data suggest that CDK5 activity is required for reduction of activated Rac1 in pRb-induced senescence and that inhibition of CDK5 results in the aberrant accumulation of active Rac1 in senescing cells. Indeed, Rac1 activity also lessened in senescent IMR90 HDF without any change in Rac1 levels, but the activity of RhoA- or Cdc42-GTP showed little change (Fig. 4C). In addition, RasV12-infected senescent fibroblasts also demonstrated a repression of Rac1 activity that was blocked when CDK5 activity was inhibited by roscovitine (Fig. 4D). Overall, these results suggest that Rac1 activity but not that of RhoA or Cdc42 decreases in the process of senescence and that this repression of Rac1 activity is regulated by CDK5.



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  		FIG. 4. Rac1 activity decreases in senescing cells. (A) Cells were collected at 2-, 4-, and 7-days posttransfection from SAOS-2 cells transfected with pSVE vector (V) or pSVE-RB or cotransfected with pBS/U6 CDK5 siRNA. GST-Pak pulldowns were separated by SDS-PAGE and immunoblotted for GTP-bound Rac1. As the control, an equal volume of lysates used for the pulldown was subjected to immunoblotting for total Rac1. (B) MDA-MB-468 cells were infected with Ad-RB for the indicated time in days. Lysates were incubated with GST-Pak fusion protein for Rac1 or Cdc42 activity or with GST-rhotekin fusion protein for RhoA activity. The amount of active GTP-bound Rac1, RhoA, or Cdc42 was analyzed by immunoblotting with anti-Rac1, -RhoA or -Cdc42 antibodies after GST precipitation. The total amount of Rac1, RhoA, or Cdc42 was determined by immunoblotting. (C) IMR90 fibroblasts were harvested at early passage (EP), middle passage (MP), or after undergoing senescence (S). The activation state of Rac1, RhoA, or Cdc42 was detected as described for panel B. (D) IMR90 cells infected with pBabe-puromycin or pBabe-RasV12 were untreated or treated with roscovitine. The Rac1 activity was determined by immunoblotting Rac1 in GST-Pak immunocomplexes. Puro, puromycin. (E) SAOS-2 cells were transfected with pSVE or pSVE-RB for the indicated number of days. IMR90 cells were passaged in culture, and cells were harvested for lysates at early passage, middle passage, or after undergoing senescence. To determine Pak1 kinase activity, cell lysates were immunoprecipitated with Pak1 antibody. The immunoprecipitates were then subjected to histone H4 kinase assays.

Previous studies have demonstrated that Pak1 kinase activity is inhibited by p35/CDK5 (27). In order to further determine the importance of CDK5 activation in the regulation of cell senescence, an in vitro Pak1 kinase assay was performed. Cell lysates from SAOS-2 cells transfected with pRb or p35/CDK5 and passaged IMR90 HDFs were immunoprecipitated with anti-Pak1 antibody. The immunocomplexes were subjected to a kinase assay using histone H4 as the substrate. The results revealed that Pak1 kinase activity was decreased in SAOS-2 cells overexpressing p35/CDK5 (Fig. 4E). Similarly, the activity of Pak1 was also reduced in senescent SAOS-2 cells 10 days after transfection and in senescent IMR90 HDFs, likely because of increased CDK5 activity in these cells (Fig. 4E). These results suggest that the upregulated CDK5 in senescent cells not only inhibits Rac1 activity but regulates its effector Pak1 as well.

Rac1 regulates actin polymerization in senescing cells. CDK5-dependent flattening of senescent cells suggests that extensive changes must occur to the cellular cytoskeleton as cells undergo senescence. Both CDK5 and Rac1 have known roles in regulating cell shape through actin reorganization. With this in mind, we investigated changes to actin structure as cells underwent senescence. We immunostained for actin in cells induced to senesce by pRb (SAOS-2) or passage in culture (IMR90 and WI38). We observed a significant increase in polymerized actin filaments during senescence (Fig. 5A), which was confirmed by staining for F-actin (data not shown). This appearance of polymerized actin structures was accompanied by an overall increase in actin levels (data not shown).



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  		FIG. 5. CDK5 and Rac1 regulate actin. (A) Indirect immunostaining and immunofluorescence following anti-actin antibody treatment in presenescent and senescent pSVE-RB-transfected SAOS-2 cells or passaged (Psg) IMR90 or WI38 cells untreated or treated with roscovitine (Rosc.) as described in the legend to Fig. 1. (B) Indirect immunostaining for actin in SAOS-2 cells transfected with pBabe-puro, pSVE-RB, and RacV12 or RacN17 following 2 and 7 days of selection.

To determine whether CDK5 could regulate these changes in actin in senescing cells, pRb-transfected SAOS-2 cells were treated 2- through 7-days posttransfection with roscovitine, and senescent HDFs were treated for 48 h with roscovitine and stained for actin. Strikingly, roscovitine inhibited most actin polymerization (Fig. 5A), suggesting that CDK5 controls senescent cytoskeletal changes by regulating actin organization. To explore the possibility that CDK5 regulation of Rac1 activity was linked to the reorganization of actin, as is the case in neuronal cells, we investigated if Rac1 also played a role in the cytoskeletal changes of senescing cells. SAOS-2 cells were cotransfected with RB and either RacV12 or RacN17 and immunostained for actin 2- and 7-days posttransfection. Both RacV12 and RacN17 altered the shape of senescent cells in a manner consistent with a role for Rac1 in the senescent shape change. Cells expressing RacV12 and pRb showed a profound disruption of actin filaments 7 days after transfection (Fig. 5B), reminiscent of cells treated with roscovitine (Fig. 5A). Thus, both inhibition of CDK5 and hyperactivity of Rac1 disrupt the senescent cytoskeleton, even though the cells remain nonproliferative (data not shown), again linking these proteins in establishment of the senescent phenotype. Consistent with this finding, RacN17 greatly enhanced the appearance of actin filaments when coexpressed with pRb (Fig. 5B). Together, these data suggest that downregulation of Rac1 activity is required for expression of SA-ß-Gal and acquisition of the senescent morphology. Moreover, these aspects of senescent cells depend on a CDK5 activity that is not required in cells with artificially blocked Rac1 activity.


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DISCUSSION
 
CDK5/Rac1 interaction as a link between early and late events in senescence. It is widely accepted that a variety of cellular stresses in culture and in vivo, including culture conditions, oncogene activation, drug treatment, and telomere shortening, can initiate the senescensce process through activation of the pRb and/or p53 tumor-suppressive pathways (4, 22, 37). The consequence of these stresses is loss of proliferative capacity and a host of physiological changes characteristic of senescent cells. Indeed, senescent cells traditionally have been identifiable by their intriguing change in appearance and expression of SA-ß-Gal, but little is known about signaling events that link these changes to senescence initiation. We propose that activation of CDK5 and subsequent loss of Rac1 activity provides such a link, acting as part of a molecular mechanism that signals to a cell that it is senescent. CDK5 activity increases in cells induced to senesce by a variety of stimuli, and inhibition of CDK5 activity reduces the expression of the senescence marker SA-ß-Gal in human cells. Further, we found in a variety of different types of senescence that CDK5-mediated repression of Rac1 activity is necessary for proper acquisition of the cytoskeletal changes accompanying senescence. Together, these observations indicate that CDK5, acting through Rac1, can function as a regulator of multiple aspects of senescence.

Discovery of this role of CDK was unexpected, as CDK5 has been thought to function primarily in developmental signaling in neurons and potentially somatic mesoderm (31). However, given that CDK5 is ubiquitously expressed, it is likely that the kinase plays additional or related roles in other tissues. Indeed, work presented here linking CDK5 to Rac1 activity and cytoskeletal changes in postmitotic, senescent cells is not dissimilar to CDK5's reported biochemical roles in neurite outgrowth. Thus, CDK5, acting in part through Rac1, may function as a general signaling molecule coordinating cellular responses to loss of mitotic capacity in both differentiation and senescence.

Regulation of CDK5 and Rac1 in senescence. The increase in CDK5 activity in senescent cells is observed following a variety of stimuli in human primary and tumor cells, suggesting that a mechanism common to all of these systems may function in CDK5 activation. Indeed, the use of model systems in which senescence is induced by pRb reexpression in tumor cells strongly implicates the pRb pathway in CDK5 upregulation independently of any role for p53. It is not clear at this time whether such a role for pRb is direct, nor is the mechanism of CDK5 activation well understood. There is a clear induction of CDK5 protein in acute systems in which pRb or activated Ras stimulates senescence within days, and this induction appears to be largely independent of transcriptional regulation (K. Alexander, unpublished data). Indeed, pRb-mediated proliferation arrest is dependent on posttranscriptional regulation of p27Kip1 in the SAOS-2 system (1), suggesting that pRb may act directly or indirectly to alter the expression of a number of proteins involved in senescence without associated transcriptional changes.

Regardless of the mechanism of CDK5 activation in senescent cells, our data strongly argue that an important consequence of this activity is downregulation of Rac1 function. Indeed, this function of CDK5 appears to be specific for Rac1, as we observed no change in the levels of active RhoA or Cdc42 in senescent cells. Thus, increased actin polymerization and accompanying cytoskeletal changes may be understood in the context of unmasked RhoA signaling in the relative absence of Rac1 activity in senescent cells. Such a phenotype is consistent with the observed effects of RhoA overexpression in a variety of systems (14, 36). Most importantly, inhibition of CDK5 activity reverses a loss of Rac1 activity that we observe in all senescent cells, and conversely, inhibition of Rac1 by a dominant-negative subunit precludes the need for CDK5 activity for SA-ß-Gal expression and cytoskeletal changes. Taken together, these results imply that CDK5 acts upstream of Rac1 to directly or indirectly inhibit its activity. In addition, our results revealed that CDK5 phosphorylated Pak1 at Thr212 and reduced Pak kinase activity in senescent cells, similar to the reported role of CDK5 in Rac1/Pak1 activity in neurons, where p35/CDK5 kinase associates with Pak1 in a Rac-GTP dependent manner, causing hyperphosphorylation and downregulation of Pak1 kinase activity (27). In theory, the specific inhibition of Rac1 we observed in senescent cells should preclude the need to regulate its effector Pak1. Therefore, we speculate that Pak1 phosphorylation by CDK5 may have additional roles in the senescent phenotype. Indeed, we note that Pak1 has recently been reported to interact with the ERM-related tumor suppressor protein NF2/merlin (18), a protein that our preliminary studies indicate might also play a role in the senescent phenotype (H. Yang, P. Santiago, and P. Hinds, unpublished observations).

Nevertheless, the requirement for CDK5 activity in downregulation of Rac1 activity suggests an additional role for CDK5 beyond regulation of the effector molecule. For example, work in our laboratory has recently identified ezrin as a direct substrate for CDK5 in senescent cells, and the postulated interaction of ezrin with Rho family regulators makes it an attractive candidate to participate in the CDK5/Rac1 pathway in senescence (45). Despite the rudimentary nature of our understanding of Rac1 regulation in senescence, our data indicate that Rac1 activity must somehow be antisenescent, which may be due to the reported proliferative effect of Rac1 in some cells (29, 44) or may imply that established, Rac1-responsive cytoskeletal regulation collaborates with other signals in senescent cells to stimulate SA-ß-Gal expression and, potentially, other aspects of senescence. As discussed below, significant additional studies of the role of cytoskeletal changes in the biochemical and proliferative aspects typical of senescence are needed to fully appreciate the role of Rac1 in these processes.

CDK5 function in senescence and tumor suppression. A prominent role for CDK5 in the senescence process and the likelihood that senescence is tumor suppressive (22, 37) raise the possibility that CDK5, like other senescence regulators, is also a tumor suppressor. In fact, CDK5 has generally been observed to be more highly expressed in many tumor cells than in normal cells, which may result from interrupted senescence signaling in immortal cells akin to elevated p16INK4a expression seen in cells lacking pRb function. Clearly, additional work is warranted to address a general antiproliferative or tumor-suppressive role of CDK5 in postmitotic cells, but our initial indications are that CDK5 at best contributes to, but is not required for, proliferative arrest in senescence. Senescent cells in which CDK5 has been inhibited remain phenotypically senescent in that they do not revert to wild-type shape or size and they do not reenter the cell cycle. This finding supports the idea that cell cycle arrest, likely the most critical characteristic in making senescence irreversible and tumor suppressive, is predominantly mediated by the established functions of pRb and p53, the primary regulators of senescence.

Despite the persistence of growth arrest in senescent cells lacking CDK5 activity, it is quite possible that CDK5 contributes to the irreversibility of senescence. First, the effect of CDK5 inhibition on cell death following senescence reversal in a variety of systems has not been tested (9, 41, 43). Second, we and others have observed that expression of p27Kip1 and p21Cip1 is critical to senescent arrest, and the effect of CDK5 expression on these regulators is unknown. Indeed, the role of CDK5 in cytoskeletal changes and a variety of observations linking cytoskeletal signaling to p27Kip1 expression suggest an intriguing link germane to the establishment of senescence. A third indication that CDK5 may contribute to, but not be required for, phenotypic aspects of senescence contributing to growth arrest derives from the observation that CDK5 repression of Rac1 activity is not the only mechanism to trigger SA-ß-Gal expression in senescent cells, particularly in MEFs. Indeed, we and others have seen that the cell cycle inhibitors p21CIP1 and p27KIP1, which are upregulated during senescence, can on their own induce SA-ß-Gal expression even in the absence of both pRb and p53 (1, 8, 13). Our preliminary data indicate that the CDK5 activity-insensitive SA-ß-Gal expression is likely due to this cell cycle inhibitor induction of SA-ß-Gal, as this pathway seems to remain mostly intact even in the absence of CDK5 activity. However, we have observed that such p27Kip1-induced small senescent cells are less stable than those displaying a morphology characteristic of senescence, providing some evidence for a role for cytoskeletal changes in long-term proliferative arrest (K. Alexander, unpublished).

Our work begins to provide a framework for the molecular events by which the process of senescence occurs downstream of senescence inducers. It seems clear that this will be found to be a complex process involving diverse signaling pathways normally observed to be used in a different context. Still, CDK5 appears to be an important regulator upon which many senescence pathways impinge, which represents a crucial role for CDK5 in normal cells. Recent evidence from mice suggests that tumors that retain an ability to undergo senescence via p16INK4a respond more readily and survive much longer after chemotherapeutic treatment (37). Hence, the fact that the CDK5 pathway to senescence seems to be intact but latent in tumor cells may represent an advantage in cancer treatments in that its activation may perhaps be used as a mechanism to induce aspects of senescence in tumors in vivo and thus impair tumor progression.


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ACKNOWLEDGMENTS
 
We thank Li-Huei Tsai and Maria Morabito for advice and critical CDK5-related reagents. We also thank Jeffery Settleman and Raffaela Sordella for advice on detecting Rac activity and plasmids. We are grateful to Yang Shi for the siRNA plasmid and instruction on constructing our siRNA plasmid of interest.

This work was supported by NIH grant AG20208 to P.W.H.


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FOOTNOTES
 
* Corresponding author. Mailing address: Molecular Oncology Research Institute, Tufts-New England Medical Center, 75 Kneeland Street, Boston, MA 02111. Phone: (617) 636-7947. Fax: (617) 636-7813. E-mail: phinds{at}tufts-nemc.org. Back

{dagger} K.A. and H.-S.Y. contributed equally to this work. Back

{ddagger} Present address: Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, MA 02111. Back


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Molecular and Cellular Biology, April 2004, p. 2808-2819, Vol. 24, No. 7
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.7.2808-2819.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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