Báo cáo khoa học: Inhibition of the NF-jB transcriptional activity by protein kinase A pot

PKA activating agents or expression of the catalytic subunit of PKA PKAc inhibited the NF-jB-dependent reporter gene expression induced by tumor nec-rosis factor a TNFa.. In addition to

Inhibition of the NF-jB transcriptional activity by protein kinase A

Naoko Takahashi, Toshifumi Tetsuka, Hiroaki Uranishi and Takashi Okamoto

Department of Molecular Genetics, Nagoya City University Medical School, Japan

The cAMP-dependent protein kinase (PKA) signaling

pathway plays a major role in a number of

pathophysio-logical conditions However, there have been conflicting

evidences regarding the action of cAMP/PKA on nuclear

factor- jB (NF-jB) In this study, we have explored the effect

of cAMP/PKA on NF-jB activity and determined its

molecular mechanism PKA activating agents or expression

of the catalytic subunit of PKA (PKAc) inhibited the

NF-jB-dependent reporter gene expression induced by tumor

nec-rosis factor a (TNFa) PKA activators affected neither IjBa

phosphorylation, IjBa degradation, nor the NF-jB/DNA

binding Expression of PKAc inhibited the transactivation

potential of Gal4-p65 (286–551) suggesting that the inhibi-tory action of PKA is through the C-terminal transactivation domain of p65 but not by phosphorylation of the consensus PKA recognition site containing serine at position 276 Overexpression of coactivators, CBP (CREB-binding pro-tein) and p300, failed to reverse the PKA-mediated inhibition

of p65 transactivation Thus, the inhibitory action of the cAMP/PKA pathway on the transcriptional activity of NF-jB appears to be exhibited by modifying the C-terminal transactivation domain of p65, either directly or indirectly Keywords: NF-jB; PKA; cAMP; signal transduction

Nuclear factor jB (NF-jB) is an inducible cellular

tran-scription factor that regulates a wide variety of cellular and

viral genes including several cytokines, cell adhesion

mole-cules and human immunodeficiency virus (HIV) [1–4]

Members of the NF-jB family in mammalian cells include

the proto-oncogene c-Rel, Rel A (p65), Rel B, NF-kB1

(p50/105), and NF-kB2 (p52/p100)

conserved 300 amino acids region known as the Rel

homology domain, which is responsible for DNA binding,

dimerization, and nuclear translocation [1–4] In most cells,

Rel family members form hetero- and homo-dimers with

distinct specificities in various combinations A common

feature of the regulation of the NF-jB family is their

sequestration in the cytoplasm as inactive complexes with a

class of inhibitory molecules known as IjBs [1–4]

Treat-ment of cells with a variety of inducers such as phorbol ester,

interleukin-1b (IL-1b) and tumor necrosis factor a (TNFa)

results in phosphorylation, ubiquitination and degradation

of the IjB proteins [3,5,6] The degradation of IjB proteins

exposes the nuclear localization sequence in the remaining

NF-jB dimers, leading to nuclear translocation and

subse-quent binding of NF-jB to the DNA cis-regulatory element

of target genes [1–4] In addition to the nuclear translocation

and DNA-binding of NF-jB, its transcriptional activity is

regulated by coactivators, CBP (CREB-binding protein) and p300, that associate with the C-terminal transactivation domain of p65 It has been demonstrated that these coactivators physically interact with p65 and promote its transcriptional activity [7,8]

On the other hand, elevation of intracellular cAMP induces the expression of numerous genes through the protein kinase A (PKA)-mediated phosphorylation of transcription factors, including the cAMP response ele-ment binding protein (CREB) [9] Interestingly, cAMP inhibits the induction of a distinct set of NF-jB regulated genes For example, cAMP inhibits IL-2 expression in activated T-lymphocytes In monocytes, pharmacological agents that elevate intracellular cAMP, such as pentoxif-ylline, iloprost, dibutyryl cAMP (db-cAMP), forskolin (FSK), and isomethylxanthine, inhibit the TNFa produc-tion and tissue factor expression that are also under the control of NF-jB [10–14] In endothelial cells, elevation of cAMP inhibits the induction of E-selectin and VCAM-1 mediated by IL-1b or TNFa [15,16] Thus, elevation of intracellular cAMP and/or activation of PKA appear to modulate the transcriptional activity of NF-jB in a negative way

In this report, we examined whether cAMP and PKA modulates NF-jB activity and found that the cAMP/PKA signaling pathway inhibits the NF-jB activity by acting on the transactivation domain of p65 Possible mechanisms for this inhibitory action are discussed

E X P E R I M E N T A L P R O C E D U R E S Reagents

Forskolin (FSK), 8-bromo-cAMP (Br-cAMP), dibutyryl cAMP (db-cAMP) were purchased from Wako (Osaka, Japan) Recombinant human TNFa and recombinant human IL-1b were purchased from Roche (Tokyo, Japan)

Correspondence to T Okamoto, Department of Molecular Genetics,

Nagoya City University Medical School, 1 Kawasumi,

Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.

Fax: + 81 52 859 1235, Tel.: + 81 52 853 8205,

E-mail: tokamoto@med.nagoya-cu.ac.jp

Abbreviations: NF-jB, nuclear factor jB; PKA, cAMP-dependent

protein kinase; PKAc, catalytic subunit of PKA; FSK, forskolin;

db-cAMP, dibutyryl cAMP; Br-cAMP, 8-Bromo-cAMP; CREB,

cAMP response element binding protein; CBP, CREB binding

pro-tein; TNFa, tumor necrosis factor a; IL-1b, interleukin-1b; C/EPBb,

CCAAT/enhancer-binding protein b.

(Received 20 May 2002, revised 9 July 2002, accepted 29 July 2002)

10% fetal bovine serum Primary culture of rat mesangial

cells were prepared as described previously [17], and

maintained in RPMI1640 medium supplemented with

15% fetal bovine serum and 0.15 IUÆmL)1insulin

Plasmids

Mammalian expression vectors, pCMV-p300, pRSV-CBP,

and pRSV-CREB were generous gifts from Drs David

Livingston (Dana-Farber Cancer Institute), Shunsuke Ishii

(Riken) and Masatoshi Hagiwara (Tokyo Medical and

Dental University), respectively The constructions of

luciferase reporter 4 jB-luc, pCMV-p65, pGal4-p65

(1–551), and pGal4-p65 (286–551) expression plasmids were

described previously [18] The pGal4-CREB (1–280),

pCRE-luc containing four tandem repeats of the

CREB-binding sites, pGal4-luc containing five tandem repeats of

Gal4-binding sites, and the mammalian expression vector

pCMV-PKAc encoding the PKA catalytic subunit were

obtained from Stratagene (La Jolla, CA)

Transfection conditions and luciferase assay

Transient transfection of Jurkat T-lymphocytes was

accomplished by using 5 lL of SuperFect transfection

reagent (Qiagen) per ml of culture medium and a total of

1 lg of plasmid DNA Blank control plasmids

pcDNA3.1(–) and pUC19 were used to equalize the

amount of DNA for each transfection SuperFect-DNA

complexes were allowed to form for 10 min at room

temperature in serum-free medium before being added to

the cells Cells were incubated with the complex for 24 h,

stimulated with 10 ngÆmL)1of human recombinant TNFa

for an additional 24 h and harvested Gene expression was

measured by luciferase activity as previously described [18]

Luciferase assays were performed and relative light units

were determined with an AutoLumat LB9507 luminometer

(EG & G Berthold, Japan) Transfection efficiency was

monitored by Renilla luciferase activity with pRL-TK

plasmid containing TK promoter as an internal control All

luciferase activities shown in transient transfection assays

are corrected by the internal control activity of Renilla

luciferase by pRL-TK

Electrophoretic mobility shift assay (EMSA)

Jurkat T-lymphocytes were pretreated with FSK (50 lM) or

vehicle (DMSO) for 30 min, and then stimulated with

TNFa (10 ngÆmL)1) for 30 min Nuclear extracts were

prepared as previously described [19–21] The double

stranded oligonucleotide probe for NF-jB was synthesized

and endlabeled by [c-32P]-ATP The jB sequence was taken

from the human immunodeficiency virus long-terminal

repeat (HIV-LTR) The jB sequence used was forward

and complement (5¢-TTTCTGGAAAGTCCCCAGGCG

GAAAGTC CCTAG-3¢) The EMSA was performed as

Jurkat T-lymphocytes were pretreated with or without FSK (50 lM) for 30 min and then stimulated with TNFa (10 ngÆmL)1) or IL-1b (10 ngÆmL)1) After stimulation, cells were washed twice with ice-cold NaCl/Pi and lysed in TOTEX buffer (20 mM Hepes/KOH, pH 7.9, 350 mM

NaCl, 20% glycerol, 1% NP-40, 1 mM MgCl2, 0.5 mM

EDTA, 0.1 mMEGTA, 1 mMdithiothreitol, 20 mM 2-gly-cerophosphate, 0.1 mM Na3VO4, 1 mM phenylmethane-sulfonyl fluoride, and 2.5 lgÆmL)1 each of aprotinin, leupeptin and pepstatin) Equal amounts of samples (20 lg of total protein) were subjected to 10% SDS/PAGE and transferred onto a nitrocellulose membrane (Hybond-C; Amersham Pharmacia Biotech) The membranes were probed with anti-(IjBa) serum, anti-(IjBa-P) serum (New England Bio Laboratories, Beverly, MA) and proteins were visualized by enhanced chemiluminescence (Amersham Life Science Inc., Cleveland, OH)

R E S U L T S cAMP/PKA inhibits TNFa-induced NF-jB activation

To examine the effect of cAMP/PKA on TNFa-induced NF-jB activity, Jurkat T-lymphocytes were treated with PKA activating agents, FSK, db-cAMP and Br-cAMP Stimulation with these agents decreased TNFa-induced NF-jB reporter gene expression in a dose dependent manner (Fig 1A–C) To test whether these inhibitory effects of PKA activating agents are not limited for 4 kB-luc, we examined the effects of FSK and db-cAMP on HIV-LTR-luc which contains two NF-jB binding sites As shown in Fig 1D, FSK and db-cAMP inhibited the TNFa-induced HIV-LTR activation To further test whether this inhibitory effect of cAMP was mediated by PKA, we transfected the catalytic subunit of PKA (PKAc) into Jurkat and HeLa cells along with the jB-dependent luciferase reporter Transfection of PKAc led to a dramatic decrease

in the TNFa-induced expression from the jB-reporter gene

in both cells (Fig 1E and F) These data indicate that elevation of intracellular cAMP and subsequent activation

of PKA lead to the inhibition of NF-jB activity

To further examine the effect of PKA on NF-jB activity,

we cotransfected Jurkat T-lymphocytes and HeLa cells with plasmids expressing PKAc and p65 subunit of NF-jB, and measured the level of NF-jB-dependent luciferase gene expression Transfection of p65 alone resulted in high levels

of luciferase expression, whereas cotransfection of p65 with PKAc led to a dramatic decrease in the expression of the reporter gene in both cells (Fig 2A and B) supporting an idea that PKA generally inhibits NF-jB-mediated tran-scription One of the major nuclear PKA substrates is the transcription factor CREB (cAMP response element-bind-ing protein) which binds to the cAMP response element (CRE) in cAMP-inducible genes [9] The phosphorylation

of CREB by PKA results in increased transactivation

To examine whether the inhibitory effect of PKAc on

transcription factor is specific for NF-jB, we cotransfected

the reporter plasmid pCRE-luc with PKAc and measured

CRE-dependent luciferase activity in Jurkat T-lymphocytes

As expected, overexpression of PKAc led to an increase in

the CRE-dependent gene expression (Fig 2C)

PKA activation does not affect IjB degradation

nor DNA-binding activity of NF-jB

In the unstimulated cells, NF-jB is sequestrated in the

cytoplasm as inactive complexes with IjBs, which are

phosphorylated and degraded upon stimulation of cells with

inducers of NF-jB, such as TNFa and IL-1b [1–4] As

Neumann et al indicated that elevation of cAMP reduces

NF-jB activity, possibly by stabilizing IjBa [22], we

investigated whether the activation of PKA could inhibit

IjBa degradation Jurkat T-lymphocytes were stimulated

with TNFa (20 ngÆmL)1) in the presence or absence of FSK

(50 lM), and IjBa were detected by immunoblotting As

demonstrated in Fig 3A, there was no difference in IjBa

degradation in the presence or absence of FSK Similar

observation was obtained with HeLa cells (data not shown)

In addition, in primary culture of rat glomerular mesangial

cells, FSK did not affect the IL-1b-induced IjBa

phospho-rylation and subsequent degradation (Fig 3B and C) These

data indicate that PKA activation by FSK does not affect

TNFa- or IL-1b-induced IjBa degradation in most cells To

confirm that PKA activation does not modify

DNA-binding activity of NF-jB, we performed EMSA using nuclear extract prepared from Jurkat cells FSK did not inhibit TNFa-induced DNA-binding of NF-jB (Fig 3D) Thus, it is likely that the inhibitory effect of PKA on NF-jB

is not due to the inhibition of DNA-binding activity of NF-jB

PKA impairs transactivation potential of p65 p65 protein has a putative consensus PKA recognition site (RRPS) located approximately 25 amino acids N-terminal from the nuclear localizing signal (NLS) in the Rel homology domain [1–4] (Fig 4A) Zhong et al reported that the phosphorylation of this serine residue (at 276 amino acid position) could modulate the transcriptional activity of p65 [23] Thus we have examined whether the phosphory-lation of p65 at Ser276 is involved in the mechanism by which PKA inhibits NF-jB activation In Fig 4B, we examined the transcriptional activity of pGal4-p65 (1–551), containing the full-length p65, and pGal4-p65 (286–551), containing the transactivation domain of p65 but lacking the Rel homology domain and the RRPS site (Ser276) of p65, in response to PKAc overexpression When we cotransfected Jurkat T-lymphocytes with pCMV-PKAc and either pGal4-p65 (1–551) or pGal4-p65 (286–551) and measured the Gal4-dependent luciferase reporter gene expression, PKAc inhibited the Gal4-dependent luciferase activity induced by either pGal4-p65 (1–551) or pGal4-p65

Fig 1 The effect of PKA activating agents and

PKAc on NF-jB-dependent transcription

induced by TNFa (A–C) Suppression of

NF-jB-dependent gene expression (4 jB-luc)

in Jurkat cells by cAMP elevating agents

including FSK (A), db-cAMP (B), and

Br-cAMP (C) (D) Inhibition of

TNFa-induced HIV-LTR-luc activation in Jurkat

cells by FSK and db-cAMP (E and F)

Inhi-bition of TNFa-induced NF-jB-dependent

gene expression (4 jB-luc) in Jurkat (E) and

HeLa cells (F) Cells were transfected with

100 ng of 4 jB-luc or HIV-LTR-luc reporter

plasmid and/or 150 ng of pCMV-PKAc

expression plasmid After 24 h of transfection,

cells were stimulated with 10 ngÆmL)1of

TNFa for 24 h in the presence or absence of

the PKA activating agent Cells were then

harvested and subjected to luciferase assay.

The data are presented as the fold-increase in

luciferase activities relative to control

trans-fection (no TNF stimulation) Values are the

mean of two independent transfections (A–C)

or the mean ± SE of three independent

transfections (D–F) Experiments were

repea-ted at least three times with the same results.

(286–551) almost to a similar extent (Fig 4B) These results

excluded the possibility that the activation of PKA inhibits

NF-jB by direct phosphorylation of the consensus PKA

recognition site at Ser276 of p65 but suggested that PKA

inhibits NF-jB by acting at the C-terminal transactivation

domain of p65 subunit These results also excluded the

possibility that PKAc directly affected the NF-jB DNA

binding In contrast, PKAc augmented the transcriptional

activity of Gal4-CREB (1–280) containing the CREB

transactivation domain and the possible PKA-mediated

phosphorylation site (Fig 4C) Thus, the inhibitory effect of

PKAc on transcription appeared to be specific for NF-jB

subunit p65

CREB and coactivator CBP/p300 are not involved in the

PKA-mediated inhibition of NF-jB

To examine if CREB, a well-known nuclear substrate of

PKA, is involved in the PKA-mediated inhibition of p65

activity, Jurkat cells were transfected with pRSV-CREB in

addition to pCMV-p65 and 4 jB-luc (Fig 5A) However,

CREB did not affect the transcriptional activity of p65 As

both CREB and p65 use coactivators CBP/p300 [7,8], the

limiting amount of CBP/p300 might account for the PKA-mediated inhibition of p65 transactivation To test this hypothesis, pRSV-CBP expressing CBP was cotransfected with pCMV-PKAc and pCMV-p65 However, overexpres-sion of CBP did not reverse the PKA-mediated inhibition of p65 transactivation (Fig 5B) Similar results were obtained with p300 overexpression (data not shown) These obser-vations indicate that PKA-mediated inhibition of p65 transactivation is not likely through the sequestration of CBP/p300

D I S C U S S I O N

In this study we have observed reproducibly that the cAMP/ PKA signaling pathway inhibits transcriptional activity of NF-jB Although a previous report by others [22] suggested that PKA inhibited NF-jB activation by stabilizing IjB, PKA activating agents such as FSK did not significantly affect the TNFa-induced IjBa degradation or DNA-binding activity of NF-jB (Fig 3) These findings clearly indicated that PKA inhibits NF-jB activation at a step downstream of IjB degradation Sequestration of CBP/ p300 coactivators by CREB is not likely because CBP/p300 overexpression did not abolish the inhibitory effect of PKA

on NF-jB (Fig 5B)

the evidence that the C-terminal transactivation domain of p65 is responsible for the inhibition of NF-jB mediated by the cAMP/PKA signaling (Fig 4)

Fig 2 Inhibition of NF-jB (p65)-dependent gene expression byPKAc.

Jurkat (A) or HeLa (B) cells were transfected with 100 ng of 4 jB-luc,

50 ng of pCMV-p65 and/or 150 ng of pCMV-PKAc (C) Activation of

CREB-dependent gene expression by PKAc Jurkat cells were

trans-fected with 100 ng of pCRE-luc and various amounts of

pCMV-PKAc The data are presented as the fold-increase in luciferase

activ-ities relative to control transfection Values are the mean ± SE of

three independent transfections.

Fig 3 Forskolin does not affect IjBa degradation or DNA-binding activityof NF-jB (A) Jurkat cells were pretreated with 50 l M of FSK for 30 min, and then stimulated with 10 ngÆmL)1of TNFa for indi-cated time periods Cells were harvested and subjected to Western blot analysis (B) The time course of IL-1b-induced IkBa degradation and phosphorylation in rat glomerular mesangial cells Mesangial cells were stimulated with IL-1b (10 ngÆmL)1) for indicated time periods Cells were harvested and subjected to Western blot analysis with anti-IjBa antibody or the antibody against phosphorylated anti-IjBa at Ser32 (IjBa-P) (C) Effect of FSK on the IL-1b-induced IjBa degradation and phosphorylation in rat mesangial cells Cells were pretreated with

50 l M of FSK for 30 min, and then stimulated with IL-1b (10 ngÆmL)1) for 5 and 15 min Cells were harvested and total cell lysates were subjected to Western blot analysis (D) Effect of FSK on the NF-jB DNA-binding activity Jurkat cells were pretreated with or without 50 l M FSK for 30 min, and then simulated with TNFa (10 ngÆmL)1) for 30 min Cells were harvested and nuclear extracts were subjected for EMSA analysis with the jB probe.

There have been conflicting evidences regarding the

action of PKA on the NF-jB activity Although the

majority of reports [10–14] including this study,

demonstra-ted the inhibitory actions of PKA on the NF-jB dependent

gene expression, some reports such as Zhong et al [23,24]

showed that PKAc, associated with the cytosolic NF-jB/

IjB complex, enhanced NF-jB-dependent gene

expres-sion by transient luciferase reporter assay This marked

difference may be explained by use of distinct reporter

plasmids It is noteworthy that several commonly used

reporter plasmids contain CRE sites that mediate induction

of reporter gene expression in cells stimulated with cAMP-increasing agents [25] It is also possible that NF-jB-dependent gene expression may be enhanced or inhibited depending on the level of PKA enzyme activity

Induction of C/EBPb

pro-tein b) by cAMP/PKA might be involved in cAMP/PKA-mediated inhibition of NF-jB activity In our preliminary experiments, cAMP elevating agents induced C/EBPb and formed a complex with NF-jB, and overexpression of C/EBPb inhibited the transactivation potential of p65, Gal4-p65 (1–551) and Gal4-p65 (286–551) (data not shown) A number of reports suggest a physiological role

of C/EBPb in mediating the PKA signaling For instance, PKA stimulates C/EBPb transcription in hepatocytes, neuronal cells and in fibroblasts [26] In addition, C/EBPb

is induced through PKA signaling pathway upon stimula-tion with human chorionic gonadotropin in rat granulosa

Fig 5 Effects of CREB and CBP on transcriptional activityof p65 (A) Effect of CREB on the p65-mediated transcription Jurkat cells were transfected with 4 jB-luc reporter plasmid (100 ng), pCMV-p65 and/

or pRSV-CREBs After 24 h of transfection, cell lysates were harves-ted and the luciferase assay was performed (B) Effect of CBP over-expression on the PKAc-mediated inhibition of p65 activity Jurkat cells were transfected with 4 jB-luc, p65 (50 ng), pCMV-PKAc (150 ng), and various amounts of pRSV-CBP (50, 150, 500 ng) The relative luciferase activity as compared with control (4 jB-luc reporter plasmid alone) is shown Values are the mean ± SE of three independent transfections.

Fig 4 Inhibition of NF-jB activitybyPKAc through the C-terminal

transactivation domain of p65 (A) Functional configuration of p65

protein Location of rel-homology domain (RHD), putative PKA

phosphorylation site (Ser276), and two transactivation domains (TA1

and TA2) are indicated (B) Inhibition of p65 transcriptional activity

by PKAc In order to evaluate the effect of PKAc in Jurkat cells, either

pGal4-p65 (1–551), encoding a fusion protein of Gal4 DNA-binding

domain and the full-length p65 (amino acids 1–551), or pGal4-p65

(286–551), that lacking the RHD and the putative PKA

phosphory-lation site was cotransfected with pCMV-PKAc and the gene

expres-sion from the pGal4-luc reporter plasmid (100 ng) was assessed (C)

Activation of CREB activity by PKAc Jurkat cells were transfected

with Gal4-CREB (1–280), pCMV-PKAc, and pGal4-luc reporter

plasmid Data are presented as percentage inhibition (B) or percentage

activation (C) relative to control transfection without pCMV-PKAc.

Values are the means of two independent transfections Similar results

were obtained repeatedly.

least in part However, the effect of C/EBPb on

NF-jB-dependent transcription may be context-NF-jB-dependent because

in some promoters such as IL-6 and IL-8, in which NF-jB

binding site and C/EBP binding site are very closely located,

C/EBPb synergistically activates the NF-jB-dependent

transcription [31] Thus, further studies are needed to clarify

the role of C/EBPb in controlling the transcriptional activity

of NF-jB

The C-terminal transactivation domain of p65 has been

shown to be phosphorylated at Ser529 and Ser536 by

various upstream stimuli [32–35] Thus, we cannot exclude a

possibility that PKA may inhibit NF-jB by modulating the

phosphorylation status of p65 with its C-terminal

transac-tivation domain, either directly or indirectly It is possible

that PKA inhibits NF-jB in a multiplicative way including

stabilization of inhibitor IjB, induction of a repressor

complex such as C/EBPb which interacts with NF-jB, and

modulation of the phosphorylation status of the

transacti-vation domain of p65

A C K N O W L E D G E M E N T

N T and T T contributed equally to this work This work was

supported in part by grants in aid from the Ministry of Health, Labor

and Welfare, the Ministry of Education, Culture, Sports, Science and

Technology, and the Japanese Health Sciences Foundation We thank

Drs Shunsuke Ishii, Masatoshi Hagiwara, and David Livingston for

plasmids.

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