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is a negative regulator of cell cycle progression in C2C12 mouse myoblasts
* Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell’Apparato Locomotore, Sezione di Anatomia, Università di Bologna, Bologna, Italy;
Servizio di Immunoematologia e Trasfusionale, Policlinico S. Orsola-Malpighi, Bologna, Italy;
Dipartimento di Anatomia e Istologia, Università di Modena e Reggio Emilia, Modena, Italy;
Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata, Japan; and
|| IGM-CNR, IOR, Bologna, Italy
1Correspondence: Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell’Apparato Locomotore, Cell Signalling Laboratory, Università di Bologna, via Irnerio 48, 40126 Bologna, Italy. E-mail: alberto.martelli{at}unibo.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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interferes with cell cycle progression. We previously reported that nuclear DGK- expression increased during myogenic differentiation, whereas its down-regulation impaired differentiation. Here, we evaluated the possible involvement of nuclear DGK- in cell cycle progression of C2C12 myoblasts. Overexpression of a wild-type DGK-, which mainly localized to the nucleus (but not of a kinase dead mutant or of a mutant that did not enter the nucleus), blocked the cells in the G1 phase of the cell cycle, as demonstrated by in situ analysis of biotinylated-16-dUTP incorporated into newly synthesized DNA and by flow cytometry. In contrast, down-regulation of endogenous DGK- by short interfering RNA (siRNA) increased the number of cells in both the S and G2/M phases of the cell cycle. Cell cycle arrest of cells overexpressing wild-type DGK- was accompanied by decreased levels of retinoblastoma protein phosphorylated on Ser-807/811. Down-regulation of endogenous DGK-, using siRNA, prevented the cell cycle block characterizing C2C12 cell myogenic differentiation. Overall, our results identify nuclear DGK- as a key determinant of cell cycle progression and differentiation of C2C12 cells. —Evangelisti, C., Tazzari, P. L., Riccio, M., Fiume, R., Hozumi, Y., Falà, F., Goto, K., Manzoli, L., Cocco, L., Martelli, A. M. Nuclear diacylglycerol kinase- is a negative regulator of cell cycle progression in C2C12 mouse myoblasts.
Key Words: nucleus • lipid-dependent signaling pathways • siRNA • phosphorylated pRB
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Diacylglycerol (DG) is a fundamental lipid second messenger that is generated within the nucleus and is involved in different processes including cell proliferation, differentiation, and apoptosis (3)
. Indeed, it has been shown that nuclear DG mass increases along cell cycle progression (4)
. Conceivably, nuclear DG attracts DG-dependent isoforms of protein kinase C (PKC), which move to the nucleus in response to different agonists (5)
. Previous results have shown that the nucleus also contains diacylglycerol kinases (DGKs), i.e., the enzymes that phosphorylate DG to yield phosphatidic acid (PA). DGKs attenuate DG signals and initiate resynthesis of phosphoinositides consumed by phospholipase C (PLC) during cellular signal transduction. PA itself is an important lipid second messenger with targets distinct from those of its precursor DG. For example, PA is involved in cytoskeletal organization by inducing actin polymerization and stress fiber formation. Moreover, it regulates important enzymes including PAK1, PKC-, Ras-GAP, and protein phosphatase 1 (6
, 7)
. Ten mammalian isoforms of DGK have been identified. DGKs have a conserved catalytic domain in the COOH-terminal region and at least a pair of cysteine-rich motifs that could bind DG. The different isoforms are also characterized both by the presence of additional motifs that have structural diversity and by a complex pattern of tissue expression. These differences suggest that they function in distinct cellular processes (7)
.
DGK- is an isoform that is detected in muscle, brain, and gut. DGK- has four tandem ankyrin-repeats near the COOH terminus and exhibits a nuclear localization signal (NLS; refs. 8
, 9
) that overlaps with a sequence similar to the myristoylated alanine-rich protein kinase C substrate (MARCKS; ref. 10
). This sequence is phosphorylated by conventional PKC-
and -
isoforms, and this facilitates DGK- export from the nucleus (11)
. Moreover, DGK- has a classic leucine-rich nuclear export signal spanning from amino acids 362–370 (9)
. In C2C12 mouse myoblasts, DGK- is localized in both the nucleus and the cytoplasm, even though in the latter compartment it is much less expressed (12)
. Furthermore, previous studies (11)
showed that overexpression of DGK- within the nucleus regulates nuclear DG levels and cell cycle progression from G1 to S phase. Nevertheless, these results were obtained by overexpressing DGK- cDNA and might not reflect a physiological role for this DGK isoform. Here, using C2C12 cells, we confirm that DGK- acts as a negative regulator of cell cycle progression when overexpressed in the nucleus. DGK--induced cell cycle arrest was characterized by a decrease in the phosphorylation levels of the retinoblastoma protein (pRB) selectively on Ser-807/811 residues. More importantly, however, we also show that endogenous DGK- acts a repressor of DNA replication, as its down-regulation, by short interfering RNA (siRNA), resulted in a higher percentage of cells being in the S and G2/M phases of the cell cycle. We also demonstrate that DGK- plays an important role in the cell cycle withdrawal, which characterizes myogenic differentiation of C2C12 cells in response to insulin. Thus, our findings for the first time identify an experimental model in which changes in the amount of endogenous nuclear DGK- are in relationship with the replicative state.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]ATP was from Amersham Biosciences (Milan, Italy). Genesilencer siRNA transfection reagent was from Gene Therapy Systems (San Diego, CA, USA). The following primary antibodies were employed in the present study: rabbit polyclonals to Ser-807/811 pRB, Ser-780 pRB, and mouse monoclonals to ß-tubulin, ß-actin, p21Waf1/Cip1, and Ser-795 pRB from Sigma-Aldrich; mouse monoclonal to green fluorescent protein (GFP) from Roche Applied Science; mouse monoclonals to cyclin D3, cyclin-dependent kinase (cdk) 4, cdk6, and rabbit polyclonals to p15INK4B and p27Kip1 from Cell Signaling Technology (Beverly, MA, USA). Goat polyclonal antibody to lamin B was from Santa Cruz Biotechnology. Rabbit polyclonal antibody to DGK-, and the rat cDNA constructs [wild-type (WT) DGK-/pEGFP, 
C DGK-/pEGFP] were as previously reported (13)
, while rabbit polyclonal antibody to DGK-
was a kind gift from Dr. F. Sakane (Sapporo Medical University School of Medicine, Sapporo, Japan). Kinase dead (KD) rat DGK- cDNA was generated by replacing the catalytic domain Gly356 with Asp using the Quickchange site-directed mutagenesis kit of Stratagene (La Jolla, CA, USA).
Cell culture and transfection
Mouse C2C12 cells were grown in Dulbecco’s modified minimum essential medium (DMEM), containing 10% heat-inactivated FCS, and 1% (v/w) penicillin/streptomycin. Cells were differentiated in DMEM supplemented with 50 nM insulin and 1% (v/w) penicillin/streptomycin in the absence of FCS. Low-passage cells were cultured in 6-well plates for 18 h before transfection. Approximately 3 x 105 cells per well were used for transfection experiments by FuGene method, according to the manufacturer’s protocols.
Flow cytometric analysis of cell cycle
C2C12 cells were harvested and fixed with 70% cold (–20°C) ethanol. Samples were washed twice with PBS, and pellets were incubated with propidium iodide (PI; 20 µg/ml) for 30 min. Samples were analyzed by a FC500 flow cytometer (Beckman Coulter, Miami, FL, USA) equipped with CXP software.
siRNA down-regulation of DGK- and DGK-
This was performed using the Genesilencer siRNA transfection reagent as described previously (12)
.
Preparation of cell homogenates and Western blot analysis
Cells were washed twice in phosphate-buffered saline (PBS, pH 7.4) containing the Complete Protease Inhibitor Cocktail supplemented with 1 mM Na3VO4. Cells were lysed at 107/ml in boiling electrophoresis sample buffer containing the protease inhibitor cocktail. Lysates were then briefly sonicated to shear DNA and reduce viscosity and boiled for 5 min to solubilized protein. Protein separated on sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was transferred to nitrocellulose membranes using a semidry blotting apparatus. Membranes were saturated for 60 min at 37°C in blocking buffer (PBS supplemented with 5% NGS, and 4% BSA) and then incubated overnight at 4°C in blocking buffer containing the primary antibody. After four washes in PBS containing 0.1% Tween 20, they were incubated for 30 min at room temperature with peroxidase-conjugated secondary antibody diluted 1:5000 in PBS-Tween 20 and washed as above. Bands were visualized by the ECL method. The level of expression of different proteins was analyzed by using the public domain software Image J [a Java image processing program inspired by National Institutes of Health (NIH) Image for Macintosh] by working in a linear range. It can calculate area and pixel value statistics of user-defined selections. Briefly, it was done as follows: X-ray films were scanned and saved as 8-bit grayscale JPEG files. The percentage of measurable pixels in the image was set (and highlighted in red) by using the adjust image threshold command. The number of square pixels in the section selected (the protein bands) was then counted by measuring the area in the binary or thresholded image.
Preparation of isolated nuclei
This was accomplished as reported elsewhere (12)
.
Protein assay
This was performed according to the instruction of the manufacturer using the Bio-Rad Protein Assay.
DGK Assay
Cells were lysed 24 h after transfection in lysis buffer (50 mM Tris, pH 8.0, 50 mM KCl, 10 mM EDTA, 1% Nonidet P-40, Complete Protease Inhibitor Cocktail, and 2 mM Na3VO4). To determine the kinase activity of overexpressed WT DGK- and mutants, the homogenate samples were first immunoblotted with an antibody to GFP to assess that they contained comparative amounts of the hybrid protein. Immunoprecipitations were performed overnight using an anti-GFP monoclonal antibody (for transfected cells) or a polyclonal antibody to DGK- in untransformed cells. Antibodies were captured using protein A/G-agarose, and immunoprecipitates were washed with lysis buffer. They were washed once with 25 mM Tris, pH 7.4, 10 mM MgCl2, 80 mM KCl, and 1 mM EGTA (kinase buffer) and then resuspended in 20 µl of 10 mM Tris (pH 7.4) for the DGK activity assay. 1-Stearoyl-2-arachidonoyl-sn-glycerol (Biomol, Plymouth Meeting, PA, USA) was used as substrate. Reactions were performed at 30°C for 10 min in kinase buffer containing 10 µM cold ATP and 5 µCi of [-32P]ATP in a final volume of 100 µl. Lipids were extracted with 0.5 ml of chloroform/methanol (1:1, v/v), followed by the addition of 125 µl of 2.4 M HCl and phase separation. Lipid extracts were dried and separated by thin layer chromatography (silica gel 60 TLC plates soaked in 1 mM EDTA and 1 mM potassium oxaloacetate and heat-activated) using chloroform/methanol/water/ammonia (45:35:7.5:2.5, v/v/v/v). Lipids were visualized by autoradiography and quantified by scintillation counting.
Immunofluorescence staining
Cells were fixed for 30 min at room temperature with 4% freshly prepared paraformaldehyde in PBS. The coverslips were blocked using PBS containing 3% BSA, 5% NGS for 1 h at room temperature. The primary antibodies were appropriately diluted (anti-pRB Ser-807/811, anti-pRB Ser-780, and anti-pRB Ser-795 1:1000, anti-DGK- 1:5000, antip21Waf1/Cip1 1:1000) in PBS with 2% BSA and incubated for 3 h at 37°C. Samples were washed with PBS, and the fluorescent secondary antibodies (1:200 for FITC-conjugated, 1:1000 for Cy3-conjugated) were incubated for 30 min in the same buffer as the primary antibody. Cy3- or FITC-conjugated anti-mouse IgG were employed to reveal p21Waf1/Cip1, while Cy3-conjugated anti-rabbit IgG was used to label pRB Ser-807/811, pRB Ser-780, and DGK-. Cy3-conjugated anti-mouse IgG was used to label pRB Ser-795. Images were taken on a Zeiss Axio Imager.Z1 microscope, equipped with 60x/NA 1.40 optics and Apotome apparatus, coupled to a computer driven Zeiss AxioCAM digital camera (MRm), using Zeiss Axio Vision (4.4) software.
In situ DNA synthesis by biotinylated-16-dUTP
DNA replication sites were labeled essentially as described by Nakayasu and Berezney (14)
with some modifications. Cells were grown on coverslips in DMEM supplied with 10% FCS. Cells were washed with TBS buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 5 mM MgCl2) and then incubated in glycerol buffer (20 mM Tris-HCl, pH 7.4, 25% glycerol, 5 mM MgCl2, 0.5 mM EGTA, and 0.5 mM PMSF) for 10 min. Washed cells were gently permeabilized with 0.04% Triton X-100 in glycerol buffer for 3 min at room temperature and further washed with glycerol buffer. Cells were treated at 37° for 15 min with 60 µl of DNA synthesis buffer (50 mM Tris HCl, pH 7.4, 10 mM MgCl2, 0.5 mM EGTA, 25% glycerol, 40 µM dATP, 40 µM dGTP, 40 µM dCTP, 16 µM biotinylated-16-dUTP, and 2 mM ATP) and washed with TBS buffer. After biotin incorporation, cells were fixed in absolute methanol at –20° for 10 min. Coverslips were washed twice with TBS buffer containing 0.2% Tween and incubated at 37° for 45 min with 60 µl of Cy3-conjugated streptavidin (1:100 in TBS-Tween buffer). Finally, samples were washed for three times with TBS containing 0.2% Tween and stained for DNA using 0.5 µg/ml 4',6-diamidino-2-phenylindole (DAPI).
Statistical evaluation
Data are shown as mean values ± SD. Data were statistically analyzed by a Dunnet test after one-way analysis of variance (ANOVA) at a level of significance of P < 0.05 vs. control samples.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/GFP forms in the control of cell cycle progression in C2C12 cells, we transiently transfected cells with WT DGK-/GFP, C DGK-/GFP, and KD DGK-/GFP.
Previous results have shown that WT DGK-/GFP predominantly localized to the nucleus of neurons and C2C12 cells (13
, 15)
. C DGK- is a truncated form that comprises amino acid residues 1–663 and did not localize to the nucleus (13)
. Indeed, the ankyrin repeats in the COOH terminus of DGK- seem to be pivotal for the nuclear localization (13)
. In KD DGK-, there is a single amino acid substitution (Gly356Asp) in the catalytic domain that negatively affects ATP binding and kinase activity. The expression, activity, and localization in C2C12 cells of the three DGK-/GFP hybrid proteins were analyzed first. Western blot analysis with a polyclonal antibody to rat DGK- (13)
showed similar levels of expression for both WT DGK-/GFP and KD DGK- 24 h after transfection (Fig. 1
A). The molecular mass of the two proteins was, as expected, around 131 kDa. Expression levels were 
3-fold over endogenous DGK-. However, the polyclonal antibody did not recognize C DGK-, since it had been raised using a fragment encoding amino acid residues 606–929 of DGK- (13)
. Nevertheless, this antibody allowed us to detect endogenous levels of DGK-, which were not affected by transfection. The molecular mass of the endogenous protein was 104 kDa, which corresponds to the deduced molecular mass of the mouse DGK- (Fig. 1A
). To analyze expression levels of C DGK-, we then employed a monoclonal antibody to GFP. In this case, Western immunoblotting revealed similar levels of expression for all the three hybrid proteins after 24 h of transfection. The molecular mass of C DGK-/GFP was 104 kDa, as expected (Fig. 1B
). In vitro DGK activity assays showed that both immunoprecipitated WT DGK-/GFP and C DGK-/GFP generated almost the same amount of PA, whereas KD DGK-/GFP retained only 10% of catalytic activity (Fig. 1C
). As a control, we also assayed endogenous DGK- activity of untransfected C2C12 cells by immunoprecipitating DGK- from cell homogenates. This activity was 30% of the DGK activity measured in cells overexpressing either WT DGK-/GFP or C DGK-/GFP (Fig. 1C
).
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Immunofluorescence staining revealed that both WT DGK-/GFP and KD DGK-/GFP mostly localized to the nucleus, whereas C DGK-/GFP was completely excluded from it (Fig. 1D
).
Taken together, these findings indicated that both WT DGK-/GFP and C DGK-/GFP retained the same catalytic activity; however, WT DGK-/GFP was mostly nuclear, whereas C DGK-/GFP was completely cytoplasmic. As to KD DGK-/GFP, it displayed a greatly reduced catalytic activity but was still capable of localizing to the nucleus.
In situ detection of DNA synthesis by biotinylated-16-dUTP
DNA synthesis was analyzed by following the incorporation of biotinylated-16-dUTP into nascent DNA of gently permeabilized C2C12 cells. Several previous studies (14
, 16
, 17)
have indicated that this is a faithful method to study DNA replication at the single cell level. Fluorescent microscopy was used to map DNA replication sites in the interphase cell nucleus after incubation with Cy3-conjugated streptavidin. The nascent DNA was located in numerous granules that were distributed throughout the nuclear interior. Twenty-four hours after seeding, 35–40% of cells were replicating their DNA (Fig. 2
A). C2C12 samples transfected with WT DGK-/GFP cDNA never showed double positive cells, despite an 35–40% transfection efficiency (Fig. 2B
). In cells overexpressing either C DGK-/GFP cDNA or KD DGK-/GFP, there were 16% of double positive cells (Fig. 2C, D
). Considering that the transfection efficiency of the constructs after 24 h was 35–40%, these results were consistent with an 40% of the cells being in the S phase of the cell cycle, in full agreement with the data for untransfected cells. These findings also showed that transfection per se did not alter DNA replication. Taken together, these results suggested that overexpressed catalytically active DGK- acts as a repressor of cell cycle progression when it localizes to the nucleus.
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Flow cytometric analysis of cell cycle
The effects of overexpressing the various hybrid proteins on exponentially growing C2C12 cells were also evaluated by flow cytometry analysis of PI-stained samples. In comparison with control (untransfected) cells, cells overexpressing WT DGK-/GFP displayed a significant decrease in the amount of cells in S phase of the cell cycle, from 38 to 23%, and a concomitant increase in the percentage of G1 cells, from 42 to 62% (Table 1
). In contrast, no changes at all were seen in samples transfected with the other cDNAs (KD DGK-/GFP or C DGK-/GFP). Thus, these findings, demonstrating a cell cycle block in G1 after overexpression of WT DGK-/GFP, were in full agreement with those obtained by means of biotinylated-16-dUTP incorporation.
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siRNA down-regulation of endogenous DGK- positively affects DNA replication
To date, the evidence that suggests that nuclear DGK- acts as a negative regulator of the cell cycle has been gathered only by overexpression experiments (11
, 18)
. To establish if also endogenous DGK- could be involved in controlling progression from G1 to S phase, DGK- levels were down-regulated by siRNA. DGK- expression was decreased by 50%, as evaluated by densitometric scanning of the blots (Fig. 3
A). As demonstrated by flow cytometric analysis, DGK- down-regulation resulted in a significant increase (P<0.05) of the cells in the S phase (from 25 to 34%) and in G2/M phase (from 17 to 24%, P<0.05) in the siRNA treated cells, whereas the quantity of G1 phase cells decreased from 50 to 40% (P<0.05, Fig. 3B
). In contrast, siRNA down-regulation of another DGK isoform expressed by C2C12 cells, DGK-, did not significantly affect cell cycle, despite a similar decrease in the DGK expression levels (Fig. 3A, B
).
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WT DGK-/GFP overexpression results in hypophosphorylated Ser-807/811 pRB
pRB is a critical regulator of the cell cycle transition from G1 to S phase (19)
. As cells progress through late G1 to S phase, pRB becomes increasingly phosphorylated. Different cyclin/cdk complexes preferentially phosphorylate particular phosphoacceptor residues in pRB, and every phosphorylation step has distinct effects (20
, 21)
. Thus, the phosphorylation state of pRB was examined by both immunofluorescence staining and Western blot in C2C12 cells overexpressing the various hybrid proteins. In cells overexpressing WT DGK-/GFP, we never observed, by immunofluorescence staining, cells double positive for GFP and Ser-807/811 pRB (Fig. 4
A). In contrast, cells overexpressing either C DGK-/GFP or KD DGK-/GFP were also positive for Ser-807/811 pRB, as revealed by immunofluorescence analysis. Indeed, we found that 15–18% of cells were double positive for both this pRB phosphorylated form and GFP, and this, assuming a transfection efficiency of 40%, was in agreement with a percentage of cells in S phase of 40% (Fig. 4A
). Overexpression of WT DGK-/GFP did not affect pRB phosphorylation on Ser-780 or Ser-795, as double positive cells were detected (Fig. 4A
).
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These results were corroborated by Western blot analysis, which demonstrated a dramatic decrease in the amount of Ser-807/811 only in samples with forced expression of WT DGK-/GFP. Levels of Ser-780 and Ser-795 did not change in these samples. Moreover, the amount of all three of the pRB phosphorylated forms did not show any variations in cells overexpressing either C DGK-/GFP or KD DGK-/GFP. Western blot analysis also showed no variations in the amount of total pRB expressed in response to transfection with any of the three types of constructs (Fig. 4B
). Overall, these experiments indicated a selective inhibition of Ser-807/811 pRB phosphorylation in samples overexpressing a functional form of DGK- that mainly localizes to the nucleus of mouse myoblasts.
Key cell cycle regulatory protein expression levels do not change in WT DGK- transfected cells
Since WT DGK- overexpression results in a G1/S phase transition block, the expression of some key regulatory proteins for progression from G1 to S phase was evaluated next. Indeed, cdk4 and cdk6 assemble with D-type cyclins (including cyclin D3) to form holoenzymes that facilitate exit from G1 phase by phosphorylating key substrates, such as pRB (22)
. Activation of the holoenzymes is antagonized by polypeptide inhibitors of cdk activity. p27Kip1 is a cdk inhibitor that plays an important role in negative regulation of the cell cycle during G0 and G1 phases. Degradation of p27Kip1 is a critical event for the G1/S transition (23)
. p15INK4B is a well-established inhibitor of cdk4 and cdk6 (24)
. However, the amount of cdk4, cdk6, cyclin D3, p15INK4B, and p27Kip1 did not significantly change in cells transduced with the three hybrid proteins (WT DGK-/GFP, C DGK-/GFP, KD DGK-/GFP) when compared with untransfected cells, as demonstrated by Western blot analysis (Fig. 5
A). We also investigated by immunocytochemistry the expression of the cdk inhibitor p21Waf1/Cip1. p21Waf1/Cip1 is not expressed in exponentially growing C2C12 cells (25)
. However, as shown in Fig. 5B
, cells overexpressing WT DGK-/GFP did not show any positivity for p21Waf1/Cip1.
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DGK- down-regulation impairs myogenic differentiation by opposing cell cycle arrest
Previous evidence had shown that, during C2C12 myogenic differentiation, nuclear DGK- increased, whereas siRNA down-regulation of DGK- negatively affected the differentiation program of C2C12 myoblasts (12)
. Considering that myogenic differentiation of C2C12 cells is characterized by a withdrawal from the cell cycle (26)
and that overexpression of DGK- blocks C2C12 cells in the G1 phase of the cell cycle (this study), we aimed at determining if siRNA down-regulation of DGK- would oppose the cell cycle arrest induced by insulin and low serum. In preliminary experiments to demonstrate that cells with an increased nuclear content of DGK- were in the G0/G1 phase of the cell cycle, we performed double immunostaining with antibodies to DGK- and p21Waf1/Cip1 antibodies. Indeed, it has been shown that cells positive for nuclear p21Waf1/Cip1 do not replicate DNA anymore (27)
. As indeed shown in Fig. 6
A, cells with enhanced expression of nuclear DGK- also exhibited strong nuclear positivity for p21Waf1/Cip1.
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Next, DGK- was down-regulated by siRNA in cells exposed to insulin for 48 h, and the cell cycle was analyzed by flow cytometry. In control cells, 48 h after insulin treatment, 92% of cells were in G0/G1. In contrast, in cells with down-regulated DGK-, 60% of cells were in G0/G1 (P<0.05), whereas cells in the S and G2/M phases accounted for 33% of the total (P<0.05; Fig. 6B
). As a control, siRNA down-regulation of DGK- did not affect at all the cell cycle of insulin-treated cells. Western blot analysis confirmed a similar decrease in the amount of both DGK- and DGK- (Fig. 6B
). Taken together, these findings demonstrated that siRNA down-regulation of endogenous DGK- opposes the G0/G1 block, which characterizes myogenic differentiation of C2C12 cells, in full agreement with the hypothesis of a negative role played by this DGK isoform in regulating G1/S phase progression. The expression of DGK- was evaluated in both whole homogenate and isolated nuclei of control and differentiated cells (Fig. 6C
). Western blot analysis demonstrated a slight increase in DGK- expression in whole cell homogenates. However, a marked up-regulation of DGK- (>3-fold) was observed in the nuclear fraction, which was similar to the increase detected in cells transfected with WT DGK-/GFP (compare Fig. 6C
with Fig. 1A
).
pRB phosphorylation was also evaluated in differentiating C2C12 cells by Western blot, which displayed a dramatic decrease in this pRB form along with an increase in total pRB (Fig. 6D
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/GFP hybrid protein, which mainly localized to the nucleus of C2C12 mouse myoblasts, blocked cells in the G1 phase of the cell cycle. In contrast, neither a KD DGK-/GFP nor a C DGK-/GFP hybrid protein had any effects on cell cycle progression. Thus, our findings strongly indicate that both the DGK catalytic activity and nuclear localization are required to negatively affect cell cycle progression, in agreement with the data of Topham et al. (11)
. Indeed, using COS-7 cells transfected with cDNAs encoding WT DGK-, a KD mutant (ATP, G355
D355), or a mutant that did not localize to the nucleus (i.e., lacking the NLS) along with GFP as a reporter, these authors showed that overexpression of WT DGK-, but not of the KD or the NLS mutants, induced an accumulation of the cells in the G0/G1 phases of the cell cycle.
However, we have shown here for the first time that endogenous DGK- also acts as a negative regulator of DNA replication, since its down-regulation by siRNA resulted in a significant increase in the percentage of cells in the S and G2/M phases of the cell cycle, along with a concomitant decrease in G1 phase cells. It is widely accepted that nuclear DGKs are involved in the control of DG/PA levels (6)
. How nuclear DG/PA levels might regulate cell cycle progression is not clear. DG is a potent activator of some PKC isoforms. For example, DG-dependent PKC- was found capable of regulating cell cycle progression indirectly by modulating the cdk inhibitor, p21Waf1/Cip1 (28)
, whereas PKC-ßII directly phosphorylated pRB (29)
. Several PKC isoforms are known to migrate to the nucleus in response to a variety of stimuli (30)
; however, the information concerning nuclear PKC isoforms in C2C12 cells is extremely limited (31)
. Nevertheless, in cells overexpressing WT DGK-, p21Waf1/Cip1 was not detected, so that it seems unlikely that DGK- may control cell cycle progression by regulating the levels of this cdk inhibitor.
In contrast to DG, there are no clear roles for PA in cell cycle progression. However, it is noteworthy that protein phosphatase PP1 is potently inhibited by its interaction with PA (32)
. Interestingly, PP1 is a nuclear protein, the nuclear location of which is regulated during cell cycle progression (33)
. Our findings showing a decreased phosphorylation level of Ser-807/811 pRB in cells with force expression of WT DGK- seem particularly intriguing in light of two studies demonstrating that these two residues are key determinants of pRB activity. In fact, it has been shown that a pRB mutant with alanine substitutions at Ser-807/811 had enhanced growth suppressing activity (34)
. Moreover, phosphorylation of Ser-807/811 led to an inactivation of pRB tumor suppressor activity in uveal melanoma (35)
. As stated above, PKC-ßII directly phosphorylates pRB; however, the phosphorylative events do not involve Ser-807/811 (29)
. Furthermore, depending on the timing of PKC activation during G1, cell proliferation can be stimulated or inhibited, and this includes either potentiation or inhibition of pRB phosphorylation (36)
.
Recently, Los et al. (18)
have shown that unphosphorylated pRB specifically interacted with DGK- in vitro and in vivo. Moreover, they demonstrated that pRB and other pocket protein family members (p107 and p130) are potent activators of DGK- activity in vitro. In a subsequent study, the same group showed that the direct physical interaction between pRB and DGK- was regulated by PKC, particularly PKC- (37)
. Since overexpression of DGK- was able to partially rescue a cell cycle arrest defect in response to -irradiation in pRB-null mouse embryonic fibroblasts, these authors hypothesized that DGK- was down-stream of pRB or in a parallel pathway leading to inhibition of G1/S phase transition (18)
. However, our findings showing a decreased pRB phosphorylation selectively on Ser-807/811 in cells overexpressing WT DGK- suggest the possibility that this DGK isoform may act up-stream of pRB, thus negatively influencing DNA replication. This hypothesis is also strengthened by our unpublished results showing that, in C2C12 cells, pRB did not coimmunoprecipitate with DGK-, at variance with the findings of Los et al. (18)
. In contrast, we have demonstrated coimmunoprecipitation of DGK- with phosphatidylinositol-specific PLC-ß1 (12)
.
Since the results of Los et al. (18)
hinted that nuclear DGK- could repress DNA replication even in the absence of pRB, we investigated other factors that could influence progression from G1 to S phase of the cell cycle. However, in cells overexpressing WT DGK-, we did not detect changes in the expression levels of cdk4, cdk6, cyclin D3, as well as of the two cdk/cyclin complex inhibitors p15 INK4B and p27Kip1. Also, p21Waf1/Cip1 expression was not influenced by WT DGK- overexpression. Clearly, given the great complexity of cell cycle regulation, we could not rule out that other proteins, important for G1/S progression, are affected by nuclear DGK-.
We have also been able to link for the first time an increased expression of nuclear DGK- with a withdrawal from the cell cycle in an in vitro model of cell differentiation. Indeed, we have demonstrated that, when C2C12 cells underwent myogenic differentiation in the presence of insulin, nuclear DGK- was up-regulated, and this correlated with a G0/G1 block and expression of p21Waf1/Cip1. Indeed, previous evidence has suggested that only when C2C12 cells express p21Waf1/Cip1 at the nuclear level do they undergo cell cycle arrest and do not replicate their DNA anymore (27)
. Remarkably, the level of DGK- expression detected in differentiated cells was similar to that observed in cells overexpressing WT DGK-. This finding suggests a physiological significance of the experiments performed by overexpressing WT DGK-/GFP.
Accordingly, when DGK- was down-regulated by siRNA, the cell cycle arrest was not complete and myogenic differentiation was severely impaired, as demonstrated by low levels of the expression of the myogenic marker myogenin. In contrast, down-regulation of DGK-, which in C2C12 cells does not localize to the nucleus (12)
, did not influence at all the cell cycle block and this fact demonstrated the specificity of action of nuclear DGK-. Remarkably, cell cycle arrest of C2C12 cells exposed to insulin was characterized by a down-regulation of Ser-807/811 pRB levels, as happens in untreated C2C12 cells overexpressing WT DGK-. It is known that cell cycle arrest of differentiating C2C12 cells is characterized by an increase in the amount of unphosphorylated pRB and cyclin D3 (38)
and by a decrease of phosphorylated pRB (26)
. Cyclin D3 sequesters unphosphorylated pRB. Among the different phosphorylated pRB forms, Ser-780 pRB formed a complex with LAP2 (39)
. However, it is not clear how dephosphorylation of pRB is regulated in this experimental model, even though it could be due in part to p21Waf1/Cip1 interacting with and blocking cdk4 activity (40)
. Our results strongly suggest that increased levels of nuclear DGK- could be responsible for dephosphorylation of pRB on Ser-807/811, even though the precise molecular mechanisms underlying this phenomenon still require identification.
The increase in nuclear DGK- might be related to the up-regulation of nuclear phosphatidylinositol-specific PLC-ß1, which also characterizes myogenic differentiation of C2C12 cells. Such an increase is responsible for increased expression of the cyclin D3 gene (41)
, as well as for the decreased amount of nuclear phosphatidylinositol 4,5-bisphosphate (the PLC-ß1 substrate and the DG precursor) that is observed in these cells in response to insulin-induced differentiation (12)
.
In conclusion, it is now clear that lipid-dependent signaling pathways are key players in cell growth and differentiation, and transient accumulation of DG, particularly in the nucleus, is important for this regulation (4
, 42
, 43)
.
Our results show that nuclear DGK- is a key player for both DNA replication and differentiation in C2C12 cells for its ability to control G1/S phase progression, most likely through the phosphorylation status of pRB on critical serine residues. Future investigations should clarify how nuclear DGK- could influence G1/S phase transition.
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ACKNOWLEDGMENTS |
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Received for publication February 13, 2007. Accepted for publication April 5, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. J. Biol. Chem. 273,29738-29744. J. Biol. Chem. 271,10230-10236. Nature 394,697-700[CrossRef][Medline] in C2C12 mouse myoblasts. J. Cell Physiol. 209,370-378[CrossRef][Medline] in neurons. Eur. J. Neurosci. 18,1448-1457[CrossRef][Medline]. J. Biol. Chem. 281,858-866-induced cell cycle progression. Mol. Cell Biol. 20,4580-4590. J. Biol. Chem. 279,39122-39131 to the retinoblastoma protein. Biochim. Biophys. Acta 1773,352-357
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