Báo cáo khoa học: Synergistic co-operation of signal transducer and activator of transcription 5B with activator protein 1 in angiotensin II-induced angiotensinogen gene activation in vascular smooth muscle cells potx

activator of transcription 5B with activator protein 1in angiotensin II-induced angiotensinogen gene activation in vascular smooth muscle cells Mei Han, Ai-Ying Li, Fang Meng, Li-Hua Don

activator of transcription 5B with activator protein 1

in angiotensin II-induced angiotensinogen gene activation

in vascular smooth muscle cells

Mei Han, Ai-Ying Li, Fang Meng, Li-Hua Dong, Bin Zheng, Hai-Juan Hu, Lei Nie and Jin-Kun Wen Department of Biochemistry and Molecular Biology, Hebei Medical University, Shijiazhuang, China

Angiotensin II (Ang II), an extensively characterized

peptide produced by successive proteolytic cleavage

reactions of its prohormone, angiotensinogen (AGT),

is an important contributor to the regulation of

vol-ume homeostasis and blood pressure in humans and to

the initiation of pathophysiological events that lead to

hypertension and cardiovascular disorders [1,2]

In human genetic studies, a clear linkage has been

established between the AGT gene and hypertension

[3] Several lines of evidence have indicated that small

variations in AGT concentration result in substantial

changes in the circulating Ang II levels [4] At the

cellular level, Ang II-mediated signaling is achieved

through its binding to the cell-surface AT1 receptor,

which causes activation of Janus kinase 2 (JAK2) [5,6] and then activates signal transducer and activator of transcription (STAT) molecules in cardiac myocytes and in rat aortic (vascular) smooth muscle cells (VSMCs) [5,7–9], resulting in the positive feedback of AGT transcription [5] The AGT gene itself is the target for the activated STAT protein in cardiac myo-cytes through the AGT promoter region [5] However, the interaction of STAT5B with the AGT gene promoter was observed in liver and cardiac myocytes [8,10], but not in the smooth muscle cell line

The molecular basis for activation of the AGT gene

is only partially understood The analysis of biological information presumes that the 500-bp region of the rat

Keywords

activator protein-1; angiotensinogen; gene

regulation; signal transducer and activator of

transcription-5; vascular smooth muscle

cells

Correspondence

J.-K Wen, Department of Biochemistry and

Molecular Biology, No 361, Zhongshan East

Road, Shijiazhuang 050017, China

Fax: +86 311 8626 6180

Tel: +86 311 8626 5563

E-mail: wjk@hebmu.edu.cn

(Received 29 October 2008, revised 29

December 2008, accepted 12 January 2009)

doi:10.1111/j.1742-4658.2009.06902.x

The binding sequences for signal transducer and activator of transcription (STAT) and activator protein 1 have been found in the promoter region of the angiotensinogen gene We examined whether the elements for activator protein 1 and STAT5B function in angiotensinogen gene activation induced

by angiotensin II in vascular smooth muscle cells Stimulation with angio-tensin II increased the level of angioangio-tensinogen mRNA by 2.1-fold in vascular smooth muscle cells The increased level of angiotensinogen mRNA occurred with concurrent elevations in the levels of STAT5B and c-Jun phosphorylation after stimulation with angiotensin II Likewise, angiotensin II resulted in similar enhancements of the DNA-binding activ-ity of STAT5B and c-Jun in angiotensin II-induced angiotensinogen expres-sion Notably, the STAT5B–DNA complex interacted with the c-Jun–DNA complex by forming a stable quaternary complex in angiotensin II-induced angiotensinogen expression Our findings support a model in which co-operative interaction of STAT5B and activator protein 1 bound to the the promoter region provides maximal activation of angiotensinogen expression by angiotensin II in vascular smooth muscle cells

Abbreviations

AGT, angiotensinogen; Ang II, angiotensin II; AP-1, activator protein 1; ChIP, chromatin immunoprecipitation; CoIP,

cross-coimmunoprecipitation; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JAK, Janus kinase; STAT, signal transducer and activator of transcription; VSMCs, vascular smooth muscle cells.

AGT gene promoter contains clusters of regulatory

elements, perfectly or partially matched to consensus

sequences, including binding sequences for STAT5B

and activator protein 1 (AP-1) Previous studies

indi-cate that transcription activation by STATs requires

activated AP-1 [11–13] AP-1 is a complex composed

of the Fos and Jun proteins [14–16] In general, Fos

and Jun family proteins function as dimeric

transcrip-tion factors that bind to AP-1 regulatory elements in

the promoter and enhancer regions of the target

[14,16] However, the role of AP-1 in AGT gene

tran-scription activation is unknown It has been

demon-strated that STAT proteins co-operate to bind to

target DNA, not only with other STAT family

mem-bers [17–19], but also with other proteins and

tran-scription factors [11,13,20,21] Recently, the physical

association between STAT and c-Jun on the a2

-macro-globulin promoter element has been shown to yield

maximal enhancer function [13]

Based on these pieces of knowledge, we

hypothe-sized that co-operative interaction between c-Jun and

STAT5B may be important in transcription activation

of the AGT gene induced by Ang II To understand

whether elements for AP-1 and STAT5B function in

AGT gene activation induced by Ang II, we tested the

effect of co-operative interaction between c-Jun and

STAT5B on the AGT promoter activity and AGT

mRNA expression in VSMCs We showed that

Ang II-induced AGT expression in VSMCs involves

co-operation between AP-1 and STAT5B We also

demonstrated that there exists a physical interaction

between AP-1 and STAT5B during AGT expression

induced by Ang II

Results

Ang II increases AGT gene expression with

concurrent increases in the phosphorylation

of STAT5B and c-Jun in VSMCs

It has been demonstrated that Ang II stimulates

AGT expression in hepatocytes [22] and in cardiac

muscle [5] The present study showed that Ang II

increased the AGT mRNA level in VSMCs

Follow-ing treatment of VSMCs with Ang II for different

periods of time, AGT mRNA was detected using

RT-PCR As shown in Fig 1A, the level of AGT

mRNA peaked 3 h after stimulation with Ang II,

showing an increase of 5.2-fold, and decreased

there-after STAT5B is necessary for expression of the

AGT gene [10], and Ang II activates JAK-STAT

and AP-1 [23] To determine the relationship

between the activation of STAT5B and c-Jun, and

the expression of the AGT gene in VSMCs stimu-lated with Ang II, the effect of Ang II on the phos-phorylation of STAT5 and c-Jun was measured Ang II stimulated the phosphorylation of STAT5B and c-Jun, with levels of phosphorylated STAT5B and c-Jun significantly increasing 1 h, and peaking

3 h, after stimulation with Ang II, whereas total c-Jun and STAT5B were not changed after treatment with Ang II for different periods of time (Fig 1B) However, the phosphorylation of STAT5B induced

by Ang II was dramatically inhibited by pretreating VSMCs with AG490 (a specific inhibitor of the JAK-STAT pathway) for 16 h [24], indicating that the activation of STAT5B and c-Jun may be involved in Ang II-induced AGT mRNA expression (Fig 1C)

DNA-binding activity of STAT5B and c-Jun increases in Ang II-induced AGT expression

To find out whether the increase in STAT5B and c-Jun phosphorylation induced by Ang II affects the binding of STAT5B and c-Jun to their cis-elements, the activity of STAT5B binding and of c-Jun binding

to DNA was detected, respectively, by electrophoretic mobility shift assays (EMSAs) using radiolabeled oli-gonucleotides containing either a STAT5B-binding site

or an AP-1-binding site in the rat AGT gene promoter

As shown in Fig 2A,B, DNA–protein complexes were formed when these two probes were incubated with nuclear extracts from VSMCs treated with Ang II for 0.5, 1, and 3 h, and the DNA-binding activity of STAT5B and of AP-1 increased in a time-dependent manner The specificities of two DNA–protein com-plexes were demonstrated by their disappearance upon the addition of a 100-fold molar excess of unlabeled probe Further to confirm whether STAT5B and AP-1 are involved in the shifted complexes, supershift assays were performed by adding antibodies against STAT5B

or c-Jun Figure 2A,B showed new supershifted bands, indicating that the complexes contained STAT5B or c-Jun Finally, to verify whether Ang II can stimulate recruitment of STAT5B and c-Jun to the AGT pro-moter in vivo, chromatin immunoprecipitation (ChIP) assays were performed using antibodies to STAT5B and to c-Jun, respectively As shown in Fig 2C, DNA fragments containing the STAT5B- and AP-1-binding sites could be detected in the immunoprecipitates pulled by anti-c-Jun or anti-STAT5B IgGs Increased binding of AP-1 or STAT5B to the AGT promoter was observed in VSMCs treated with Ang II for 3 h However, AG490 decreased the recruitment of STAT5B to the AGT promoter region with the

inhibition of STAT5B phosphorylation (Fig 1C and Fig 2C), suggesting that STAT5B phosphorylation is necessary for its binding to the AGT promoter

Co-operation of STAT5B with AP-1 activates the AGT promoter

To determine whether the binding of STAT5B and AP-1 with the AGT promoter is essential to AGT expression induced by Ang II, 293A cells were cotrans-fected with the pGL3-AGT-Luc reporter plasmid, which contains both AP-1- and STAT5B-binding sequences in the AGT promoter from )545 to 39 bp (Fig 3A), and pcDNA3.1-STAT5B and⁄ or pcDNA3.1-c-Jun expres-sion plasmids Overexpresexpres-sion of STAT5B or c-Jun alone modestly increased the reporter activity following stimulation with Ang II (Fig 3B) On the other hand, the cotransfection of STAT5B with c-Jun expression vectors significantly increased the AGT reporter activity

by 6.8-fold over that seen with the reporter alone These results indicate that STAT5B and c-Jun synergistically activate AGT gene transcription

STAT5B and AP-1 form a stable complex in the AGT promoter in Ang II-induced AGT expression

To establish whether there is a direct interaction between STAT5B and c-Jun in the expression of AGT induced by Ang II stimulation, DNA–protein inter-actions were investigated by cross-supershift assays As seen in EMSAs, DNA–protein complexes formed by

0 2 4 6 8 10

p-c-Jun pSTAT5B

IP: c-Jun/ IB: p-Ser

IP:c-Jun/ IB: c-Jun

IP:STAT5B/IB: PY99

*

*

*

*

*

*

Ang II

A

B

C

AGT

GAPDH

*

*

*

*

0

2

4

6

8

IP: STAT5B/IB: PY99

IP:STAT5B/IB: STAT5B

– 3 3 6 6 h

* 0

2 4 6 8 10

Ang II

AG490

*

IP:STAT5B/IB: STAT5B

Fig 1 Ang II induces AGT gene expression with concurrent increases in the phosphorylation of c-Jun and STAT5B in VSMCs (A) VSMCs were treated with Ang II (10)7M ) for 0, 3, 6, 12 and

24 h Total RNA was isolated from VSMCs and subjected to RT-PCR analysis using specific primers of the AGT gene GAPDH was used as an internal control Bar graphs show the relative level

of AGT mRNA for four independent experiments *P < 0.05, com-pared with 0 h (n = 3) (B) VSMCs were treated with Ang II (10)7M ) for the indicated periods of time Cell extracts were immunoprecipitated with antibodies to c-Jun or to STAT5B and immunoblotted with anti-phospho-Ser IgG or anti-PY99 IgG by western blot analysis Bar graphs show the relative level of phos-phorylated c-Jun or phosphos-phorylated STAT5B for four independent experiments *P < 0.05, compared with 0 h (n = 3) (C) VSMCs were pretreated with or without AG490 (10)5M ) for 16 h before stimulation with Ang II (10)7M ) for 3 and 6 h Cell extracts were immunoprecipitated with anti-STAT5B IgG and analyzed by western blotting using anti-PY99 and anti-STAT5B IgGs, respectively Bar graphs show the relative level of phosphorylated STAT5B for four independent experiments *P < 0.05, compared with treatment without AG490 in Ang II-treated cells for 3 and 6 h, respectively (n = 3).

Ang II Nuclear extract STAT5B probe Cold STAT5B probe Anti-STAT5B IgG Anti-c-Jun IgG Rabbit IgG

Supershift Shift

Free probe

Ang II Nuclear extracts AP-1 probe Cold AP-1 probe Anti-c-Jun IgG Anti-STAT5B IgG Rabbit IgG

Supershift Shift

Free probe

B

C

Ang II AG490

IP: STAT5B

No antibody

Input

IP: c-Jun

No antibody

Input

STAT5B binding sequence

AP-1 binding sequence

Fig 2 Ang II increases the DNA-binding

activity of AP-1 and STAT5B (A and B)

VSMCs were treated with Ang II (10)7M )

for 0.5, 1 and 3 h Nuclear extracts were

analyzed by EMSA using oligonucleotide

probes containing the AP-1-binding site (A)

and the STAT5B-binding site (B) in the AGT

gene promoter Protein–DNA complexes

were separated by nondenaturing PAGE and

then visualized by autoradiography

Super-shift assays were performed by adding

anti-bodies against c-Jun or STAT5B Rabbit IgG

was used as negative control The data

shown represent the best of three

indepen-dent experiments (C) VSMCs pretreated

with or without AG490 were treated with

Ang II (10)7M ) for the indicated periods of

time Chromatin fragments were

immuno-precipitated by anti-c-Jun and anti-STAT5B

IgG and the AGT promoter region containing

the AP-1 ( )644 to )381 bp) or the STAT5B

( )200 to )60 bp) binding sequence was

amplified by PCR, respectively The data

shown represent the best of three

indepen-dent experiments.

nuclear protein with the AP-1 probe were supershifted

by antibody to STAT5B Similarly, the STAT5B probe–

protein complexes were supershifted by antibody to

c-Jun (Fig 2A,B) These findings indicate that AP-1

interacts with STAT5B in the AGT expression

stimu-lated by Ang II The interaction between STAT5B and

AP-1 was also tested by cross-coimmunoprecipitation

(CoIP) of the nuclear extracts As shown in Fig 4A

c-Jun protein was detected in the pellets

immunoprecipi-tated with antibody to STAT5B, suggesting that

STAT5B interacts with AP-1 Treatment of VSMCs

with Ang II for 1 and 3 h resulted in an increase in the

interaction of STAT5B with c-Jun The interaction of

STAT5B with c-Jun induced by Ang II was significantly

decreased by pretreating VSMCs with AG490,

suggest-ing that STAT5B phosphorylation is required for the

interaction of STAT5B with AP-1 To verify this further

in vivo, ChIP was performed by using antibodies to

c-Jun or to STAT5B STAT5B protein was found

in protein eluates from anti-c-Jun IgG-precipitated

chromatin, whereas the eluates from anti-STAT5B

IgG-precipitated chromatin contained c-Jun protein

(Fig 4B) Furthermore, ChIP assays showed that the STAT5B-binding sequence could be amplified by PCR

in the immunoprecipitates formed with anti-c-Jun IgG, and the AP-1-binding sequence was similarly produced from the STAT5B–chromatin complexes immunopre-cipitated by anti-STAT5B IgG (Fig 4C), indicating that STAT5B physically interacts with c-Jun by forming a stable complex with the AGT promoter in Ang II-induced AGT expression

Discussion

In this report, we demonstrated, for the first time, that,

in addition to STAT5, AP-1 is an important transcrip-tion factor which maintains the transcriptranscrip-tion of AGT mRNA in VSMCs, and that the activation of AP-1 participates in transcription activation of the AGT gene

to modulate the autocrine Ang II loop in the local renin-angiotensin system Jun and Fos family proteins usually function as dimeric transcription factors that bind to AP-1 regulatory elements in the promoter of numerous genes Jun proteins can form stable homo-dimers or heterhomo-dimers with Fos proteins Recent study has indicated that Ang II activates AP-1 to regulate sev-eral inflammatory genes in VSMCs [23] We showed that Ang II could activate AP-1 through enhancement

of the phosphorylation and association to DNA of Jun proteins in the induction of the AGT gene by Ang II in VSMCs ChIP assays confirmed that Ang II increased the recruitment of AP-1 to the AGT gene promoter Overexpression of c-Jun increased AGT-Luc reporter activity in A293 cells These findings indicate that AP-1 activation is involved in regulatory mechanisms of Ang II-induced AGT gene expression in VSMCs Ang II is known to activate the JAK-STAT pathway

in several cells [9], STAT1, STAT2 and STAT3 in VSMCs [9,23,25,26] and STAT5 in cardiac myocytes [8], whereas the activity of STAT5 is unknown in Ang II-induced VSMCs under the same conditions [9,25,27] However, we demonstrated that Ang II enhances the phosphorylation of STAT5B and its association with DNA, and consequently the transacti-vation transcription of the AGT gene in VSMCs Super-EMSA and ChIP confirmed that Ang II could increase the binding activity of STAT5B to the cis-element and the recruitment of STAT5B to the promoter of the AGT gene in vitro and in vivo [28–32]

It was previously demonstrated that the activation

of STAT5B in the liver, and of STAT3 and STAT5A

in the heart, participates in transcription activation of the AGT gene to modulate the autocrine Ang II loop, and that Ang II-mediated activation of JAK2 triggers

a pattern of tissue-specific phosphorylation of the

pGL3-AGT-Luc

pcDNA3.1-STAT5B

pcDNA3.1-c-Jun

pcDNA3.1

0

10

20

30

40

50

60

70

80

90

*

A

B

STAT5B

c-Jun

AP-1

–419~–412 –282~–277 –172~–163 TATA

+1

Fig 3 Co-operation of STAT5B with AP-1 activates the AGT

pro-moter (A) Schematic representation of the AP-1-binding site and

the STAT5B-binding site in the AGT promoter region (B) 293A cells

were co-transfected with the pGL3-AGT-Luc reporter and with an

expression vector for c-Jun, STAT5B or c-Jun + STAT5B,

respec-tively Cell lysates were subjected to luciferase activity assays and

western blotting using anti-STAT5B and anti-c-Jun IgG, respectively.

Bar graphs are expressed as the relative luciferase activity.

*P < 0.05 versus pcDNA3.1-transfected cells (n = 3).

STAT protein in different tissues [10] Ang II causes

activation of JAK via its interaction with the AT1

receptor and then activates STAT1, STAT2, STAT3,

STAT5A, STAT5B and STAT6 in heart tissues under

different experimental conditions [6,8] In the present

study, we provided evidence indicating that stimulation

with Ang II results in JAK2 activation, which then

triggers STAT5B phosphorylation and maintains the

transcription of AGT mRNA in VSMCs Treating

VSMCs with AG490 (10)5m), a potent and selective

inhibitor of JAK2 phosphorylation [24], inhibited

STAT5B activation and interaction with DNA, and

consequently caused a decrease in the transcription of

AGT mRNA There is an elevated level of AGT

mRNA that correlated well with the enhanced

STAT5B phosphorylation These observations

there-fore lend support to the notion that the activation of

STAT5B and the expression of the AGT gene in

VSMCs are causally linked STAT5B may be an

important upstream component in the Ang II feedback

circuit that regulates the transcription of AGT mRNA

in VSMCs

In the context that STAT5B and c-Jun are present

in the binding complexes with the AGT gene pro-moter, we investigated whether there is any cross-talk between STAT5B and c-Jun in the induction of expres-sion of AGT mRNA by Ang II, using super-EMSA, ChIP, CoIP and western blot analysis Co-operative DNA binding of proteins usually involves regions in close proximity, which functionally represent a com-posite regulatory element [13,14] In this study, the

450 bp region encompassing the one STAT5B site and the two AP-1 sites of the AGT gene promoter may serve as composite binding elements These closely located sites support that the c-Jun interaction with STAT5B in binding complexes on DNA elements is important for maximal gene activation Experimental support of this is provided by the increased AGT pro-moter⁄ luciferase reporter activity with co-transfection

of c-Jun and STAT5B expression vectors Definitive evidence of physical association between c-Jun and STAT5B is provided by the results of the CoIP and super-EMSA of nuclear extracts, and by the results of the ChIP assay of immunoprecipitated chromatin from

IP: STAT5B/IB: c-Jun IP: STAT5B/IB: STAT5B

Ang II AG490

A

Ang II 3 h

IP Input STAT5B c-Jun No Ig IB: STAT5B

IB: c-Jun

IP: STAT5B Con Ang II

B

STAT5B binding sequence

AP-1 binding sequence

C

Fig 4 STAT5B and AP-1 form a stable complex with the AGT promoter in Ang II-induced AGT expression (A) VSMCs were treated with Ang II (10)7M ) for the indicated periods of time after pretreatment with or without AG490 (10)5M ) for 16 h Nuclear extracts were immuno-precipitated with anti-STAT5B IgG and analyzed by western blotting using anti-c-Jun and anti-STAT5B IgG, respectively (B) VSMCs were treated with Ang II (10)7M ) for 3 h The protein eluates from chromatin precipitated with anti-c-Jun IgG or anti-STAT5B IgG were analyzed

by western blot using anti-STAT5B and anti-c-Jun IgG, respectively (C) The chromatin fragments immunoprecipitated with anti-c-Jun and anti-STAT5B IgG were used as templates to amplify the AGT promoter regions containing the AP-1 and STAT5B binding sequences, respec-tively.

VSMCs using antibodies to c-Jun and STAT5B

Previ-ous studies have demonstrated that administration of

Ang II stimulates the interaction between p300 and

STAT5B in the liver, and with STAT3 and STAT5A

in the heart, under similar conditions [10] Taken

together, these observations emphasize the differences

in binding of STAT proteins to the same target

sequence in response to stimulus The previous reports

and our data suggest a role of JAK2 phosphorylation

in tissue-specific mobilization of STATs, as Ang

II-mediated stimulation of physical interaction between

STATs and c-Jun or the other transcription factors

was effectively reduced by AG490 [10] Yet, the

selec-tive activation of STATs might require a tissue-specific

factor(s), the identity of which is hitherto unknown

Previously, it has been shown that the cis-elements

in the AGT promoter, well characterized for their

func-tions in vitro, were dispensable in vivo [33] We

there-fore complemented our in vitro data with the studies

on protein–protein and protein–DNA interactions and

demonstrated that the requirement for the interaction

of AP-1 with STATs for the AGT promoter activity is

the same as in vitro transient transfection assays in

293A cells Our study demonstrated that, in addition

to STAT5B, AP-1 is also involved in the signal

trans-duction triggered by Ang II, and pointed to the

under-lying complexity in the regulation of the Ang II

autocrine loop We speculate that AP-1 and STAT5B

bind to their elements in the AGT gene promoter,

respectively, and meanwhile interact with each other in

Ang II-stimulated VSMCs Taken together, the

inter-action of STAT5B and c-Jun bound to the promoter

provides maximal activation of AGT expression by

Ang II in VSMCs

Experimental procedures

Cell culture

Rat VSMCs were isolated and subcultured as described

previously [34] Cells used in the experiments were from

passages 3–5 VSMCs were allowed to attach to the plate

wall and were then serum-deprived for 24 h in DMEM

(Gibco, Grand Island, NY, USA) containing 0.1% BSA

Cells were then stimulated with Ang II (10)7m; Sigma,

St Louis, MO, USA) dissolved in serum-free DMEM

con-taining 0.1% BSA, with or without pretreatment with AG490

(10)5m, Sigma,) for 16 h before the addition of Ang II

RNA isolation and RT-PCR

Total RNA was isolated from cells using TRizol reagent

according to the manufacturer’s instructions (Invitrogen,

Carlsbad, CA, USA) Reverse transcription was performed using the Superscript First Stand Synthesis System for RT-PCR (Invitrogen) The cDNA was then used as a tem-plate for PCR using specific primers for AGT (forward, 5¢-ACCTTTGAGCCTGTGCCCAT-3¢; reverse, 5¢-GCTACA

dehydrogenase (GAPDH) (forward, 5¢-CAGGGTGTGATG

The amplified RT-PCR products were separated on a 2% agarose gel containing ethidium bromide and the band intensities were quantified using NIH image j software

Nuclear protein extraction

The cells were scraped into cold NaCl⁄ Pi and centrifuged (14 000 g, 4C, 10 min) After the supernatant was dis-carded, nuclear extracts were prepared by lysing the cells in ice-cold buffer containing 10 mm Hepes-KOH (pH 7.9),

for 15 min, and then centrifuged at 1500 g to obtain cellu-lar nuclei The nuclei were washed in lysis buffer without Nonidet P-40 and centrifuged again at 1500 g for 5 min The supernatant was removed and the pellet was resus-pended in nuclear resuspension buffer (20 mm Hepes-KOH,

pH 7.9, 400 mm NaCl, 1 mm EDTA, 0.1 mm EGTA, 1 mm phenylmethanesulfonyl fluoride, 1 mm Na3VO4, 1 mm dith-iothreitol), vigorously vortexed for 10 s and then centri-fuged at 1500 g for 5 min The supernatant was separated into aliquots for use in western blotting and EMSAs

Immunoprecipitation (CoIP) and western blot analysis

Equal amounts of proteins were incubated overnight at

(Santa Cruz Biotechnologies, Santa Cruz, CA, USA)

washed, separated by electrophoresis on an 8% SDS-poly-acrylamide gel and then electrophoretically transferred to

Billerica, MA, USA) The membrane was incubated with anti-phospho-Ser, anti-PY99, anti-STAT5B or anti-c-Jun IgGs (1 : 1000; Santa Cruz), followed by a secondary anti-rabbit IgG (1 : 20 000; Santa Cruz), using the chemi-luminescence protocol (Santa Cruz)

EMSA

The sequences of double-strand oligonucleotide fragments

GACCC-3¢, and 5¢-AGAGCCGCTGATGACTTATGAGA

GGT-3¢, respectively Nuclear extracts (10 lg) were

incu-bated for 30 min with oligonucleotide probes end-labeled

with [32P]dATP[cS] and then loaded onto a 6%

nondenatu-rating polyacrylamide gel, as described previously [21,35]

When specified, the reaction proceeded in the presence of

2 lg of anti-c-Jun or anti-STAT5B IgGs

Chromatin immunoprecipitation assay

VSMCs were treated with Ang II (10)7m) for the time

peri-ods indicated and were then fixed with 1% formaldehyde

ChIP assays were performed using 2 lL of anti-c-Jun or

anti-STAT5B IgGs, as described previously [35] An aliquot

of the cell lysates was used to isolate total input DNA PCR

amplification of the immunoprecipitated DNA was

per-formed using primers specific for the AP-1-binding site or

the STAT5B-binding site in the AGT gene promoter The

sequences of the PCR primers were as follows: AP-1-binding

GA-3¢ and 5¢-TGGCAGATGAGCTTCAGGCA-3¢; and

AGCTCATCTGCCACTAG-3¢ and 5¢-TAGCTCCAGCCC

AGACAAGCACAG-3¢ The proteins from the

immuno-precipitated chromatin fragment were eluted for western blot

analysis using anti-c-Jun or anti-STAT5B IgGs

Plasmid construction

[5] was obtained by PCR using the following primers:

ATGTGC-3¢ (KpnI site underlined); reverse, 5¢-GCCA

site underlined), digested by KpnI and HindIII, and then

inserted into the pGL3-basic luciferase reporter gene vector

(Promega, Madison, WI, USA) linearized by KpnI–HindIII

and named pGL3-AGT-Luc For the c-Jun expression

plas-mid, c-Jun cDNA was obtained from the pBIISK(-)-Jun

plasmid by EcoRI digestion, inserted into the pcDNA3.1

vector and sequenced

Transient transfection and luciferase assay

293A cells were grown to 60% confluence in six-well plates

and transfected with Lipofectamine 2000 Reagent

(Invitro-gen), as described by the manufacturer The transfection

was performed using 1 lg of pGL3-AGT-Luc reporter

plas-mid and c-Jun expression plasplas-mid or STAT5B expression

plasmid (gifted by Yu-Lee, Baylor College of Medicine) or

c-Jun plus the STAT5B expression plasmid or control

plas-mid pcDNA3.1 In addition, 10 ng of the renilla luciferase

reporter plasmid pRL-TK (Promega) was included in each

sample as an internal standard for transfection efficiency

Firefly and renilla luciferase activities were determined 48 h

after the initial transfection using the Dual-Luciferase Reporter Assay System (Santa Cruz) and Flash & Glow

LB 955 Tube Luminometer (Alpha Innotech HD2, San Lenndro, CA, USA) Firefly luciferase values were normal-ized on the basis of the renilla luciferase values

Statistical analysis

Results are expressed as means ± SD, and an analysis of variance with Bonferroni’s test was used for the statistical analysis of multiple comparisons of data P-values of less than 0.05 were considered statistically significant

Acknowledgements This work was supported by the National Natural Science Foundation of China (nos 30670845 and 30770787), the ‘973’ Program of China (nos 2008CB517402) and the Hebei Province Natural Science Foundation (nos C2006000814 and C2005000722)

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