Immunocytochemistry (ICC) - Regulation of TPA-induced c-fos expression by nuclear Erk1/2 in HDF

F. Regulation of TPA-induced c-fos expression by nuclear Erk1/2 in HDF old cells

I. INTRODUCTION

5. Immunocytochemistry (ICC)

Cells on cover slips were washed with PBS before fixation with 4% paraformaldehyde for 15 min, permeabilized with 0.05% Triton X-100, and then incubated with 3% bovine serum albumin in 0.05% Triton X-100 for 2 h. Primary antibody was applied overnight at 4 °C, secondary antibody at 4 °C for 2 h, and then stained with 4% 6-diamidino-2-phenylindole (2.0 mg/ml) for 10 min at RT before mounting. Expressions of pErk1/2 and PEA-15 were detected using polyclonal primary antibodies along with Alexa 488 or Alexa 555 conjugated goat-anti rabbit IgG as a secondary antibody. Data visualized under fluorescence microscope were photographed by AxioVision image acquisition and analyzed by software package (Carl Zeiss MicroImaging GmbH).

6. BrdU incorporation assay

Cells were treated with TPA for 30 min after transfection with either siControl or siPEA-15 for 48 h, pre-labeled with 10 μM BrdU (Sigma) for 2 h, fixed in 4% paraformaldehyde solution, and incubated in 2 M HCl for 30 min. pH was raised with 0.1 M sodium borate (pH 8.5) for 2 min. Immunocytochemistry steps were followed by using anti-BrdU FITC-labeled antibody

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(eBioscience, San 125 Diego, CA, USA), and positive cells were counted under fluorescence microscope.

7. Cell cycle analysis

Cells were treated with TPA for 30 min after transfection with siPEA-15 for 48 h. They were then trypsinized and fixed before re-suspended in propidium iodide solution. DNA analysis was performed by flow cytometry (BD Biosciences, San Jose, CA, USA).

8. Cell proliferation assay

HDF mid-old cells (1.0 x 10⁵) were plated in 60 mm dish 24 hours before transfection for 4 h with siRNAs (40nM) against PEA-15 or its scrambled sequences (siControl), and then the culture medium was changed with fresh one before incubation for 4 more days. Treatment of HDF cells with either DMSO (0.01%) or TPA (50 ng/ml) before the 4 day incubation was employed as the negative and positive controls of the experiment, respectively. All of the cells were harvested in 2 and 4 days after the treatment, and the numbers of cells were then counted by using Hemocytometer (Marienfeld-Superior, Germany). Changes of cell numbers were evaluated by Student's t-test. To monitor cell proliferation assay, HDF young cells (0.8 x 10⁵) were plated in 60 mm dish, and then their proliferation activity was also monitored every 2 days.

9. Real-time PCR analysis

Total cellular RNAs were extracted with RNAiso Plus (TaKaRa Bio, Ohtsu, Japan), and cDNAs were synthesized with 1.0 μg of RNA and reverse transcription kit (Invitrogen). The cDNAs were used for real time PCR analysis with specific primers and SYBR Green PCR Master Mix (Applied Biosystems) under the conditions: Initial activation at 95 °C for 15 min, followed by 40 cycles at 95 °C for 20s and 60 °C for 40s. Primers used for c-fos amplification were 5’-TGACTGATACACTCCAAGCGGA-3 and 5’-CAGGTCATCAGGGATCTTGCA-3’

and for β-actin were 5’-CCCTGGCACCCAGCAC-3’ and 5’-GCCGATCCACACGGAGTAC-3’

10. siRNA transfection

siRNAs against PEA-15 (siPEA-15) were purchased from Santa Cruz Biotechnology and control siRNAs from DHARMACON (Lafayette, CO, USA). Cells on a cover slip were transfected with siRNAs and Oligofectamine (Invitrogen) for 4-6 h. In 48 h, the cells were

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treated with mitogen for 30 min before subjected to immunocytochemistry or immunoblot analyses.

11. Statistical analysis

All data were presented as means ±S.D and analyzed by one-way ANOVA for comparison between multiple groups or Student’s t-test using SPSS. Probability value less than 0.05 was considered as statistically significant.

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III. RESULTS

A. Nuclear translocation of pErk1/2 apart from cytoplasmic PEA-15 upon TPA treatment To explore changes of PEA-15 expression and its interaction with pErk1/2 in cellular senescence, immunoblot and immunoprecipitaion analyses were carried out in HDF cells (Fig.

2A); expressions of PEA-15 in the young and old cells were same (upper panel), while their interaction with pErk1/2 was significantly higher in the old than young cells (lower panel).

Moreover, pErk1/2 and PEA-15 were co-localized in the cytoplasm of the old cells, examined by immunocytochemistry (Fig. 2B). Nuclear translocation of pErk1/2 was significantly induced in young cells upon by either EGF or TPA treatment, however, old cells responded only to TPA, but not to insulin or EGF (Fig. 2C). Therefore, SA-pErk1/2 observed in the cytoplasm was accordingly reduced. Cell fractionation (Fig. 2D) and immunocytochemistry (Fig. 2E) revealed evident nuclear translocation of pErk1/2 apart from PEA-15 in HDF old cells after TPA stimulation.

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Fig. 2. Nuclear translocation of SA-pErk1/2 apart from PEA-15 upon TPA treatment. (A) Interaction of PEA-15 with SA-pErk1/2 in HDF old, but not young cells. HDF young and old cells were subjected to immunoblot and immunoprecipitation analyses by using anti-PEA-15 antibody. β-actin and H-chain were used as loading controls, respectively. Expression of 15 was not different between young and old cells (upper panel), whereas the interaction of PEA-15 with pErk1/2 was significant only in old cells (lower panel). (B) Immunocytochemistry:

Cytoplasmic co-location of SA-pErk1/2 and PEA-15 in the cytoplasm of HDF old cells. (C) Nuclear translocation of pErk1/2 in HDF old cells only by TPA treatment. Young and old cells were treated with either vehicle (0.01% DMSO), insulin (10 μg/ml), EGF (10 ng/ml) or TPA (50 ng/ml) for 30 min, and then subjected to immunocytochemical analysis with anti-pErk1/2 antibody. More than 100 cells were counted in randomly chosen fields, and the cells with pErk1/2 in nuclei were counted. Data indicate means ± SD after 3 independent experiments.

TPA significantly increased nuclear translocation of pErk1/2 in both HDF young and old cells, while EGF was effective only in young cells. (D) Immunoblot anlsysis showing nuclear translocation of pErk1/2 without PEA-15 upon TPA treatment. To evaluate whether pErk1/2 translocation to nuclei is accompanied with PEA-15 or not, HDF old cells treated with either vehicle or TPA for 30 min were fractionated into nucleus (Nu) and cytoplasm (Cyt), and hydridized with anti-pErk1/2 and anti-PEA-15 antibodies. Lamin B1 and α-tubulin were used to exclude reciprocal contamination of the factions and loading controls. Note absence of PEA-15 in the nuclear fraction, despite translocation of pErk1/2 into nuclei, of old cells after TPA treatment. Cytoplasmic pErk1/2, but not PEA-15, was increased by TPA treatment. (E) Immunocytochemistry; No nuclear translocation of PEA-15 in old cells treated with TPA for 30 min.

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B. Knockdown of PEA-15 expression induced pErk1/2 translocation to nuclei

To further explore the role of PEA-15 in the pErk1/2 translocation, PEA-15 phosphorylation at S¹⁰⁴ and S¹¹⁶ residues was examined. TPA treatment induced PEA-15 phosphorylation at S¹⁰⁴ in both young and old cells, whereas insulin and EGF induced PEA-15 phosphorylation at S¹¹⁶ along with Akt activation (Fig. 3A). Despite increased pErk1/2 expression in HDF old cells (shown in Fig. 3B, 10% input data), IP-immunoblot analysis revealed that TPA treatment reduced the interaction of PEA-15 with pErk1/2 about 50% in the old cells compared with that of the vehicle treatment, whereas there was almost no interaction in the young cells (Fig. 3B).

Moreover, knockdown of PEA-15 by siPEA-15 transfection (Fig. 3C, left panel) significantly induced pErk1/2 translocation, regardless of insulin or EGF co-treatment (Fig. 3C, right panel,

#p<0.04). However, TPA co-treatment further increased the effect of siPEA-15 transfection compared with other treatments (†p<0.05), suggesting dissociation of pErk1/2 from PEA-15 by phosphorylating S¹⁰⁴ residue and knockdown of PEA-15 induced nuclear translocation of SA-pErk1/2 in HDF old cells.

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Fig. 3. Knockdown of PEA-15 induced nuclear translocation of pErk1/2. (A) TPA induced phosphorylation of PEA-15 at S¹⁰⁴ residue. When cells were treated with either vehicle, insulin, EGF or TPA for 30 min, TPA induced phosphorylation of PEA-15 at S¹⁰⁴ in both young and old cells, whereas insulin and EGF induced phosphorylation of PEA-15 at S¹¹⁶ and Akt at S⁴⁷³ residues. (B) Immunoprecipitation and immunoblot analyses. Treatment of old cells with TPA significantly reduced PEA-15 bound to pErk1/2 compared with the vehicle treatment (0.5 in the TPA vs. 1.0 in the vehicle), despite the presence of much higher amount of pErk1/2 in the TPA-treated sample (right panel). In contrast, there was no interaction of pErk1/2 with PEA-15 in young cells, despite the high induction of pErk1/2 in young cells (left panel). (C) Induction of nuclear translocation of pErk1/2 by knockdown of PEA-15 in HDF old cells. Cells were transfected with either scrambled siRNAs (siControl) or siPEA-15 for 48 h (left panel). To explore the effect of PEA-15 knockdown on pErk1/2 translocation, cells were treated with either vehicle, insulin, EGF or TPA for 30 min during 48 h of transfection and then subjected to immunocytochemical analysis with anti-pErk1/2 antibody (right panel). Knockdown of PEA-15 itself significantly increased pErk1/2 translocation to nuclei of old cells (^#p<0.04), however, TPA co-treatment further increased pErk1/2 translocation compared with any single treatment (^†p<0.05). Data indicate mean ± SD from the duplicates of two independent experiments.

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C. Knockdown of PEA-15 and TPA treatment reduced senescence phenotype. Nuclear translocation of pErk1/2 by either siPEA-15 or TPA treatment was accompanied with significant reductions of senescence markers in HDF old cells; Over 10% decrease of SA-β-gal activity (Figs. 4A and 4C), and 50% decrease of p53 and p21^WAF1 expressions (Figs. 4B and 4D).

Furthermore, treatment of old cells with either siPEA-15 (Fig. 4E) or TPA (Fig. 4F) significantly reduced senescence markers such as PML body formation, and expressions of 53BP1 and H3K9me2, as opposed to no response in young cells. Taken together, siPEA-15 transfection or TPA treatment significantly reduced heterochromatin changes and inactive histone methylations in senescent cells, reversing senescence phenotypes along with nuclear translocation of pErk1/2.

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Fig. 4. Reversal of senescence phenotypes by either knockdown of PEA-15 or TPA treatment. (A) Significant inhibition of SA-β-gal activity by transfection of siPEA-15 for 48 h.

Knockdown of PEA-15 in HDF old cells significantly inhibited SA-β-gal activity in total 800 cells in randomly chosen field. Data indicate percentage of total cells (means ± SD) after 3 independent experiments. (B) HDF old cells transfected with indicated siRNAs were harvested, and expressions of p53 and p21^WAF1 were measured by immunoblot analysis. Densitometric analysis of band intensity was performed by image J software. Targeted bands were normalized to the expression of α-tubulin. Note significant inhibition of p53 and p21^WAF1 expressions by transfection with siPEA-15. (C) To evaluate the effect of TPA on SA-β-gal activity, HDF old cells were treated with TPA for 48 h, and the activity was assayed along with its quantification.

The activity was also significantly reduced 24 h after TPA treatment. (D) Under the same condition, cells were harvested and immunoblotted with anti-p53 and anti-p21^WAF1 antibodies.

Targeted bands were normalized to the expression of α-tubulin. Note significant reductions of p53 and p21^WAF1 expression after TPA treatment. Data are presented as means ± SD after 3 independent experiments. (E) Chromatin changes of HDF old cells in response to knockdown of PEA-15 by transfection with siPEA-15, and examined by immunocytochemistry before Image J software quantification. Markers of senescence were all significantly reduced by knockdown of PEA-15 expression as compared with the siControl transfection. (F) Treatment of old cells with TPA for 48 h significantly reduced the average size of PML bodies, number of 53BP1 foci and fluorescence intensity of H3K9me2 obtained from at least 500 nuclei in each group compared with the untreated control. The above data showed the reversal of senescence phenotype, whereas there was no significant difference in HDF young cells after TPA treatment.

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D. Progression of G1/S phase by either TPA or siPEA-15 transfection. To elucidate cell cycle progression upon pErk1/2 translocation, senescent cells were treated with either TPA or siPEA-15 transfection. Both BrdU incorporation (Fig. 5A) and FACS analysis (Fig. 5B) revealed significantly increased BrdU positive cells entering into S-phase in the senescent cells compared with the control. The phenomena were further evidenced by immunoblot analyses;

Both TPA treatment (Fig. 5C) and siPEA-15 transfection (Fig. 5D) significantly induced the loss of p21^WAF1 expression and pRb phosphorylation along with cyclin A synthesis. Above data strongly support that nuclear translocation of pErk1/2 apart from PEA-15 triggers the reversal of senescence phenotype.

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Fig. 5. Progression of G1 to S phase by either TPA treatment or knockdown of PEA-15 in HDF senescent cells. (A) Immunocytochemistry with anti-BrdU antibody. Senescent cells were pre-labelled with 10 μM BrdU for 2 h and fixed in 4% paraformaldehyde solution for 15 min before incubation in 2 M HCl for 30 min, and then pH was restored to 8.5 with 0.1 M sodium borate for 2 min. The steps were followed by using anti-BrdU FITC-labeled antibody. Note less BrdU positive in the mid-old cells than young cells, whereas it was significantly increased by either TPA treatment or siPEA-15 transfection. (B) Cell cycle analysis by FACS. To further confirm the above data, flow cytometry analysis was performed. Note significant induction of S phase cells by treatment with TPA and siPEA-15 transfection. To further confirm the escape of G1 arrest at molecule levels, senescent cells treated with either TPA (C) or siPEA-15 transfection (D) were subjected to immunoblot analysis. Note significant increase of cyclin A and pRb phosphorylation by TPA treatment and by siPEA-15 transfection in 24 and 48 h, respectively, indicating the cell cycle progression from G1 arrest to S phase along with reduction of p21^WAF1 expression. The reason why there was no change of cyclin D1 and cyclin E expression until 48 h might be due to long doubling time of the mid-old cells (over 7 days).

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E. Increase of cell proliferation by either knockdown of PEA-15 or TPA treatment. To examine whether senescent cells accelerate cell division cycle or not in response to knockdown of PEA-15 or TPA treatment, HDF mid-old cells were transfected with either siRNAs against PEA-15 or siControl, along with TPA treatment as the control. As shown in Fig. 6A and B, the senescent cells transfected with siPEA-15 showed significant increase of cell proliferation in 2 days as compared with the siControl transfected cells (P=0.04), and the difference was more significant in 4 days after the transfection (Fig. 6A, P=0.002). At the same time, treatment of HDF mid-old cells with TPA exhibited much more significant cell proliferation than that of the DMSO treated cells (Fig. 6B, P=0.017 in 2 days and P<0.0001 in 4 days), HDF young cells actively proliferated every day until 4 days (data not shown), indicating the reversal of senescence phenotype along with active cell proliferation.

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Fig. 6. Increase of cell proliferation by either knockdown of PEA-15 or TPA treatment. (A) To determine the change of cell proliferation by knockdown of PEA-15 or TPA treatment, 1x10⁵ HDF mid-old cells (doubling time 6-7 d) were transfected with either 40 nM siPEA-15 or siControl for 4 h, and then the cells were harvested at the indicated times. Transfection of senescent cells with siPEA-15 revealed significant increase of cell proliferation in 2 days as compared with the siControl transfected cells (P=0.04), and the difference became larger in 4 days after the transfection (P=0.002). (B) Treatment of HDF mid-old cells with TPA exhibited

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significant cell proliferation much more than that of the DMSO treated cells in 2 days (P=0.017) and the difference was also larger in 4 days (P<0.0001).

F. Regulation of TPA-induced c-fos expression by nuclear Erk1/2 in HDF old cells. In the above experiment, we showed active cell proliferation in senescent cells after knockdown of PEA-15 and TPA treatment, indicating the presence of further signal to stimulate cell proliferation after escape from G1 arrest to S phase. Therefore, the effect of pErk1/2 on the regulation of cell proliferation was evaluated by knockdown of Erk1/2 expression in HDF old cells. Transfection of HDF old cells with siRNAs to Erk1/2 (siErk1/2) for 48 h significantly inhibited TPA-induced c-fos expression compared with that of the siControl transfected cells, when examined by real-time RT-PCR analysis (p<0.01, Fig.7). DMSO (vehicle) treatment and parent cells were used as the controls for TPA treatment and the siErk1/2 transfection assay.

Inset shows immunoblot analysis of HDF old cells after transfection with siControl and siErk1/2 RNAs. The data strongly suggest the role of pErk1/2 translocation to nuclei by either knockdown of PEA-15 or TPA treatment in the proliferation of senescent cells.

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Fig. 7. Regulation of TPA-induced c-fos expression by nuclear Erk1/2 in HDF old cells. To explore whether pErk1/2 translocated to nuclei has any effect on cell proliferation, HDF old cells were transfected with either siRNA to Erk1/2 (siErk1/2) or scrambled siRNAs (siControl) for 48 h, and then treated with either DMSO (vehicle) or TPA for 30 min before subjected to real-time RT-PCR analysis. Degree of c-fos expressions was normalized based on the level of β-actin expression. Note that c-fos expression was markedly increased in the parent cells and the siControl transfected cells in response to TPA treatment, whereas the expression was significantly reduced by knockdown of Erk1/2 (p<0.01). Data represents mean ± SD of 3 independent experiments. Inset shows immunoblot analysis of HDF old cells after transfection with siControl and siErk1/2 RNAs.

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IV. Discussion

Replicative senescence exhibits two characteristic phenomena, predominant retention of Erk1/2 in cytoplasm (SA-Erk1/2) and nuclear accumulation of G-actin, which represent translocational inefficiency of intracellular proteins (Lim et al., 2000; Lim et al., 2001).

However, the characteristic morphology of replicative senescence can be reversed by TPA, when monitored by actin cytoskeleton staining (Kwak et al., 2004). We described herein that knockdown of PEA-15 reversed senescence phenotypes along with pErk1/2 translocation from cytoplasm to nucleus, and that TPA treatment dissociated SA-pErk1/2 from PEA-15 via induction of PEA-15 S¹⁰⁴phosphorylation in old cells (Figs. 2 and 3). siPEA-15 itself significantly increased pErk1/2 translocation to nuclei (Fig. 3C), and TPA further increased the translocation. The findings are well accordant with eariler studies that PEA-15 anchors Erk1/2 in cytoplasm (Formstecher et al., 2001) and PEA-15S¹⁰⁴ can be phosphorylated by PKC (Kubes et al., 1998), and are further supported by the study that downregulation of PEA-15 expression by oncogene E1A restores pErk1/2 in nuclei which avert murine embryo fibroblasts from senescence (Gaumont-Leclerc et al., 2004). Although insulin and EGF induce mitogenic and metabolic effects by regulating pErk1/2 in most cell types (Boulton et al., 1991; Wilson et al.,1994), senescent cells in the present study showed altered response to mitogens; e.g. failure of pErk1/2 translocation from cytoplasm to nuclei by insulin and EGF, but not TPA, stimulation.

It is highly possible that the failure might be due to the phosphorylation of PEA-15 at S¹⁰⁶ by pAkt (Trencia et al., 2003), because there is no difference in the expression of PEA-15 between young and old cells, despite more interaction of pErk1/2 with PEA-15 (Fig. 2A) and the TPA-induced dissociation of PEA-15 from pErk1/2 in the old cells (Fig. 3B). Therefore, cytoplasmic sequestration of pErk1/2 by PEA-15 appears to be an important mechanism to maintain cellular senescence and resistance to mitogenic signals in senescent cells.

A recent study described nuclear translocation of pErk1/2 by hBVR (Lerner-Marmarosh et al., 2008), however, we were unable to observe in vivo interaction as well as any correlation between nuclear translocation of pErk1/2 and hBVR upon TPA treatment (data not shown).

Three other mechanisms have also been suggested to regulate nuclear import of pErk1/2;

passive diffusion of a monomer, active transport of a pErk2 dimer (Khokhlatchev et al., 1998;

Adachi et al., 1999) and direct interaction with nuclear pore complex independent of

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phosphorylation, energy, and transport factors (Matsubayashi et al., 2001).

When senescent cells were treated once with TPA or with siPEA-15, senescence markers of heterochromatin foci such as PML body and 53BP1 expression were significantly reduced along with histone modification (Fig. 4), whereas there was no change in the HDF young cells after TPA treatment (data not shown). Moreover, cell cycle progression from G1 arrest to S phase, increased pRb phosphorylation with cyclin A synthesis, and reduction of p21^WAF1expression (Figs. 5C and 5D) strongly supported the reversal of senescence phenotype by the knockdown of PEA-15 expression and TPA treatment via pErk1/2 translocation to nuclei. TPA has been known as the most potent tumor promoter and PKC activator which leads to proliferation of initiated (transformed) cells under carcinogenesis. To accomplish the reversal of senescence phenotype, the cells require further stimulation for cell proliferation. Knockdown of SA-pErk1/2 by transfection of siErk1/2 significantly reduced TPA-stimulated c-fos expression in the HDF old cells (Fig. 7), implicating that induction of nuclear translocation of pErk1/2 by knockdown of PEA-15 may regulate the expression of transcription factors which stimulate cell proliferation in the senescent cells. Indeed, transfection of HDF mid-old cells with siPEA-15 RNA (Fig. 6A) as well as the treatment of the cells with TPA (Fig. 6B) significantly increased cell proliferation in 2 days after transfection. Furthermore, our previous finding that knockdown of PKCa increased proliferation of senescent cells (Kim et al., 2009) also strongly support that TPA treatment overcomes growth arrest of HDF senescent cells. In summary, therefore, translocation of pErk1/2 to nuclei of senescent cells induces further stimulation of cell proliferation, and we suggest the nuclear translocation of SA-pErk1/2 apart from PEA-15 as an

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