J Mol Med (2009) 87:1221–1239 DOI 10.1007/s00109-009-0525-5
ORIGINAL ARTICLE
Sgk1 activates MDM2-dependent p53 degradation and affects cell proliferation, survival, and differentiation Rosario Amato & Lucia D’Antona & Giovanni Porciatti & Valter Agosti & Miranda Menniti & Cinzia Rinaldo & Nicola Costa & Emanuele Bellacchio & Stefano Mattarocci & Giorgio Fuiano & Silvia Soddu & Marco G. Paggi & Florian Lang & Nicola Perrotti
Received: 26 February 2009 / Revised: 5 August 2009 / Accepted: 20 August 2009 / Published online: 11 September 2009 # Springer-Verlag 2009
Abstract Serum and glucocorticoid regulated kinase 1 (Sgk1) is a serine–threonine kinase that is activated by serum, steroids, insulin, vasopressin, and interleukin 2 at the transcriptional and post-translational levels. Sgk1 is also important in transduction of growth factors and steroiddependent survival signals and may have a role in the development of resistance to cancer chemotherapy. In the Electronic supplementary material The online version of this article (doi:10.1007/s00109-009-0525-5) contains supplementary material, which is available to authorized users. R. Amato : L. D’Antona : G. Porciatti : V. Agosti : M. Menniti : G. Fuiano : N. Perrotti (*) Department of Experimental and clinical Medicine “G. Salvatore”, Faculty of Medicine, University Magna Graecia at Catanzaro, Catanzaro, Italy e-mail:
[email protected] N. Costa Department of Pharmacobiology, University “Magna Graecia” Catanzaro, Catanzaro, Italy E. Bellacchio CSS-Mendel Institute, Rome, Italy C. Rinaldo : S. Soddu Department of Experimental Oncology, Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, Rome, Italy E. Bellacchio : S. Mattarocci : M. G. Paggi Department for the Development of Therapeutic Programs, Regina Elena Cancer Institute, Rome, Italy F. Lang Department of Physiology, University of Tübingen, Tübingen, Germany
present paper, we demonstrate that Sgk1 activates MDM2dependent p53 ubiquitylation. The results were obtained in RKO cells and other cell lines by Sgk1-specific RNA silencing and were corroborated in an original mouse model as well as in transiently and in stably transfected HeLa cells expressing wild-type or dominant negative Sgk1 mutant. Sgk1 contributes to cell survival, cell-cycle progression, and epithelial de-differentiation. We also show that the effects of Sgk1 on the clonogenic potential of different cancer cells depend on the expression of wild-type p53. Since transcription of Sgk1 is activated by p53, we propose a finely tuned feedback model where Sgk1 down-regulates the expression of p53 by enhancing its mono- and polyubiquitylation. Keywords Sgk1 . p53 . Cell death . Cell signaling . MDM2
Introduction The serum and glucocorticoid regulated kinase 1 (Sgk1) is a serine–threonine kinase finely regulated by steroids, insulin, and vasopressin at the transcriptional and posttranslational levels, through PDK1/2 [1, 2]. PDK1 binds phospho-S422 in the hydrophobic motif (H-motif) of SGK1 to phosphorylate T256. The H-motif kinase that phosphorylates SGK1 at S422 to prime it for phosphorylation by PDK1 has been recently identified as mTOR [3]. Activation of the kinase contributes to the Na+retaining effects of insulin, vasopressin, and aldosterone [4] and is critical for the hypertensive effects of hyperinsulinism [5–7]. Sgk1 is also involved in mediating growth factor and steroid-dependent survival signals [8, 9]. We have recently demonstrated that Sgk1 is essential for transducing inter-
1222
leukin 2-dependent survival signals and that ectopic expression of the IL-2 receptor in kidney cancer cells protects the cells from doxorubicin-induced apoptosis, thus demonstrating a possible mechanism underlying drug resistance [10]. A role for Sgk1 in tumor development is supported by its capability to promote G1 cellcycle progression through p27 phosphorylation [3] and by its over-expression in several human tumor cells, even in tissues where the cognate kinase Akt is not significantly activated [11, 12]. More recently, Sgk1 has been found to be essential and limiting in the inhibition of Gsk3, thus allowing the stabilization of c-myc in tumor models lacking a functional RhoB [13]. Based on these recent observations, Sgk1 antagonistic molecules are considered in the experimental therapy of prostatic cancer [14], a tumor that is also finally regulated by steroid hormones [15]. Some of these Sgk1-dependent functions might be explained by the modulation of the activity of p53, a critical player in cell survival and cell-cycle progression. As a matter of fact, a link between Sgk1 and p53 has been suggested [16]. Sgk1 is regulated by p53 at the transcriptional level. The promoter region of the gene coding for Sgk1 contains p53 DNA binding elements, and Sgk1, but not Akt, is necessary for p53-dependent suppression of FKHRL1 in mouse embryo fibroblasts [17, 18]. p53 is mutated in at least 50% of human tumors [19]. The protein can be inactivated by ubiquitylation [20, 21]. The cellular p53 protein abundance is mainly modulated by MDM2, a negative regulator of p53 that binds p53 within its N-terminal transactivation domain [22, 23]. Binding triggers the MDM2-dependent E3 ligase activity that catalyzes the ubiquitylation of p53, leading to its proteasome-mediated degradation [24, 25]. p53 is able to regulate several caretaker functions, like cell-cycle exit at the G1-S checkpoint [26], or cyclin B1 and related cdks inhibition at the G2-M checkpoint [27]. Moreover, p53 is one of the principal regulators of apoptosis since it integrates both intrinsic/ mitochondrial and extrinsic apoptosis pathways [28]. More recently, p53 has been found capable of regulating limiting genes in differentiation and in cellular architectural control [29, 30]. The present paper aims to investigate the functional and structural relationship between Sgk1 and p53 by means of Sgk1-specific RNA silencing and dominant negative [4, 10, 31] technologies. We present evidence that Sgk1, a gene regulated by p53 at the transcriptional level [32], affects cell proliferation and differentiation and that the expression and the half-life of p53 is regulated by Sgk1 through MDM2 that directs p53 to ubiquitylation and proteosomal degradation.
J Mol Med (2009) 87:1221–1239
Experimental procedures Commercially available reagents and antibodies were obtained as indicated in the Electronic supplementary materials. Recombinant DNAs The sequences coding for myc-tagged wild-type Sgk1 and dominant negative D222A Sgk1 were cloned into the PcDNA4TO expression vector (Invitrogen, Milan, Italy) as previously described [10]. pGEX4T3-MDM2 (full length) was produced by one of us [33]. The CMV-driven ubiquitin expression construct pMT107 (His6-ubiquitin) has been described [34] and was kindly provided by D. Bohmann. pCMV-Mdm2, GST-MDM2, GST-MDM2 S166A, and GST-MDM2 S188A were kindly provided by Dr. Karen Voudsen and pCMV-Mdm2ΔRing by Dr. Moshe Oren. pBABE empty vector was used together with PcDNA4-TO (empty vector) or PcDNA4-TO (myc) Sgk1 wild type (WT) or PcDNA4-TO (myc) Sgk1 D222A (DN) for colony assay experiments. pcDNA3 UB-Ha, coding for the HA-tagged ubiquitin protein, was obtained through the courtesy of Dr. Luca Ulianich. Cell culture, transient transfection, and establishment of continuous cell lines The methods for cell culture, transient transfection, and establishment of continuous cell lines are detailed in the Electronic supplementary materials, according to a previously published paper [10]. Continuous cell lines were obtained from HeLa cells stably transfected with vectors PcDNA4-TO (empty vector), PcDNA4-TO (myc) Sgk1 WT, or PcDNA4-TO (myc) Sgk1 D222A (DN) and named empty vector cells, Sgk1WT, and Sgk1 D222A DN cells. Virus generation and infection for RNA interference experiments The human sgk1 (NM_005627 GenBank) MISSION shRNA set (five individual hairpins individually cloned into pLKO.1-puro; Sigma) were used to generate lentiviral particles in HEK293T packaging cells. Subconfluent HEK293T cells were cotransfected with 13 µg p27 MISSION shRNA set, 18 µg pCMV-deltaR8.91, and 12 µg pCMV-VSVG per 100-mm tissue culture plate by Lipofectamine™ 2000. After 48 h from transfection, supernatants were collected at 8-h intervals, filtered, and used for three rounds of transduction of HeLa and RKO cell lines in the presence of 8 µg/mL polybrene (Sigma).
J Mol Med (2009) 87:1221–1239
Transduced cells were selected by puromycin (1.2 μg/mL), lysed 72 h after infection, and analyzed by immunoblotting with anti-Sgk1, anti-p53 (DO-1), and anti-MDM2 antibodies. The Sgk1 knockdown in transduced HeLa and RKO cells was verified by comparison with HeLa and RKO cells, transduced with Mission non-target control transduction virus (scrambled RNA; Sigma SHC002V). Both HeLa and RKO cells were transduced with each of the five clones expressing different shRNAs. As indicated in the “Results” section, clones 2294-Sgk1 and 458-Sgk1 were effective in HeLa cells, whereas clone 458-Sgk1 was effective in RKO cells. Colony assays Cervical carcinoma HeLa cells, human RKO cells, transformed human embryonic kidney Hek293A cells, and human non-small cell lung carcinoma H1299 cells were resuspended at a density of 2 ×106 cells/mL and cotransfected with either PcDNA4-TO (empty vector), PcDNA4-TO (myc) Sgk1 WT, or PcDNA4-TO (myc) Sgk1 D222A (DN) and with pBABE empty vector carrying the puromycin resistance gene by means of Lipofectamine 2000. After 3 weeks of selection in the presence of puromycin (5 μg/mL), the colonies obtained were washed three times with PBS and stained with crystal violet 0.5% W/V solution in methanol. Single colonies in every plate were counted and captured by digital camera. Cell stimulation and treatment with inhibitors Cell cultures were serum-starved 24 h before stimulation with any effector, unless otherwise specified in the figure legends. Hormones, buffer, and inhibitors are detailed in the Electronic supplementary materials. Immunofluorescence Empty vector cells, Sgk1 WT cells, and Sgk1 D222A DN cells, grown on cover slips, were fixed with 3.7% formaldehyde for 20 min. Fixed cells were then permeabilized with 0.5% Triton X-100 for 1 min and washed with PBS (pH 7.4). The prepared samples were incubated with primary antibodies to Sgk1, p53, MDM2, ZO-1, alpha catenin, vimentin, and myc-tag and stained as detailed in the Electronic supplementary materials. FACS analysis of synchronized and unsynchronized cells Cell-cycle analysis was performed according to standard methods as indicated in the Electronic supplementary materials.
1223
Mice models and animal procedures For cellular inoculations in mice, a cohort of six male, 9-week-old BALB/c mice were inoculated subcutaneously with either 2×106 cells of empty vector or Sgk1 D222A DN cells on the back. The mice were killed after 17 days. The inoculated cells were recovered, lysed, and analyzed by Western blot with anti-p53 DO-1 antibody, anti-α-catenin, and anti-c-myc tag antibodies. Transgenic animals Tet-CMV Sgk1 D222A DN single transgenic founders were generated in collaboration with Murinus GmbH| Falkenried 88|D-20251 Hamburg as indicated in the Electronic supplementary materials. The single transgenic founders were crossed with MUP-tTA mice obtained by Dr. T. Jake Liang [35]. The experiments were performed on double transgenic, single transgenic, and wild-type animals as indicated in the Electronic supplementary materials. p53 ubiquitylation experiments In vitro and in vivo assays were carried out as previously published [33, 36] with minor modifications, detailed in the Electronic supplementary materials. p53 half-life experiments Cells were pulse labeled with [35S]methionine and [35S] cysteine, as detailed in the Electronic supplementary materials.
Results Sgk1-specific RNA silencing in RKO cells: effects on the level of expression, mono- and polyubiquitylation of p53 Sgk1 silencing was achieved by means of retrovirally encoded small RNAs. In order to obtain an efficient silencing of the endogenous Sgk1, RKO cells were infected twice. A significant silencing of the endogenous Sgk1 was achieved using the 458-Sgk1 siRNA, and a concomitant increase of p53 expression was observed (Fig. 1a, lane 2, top). In a separate experiment, depletion of endogenous Sgk1 was almost complete after the first infection (Fig. 1b, lane 2, middle) and was associated with a significant increase of p53 expression (Fig. 1b, lane 2, top). After a second round of infection with the
1224
J Mol Med (2009) 87:1221–1239
Fig. 1 Effects of Sgk1-specific RNA silencing in RKO cells. RKO cells were infected with a retrovirus encoding Sgk1-specific RNA silencer. a p53, Sgk1, and β-tubulin were detected by immunoblotting with specific antibodies in RKO cells infected with retrovirus coding for scrambled RNA (lane 1) or Sgk1 silencing RNA 458 (lane 2). b p53, Sgk1, and GAPDH were detected by immunoblotting with specific antibodies in RKO cells infected twice with retrovirus coding for scrambled (lane 1) or subjected to a single (lane 2) or double (lane 3) infection of the retrovirus encoding Sgk1 silencing RNA 458.
c RKO cells infected with retrovirus coding for scrambled RNA (lane 1) or Sgk1 silencing RNA 458 (lane 2) were transfected with His–tagged ubiquitin. Cell extracts were subjected to nickel agarose purification. Proteins were analyzed by immunoblotting with an antibody to p53. d Cells expressing either Sgk1-specific silencing RNA 458 or scrambled RNA were plated at a density of 250,000 cells per plate and incubated in the presence of FBS 10% for 48 and 72 h before counting. Dead cells were detected and counted manually by Trypan blue exclusion. Data are means ± SE obtained from triplicates
458-Sgk1 silencer, endogenous Sgk1 was virtually undetectable (Fig. 1b, lane 3, middle), whereas a further increase in the expression of p53 was achieved (Fig. 1b, lane 3, top). The expression of p53 can be modified through its proteosomal degradation driven by mono- and polyubiquitylation. Thus, we compared the p53 ubiquitylation status in RKO cells in the presence or absence of endogenous Sgk1. As shown in Fig. 1c (lane 2, upper panel), Sgk1 depletion significantly decreased both mono- and polyubiquitylation of p53. This decrease in p53 ubiquitylation was associated with the expected increase in expression levels of p53 (Fig. 1c, lane 2, lower panel).
vitro assay was assembled where in vitro ubiquitylation reactions were performed in the presence of recombinant E1, E2, and ubiquitin (Fig. 2a, lanes 1–8) with addition of either GST (Fig. 2a, lanes 1 and 2) or GST-p53 (Fig. 2a, lanes 4–7) as substrates, Sgk1 active kinase (Fig. 2a, lanes 3, 5, and 6), and the immunocomplexes obtained by dKO MEFs transduced with either MDM2 (Fig. 2a, lanes 1, 4, and 5), or its Δ-RING deletion mutant (Fig. 2a, lanes 6 and 7) as source of WT and defective E3 activity. As shown in Fig. 2a, slowly migrating bands recognized by anti-Ub Abs were detectable only in the presence of WT MDM2 and the active kinase Sgk1 (lane 5), while the Δ-RING deletion mutant was unable to modify GST-p53 (lane 6), indicating that full-length MDM2, not its deletion mutant, catalyzes p53 ubiquitylation in vitro in the presence of active Sgk1.
Sgk1-specific RNA silencing in RKO cells: effects on cell proliferation The inhibition of the endogenous kinase in RKO cells was also associated with a significant decrease of proliferation paralleled by an increase in the number of dead cells, as assessed by direct cell counting and Trypan blue exclusion, 48 and 72 h after cell plating (Fig. 1d). Sgk1 increases MDM2-dependent monoand polyubiquitylation of p53 in vitro In order to verify whether Sgk1 directly affected MDM2, thus causing mono- and polyubiquitylation of p53, an in
Sgk1 phosphorylates MDM2 at serine 166 The amount of MDM2 can be regulated by phosphorylationdependent protein stability, and serine 166 is considered a crucial residue in this regulation. Thus, we asked whether Sgk1 might regulate p53 expression through direct phosphorylation of its negative regulator, MDM2. Phosphorylation of MDM2 by Sgk1 was clearly detected in an in vitro kinase assay using GST-MDM2 as a substrate in the presence of the active kinase and γ32P-ATP (Fig. 2b, lane 5). In order to identify the residue(s) phosphorylated
J Mol Med (2009) 87:1221–1239
1225
by Sgk1, mutant GST-MDM2 proteins (S166A and S188A) were used as substrates. As shown in Fig. 2c, serine 166 appeared to be the main substrate of Sgk1 since the GSTMDM2 S166A mutant (Fig. 2c, lane 8) was clearly less phosphorylated than the wild-type molecule (Fig. 2c, lane 7), although some phosphorylation might still occur on other residues. The phosphorylation of MDM2 at serine
166 by the active Sgk1 was also confirmed by blotting with a specific phospho-serine 166 MDM2 antibody (Fig. 2d, lane 3). Interestingly, a GST-dominant negative D222A Sgk1 mutant fusion protein was unable to phosphorylate MDM2 (Fig. 2d, lane 5) and completely inhibited the phosphorylation of MDM2 induced by equimolar amounts of the active kinase (Fig. 2d, lane 4), confirming its
Fig. 2 Sgk1 activates MDM2-dependent ubiquitylation of p53 and phosphorylates MDM2 on serine 166 in vitro. a In vitro ubiquitylation reactions were performed in the presence of recombinant E1, E2, and Ub (lanes 1–8) with addition of GST (lanes 1 and 2), GST-p53 (lanes 4–7), as substrates, recombinant active Sgk1 kinase (lanes 3, 5, and 6), and the immunocomplexes obtained by dKO MEFs transduced with MDM2 or its D-RING deletion mutant as source of WT and defective E3 activity. Reactions were performed for 2 h at 30_C (lanes 1–8). Reaction products were subdivided in two aliquots and resolved by NuPAGE (3–8%) followed by WB with anti-Ub Ab (top panel) and by SDS-PAGE (10%) followed by WB with anti-p53 (bottom panel). The arrow indicates the position of the hypothetically unmodified GST-p53 protein. b In vitro kinase assay. Recombinant active Sgk1 phosphorylates bacterially expressed GST-MDM2 in the presence of [γ-32P]ATP (lane 5). Proteins were separated by SDS-PAGE and
detected by autoradiography (upper gel) or by specific Sgk1 and MDM2 antibodies as indicated (bottom gels). c Wild-type (lanes 2 and 7) mutant, S166A GST-MDM2 (3 and 8), and mutant S188A GST-MDM2 (4 and 9) were used as substrates of the recombinant active Sgk1 kinase as above. The phosphorylation of the S166A MDM2 mutant was greatly reduced (lane 8), whereas the phosphorylation of the S188A mutant (lane 9) was similar to that observed when wild-type MDM2 was used (lane 7). d Recombinant active Sgk1 was used to phosphorylate bacterially expressed GST-MDM2, in the presence of GST (lane 3) or bacterially expressed GST DN (D222A) Sgk1 (lane 4). Bacterially expressed GST DN (D222A) Sgk1 is an inactive kinase (lane 5). Phosphorylated MDM2 was detected by immunoglobulins specific for phospho-serine 166 MDM2 (top panel). Total MDM2 and Sgk1 wild type and mutant were detected by specific antibodies, as indicated (bottom panel)
1226
function as a powerful inhibitor of Sgk1 in several systems. Effects of the inhibition of the endogenous Sgk1 kinase in different tissues and cell lines The results obtained in RKO cells were confirmed in different cell lines with different approaches. HeLa cells represent a widely used tool to study cell proliferation and differentiation [37] with the caveat that they the express the high risk human papillomavirus E6 protein that binds p53 and targets it for accelerated degradation and is expected to confer resistance to the inhibition of the Sgk1 and MDM2dependent degradation of p53 [38]. In our hands, RKO and HeLa cells express comparable amounts of p53 although both cell lines show a significantly reduced expression of p53 when compared with Hek293 cells, that express a wildtype p53, not inhibited by papillomavirus and characterized by a completely different mechanism of cell-cycle regulation [39] (Fig. 3a). Sgk1 silencing in HeLa cells In HeLa cells, two RNA molecules resulted in complete (2294-Sgk1) or almost complete (458-Sgk1) Sgk1 silencing (Fig. 3b, lanes 2 and 3). Even in HeLa cells, characterized by papillomavirus-induced down-regulation of p53, the inhibition of endogenous Sgk1 was associated with an impressive increase in the expression of p53. The enhancement of p53 was correlated with the efficiency of Sgk1 inhibition since it was more evident when 2294Sgk1 was used. Interestingly, the inhibition of Sgk1 was paralleled by a marked decrease in the expression of MDM2, the E3 ubiquitin ligase that is considered the main regulator of p53. Dominant negative technology is an alternative tool to Sgk1-specific RNA silencing Expression of p53 protein was detected by Western blotting in HeLa cells transiently transfected with an empty vector (PcDNA4-TO), or with vectors coding for either Sgk1 wild-type (PcDNA4-TO (myc) Sgk1 wild type) or the dominant negative mutant of Sgk1 (PcDNA4-TO (myc) Sgk1 D222A). p53 was clearly detectable in cells transfected with the empty control vector (Fig. 3c, lane 2, top), virtually undetectable in cells over-expressing wild-type Sgk1 (Fig. 3c lane 1 top), and clearly increased in cells over-expressing the dominant negative mutant of Sgk1 (Fig. 3c, lane 3, top), demonstrating again that even in HeLa cells, with the dominant negative technology, the inhibition of the endogenous Sgk1 induces accumulation of p53.
J Mol Med (2009) 87:1221–1239
Expression of the dominant negative mutant of Sgk1 (Sgk1-DN) in livers of double transgenic mice (MUP1-TA/ pTRE-D222A) enhances p53 levels The effect of endogenous Sgk1 inhibition on p53 levels was studied in a double transgenic mouse model expressing the dominant negative D222A Sgk1 mutant under the control of a Tet-regulated promoter (Tet-off). A small cohort of five 25-week-old male mice (three double transgenics not treated with doxicycline, one single transgenic for the tTA, and one wild-type mouse) were selected (Fig. 3d, top panels). On the day of the experiment, mice were killed, and the expression of the transgene was detected in lysed livers by Western blotting using an anti-Myc antibody (Fig. 3d, bottom panel). The liver expression of the dominant negative D222A Sgk1 was associated with a dramatic increase of p53 (Fig. 3d, lanes 3,4, and 5) when compared with the wild-type (Fig. 3d, lane 1) and the single transgenic mice (Fig. 3d, lane 2). The increased expression of p53 in the double transgenic mice paralleled the increased expression of p21, indicating that the p53 protein expressed in livers under these conditions is transcriptionally active and functional (Fig. 3d, lanes 3, 4, and 5 vs. 1 and 2). Figure 3e shows a representative gel that demonstrates, by reverse PCR, that the expression of cDNA coding for the myc-Sgk1 D222A DN mutant transgene was detected only in double transgenic animals in the absence of doxicycline (lane 3), whereas it was not detected in double transgenic mice treated with doxicycline (lane 1) as well as in single transgenic (lane 2) and in wild-type animals (lane 4). Establishment of stably transfected HeLa cell lines expressing wild-type and dominant negative mutants of Sgk1 Expression of the transgenes in empty vector cells, Sgk1WT cells, and Sgk1 D222A DN cells was detected by Western blot, reverse PCR, and immunofluorescence (Suppl. file Fig. 1). Anti-myc antibodies were able to detect a specific ∼50 kDa migrating band only in Sgk1 WT cells (Suppl. file Fig. 1 A, lane 1) and in Sgk1 D222A DN cells (Suppl. file Fig. 1 A, lane 2), not in the empty vector cells (Suppl. file Fig. 1 A, lane 3). Similar results were obtained when the expression of the transgene was assessed by reverse PCR. The expected 450 bp PCR product, identified by direct sequencing, was detected only when primers specific for myc Sgk1 were used to amplify cDNA from Sgk1WT and Sgk1 D222A DN cells (Suppl. file Fig. 1 B, lanes 3 and 4). No amplification product was detected in the presence of cDNA obtained from empty vector cells (Suppl. file Fig. 1 B, lane 2) or in the absence of cDNA (Suppl. file Fig. 1 B, lane 1).
J Mol Med (2009) 87:1221–1239
Fig. 3 Effects of the inhibition of the endogenous Sgk1 kinase in different tissues and cell lines. a The expression of p53 protein in HeLa cells (lane 1), RKO cells (lane 2), and Hek 293T cells (lane 3) was detected by rabbit immunoglobulins FL-393. Equal loading is demonstrated by red Ponceau staining (bottom panel). b Sgk1-specific RNA silencing in HeLa cells. MDM2, p53, Sgk1, and β-tubulin were detected by immunoblotting with specific antibodies in HeLa cells infected with retrovirus coding for scrambled RNA (lane 1) or Sgk1 silencing RNA 458 (lane 2) or 2294 (lane 3). c p53 was detected by Western blotting in empty vector cells (lane 2), Sgk1 WT cells (lane 1), or Sgk1 D222A DN cells (lane 3; top panel). The expression of the Sgk1 transgenes was detected by myc
1227
immunoglobulins (middle panel). Equal loading is demonstrated by red Ponceau staining (bottom panel). d Three double transgenic mice (Myc DN D222A–Sgk1/MUP) tTA (lanes 3, 4, and 5), one single transgenic tTA mouse (lane 2), and one wild-type mouse (lane 1) were genotyped and killed as described in the “Experimental procedures” section. p53 and p21 were detected in solubilized livers by Western blotting. e The expression of cDNA coding for the myc-Sgk1 D222A DN mutant transgene was detectable by reverse PCR only in double transgenic animals in the absence of doxicycline (lane 3), whereas it was not detected in double transgenic mice treated with doxicycline (lane 1) as well as in single transgenic (lane 2) and in wild-type animals (lane 4)
1228
Expression of wild-type and dominant negative (D222A) Sgk1 transgenes in the cells was confirmed by immunofluorescence with anti-myc tag immunoglobulins (Suppl. file Fig. 1 C). The morphology of the Sgk1WT cells (Suppl. file Fig. 1 C, panel 1) was different from that of Sgk1 D222A DN cells (Suppl. file Fig. 1 C, panel 2). The former cells appeared swollen with a Sgk1 preferentially localized at the cytoplasm, whereas the latter cells appeared smaller for cytoplasmic regression. The findings were confirmed when cells were observed by light transmission (Suppl. file Fig. 1 D). Parental and empty vector cells showed the typical HeLa cell morphology with a fusiform, fibroblast-like appearance, with poor contact between cells (Suppl. file Fig. 1 D, panels 3 and 4). The Sgk1 WT cells appeared swollen with abundant cytoplasm (Suppl. file Fig. 1D, panel 1), whereas the Sgk1 D222A DN cells (Suppl. file Fig. 1D, panel 2) formed a more coherent epithelial monolayer with several contacts among neighboring cells. Sgk1 down-regulates p53 in stably transfected HeLa cell lines The p53 protein expression was evaluated by Western blotting in empty vector, Sgk1 WT, and Sgk1 D222A DN cells. In Sgk1 WT cells, the expression of p53 was reduced compared with the empty vector cells. On the contrary, in Sgk1 D222A DN cells, the expression of p53 greatly increased. The difference in p53 expression was maintained in culture and detected at different passages (Fig. 4a). Comparable results were obtained when p53 expression was assessed by immunofluorescence in empty vector, Sgk1 WT, and Sgk1 D222A DN cells (Fig. 4b). Expression of p53 protein was detected in cells cultured in the presence of 10% serum and clearly increased when cells were exposed to genotoxic stress, as expected [20] (Fig. 4b, left panels). The increased expression of wild-type Sgk1 was associated with a decreased expression of p53, even in cells exposed to genotoxic stress (Fig. 4 b, middle panels), whereas inhibition of the endogenous kinase was associated with a significant increase in the expression of p53, in cells grown in basal conditions as well as in cells exposed to genotoxic stress (Fig. 4b, right panels). p53 expression in subcutaneous tumors obtained by inoculation with HeLa cells
J Mol Med (2009) 87:1221–1239
tumors obtained by inoculating empty vector cells (Fig. 4d). Myc Sgk1 was also detectable in the epithelial component of the Sgk1 D222A DN tumor cells (Fig. 5e). Interestingly, the expression of α-catenin was increased in the epithelial component of Sgk1 D222A DN cells compared with the empty vector cells (Fig. 4e), a result consistent with the epithelial appearance of the Sgk1 D222A DN cells described above (Suppl. File Fig. 1 D, panel 2). The over-expression of wild-type Sgk1 and the inhibition of the endogenous kinase by the dominant negative mutant (Sgk1-DN) affect the abundance of MDM2 in stably transfected HeLa cell lines The expression of p53 can be increased by regulating either the transcription of the gene or the degradation of the protein through MDM2 or other E3 ubiquitin ligases. An evident decrease in the expression of MDM2, the main regulator of p53, was observed upon inhibition of Sgk1 by specific RNA silencing (Fig. 3b). An effect of Sgk1 on the p53 gene transcription was ruled out since no significant difference in p53 RNA levels was detected by quantitative PCR on cDNA prepared from empty vector [1], Sgk1 WT (1.5±0.6) and Sgk1 DN D222A cells (1.5±0.1). In the attempt to elucidate the mechanism by which Sgk1 affects the expression of p53 and MDM2, the cellular localization of MDM2 and p53 was studied by immunofluorescence. p53 expression was slightly decreased in Sgk1 WT cells (Fig. 5a, middle panel) and clearly increased in Sgk1 DN D222A cells (Fig. 5a, bottom panel), as expected. The expression of MDM2 followed the opposite pattern. It was slightly increased in Sgk1 WT cells (Fig. 5a, middle panel), whereas it was clearly decreased or even abolished in D222A Sgk1cells (Fig. 5a, bottom panel), supporting the idea that a functional Sgk1 is necessary to preserve the abundance of MDM2, presumably by regulating the stability of the protein. Nuclear colocalization of p53 and MDM2 was evident in the control empty vector cells as well as in Sgk1 WT cells (Fig. 5a, upper and middle panels), whereas no colocalization could be detected in D222A Sgk1 cells since MDM2 was virtually undetectable (Fig 5a, bottom panel). MDM2 phosphorylation in intact cells
The expression of p53 was evaluated in tumors that developed 17 days after the subcutaneous inoculation of empty vector and Sgk1 D222A DN cells in a small cohort of BALB/c mice (Fig. 4c; three animals for each group). In the tumors obtained by Sgk1 D222A DN cells, the expression of p53 was increased compared with the
Sgk1-dependent phosphorylation of MDM2 was finally demonstrated in intact empty vector, Sgk1 WT cells and Sgk1 D222A DN cells, before and after insulin stimulation. Insulin increased MDM2 phosphorylation in empty vector cells, as expected (Fig. 5b, lanes 1 and 2). The
J Mol Med (2009) 87:1221–1239
1229
Fig. 4 Sgk1 down-regulates p53 in stably transfected HeLa cell lines. a Extracts from empty vector (lane 1), Sgk1 WT (lane 2), and Sgk1 D222A DN cells (lane 3) at passage 3 (upper gels) and 7 (bottom gels) were immunoblotted using anti-p53 (DO1) antibodies or β-tubulin antibodies, as indicated. b p53 was also detected by immunofluorescence before (top panels) and after DNA stress (bottom panels). The expression of p53 is increased by DNA stress in empty vector cells (left panels), is greatly reduced in Sgk1 WT cells (middle panels), and
is increased in Sgk1 D222A DN cells, both in basal condition and after DNA stress (right panels). c Two groups of three BALB/c male mice, 7 weeks old, were inoculated with 2×106 empty vector or Sgk1 D222A DN cells. d p53 was detected in the inoculated tumor cell lysates. e The expression of the Myc tag and α-catenin was detected in the cell lysates from inoculated empty vector and Sgk1 D222A DN cells as well as in cell lysates from contiguous, unrelated tissues
insulin effect was clearly increased in cells over-expressing wild-type Sgk1, suggesting that Sgk1 is limiting in insulindependent MDM2 phosphorylation (Fig. 5b, lane 3 and 4). Finally, no insulin effect was observed when the endogenous Sgk1 was inhibited by the dominant negative mutant, indicating that Sgk1 is necessary for MDM2 phosphorylation (Fig. 5b, lanes 5 and 6).
Ubiquitylation of p53 is increased in Sgk1 WT cells and decreased in D222A Sgk1 DN cells Stabilization of p53 and inhibition of MDM2 phosphorylation by the dominant negative D222A Sgk1 mutant indicated that MDM2-mediated degradation mechanisms were inhibited by the dominant negative mutant of Sgk1.
1230
J Mol Med (2009) 87:1221–1239
Fig. 5 The expression of a functional Sgk1 regulates the abundance and the activity of MDM2 in stably transfected HeLa cells. a p53 and MDM2 were detected by immunofluorescence in empty vector (top panels), Sgk1 WT (middle panels), and Sgk1 D222A DN cells (bottom panels) as indicated in the “Experimental procedures”. Single channels and merging are provided for each condition. b Phosphoserine 166 MDM2 was detected by Western blotting in MDM2 immunoprecipitates from empty vector (lanes 1 and 2), Sgk1 WT (3 and 4), and Sgk1 D222A DN cells (5 and 6), before (lanes 1, 3, and 5) and after insulin stimulation (lanes 2, 4, and 6). Antibodies to total MDM2 were used as control (bottom panel). c Ubiquitylation of p53 in Sgk1 WT cells and in D222A Sgk1 DN cells. p53 immunoprecipitates from empty vector (1), Sgk1 WT (2), and DN D222A Sgk1 (3) cells, transiently transfected with a vector coding for HA-tagged
ubiquitin and treated with MG132, were immunoblotted with antiHA-immunoglobulins to show the amount of ubiquitinated p53 (top panel). Cell extracts were also examined by immunoblotting with antibody to total p53 (middle panel) and β-tubulin (bottom panel). d Empty vector cells, Sgk1 WT cells, and D222A Sgk1 DN cells were pulse labeled with [35S]methionine and [35S]cysteine, then chased with cold methionine and cysteine as indicated in the “Experimental procedures” section. Endogenous p53 was immunoprecipitated by with DO-1 antibody, and the half-life was analyzed after SDS-PAGE. e The intensity of the bands corresponding to p53 was analyzed by scanning densitometry and expressed as arbitrary units as the percentage of the intensity at time 0 of the chase. Diamonds Empty vector cells, triangles Sgk1 WT cells, squares D222A Sgk1 DN cells
To investigate this issue, we examined whether p53 was ubiquitylated and targeted for degradation by MDM2 in empty vector cells, Sgk1 WT cells, and D222A Sgk1 DN cells, transiently transfected with a vector coding for HA-tagged ubiquitin and treated with MG132 10 μM. p53
immunocomplexes precipitated from cellular extracts were immunoblotted with anti-HA antibodies (Fig. 5c). The ability of p53 to become mono- and polyubiquitylated was clearly increased in Sgk1 WT cells when compared with the empty vector cells (Fig. 5c, lane 2, upper panel), consistent
J Mol Med (2009) 87:1221–1239
1231
Fig. 6 Importance of p53 in the Sgk1-dependent clonogenic potential of cancer cells. a–d A colony assay representative experiment in HeLa cells (a), RKO cells (b), Hek293T cells (c), and H1299 cells (d). Each cell line was treated as indicated in the “Experimental procedures”
section. The clones of several independent experiments were counted and graphically represented as histograms (bottom panel). Data are the average ± SEM of five independent experiments. The differences were evaluated by t test. P values are presented at the bottom of each panel
with the decrease in total p53 (Fig. 5c, lane 2, bottom panel). Interestingly, in D222A Sgk1 DN cells, a decrease in p53 mono- and polyubiquitylation was observed (Fig. 5c, lane 3, upper panel), consistent with the increase in total p53 (Fig. 5c, lane 3, bottom panel).
Figure 5d shows a representative result of two independent experiments. In comparison with the empty vector cells (Fig. 5d, top gel), the half-life of p53 was shortened, and the abundance of p53 was clearly reduced in cells expressing wild-type Sgk1 (Fig. 5d middle gel), whereas the half-life was prolonged and the abundance of p53 was increased when the endogenous kinase was inhibited by the expression of the dominant negative mutant Sgk1 (Fig. 5d, bottom panel). Figure 5e shows a graphical representation of the result where the intensity of the bands corresponding to p53 was analyzed by scanning densitometry and expressed in arbitrary units as the percentage of the intensity at time 0 of the chase.
Sgk1 affects the half-life of p53 The final result of the activation of MDM2-dependent p53 ubiquitylation is predicted to be a decrease in the half-life of p53. Empty vector cells, Sgk1 WT cells, and Sgk1 D222A DN cells were pulse-labeled by [35S]methionine and [35S] cysteine and chased as indicated in the method section.
1232
Sgk1 and clonogenic capacity of cancer cells Since p53 is a crucial regulator of cell proliferation and survival, we evaluated the consequences of the transient overexpression of wild-type and mutant Sgk1 on both cell cycle and survival by colony assay. The clonogenic capacity of p53 expressing cells, i.e., RKO, HeLa, and Hek293 cells, was significantly enhanced by the expression of Sgk1 wild type, whereas it was inhibited by the expression of the dominant negative D222A Sgk1 mutant (Fig. 6a–c), demonstrating that the effect of Sgk1 on the clonogenic capacity of cancer cells is independent from the mechanism of transformation. Interestingly, the dominant negative D222A mutant of Sgk1 had no effect in the p53-null H1299 cells, suggesting that p53 expression is necessary for inhibition of the clonogenic capacity (Fig. 6d). Moreover, in these cells, Sgk1 wild type appeared to inhibit clonogenesis, suggesting the possibility that Sgk1 also acts on a secondary pathway with growth inhibition potentiality, and this pathway is prevalent in the absence of p53. These results suggest that the effects of Sgk1 on the clonogenic potential of cancer cells may be explained in light of the ability of Sgk1 to inhibit p53. Apoptosis and proliferation The peculiar morphology of the Sgk1 WT cells and the Sgk1 D222A DN cells, compared with the empty vector cells, might reflect specific phenotypes related to apoptosis and proliferation. Cell proliferation in stably transfected cell lines was first assessed by cell counting and Trypan blue exclusion. The number of living cells at 48 and 72 h was significantly reduced in Sgk1 D222A DN cells when compared with the empty vector and the Sgk1 WT cells that showed, instead, a significantly increased number of living cells. Conversely, an increased number of dead cells was detected in Sgk1 D222A DN cells, at the same time points (Fig. 7a). The profiles of cell-cycle distribution of unsynchronized cells were further studied by fluorescence-activated cell sorting (FACS) analysis. The Sgk1 WT cells showed a significant reduction in the percentage of apoptotic cells and an increase in the percentage of cells in the G2-M phase, when compared with the empty vector cells. Inhibition of endogenous Sgk1 by dominant negative mutant D222A resulted in a significant increase of basal apoptosis and in a clear-cut reduction in the percentage of cells in the G2-M phase, suggesting that the expression of a functional Sgk1 is essential for cell-cycle progression and survival (Fig. 7b, c). Accordingly, the expression of cyclin B1, an accepted hallmark of the G2-M phase, was inhibited in the Sgk1 D222A DN cells, whereas it was not modified in Sgk1 WT cells (Fig. 7d).
J Mol Med (2009) 87:1221–1239
The inhibition of Sgk1 by its dominant negative mutant is responsible for a consistent delay in the G2-M entry in a synchronized HeLa continuous cell line In order to analyze the timing of cell-cycle progression for the Sgk1 D222A DN cells, compared with the empty vector cells and the Sgk1WT cells, cell cycle was studied in cells synchronized by single hit thymidine and serum starvation. The analysis of cell cycle in synchronized cells revealed that cell-cycle progression was similar in the empty vector and in the Sgk1 WT cells, with a trend to a faster entrance in the G2-M for the Sgk1 WT cells already at 8 h. On the other hand, the Sgk1 D222A DN cells showed a striking delay in the G2-M entry with only 23% of cells in the G2-M phase after 12 h (Fig. 7e). The difference in the percentage of cells in the G2-M phase between empty vector cells and Sgk1 D222A DN, as well as between Sgk1WT cells and Sgk1 D222A DN at 8 and 12 h, was statistically significant (Fig. 7f), thus confirming the crucial role of a functional endogenous Sgk1 in cell-cycle progression. Sgk1 positively modulates epithelial dedifferentiation: the inhibition of the endogenous kinase by the dominant negative mutant (Sgk1 DN) induces re-expression of epithelial hallmarks The different morphology described for the cell lines stably transfected with vectors coding for wild-type and mutant Sgk1 (Suppl. File Fig. 1 D) can be related to the differential expression of epithelial or mesenchymal markers, as was also suggested by the increased expression of α-catenin in D222A dominant negative tumors subcutaneously inoculated in mice (Fig. 4e). The transition from the expression of epithelial to mesenchymal markers (EMT) has been associated with tumor formation and progression. We focused our attention on the expression of ZO-1, α-catenin, and vimentin, detected by immunofluorescence in empty vector, Sgk1WT, and in Sgk1 D222A DN cells (Fig. 8a–c). We observed that the epithelial markers ZO-1 and α-catenin were completely down-regulated in Sgk1 WT cells, when compared with the empty vector cells, whereas the inhibition of the endogenous Sgk1 in the Sgk1 D222A DN cells resulted in the re-expression of these two markers, suggesting a role of Sgk1 in down-regulating epithelial differentiation (Fig. 8a, b). Besides the epithelial markers, the expression of the mesenchymal marker vimentin was studied in the same cells. We found that vimentin was increased in the Sgk1WT cells, compared with the empty vector cells, whereas it was clearly downregulated in the Sgk1 D222A DN cells, thus confirming the role of Sgk1 in inducing mesenchymal transition (Fig. 8c).
J Mol Med (2009) 87:1221–1239
Fig. 7 Sgk1 and cell proliferation in stably transfected HeLa cells. a Empty vector cells, Sgk1 WT cells, or Sgk1 D222A DN cells were plated at a density of 250,000 cells per plate and incubated in the presence of FBS 10% for 48 and 72 h before counting. Dead cells were detected and counted manually by Trypan blue exclusion. Data are means ± SE obtained from triplicates. b Cell-cycle and apoptosis analyses were performed on WT-Sgk1 cells, Sgk1 D222A DN cells, and empty vector cells. c Graphic representation of the mean of several independent experiments of cell cycle and apoptosis evaluations. Data are presented as the relative contribution of each cell-cycle subpopulation in the cell lines shown in a. The differences were analyzed by t test. P values are presented at the bottom of the panel. The percentage of cells in G2-M was significantly increased in Sgk1 WT cells, whereas it was significantly decreased in Sgk1 D222A DN cells. On the contrary, Sgk1 WT cells were characterized by a reduced percentage of apoptotic cells that was increased, instead, in Sgk1 D222A DN cells. d Cell lysates from empty
1233
vector (lane 1), Sgk1 D222A DN (lane 2, top panels), and Sgk1 WT cells (lane 2, bottom panels) were examined by immunoblotting with antibodies to cyclin B1 anti-β-tubulin, as indicated. When compared with empty vector cells, the expression of cyclin B1 appears to be decreased in Sgk1 D222A DN cells (top panels), whereas it is slightly increased in Sgk1 WT cells (bottom panels). e Cell-cycle analysis of synchronized empty vector, Sgk1 WT, and Sgk1 D222A DN cells, 4, 8, and 12 h after the serum pulse, as indicated in the “Experimental procedures” section. The panel shows a representative experiment where the distribution of the cells at times 4, 8, and 12 h is compared with the distribution of the cells at time 0. f Statistical representation of the timedependent distribution of the cellular subpopulations (G0-G1, S-Phase, and G2-M) in the cell lines; the results are expressed as mean ± SE of six independent experiments. The differences were analyzed by t test. P values are presented at the bottom of the panel
1234
Fig. 7 (continued)
J Mol Med (2009) 87:1221–1239
J Mol Med (2009) 87:1221–1239
1235
The results obtained by immunofluorescence for the three markers examined were confirmed by Western blot. Again, over-expression of wild-type Sgk1 was associated with a clear-cut decrease in the expression of the epithelial markers ZO-1 and α-catenin and an increase in the expression of the mesenchymal marker vimentin. On the other hand, inhibition of endogenous Sgk1 by the dominant negative mutant was associated with an increase of the
epithelial markers ZO-1 and α-catenin and a decrease of the mesenchymal marker vimentin (Fig. 8d).
Fig. 8 The expression of mesenchymal markers (vimentin) is enhanced in Sgk1 WT cells. The inhibition of the endogenous kinase by the dominant negative D222A Sgk1 mutant promotes the reexpression of epithelial markers (α-catenin and ZO-1). Sgk1 D222A DN (lane 1), empty vector (lane 2), and Sgk1 1WT cells (lane 3) were stained with immunoglobulins specific for a ZO-1 (FITC-detected), b α-catenin (FITC-detected), and c vimentin (TRITC-detected). The
panel shows two representative fields for each condition at identical magnifications. The cells decorated with anti-catenin antibodies were also stained with ToPro 3 (a). d Cell lysates from empty vector (1), Sgk1 1WTA (2), and Sgk1 D222A DN cells (3) were examined by immunoblotting with antibodies to ZO-1, α -catenin, vimentin, and β-tubulin. Red Ponceau staining is also shown for equal loading
Discussion The results described in the present paper focus on the regulation of p53 as an important mechanism by which
1236
Sgk1 controls cell proliferation, survival, and differentiation. This regulation can take place at the transcriptional or post-translational levels. We were unable to demonstrate an effect of wild-type and mutant Sgk1 on the transcription of p53 by quantitative PCR. On the other hand, we presented evidence demonstrating that Sgk1 regulated the expression of p53 at the post-translational level. The results were obtained in RKO cells, a not virally transformed human colon carcinoma, by Sgk1-specific RNA silencing. The experiments in RKO cells, corroborated by in vitro reconstituted kinase and ubiquitylation assays, led to the original conclusion that Sgk1 kinase activates MDM2-dependent mono- and polyubiquitylation of p53 protein and that the inhibition of the endogenous kinase increases the expression of p53 protein. The E3 ubiquitin ligase MDM2 is considered the main regulator of p53. Even subtle perturbations in MDM2 stoichiometry have profound effects on p53 activity [40]. The interaction between p53 and MDM2 is probably the limiting factor in p53 degradation. For instance, upon genotoxic and oxidative stress, p53 can be stabilized by a series of interdependent phosphorylations on serine and threonine residues at its N-terminal side that contribute to the disruption of the interaction with MDM2 [41]. On the other hand, MDM2 can be activated and stabilized by phosphorylation on several serine residues (serines 166, 186, and 473) by Akt [42, 43] or other kinases including mitogen-activated protein kinase-activated kinase 2 [44]. We believe that modifications in the activity of MDM2 can be explained by the specific phosphorylation of MDM2 on serine 166, a reaction that was effectively inhibited by the dominant negative mutant D222A of Sgk1, in our cell-free experiments. This phosphorylation induces activation and stabilization of MDM2, increasing the E3 ligase function required for p53 ubiquitylation [45]. In fact the inhibition of the endogenous kinase in RKO cells resulted in a significant decrease in the mono- and polyubiquitylation of p53 that paralleled the increase in the expression of the protein. The results obtained in RKO cells were confirmed by dominant negative technology in different cell lines, even in HeLa cells, which are widely used cells [38], that are expected to be more resistant to the inhibition of proteosomal degradation since they express the E6 protein of papillomavirus. In fact the induction of p53 by proteasome inhibition is attenuated in E6 expressing RKO cells, when compared with the parental RKO cells [38]. The results were confirmed in stably transfected HeLa cell lines and in transiently transfected cells, where the effect of the genetic background is generally considered negligible, given the stoichiometry of the high level of expression of the transgene. These observations pointed to Sgk1 as a molecule that is limiting and necessary for the protein expression of p53. In fact the expression of p53 is increased
J Mol Med (2009) 87:1221–1239
by the inhibition of the endogenous kinase by both Sgk1specific RNA silencing and dominant negative technology. On the contrary the expression of p53 was decreased by the over-expression of exogenous kinase. Ubiquitylation experiments in intact HeLa cell lines confirmed the results obtained in vitro and in intact in RKO cells. Wild-type Sgk1 over-expression appeared to be associated with mono- and polyubiquitylation of p53. Conversely, in cells expressing the dominant negative mutant of Sgk1, p53 ubiquitylation was greatly reduced. The overall result of the Sgk1-dependent activation of MDM2 is expected to be decreased in the half-life of p53. Accordingly, in our stably transfected HeLa cells, the halflife of p53 was clearly shortened in Sgk1 WT cells, whereas it was clearly prolonged in Sgk1 D222A DN cells, when compared with the empty vector cells, thus confirming the functional consequences of the hyper-expression of Sgk1 or the inhibition of the endogenous kinase. Finally the findings were confirmed in vivo, in tumors obtained in BALB/c mice 17 days after the subcutaneous inoculation of the cells expressing the dominant negative mutant of Sgk1 and in liver extracts from MUP1-TA D222A DN Sgk1 double transgenic mice characterized by tissue-specific and inducible expression of the transgene. We must here report that, despite the fundamental role of Sgk1 in cell-cycle progression, our MUP1-TA D222A DN Sgk1 double transgenic mice have virtually no phenotype. This is not at all surprising if we consider that even in SGK1 knockout mice the phenotype is mild [46]. Apparently, Sgk1 plays a critical role in cancer cells with upregulated Sgk1 but can be circumvented in normal proliferating tissues. Mice that either are made knockout or harbor null mutations for cancer-related genes do not indeed always show a phenotype [47]. The reproducibility of the observation in different settings proves that the effect of Sgk1 on the expression of p53 can have a general meaning, not limited to a specific cell line or a single methodology. The importance of p53 in Sgk1 signaling was further emphasized by colony assays, a tool designed to study the overall result of both proliferation and survival. The increase in the expression of wild-type Sgk1 was associated with an enhanced clonogenic potential in each cell line expressing wild-type p53, both virus-transformed (HeLa cells and Hek293A) and not virus-transformed (RKO cells). In these cells, the inhibition of the endogenous Sgk1 by a dominant negative mutant greatly reduced the clonogenic potential, indicating that Sgk1 is limiting and necessary for the full expression of the clonogenic phenotype in these cancer cells. On the other hand, inhibition of the endogenous Sgk1 had no effect on the clonogenic potential of cells that did not express p53 (HI299 cells) showing that the clonogenic potential regulated by Sgk1 may depend on the expression of p53.
J Mol Med (2009) 87:1221–1239
Moreover, in these cells, the increased expression of Sgk1 seemed to decrease the clonogenic potential, suggesting that Sgk1 might activate different pathways leading to growth inhibition, in the absence of p53. As a matter of fact Sgk1 has been demonstrated by others to inhibit Raf signaling, thus down-regulating MAP and MAP kinasedependent proliferation [48]. FACS analysis of our stably transfected cells was particularly informative. When compared with the empty vector control cells, the number of hypo-diploid cells, commonly identified as apoptotic cells, was significantly decreased in Sgk1WT cells, whereas it was significantly increased in Sgk1 DN D222A cells, a result that was consistent with what we have previously shown in different cell lines [10]. Sgk1WT cells showed a significant increase in the relative percentage in the number of cells in G2/M, whereas in Sgk1 DN D222A cells, this percentage was significantly decreased together with the expression of cyclin B1, an important marker for G2/M. Cell-cycle analysis of synchronized cells demonstrated that the expression of a functional Sgk1 was essential for cellcycle progression. In fact, the inhibition of the endogenous Sgk1 by the dominant negative mutant was associated with a delay in the entrance to G2/M and an increase in the number of apoptotic cells. This is, per se, a novel observation since both Sgk1 and Akt have already been described as proteins involved in the transduction of survival signals, but neither of them had been associated with regulation of the cell-cycle progression. Interestingly, over-expression of either wild-type Sgk1 or dominant negative mutant had significant consequences on the cellular phenotype. Compared with the empty vector control cells, the Sgk1 WT cells appeared swollen, with a large cytoplasm that we interpreted in the light of the ability of Sgk1 to regulate a wide variety of Na+ coupled transporters [9], which are expected to swell the cells [49]. The Sgk1 DN D222A cells were small, with a high nuclear cytoplasmic ratio and several contacts between neighboring cells, like an epithelial monolayer. As a matter of fact, the expression of mesenchymal markers was found to be increased in Sgk1 WT cells, whereas the expression of epithelial markers was increased, instead, in the Sgk1 DN D222A cells. This observation was confirmed both by immunofluorescence and Western blotting that support a crucial role of Sgk1 in EMT, at least in HeLa cells. Sgk1 was limiting for EMT since the expression of mesenchymal markers was increased in cells over-expressing the kinase. Sgk1 was also essential for EMT since the expression of epithelial markers was increased when the endogenous kinase was inhibited by the dominant negative mutant. The loss of epithelial markers like α-catenin and ZO-1, necessary for monolayer adhesion and cellular polarization,
1237
together with the increase in the expression of mesenchymal markers, like vimentin, can be interpreted in the light of epithelial de-differentiation and transformation, a result that was consistent with the increased expression of Sgk1 detected in several epithelial tumors [50, 51]. Taken together, our data allowed us to formulate a model by which Sgk1 activates MDM2 by phosphorylation, thus driving p53 to proteosomal degradation. According to this model, a novel auto-regulative feedback links Sgk1, p53, and MDM2, whereby p53 exerts a transcriptional regulation of Sgk1, whereas MDM2 and Sgk1 down-regulate the expression of p53 by means of MDM2-mediated ubiquitylation. Since Sgk1 is also regulated by mTOR [3] that is involved in a p53/MDM2 cross talk [52, 53], it is possible that this regulatory loop is part of a more complicated network that includes mTOR, MDM2, and p53. Acknowledgments We thank the American Journal Experts for the editorial revision of the manuscript. This work was supported by the Fondazione Carical, Cofin 2005/068017_004, Interlink II04C0G4EM, Cofin 2006/prot. 2006065339_005, and Firb 2001/RBNE01724C_007. Rosario Amato was partially supported by the Fondazione “Lilli Funaro”. Conflict of interest statement conflict of interests.
The authors declare that they have no
References 1. Perrotti N, He RA, Phillips SA, Haft CR, Taylor SI (2001) Activation of serum- and glucocorticoid-induced protein kinase (Sgk) by cyclic AMP and insulin. J Biol Chem 276:9406–9412 2. Webster MK, Goya L, Ge Y, Maiyar AC, Firestone GL (1993) Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol Cell Biol 13:2031–2040 3. Hong F, Larrea MD, Doughty C, Kwiatkowski DJ, Squillace R, Slingerland JM (2008) mTOR-Raptor binds and activates SGK1 to regulate p27 phosphorylation. Mol Cell 30:701–711 4. Faletti CJ, Perrotti N, Taylor SI, Blazer-Yost BL (2002) Sgk: an essential convergence point for peptide and steroid hormone regulation of ENaC-mediated Na+ transport. Am J Physiol Cell Physiol 282:C494–C500 5. Busjahn A, Aydin A, Uhlmann R, Krasko C, Bahring S, Szelestei T, Feng Y, Dahm S, Sharma AM, Luft FC, Lang F (2002) Serumand glucocorticoid-regulated kinase (SGK1) gene and blood pressure. Hypertension 40:256–260 6. von Wowern F, Berglund G, Carlson J, Mansson H, Hedblad B, Melander O (2005) Genetic variance of SGK-1 is associated with blood pressure, blood pressure change over time and strength of the insulin-diastolic blood pressure relationship. Kidney Int 68:2164–2172 7. Huang DY, Boini KM, Osswald H, Friedrich B, Artunc F, Ullrich S, Rajamanickam J, Palmada M, Wulff P, Kuhl D, Vallon V, Lang F (2006) Resistance of mice lacking the serum- and glucocorticoid-inducible kinase SGK1 against salt-sensitive hypertension induced by a high-fat diet. Am J Physiol Renal Physiol 291:F1264–F1273 8. Mikosz CA, Brickley DR, Sharkey MS, Moran TW, Conzen SD (2001) Glucocorticoid receptor-mediated protection from apopto-
1238
9.
10.
11.
12.
13.
14.
15.
16. 17.
18.
19. 20.
21.
22.
23. 24.
25.
26.
sis is associated with induction of the serine/threonine survival kinase gene, sgk-1. J Biol Chem 276:16649–16654 Lang F, Böhmer C, Palmada M, Seebohm G, Strutz-Seebohm N, Vallon V (2006) (Patho)physiological significance of the serumand glucocorticoid-inducible kinase isoforms. Physiol Rev 286:1151–1178. Review Amato R, Menniti M, Agosti V, Boito R, Costa N, Bond HM, Barbieri V, Tagliaferri P, Venuta S, Perrotti N (2007) IL-2 signals through Sgk1 and inhibits proliferation and apoptosis in kidney cancer cells. J Mol Med 85:707–721 Iwata S, Nomoto M, Morioka H, Miyata A (2004) Gene expression profiling in the midbrain of striatal 6hydroxydopamine-injected mice. Synapse 51:279–286 Simon P, Schneck M, Hochstetter T, Koutsouki E, Mittelbronn M, Merseburger A, Weigert C, Niess A, Lang F (2007) Differential regulation of serum- and glucocorticoid-inducible kinase 1 (SGK1) splice variants based on alternative initiation of transcription. Cell Physiol Biochem 20:715–728 Huang M, Kamasani U, Prendergast GC (2006) RhoB facilitates c-Myc turnover by supporting efficient nuclear accumulation of GSK-3. Oncogene 25:1281–1289 Sherk AB, Frigo DE, Schnackenberg CG, Bray JD, Laping NJ, Trizna W, Hammond M, Patterson JR, Thompson SK, Kazmin D, Norris JD, McDonnell DP (2008) Development of a smallmolecule serum- and glucocorticoid- regulated kinase-1 antagonist and its evaluation as a prostate cancer therapeutic. Cancer Res 68:7475–7483 Papadopoulou N, Papakonstanti EA, Kallergi G, Alevizopoulos K, Stournaras C (2009) Membrane androgen receptor activation in prostate and breast tumor cells: molecular signaling and clinical impact. IUBMB Life 61:56–61 Luft FC (2007) SGK1 survival through various lives may save us all. J Mol Med 85:657–659 You H, Jang Y, You-Ten AI, Okada H, Liepa J, Wakeham A, Zaugg K, Mak TW (2004) p53-dependent inhibition of FKHRL1 in response to DNA damage through protein kinase SGK1. Proc Natl Acad Sci U S A 01:14057–14062 Cornez I, Creppe C, Gillard M, Hennuy B, Chapelle JP, Dejardin E, Merville MP, Close P, Chariot A (2008) Deregulated expression of pro-survival and pro-apoptotic p53-dependent genes upon Elongator deficiency in colon cancer cells. Biochem Pharmacol 75:2122–2134 Hollstein M, Sidransky D, Vogelstein B, Harris CC (1991) p53 mutations in human cancers. Science 253:49–53 Jimenez GS, Khan SH, Stommel JM, Wahl GM (1999) p53 regulation by post translational modification and nuclear retention in response to diverse stresses. Oncogene 18:7656–7665 Li M, Brooks CL, Wu-Baer F, Chen D, Baer R, Wei Gu (2003) Mono- versus polyubiquitination: differential control of p53 fate by Mdm2. Science 302:1972–1975 Midgley CA, Lane DP (1997) p53 protein stability in tumour cells is not determined by mutation but is dependent on Mdm2 binding. Oncogene 15:1179–1189 Moll UM, Petrenko O (2003) The MDM2–p53 interaction. Mol Cancer Res 1:1001–1008. Review Roth J, Dobbelstein M, Freedman DA, Shenk T, Levine AJ (1998) Nucleo-cytoplasmic shuttling of the mdm2 oncoprotein regulates the levels of the p53 protein via a pathway used by the human immunodeficiency virus rev protein. EMBO J 17:554–564 Chen Z, Knutson E, Wang S, Martinez LA, Albrecht T (2007) Stabilization of p53 in human cytomegalovirus-initiated cells is associated with sequestration of MDM2 and decreased p53 ubiquitination. J Biol Chem 282:29284–29295 Li Y, Jenkins CW, Nichols MA, Xiong Y (1994) Cell cycle expression and p53 regulation of the cyclin-dependent kinase inhibitor p21. Oncogene 9:2261–2268
J Mol Med (2009) 87:1221–1239 27. Taylor WR, Stark GR (2001) Regulation of the G2/M transition by p53. Oncogene 20:1803–1815 28. Soengas MS, Alarcón RM, Yoshida H, Giaccia AJ, Hakem R, Mak TW, Lowe SW (1999) Apaf-1 and caspase-9 in p53dependent apoptosis and tumor inhibition. Science 284:156–159 29. Porrello A, Cerone MA, Coen S, Gurtner A, Fontemaggi G, Cimino L, Piaggio G, Sacchi A, Soddu S (2000) p53 regulates myogenesis by triggering the differentiation activity of pRb. J Cell Biol 6:1295–1304 30. Cam H, Griesmann H, Beitzinger M, Hofmann L, BeinoraviciuteKellner R, Sauer M, Hüttinger-Kirchhof N, Oswald C, Friedl P, Gattenlöhner S, Burek C, Rosenwald A, Stiewe T (2006) p53 family members in myogenic differentiation and rhabdomyosarcoma development. Cancer Cell 10:281–293 31. Mies F, Spriet C, Heliot L, Sariban-Sohraby S (2007) Epithelial Na channel stimulation by n-3 fatty acids requires proximity to a membrane-bound A-kinase-anchoring protein complexed with protein kinase A and phosphodiesterase. J Biol Chem 282:18339–18347 32. Maiyar AC, Phu PT, Huang AJ, Firestone GL (1997) Repression of glucocorticoid receptor transactivation and DNA binding of a glucocorticoid response element within the serum/glucocorticoidinducible protein kinase (sgk) gene promoter by the p53 tumor suppressor protein. Mol Endocrinol 11:312–329 33. Rinaldo C, Prodosmo A, Mancini F, Iacovelli S, Sacchi A, Moretti F, Soddu S (2007) MDM2-regulated degradation of HIPK2 prevents p53Ser46 phosphorylation and DNA damage-induced apoptosis. Mol Cell 25:739–750 34. Treier M, Staszewski LM, Bohmann D (1994) Ubiquitindependent c-Jun degradation in vivo is mediated by the delta domain. Cell 78:787–798 35. Manickan E, Satoi J, Wang TC, Liang TJ (2001) Conditional liver-specific expression of simian virus 40 T antigen leads to regulatable development of hepatic neoplasm in transgenic mice. J Biol Chem 276:13989–13994 36. Bloom J, Pagano M (2005) Experimental tests to definitively determine ubiquitylation of a substrate. Methods Enzymol 399:249–266 37. Bhushan S, Malik F, Kumar A, Kaur Isher H, Pal Kaur I, Taneja SC, Singh J (2009) Activation of p53/p21/PUMA alliance and disruption of PI-3/Akt in multimodal targeting of apoptotic signaling cascades in cervical cancer cells by a pentacyclic triterpenediol from Boswellia serrata. Mol Carcinog. doi:10.1002/mc.20559 38. Chen F, Chang D, Goh M, Klibanov SA, Ljungman M (2000) Role of p53 in cell cycle regulation and apoptosis following exposure to proteasome inhibitors. Cell Growth Differ 11:239– 246 39. Lober C, Lenz-Stoppler C, Dobbelstein M (2002) Adenovirus E1transformed cells grow despite the continuous presence of transcriptionally active p53. J Gen Virol 83:2047–2057 40. Geyer RK, Yu ZK, Maki CG (2000) The MDM2 RING-finger domain is required to promote p53 nuclear export. Nat Cell Biol 2:569–573 41. Lee JH, Kim HS, Lee SJ, Kim KT (2007) Stabilization and activation of p53 induced by Cdk5 contributes to neuronal cell death. J Cell Sci 120:2259–2271 42. Feng J, Tamaskovic R, Yang Z, Brazil DP, Merlo A, Hess D, Hemmings BA (2004) Stabilization of Mdm2 via decreased ubiquitination is mediated by protein kinase B/Akt-dependent phosphorylation. J Biol Chem 279:35510–35517 43. Zhou BP, Liao Y, Xia W, Zou Y, Spohn B, Hung MC (2001) HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nat Cell Biol 3:973–982 44. Weber HO, Ludwig RL, Morrison D, Kotlyarov A, Gaestel M, Vousden KH (2005) HDM2 phosphorylation by MAPKAP kinase 2. Oncogene 24:1965–1972
J Mol Med (2009) 87:1221–1239 45. Ashcroft M, Ludwig RL, Woods DB, Copeland TD, Weber HO, MacRae EJ, Vousden KH (2002) Phosphorylation of MDM2 by Akt. Oncogene 21:1955–1962 46. Wulff P, Vallon V, Huang DY, Völkl H, Yu F, Richter K, Jansen M, Schlünz M, Klingel K, Loffing J, Kauselmann G, Bösl MR, Lang F, Kuhl D (2002) Impaired renal Na(+) retention in the sgk1knockout mouse. J Clin Invest 110:1263–1268 47. Bera TK, Pastan I (2000) Mesothelin is not required for normal mouse development or reproduction. Mol Cell Biol 20:2902–2906 48. Zhang BH, Tang ED, Zhu T, Greenberg ME, Vojtek AB, Guan KL (2001) Serum- and glucocorticoid-inducible kinase SGK phosphorylates and negatively regulates B-Raf. J Biol Chem 276:31620–31626 49. Waldegger S, Barth P, Raber G, Lang F (1997) Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc Natl Acad Sci 94:4440–4445
1239 50. Chung EJ, Sung YK, Farooq M, Kim Y, Im S, Tak WY, Hwang YJ, Kim YI, Han HS, Kim JC, Kim MK (2002) Gene expression profile analysis in human hepatocellular carcinoma by cDNA microarray. Mol Cell 14:382–387 51. Leong ML, Maiyar AC, Kim B, O'Keeffe BA, Firestone GL (2003) Expression of the serum- and glucocorticoid-inducible protein kinase, Sgk, is a cell survival response to multiple types of environmental stress stimuli in mammary epithelial cells. J Biol Chem 278:5871–5882 52. Kojima K, Shimanuki M, Shikami M, Samudio IJ, Ruvolo V, Corn P, Hanaoka N, Konopleva M, Andreeff M, Nakakuma H (2008) The dual PI3 kinase/mTOR inhibitor PI-103 prevents p53 induction by Mdm2 inhibition but enhances p53-mediated mitochondrial apoptosis in p53 wild-type AML. Leukemia 22:1728–1736 53. Feng Z, Zhang H, Levine AJ, Jin S (2005) The coordinate regulation of the p53 and mTOR pathways in cells. Proc Natl Acad Sci U S A 102:8204–8209
