Báo cáo khoa học: PKCd-dependent cleavage and nuclear translocation of annexin A1 by phorbol 12-myristate 13-acetate pdf

We have found that PMA induces the cleavage ofANX-1 in human embryonic kidney HEK 293 cells, and that the cleaved form of ANX-1 translocates to the nucleus.. The PMA-induced nuclear tran

PKCd-dependent cleavage and nuclear translocation of annexin A1

by phorbol 12-myristate 13-acetate

Yoon S Kim1,*, Jesang Ko2,*, In S Kim1, Sung-Wuk Jang2, Ho J Sung2, Hye J Lee1, Si Y Lee1, Youngho Kim3 and Doe S Na1

1

1

Department of Biochemistry and Molecular Biology;2Asan Institute for Life Sciences, and3Genome Research Center for Birth Defects and Genetic Diseases, University of Ulsan College of Medicine, Seoul, Korea

Annexin A1 (ANX-1), a calcium-dependent, phospholipid

binding protein, is known to be involved in diverse cellular

processes, including regulation ofcell growth and

differen-tiation, apoptosis, and inflammation The mitogen phorbol

12-myristate 13-acetate (PMA) induces expression and

phosphorylation ofANX-1 However, the roles ofANX-1

in PMA-induced signal transduction is unknown Here,

we study the cellular localization ofANX-1 in the

PMA-induced signal transduction process We have found

that PMA induces the cleavage ofANX-1 in human

embryonic kidney (HEK) 293 cells, and that the cleaved

form of ANX-1 translocates to the nucleus The PMA-induced nuclear translocation ofANX-1 was inhibited by the protein kinase C (PKC)d-specific inhibitor rottlerin, indicating that PKCd plays a role in nuclear translocation of the cleaved ANX-1 We propose a novel mechanism of PMA-induced translocation ofANX-1 to the nucleus that may participate in the regulation ofcell proliferation and differentiation

Keywords: annexin A1; PMA; cleavage; nuclear transloca-tion; PKCd

Annexins (ANXs) are a family of calcium-dependent,

phospholipid-binding proteins Several members ofthe

ANX family are known to be involved in various

physio-logical functions including anti-inflammatory processes, cell

signaling, regulation ofcell growth and differentiation,

apoptosis, membrane fusion, exocytosis, and interaction

with cytoskeletal proteins [1–3] Although there have been

recent advances in understanding the molecular mechanisms

by which ANXs play a role in these cellular processes, the

regulatory mechanism in cell proliferation and

differenti-ation remains to be characterized It has been reported that

ANX-1 is involved in regulation ofthe mitogenic signal

transduction pathways including the mitogen-activated

protein (MAP) kinase-, the epidermal growth factor receptor

(EGFR)-, and the hepatocyte growth factor receptor

(HGFR) kinase-mediated signaling pathways [4–6] The

expression level ofANX-1 increases in response to phorbol

12-myristate 13-acetate (PMA) and interleukin (IL)-6 [7,8],

and dys-regulation ofANX-1 results in development of various cancers [9,10] Mitogens such as EGF and PMA induce phosphorylation, cleavage, and translocation of ANX-1 to the membrane [11–13] and this phosphorylation event is mediated by protein kinase C (PKC) [14]

ANX-1 mainly exists in the cytosol, but also exists in the membrane or the nucleus [15] Recent reports suggest that subcellular localization ofANX-1 can be redistributed by treatment with specific stimuli ANX-1 translocates to the membrane and is secreted to the extracellular surface of the cell membrane in response to glucocorticoid and PMA [16,17] Although we have previously proposed that EGF, oxidative stress and heat shock induce translocation of ANX-1 to the nucleus [18,19], the mechanism for nuclear translocation ofANX-1 is still unknown

PKCs are serine-threonine kinases that are activated by diverse stimuli including mitogens and participate in a variety ofcellular processes such as cell proliferaction and differentiation, and apoptosis [20,21] The PKC family consists of12 isoforms that are grouped into three subfamilies: the classical PKCs (a, b1, b2, v), the novel PKCs (d, e, g, h), and the atypical PKCs (n, k/i) PKCd, a member ofthe novel PKC subfamily, is activated by diacylglycerol (DAG) and phorbol esters in a calcium-independent manner and plays a critical role in the control ofcell growth and apoptosis [22]

In this study, we aimed to elucidate whether PMA induces the translocation ofANX-1 to the nucleus in human embryonic kidney (HEK) cells and the roles of PKCd in the nuclear translocation ofANX-1 We propose

a mechanism for the nuclear translocation of ANX-1 in response to PMA that may be involved in cellular processes such as cell proliferation and differentiation

Correspondence to D S Na, Department ofBiochemistry and

Molecular Biology, University ofUlsan College ofMedicine,

388–1 Poongnap-dong, Songpa-gu, Seoul 138–736, Korea.

Fax: + 82 2 477 9715, Tel.: + 82 2 3010 4275,

E-mail: dsna@amc.seoul.kr

Abbreviations: ANX, annexin; PMA, phorbol 12-myristate 13-acetate;

HEK, human embryonic kidney; PKC, protein kinase C; MAP kinase,

mitogen-activated protein kinase; EGFR, epidermal growth factor

receptor; HGFR, hepatocyte growth factor receptor; DAG,

diacylglycerol; DMEM, Dulbecco’s modified Eagle’s medium.

*Note: the first two authors contributed equally to this work.

(Received 15 July 2003, revised 17 August 2003,

accepted 21 August 2003)

Materials and methods

Materials

Dulbecco’s modified Eagle’s medium (DMEM), and fetal

bovine serum (FBS) were purchased from Life

Technolo-gies, Inc (Gaithersburg, MD, USA) Rottlerin, Ro-31-8425,

PD98059, Ly294002, SB202190 were from Calbiochem

(San Diego, CA, USA) PMA and goat anti-(mouse IgG) Ig

conjugated with tetramethylrhodamine isothiocyanate

(TRITC) were products ofSigma (St Louis, MO, USA)

Anti-ANX-1 monoclonal antibody was purchased from

Transduction Laboratories (Lexington, KY, USA)

Cell culture

HEK 293 cells were maintained in DMEM supplemented

with 10% heat-inactivated FBS, penicillin (100 UÆmL)1),

and streptomycin (100 lgÆmL)1) at 37C under 5% CO2

atmosphere For Western blot analysis, cells were

seeded into 60 mm dishes at 1· 106 cells per dish After

18–24 h, cells were further grown in the same medium

supplemented without FBS for 24 h Serum-starved cells

were treated with PMA for the indicated times For

immunostaining, 2· 105 cells grown on cover slides

(22· 22 mm) were starved for 24 h before stimulation

with PMA

Immunocytochemistry

HEK 293 cells grown on cover slides were fixed with

3.7% paraformaldehyde for 15 min and permeabilized

with 0.2% Triton X-100 in NaCl/KCl/Pi (NaCl/Pi,

137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.5 mM

KH2PO4) for 5 min After washing the cells with NaCl/

KCl/Pi three times, the cells were blocked for 30 min in

NaCl/Picontaining 1% bovine serum albumin

Immuno-staining was performed by incubation with anti-ANX-1

monoclonal antibody (0.05 lgÆmL)1) f or 2 h Af ter

washing the cells with NaCl/Pi three times, the cells

were incubated with TRITC-conjugated goat anti-(mouse

IgG) Ig for 1 h Cover slides were washed with NaCl/Pi,

mounted, and examined using a Leica TCS SP2

Confo-cal microscope (Leica Microsystems, Wetzlar GmBH,

Germany)

Cell fractionation

HEK 293 cells were seeded into 60 mm dishes at 1· 106

cells/dish and cultured in DMEM for 18–24 h The cells

were starved for 24 h in serum-free media After

treatment with PMA for a given time, the cells were

harvested and washed with ice-cold NaCl/Pi The cells

were then resuspended in 100 lL oflysis buffer (10 mM

Hepes, 10 mMNaCl, 0.1 mMEDTA, 0.1 mMEGTA, 1%

NP-40, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 mM

dithiothreitol, 0.1 mM sodium orthovanadate, and

prote-ase inhibitors) and incubated on ice for 10 min The

nuclei were collected by centrifugation at 2000 g for

5 min at 4C The supernatant was collected as a

cytosolic fraction Protein concentration of each sample

was determined

Western blot analysis For preparing whole cell lysates, 1· 106cells were lysed in

1· SDS gel-loading buffer (50 mM Tris/HCl, pH 6.8,

100 mMdithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) Protein samples were separated on 12% SDS/polyacrylamide gels and transferred to nitrocellulose filters The blots were incubated with anti-ANX-1 mono-clonal antibody for 1 h After washing three times with NaCl/Pi containing 0.05% Tween 20, the blots were incubated with goat anti-(mouse IgG) Ig conjugated with horseradish peroxidase (HRP) for 1 h The blots were washed three times with NaCl/Picontaining 0.05% Tween

20 and developed with the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech., Piscata-way, NJ, USA)

Results

PMA induces the cleavage of ANX-1

As ANX-1 is cleaved by treatment with PMA in epithelial A549 cells [7], we first examined whether PMA induces the cleavage ofANX-1 in HEK 293 cells Cells were treated with 10 nMPMA for 30 min and cell lysates were subjected

to Western blot analysis using ANX-1 antibody As shown

in Fig 1, the cleaved form of ANX-1 was detected in PMA-stimulated HEK 293 cells, whereas only the intact form of ANX-1 was found in control cells ANX-1 was partially cleaved by the treatment ofPMA and most ofANX-1 remained in the intact form This result indicates that PMA induces the cleavage ofANX-1 in HEK 293 cells

ANX-1 translocates to the nucleus by PMA stimulation There have been reports that the cleaved form of ANX-1 is secreted and presents mainly on the outer surface of cell membrane [17] To examine the subcellular localization of ANX-1 in response to PMA in HEK 293 cells, cells were incubated in the absence or presence of10 nM PMA for

30 min and immunostained with anti-ANX-1 monoclonal antibody In unstimulated cells, ANX-1 was evenly detected throughout the cells including cytosol and nucleus (Fig 2) However, the level ofANX-1 significantly increased in the nucleus in about 20–30% ofPMA-treated cells observed (Fig 2), indicating that ANX-1 translocates to the nucleus

by PMA stimulation

Fig 1 PMA induces the cleavage of ANX-1 Serum-starved HEK 293 cells were incubated in the absence or presence of10 n M PMA for

30 min and lysed with 1 · SDS-loading dye Whole cell lysates were separated in a 12% SDS/polyacrylamide gel and transferred to nitro-cellulose membrane Cleavage ofANX-1 was detected by Western blotting with anti-ANX-1 monoclonal antibody.

PMA induces the nuclear translocation of the cleaved

form of ANX-1

As PMA induced the cleavage ofANX-1 and accumulation

ofANX-1 in the nucleus, we wondered ifthe cleaved

ANX-1 is responsible for the increase of ANX-1 in the

nucleus To determine this possibility, HEK 293 cells were

treated with 10 nMPMA for 30 min, and fractionated into

cytosolic and nuclear fractions As shown in Fig 3A, the

cleaved ANX-1 was detected in the nuclear fraction of

PMA-treated cells and the amount ofthe intact ANX-1

level was not changed between the nuclear fractions of

treated and untreated cells These results indicate that

accumulation ofANX-1 in the nucleus is a result of

translocation ofthe cleaved ANX-1 to the nucleus

We next examined dose- and time-dependency ofthe

nuclear translocation ofthe cleaved ANX-1 HEK 293 cells

were treated with the indicated concentrations ofPMA for

30 min and subjected to Western blot analysis The cleavage

and translocation ofANX-1 was induced at a concentration

ofmore than 1 nM (Fig 3B) Figure 3C shows that the

cleaved ANX-1 in the nuclear fraction began to be found

after 15 min of exposure to PMA These results indicate

that the cleavage and translocation ofANX-1 to the nucleus

in response to PMA are immediate early events

PMA-induced nuclear translocation of ANX-1

is mediated via PKCd

It has been reported that PKC induces phosphorylation and cleavage ofANX-1 [12] Therefore, we examined whether PKC is involved in the cleavage and nuclear translocation of ANX-1 HEK 293 cells were preincubated in the presence ofrottlerin or Ro-31-8425, and were stimulated with PMA The cleavage and nuclear translocation ofANX-1 were inhibited by a PKCd-specific inhibitor rottlerin, but not by Ro-31-8425 (Fig 4A) To investigate whether other signa-ling molecules are involved in the cleavage and nuclear translocation ofANX-1, Western blot analysis was con-ducted in the absence or presence ofinhibitors ofERK (PD98059), p38 (SB202190), or PI-3 kinase (LY294002) Figure 4A shows that the PMA-induced cleavage and

Fig 2 ANX-1 accumulates in the nucleus by PMA stimulation

Serum-starved HEK 293 cells were incubated in the absence or presence of

10 n M PMA for 30 min and processed for immunocytochemical

detection ofendogenous ANX-1 using ANX-1 monoclonal

anti-body as described in Materials and methods Confocal images of

untreated (A) and PMA-treated (B) cells are representatives ofthe cells

observed in three independent experiments.

2

Fig 3 PMA induces the nucleartranslocation of the cleaved ANX-1 (A) Serum-starved HEK 293 cells were incubated in the absence or presence of10 n M PMA for 30 min Cells were then fractionated into cytosol and nucleus, and separated in a 12% SDS/polyacrylamide gel and subjected to immunoblotting using anti-ANX-1 monoclonal antibody (B) HEK 293 cells were incubated in serum-free DMEM for

24 h and stimulated with PMA at indicated concentrations for 30 min Cells were harvested and separated into cytosolic and nuclear frac-tions, then samples were resolved by 12% SDS/polyacrylamide gels and transferred to nitrocellulose membrane Translocation of ANX-1 was detected by Western blotting (C) Serum-starved HEK 293 cells were treated with 10 n M PMA for indicated times, then harvested and fractionated into cytosol and nucleus Samples were separated in 12% SDS/polyacrylamide gels Time-dependent translocation ofANX-1 to the nucleus was detected by Western blotting with anti-ANX-1 monoclonal antibody.

nuclear translocation ofANX-1 were not inhibited by the

addition ofthese inhibitors suggesting that these molecules

are not involved in the PMA-induced cleavage and nuclear

translocation ofANX-1

We next confirmed the inhibitory effect of rottlerin on

the cleavage and nuclear translocation ofANX-1 using

immunocytochemical analysis (Fig 4B) In cells

preincu-bated with rotterin, PMA was not able to induce the

increase ofANX-1 in the nucleus These results indicate that

PKCd plays a role in the PMA-induced cleavage and

nuclear translocation ofANX-1

Discussion

Despite recent advances in our understanding ofthe roles

ofANX-1 in mitogenic signal transduction, the exact

mechanism through which ANX-1 functions in response

to mitogenic stimuli remains unclear In this work, we

attempted to elucidate the regulatory mechanism ofANX-1

in the PMA signaling pathway in HEK 293 cells We have

demonstrated that (a) PMA induces the cleavage ofANX-1

(b) the cleaved ANX-1 translocates to the nucleus in a

time-dependent manner, and (c) the cleavage and nuclear

translocation ofANX-1 is mediated by a PKCd-dependent

mechanism

Several lines ofevidences suggest that ANX-1 is cleaved

in response to IL-6 and PMA in different cell lines [7,12] and

that the cleaved ANX-1 is translocated to the extracellular

surface of the cell membrane [16,17] To investigate the role

ofANX-1 in the PMA-induced signal transduction

path-way, we first examined whether PMA induces the cleavage

ofANX-1 in HEK 293 cells Our data showed that PMA

induced the cleavage ofANX-1 and the cleavage is comparable to that reported previously in other cell lines [7,12] It has been reported that truncated ANX-1 that is missing the first 29 N-terminal amino acids is secreted from the prostate cancer cells [23] Whether the same type of cleavage occurs in PMA treated cells is not clear Never-theless it is reasonable to assume that the cleavage site of ANX-1 in response to PMA is at the N-terminal region of ANX-1 The cleavage site ofANX-1 and the enzyme that is responsible for the cleavage is under investigation in this laboratory

Interestingly, data from confocal microscopy show that ANX-1 is accumulated in the nucleus We have previously reported that ANX-1 translocates to the nucleus by EGF, oxidative stress, and heat shock [18,19] Our results suggest that PMA also induces the translocation ofANX-1 to the nucleus However, the nuclear translocation ofANX-1 was observed in about 20–30% ofPMA-treated cells indicating that this may be cell cycle-dependent event When PMA-treated HEK 293 cells were fractionated into cytosolic and nuclear fractions, the cleaved form of ANX-1 was found in the nuclear fraction, indicating that the cleaved form but not the intact form of ANX-1 translocates to the nucleus The cleaved ANX-1 was detected in the cytosolic fraction with a longer exposure, indicating that ANX-1 is cleaved in the cytosol, then the cleaved ANX-1 translocates to the nucleus However, results from the Western blotting with a longer exposure and confocal microscopy demonstrate that the minimal level ofthe cleaved ANX-1 was also found in the nuclear fraction of control cells (data not shown) Therefore,

it cannot be ruled out the possibility that the cleavage of ANX-1 occurs both in the cytosol and the nucleus

Fig 4 PMA-induced nucleartranslocation of ANX-1 is mediated via PKCd (A) Serum-starved HEK 293 cells were preincubated in the absence or presence ofrottlerin (5 l M ), Ro-31-8425 (50 l M ), PD98059 (50 l M ), LY294002 (10 l M ), and SB202190 (20 l M ) f or

30 min and stimulated with 10 n M PMA for

30 min Cells were fractionated into cytosolic and nuclear fractions, and analyzed by 12% SDS/polyacrylamide gels and transferred to nitrocellulose membrane Translocation of ANX-1 was probed with anti-ANX-1 mono-clonal antibody (B) HEK 293 cells were incubated in serum-f ree DMEM f or 24 h Serum-starved cells were preincubated in the absence or presence ofPKCd-specific inhibitor rottlerin (5 l M ) for 30 min and treated with

10 n M PMA for 30 min Cells were then immunostained with anti-ANX-1 monoclonal antibody as described in Materials and methods The micrographs are representatives ofthe cells observed in three independent experiments.

3

PMA induced the cleavage and nuclear translocation of

ANX-1 in a time-dependent manner The cleaved ANX-1

began to be detected in the cytosolic fraction at 10 min of

PMA treatment After 15 min of exposure to PMA, the

cleaved ANX-1 began to translocate to the nucleus This

result confirms the data that ANX-1 is cleaved first in the

cytosol, then translocates to the nucleus The nuclear

translocation ofANX-1 seems to be an immediate early

process and is comparable to that ofother signaling

molecules such as MAP kinase and NF-jB [24,25] As

ANX-1 binds both DNA and RNA [26], there is a

possibility that the cleaved ANX-1 translocates to the

nucleus and participates in cell proliferation and

differen-tiation processes by regulating transcription Taken

together, the cleavage and translocation ofANX-1 to the

nucleus by PMA may be a physiological process involved in

cell proliferation and differentiation

The PKC family, which comprises several isoforms, plays

an important role in cell proliferation and differentiation

[20,21] PKC is known to be involved in the cleavage and

secretion ofANX-1 [12] However, it is not clear which

isoform of PKC mediates the cleavage of ANX-1 PKCd is a

member ofthe novel PKC subfamily and is known to play

a critical role in the regulation ofcell proliferation and

apoptosis [22] Our data indicate that PKCd is responsible

for the cleavage and nuclear translocation of ANX-1 It has

been known that the major phosphorylation sites ofANX-1

by PKC are Ser-27 and Thr-41, and the phosphorylation of

ANX-1 at Ser-27, Ser-28, and Thr-24 has also been

identified [27] PKCd probably phosphorylates ANX-1 at

one or several ofthese sites directly or indirectly and induces

the cleavage and nuclear translocation ofANX-1 We also

investigated the involvement ofother signaling molecules

such as MEK, PI-3 kinase, and p38 in PMA-induced

translocation ofANX-1 Inhibition ofthese molecules did

not affect the cleavage and nuclear translocation of ANX-1

in response to PMA, indicating that these molecules are not

required for the PMA-induced cleavage and nuclear

trans-location ofANX-1

ANX-1 is involved in the regulation ofcell proliferation

and differentiation [28,29] Recently, ANX-1–/–mice were

generated and partially characterized [30] and it has been

confirmed that ANX-1 is a mediator

ofglucocorticoid-induced growth inhibition [31] However, the exact

mech-anism by which ANX-1 plays a role in cell growth is not

known In the present study, we propose the mechanism by

which PMA induces the translocation ofANX-1 to the

nucleus We have demonstrated that PMA induces the

cleavage ofANX-1 leading to the nuclear translocation of

ANX-1, and that PKCd plays a critical role in this process

While further studies are required to characterize the exact

functions of ANX-1 in the nucleus, from this study we can

begin to understand the role ofANX-1 in the PMA-induced

signal transduction, which may provide an important

clue for understanding the molecular mechanism of cell

proliferation and differentiation

Acknowledgements

This work was supported by a Korea Research Foundation Grant

(KRF-2002-042-C00076) (to D S N and J K).

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