Modulation of immediate early gene expression by tristetraprolin in the differentiation of 3T3-L1 cells

Modulation of immediate early gene expression by tristetraprolin

in the differentiation of 3T3-L1 cells

Nien-Yi Lin

, Chung-Tien Lin

, Ching-Jin Chang

a_{Department and Graduate Institute of Veterinary Medicine, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan} b_{Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, No. 1 Sec 4 Roosevelt Road, Taipei 106, Taiwan}

c

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Received 18 October 2007

Available online 29 October 2007

Abstract

Tristetraprolin (TTP) is a zinc-finger-containing AU-rich elements (ARE)-binding protein. AREs presented in the 3

_untranslated

region (UTR) of mRNAs from many proto-oncogenes, cytokines, and growth factors may be targets for regulation of messenger

RNA stability. In this study, we observed that many immediate early genes (IEGs) were induced during the early differentiation of

3T3-L1 preadipocytes and their ARE-containing transcripts were degraded rapidly. Immunoprecipitation followed by RT-PCR analysis

showed that two of IEG mRNAs, COX-2 (cyclooxygenase-2) and MKP-1 (mitogen-activated protein kinase phosphatase), were the

tar-get of TTP. Biotinylated MKP-1 AREs also could bring down TTP and the other ARE-binding protein HuR. RNA EMSA and

com-petition assays showed that each of three AREs located in 3

_{UTR of MKP-1 mRNA has differential binding affinity to TTP. Sequence}

analysis of 3

_{UTR of IEG mRNAs suggested that TTP may prefer binding to UUAUUUAUU sequence. Taken together, our results}

implied that TTP may target specific ARE-containing IEGs’ mRNAs such as COX-2 and MKP-1 mRNAs to modulate their expression

post-transcriptionally.

 2007 Elsevier Inc. All rights reserved.

Keywords: Immediate early gene; Tristetraprolin; 3T3-L1; AU-rich element; MKP-1

The established preadipocyte cell line 3T3-L1 has been

used in examining the process of adipogenesis in vitro.

When treated with an empirically-derived prodifferentiative

regimen that includes cAMP, insulin, and glucocorticoids

in the presence of fetal bovine serum, they undergo

differ-entiation to mature fat cells over a period of 4–6 days.

The first step in the process of adipogenesis is the re-entry

of growth-arrested preadipocytes into the cell cycle and the

completion of several rounds of clonal expansion

[1–3]

.

Several transcriptional factors are expressed coordinately

to exert the terminal differentiation. Many immediate early

genes (IEGs) such as c-jun, c-fos, egr-1, egr-2, nur77, cox-2,

cyr61, pip92, btg2, ttp, and mkp-1, which expressed briefly

in the trigger of differentiation hormones, have been

observed

[4]

.

Most IEGs contain adenylate/uridylate-rich elements

(AREs) in the 3

_{UTR of their mRNAs to control their}

RNA turnover

[5]

. AREs can range in size and generally

contain one or more copies of the pentameric sequence

AUUUA, and have been divided into three classes

[6]

.

Sev-eral ARE-binding proteins have been identified to regulate

mRNA turnover

[7]

. HuR can respond to certain

extracel-lular stimuli to mediate specific mRNAs stabilization

[8]

.

Knockdown of HuR could attenuate the differentiation

process in 3T3-L1 cells

[9]

. In contrast, TTP is important

for the destabilization of tumor necrosis factor and

GM-CSF mRNAs, as shown in knockout mice

[10,11]

and in

0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.10.119

*

Corresponding authors. Address: Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, No. 1 Sec 4 Roosevelt Road, Taipei 106, Taiwan (C.-J. Chang). Fax: +886 2 7359931 (C.-T. Lin), +886 2 23635038 (C.-J. Chang).

E-mail addresses: [email protected] (C.-T. Lin), chingjin@gate. sinica.edu.tw(C.-J. Chang).

www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 365 (2008) 69–74

tissue culture by ectopic-overexpression studies

[12]

. It

binds AREs of target mRNAs and induces deadenylation

[13,14]

, or directs them to the exosome

[15–17]

, or

associ-ates with RISC-microRNA complexes

[18]

for rapid

degra-dation of target mRNAs. Recent reports showed that TTP

could interact with mRNA decapping enzymes and

nucle-ate processing body (PB) formation to deliver

ARE-mRNAs to PBs

[19,20]

. Our study showed that TTP could

be induced as an IEG during 3T3-L1 differentiation and its

expression was controlled by negative autoregulation

[21]

.

In this study, we detected the expression and half-lives

of IEG mRNAs during early differentiation of 3T3-L1 cells

by using real-time PCR analysis. A novel TTP

target-mRNA, MKP-1 target-mRNA, was identified. Sequence analysis

of 3

_{UTR of IEG mRNAs and RNA EMSA using MKP-1}

AREs as probes suggested that TTP may prefer binding to

UUAUUUAUU sequence.

Materials and methods

Cell culture. 3T3-L1 cells are grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco-BRL) containing 1.5 g/l NaHCO3and

supple-mented with 10% Bovine serum (BS, Gibco-BRL), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco-BRL) in 5% CO2humidified atmosphere

(37C). Two-day postconfluent cells (day 0) are stimulated to differentia-tion by change fresh medium containing 10% fetal bovine serum (FBS, Hyclone-Characterized) and addition of hormone cocktail (5 lM dexa-methasone (Sigma–Aldrich), 1.7 lM insulin (Sigma–Aldrich, from bovine), and 0.5 mM 1-methyl-3-isobutylmethylxanthine (MIX, Sigma–Aldrich).

Plasmid constructs. The 30_{UTR of MKP-1 (from cDNA 1254–1887 nt)}

was PCR cloned by using primers 50

-AGGTGTGGAGTTTCACTTGCC-30_{and 5}0_{-CCCAGTAACAAAATGTCTTCAC-3}0_{, and the cDNAs from}

1 h differentiation-triggered 3T3-L1 as templates. The PCR fragment was cloned into pCRII-TOPO vector (Invitrogen) and sequence confirmed to prepare riboprobe.

RNA isolation and reverse trasncription-PCR. Total RNAs were extracted from the cell cultures by using Blue extract reagent (LTK, Inc., Taiwan) following the procedures recommended by the manufacturer. Five micrograms of total RNAs extracted from 3T3-L1 cells treated with differentiation inducers for different time intervals was reverse-transcribed to produce cDNA using reverse transcriptase and oligo dT (Promega) as a primer.

Real-time PCR. Real-time PCR was performed with the Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems) in a total volume of 20 ll. Expression of IEGs was analyzed using SYBR Green PCR Master Mix (Applied Biosystems) containing 50 ng of cDNAs and 160 nM of each specific primers showed inTable 1. The real-time PCR amplification conditions were 40 cycles of 95C for 15 s and 60 C for 1 min. The real-time PCR data were analyzed using the 2DDCTrelative quantitation method, according to the manufacturer’s directions.

RNA-Immunopreciptation assays. One milligram cytoplasmic extracts from 3T3-L1 cells were incubated with pre-immune serum or anti-HuR (Santa Cruz), or anti-TTP antibody to precipitate the associated RNAs as described previously[21]. RNAs were extracted for RT-PCR analysis. The specific cDNA of IEGs was amplified using 5% of the RT reaction in 20 ll containing 10 pmol of forward and reverse primer as shown inTable 1, and lypholized Taq DNA polymerase, buffer and dNTPs (LTK, Inc.). PCR was performed in a Robocycler gradient 96 PCR thermal machine (Stratagene) using the following conditions: 94C (3 min) for one cycle, 94C (40 s), 55 C (40 s), 72 C (depending on the product length, 1 min/ 1 kb) for 25–30 cycles, and a final incubation at 72C for 3 min. The PCR products were separated in 2% agarose gel.

RNA pull-down assay. Cytoplasmic extracts from 107_{3T3-L1 cells were}

isolated and pre-treated as described previously[21]. Four micrograms of

in vitro transcribed biotinylated MKP-1 ARE or control RNA (T7-MEGA shortscript, Ambion) was added to the extract and the mixture was incubated for 1 h at 4C. The protein and biotinylated RNA com-plexes were recovered by addition of 12 ll Streptavidin Sepharose at 4C for 2 h with rotation. After extensive washes, the brought-down complexes were analyzed by Western blotting with anti-HuR and anti-TTP antibodies.

REMSA (RNA electrophoretic mobility shift assay). Three MKP-1 AREs were PCR cloned in pCRII-TOPO (Invitrogen) by using following primers: F1: 50_{-AGGTGTGGAGTTTCACTTGCC-3}0 _{and R1: 5}0_-T

TGGTCCCGAATGTGCCGAG-30 _{for ARE1; F2: 5}0_{- GATGACAT}

GCGCGTATGAGAG-30 _{and R2: 5}0_{-CCTGCTCTGGGTCTATTTAC-3}0

for ARE2; F3: 50_{-GTAAATAGACCCAGAGCAGG-3}0 _{and R3: 5}0_-CC

CAGTAACAAAATGTCTTCTC-30_{for ARE3. The plasmids were}

line-arized with restriction enzyme and in vitro transcribed by T7 or SP6 RNA polymerase in the presence of (a-32P)-UTP for REMSA. One picomole of radiolabeled probe was incubated with recombinant GST-TTP proteins at room temperature for 40 min in a final volume of 10 ll containing 15 mM Hepes (pH 7.9), 10 mM KCl, 5 mM MgCl2, 10% glycerol, 0.2 mM DTT,

0.5 lg heparine sulfate, and 5 lg of yeast total RNA. Binding mixtures were then loaded onto native 5% polyacryamide gel (acryl:bis = 40:1) containing 2.5% glycerol in 0.25· Tris–borate–EDTA buffer. After elec-trophoresis at 15 V/cm for 60 min, gel was dried and exposed to Kodak XAR film at70 C for appropriate time.

Result and discussion

mRNA Expression profiles and half-life analysis of IEGs

during early differentiation of 3T3-L1 cells

To verify the induction of IEGs during the

differentia-tion of preadipocytes, confluent 3T3-L1 cells were treated

Table 1

Primers used to characterize the IEGs expression in differentiation of 3T3-L1 cells by real-time PCR IEGs Primers c-jun F: 50_{-GCAGAGAGGAAGCGCATGAG-3}0 R: 50-AGCATGTTGGCCGTGGAT-30 c-fos F: 50_{-CTTCTTGTTTCCGGCATCATC-3}0 R: 50_{-GCTCCCAGTCTGCTGCATAGA-3}0 egr-1 F:50_{-ACTCCCAACACTGACATTTTTCCT-3}0 R: 50_{-GGGAACCTGGAAACCACCTT-3}0 egr-2 F: 50_{-ACGGGACCAGGAGCAAGTG-3}0 R: 50_{-CGTTTTGCTGGGCCTGTTAG-3}0 nur77 F: 50_{-CCTGGGACGGCTCATTTG-3}0 R: 50_{-GTGGGAGGACTGAAGGAGAAGA-3}0 cox-2 F: 50_{-TGGAGGCGAAGTGGGTTTTA-3}0 R: 50-GTTTTGGTAGGCTGTGGATCTTG-30 cyr61 F: 50_{-GCTCCACCGCTCTGAAAGG-3}0 R: 50_{-CGGCGCCATCAATACATGT-3}0 pip92 F: 50_{-GCGATTTGAGCGACAGTAGTGA-3}0 R: 50_{-AGACTGGAGAAGCGCCTTTG-3}0 btg2 F: 50_{-TGTGGGTTGATCCCTATGAAGTG-3}0 R: 50_{-CAGGTGAGGAGCCCATAGGA-3}0 ttp F: 50_{-GGATCTCTCTGCCATCTACGA-3}0 R: 50_{-CAGTCAGGCGAGAGGTGAC-3}0 mkp-1 F: 50_{-TAGACTCCATCAAGGATGCTGG-3}0 R: 50_{-GCAGCTTGGAGAGGTGGTGAT-3}0

with a cocktail of fetal bovine serum, MIX,

dexametha-sone, and insulin (MDI) to induce their differentiation.

RNAs were isolated for real-time PCR by using specific

primers for IEGs including c-jun, c-fos, egr-1, egr-2,

nur77, cox2, cyr61, pip 92, btg2, ttp, and mkp-1.

Fig. 1

A

shows their mRNA expression profiles from 0 to 16 h after

the trigger of differentiation. All IEG mRNAs presented

the similar expression kinetics, they rapidly increased to

the highest level at 1-h induction that resulted in activation

ranging from 3.2 folds (for c-jun) to 135 folds (for nur77),

and then decreased dramatically. This observation was

consistent with the result of previous report by using

Northern blotting analysis

[4]

. The transient expression

may

imply

that

post-transcriptional

regulation

was

involved in the control of IEG mRNAs.

The half-lives of these IEG mRNAs were determined

after hormone induction for 1 h (

Fig. 1

B). The results

showed that all detected half-lives were shorter than

20 min, raging from 6.6 min (TTP) to 18.6 min (Egr-1).

The short half-lives may reflect some specific sequences

or structures in their mRNAs. c-Jun, c-Fos, TTP and

COX-2 mRNAs have been reported having AREs located

0% 20% 40% 60% 80% 100%

0

10

20

min after Act. D

c-jun c-fos Egr-1 Egr-2 COX-2 Nur77 MKP-1 TTP Cyr61 PIP92 Btg2 0 2 4 0 1 2 4 8 16 fold h 0 1 2 4 8 16 h 0 1 2 4 8 16 h 0 1 2 4 8 16 h 0 1 2 4 8 16 h 0 1 2 4 8 16 h 0 1 2 4 8 16 h 0 1 2 4 8 16 h 0 1 2 4 8 16 h 0 1 2 4 8 16 h 0 1 2 4 8 16 h c-jun 0 10 20 30 fold c-fos 0 5 10 15 fold Egr-1 0 5 10 15 fold Egr-2 0 50 100 150 fold Nur77 0 30 60 90 fold COX-2 0 20 40 60 fold Cyr61 0 10 20 fold PIP92 0 20 40 60 fold Btg2 0 10 20 30 fold TTP 0 30 60 90 fold MKP-1

A

B

Fig. 1. mRNA metabolism of the IEGs during the early differentiation of 3T3-L1 cells. (A) mRNA expression profiling. Two-day postconfluent 3T3-L1 preadipocytes were induced to differentiation with MDI and 10% FBS for 0, 1, 2, 4, 8, and 16 h, and RNAs were isolated and reverse-transcribed to produce cDNAs for real-time PCR with specific primers for IEGs. Two independent experiments were performed and consistent results were gotten. (B) mRNA half-life of IEGs. Two-day postconfluent 3T3-L1 preadipocytes were induced to differentiation for 1 h. Transcription was then stopped by adding 10 lg/ml of actinomycin D (Act.D) for 0, 10, and 20 min. The following experiments were as described in (A).

in their 3

_UTR

_[7,21–23]

_{. Analysis of 3}

_{UTR of all these}

IEG mRNAs showed that most of them were

ARE-con-taining mRNAs (

Table 2

). Frevel et al. provided evidence

that the number of the overlapping pentamer AUUUA

may contribute to the mRNA half-life, and the mRNA

half-lives in the class II category (overlapping AUUUA)

were significantly shorter than those of class I (separated

AUUUA)

[24]

. Our analysis showed the class II mRNAs

such as TTP and MKP-1 had the shortest half-life, and this

result

seemed

to

partially

correlate

with

previous

description.

c-jun, c-fos, egr-1, and egr-2 encode transcription

fac-tors. Egr-2 has been identified to be acting early in the

adi-pogenic program and appearing to contribute to induction

of C/EBPb expression

[25,26]

. Orphan nuclear receptor

Nur77 has the effect to promote mitotic clonal expansion

[27]

. COX-2 is required for conversion of arachidonic acid

to prostaglandins which may serve as a ligand for PPARc

(the key regulator of adipogenesis)

[28]

. Cyr61 could

pro-mote cell proliferation

[29]

. A cofactor of transcription,

BTG2, is known to have antiproliferative effect

[30]

.

MKP-1 (also named Dusp1) is a mitogen-activated protein

kinase phosphatase. It has been reported that MKP-1 plays

an essential role in adipocyte differentiation through

down-regulation of ERKs activity

[31]

. ttp encodes a zinc-finger

containing ARE-binding protein. The tight expression

con-trol of these IEG transcripts may implicate their critical

function in early differentiation of 3T3-L1 cells.

MKP-1 and COX-2 mRNAs interact with TTP protein

Our previous report showed that ARE-binding protein

TTP could bind to its own mRNA and cause its mRNA

destabilized

[21]

. To elucidate whether TTP could target

other IEG mRNAs, immunoprecipitation (IP) reactions

were performed to isolate mRNA subsets bound to TTP

using anti-TTP antibody. RNAs present in IP reactions were

reverse transcribed and PCR was performed with IEGs

spe-cific primers.

Fig. 2

A showed that COX-2 and

MKP-1mRNA could interact with TTP, whereas the other IEG

mRNAs were not detectable in TTP brought-down

com-Table 2

Characteristics of IEG’s AREs Gene name (Accession No.) Length of 30_UTR (b) No. of UUAUUUAUU No. of UAUUUAU No. of AUUUA ARE classa

mRNA half-life (min)

mkp-1 (NM_013642) 633 2 1 1 II 6.8 cox-2 (M64291) 2047 1 4 6 II 8.9 ttp (NM_011756) 773 1 2 0 II 6.6 nur77 (J04113) 539 0 0 2 I 18.4 c-jun (NM_010591) 1214 0 0 4 I 9.8 c-fos (V00727) 846 0 1 1 I 8.3 egr-1 (M20157) 1208 0 0 2 I 18.6 egr-2 (NM_010118) 1200 0 0 2 I 11.9 cyr61 (NM_010516) 689 0 2 3 II 14.4 pip92 (L26490) 966 0 0 0 III 13.5 btg2 (M64292) 2040 0 0 4 I 17.7 a

According to the rule of Chen and Shyu[6].

Fig. 2. TTP could interact with MKP-1 mRNA. (A) RNA immunopre-cipitation analysis. The cytoplasmic extracts from MDI and FBS treated (+) or non-treated () 3T3-L1 cells were immunoprecipitated using pre-immune serum, or anti-TTP or anti-HuR antibody. After extensive washes, the protein-associated RNAs were extracted for RT-PCR with IEGs specific primers. (B) Nucleotide sequences of 30_{UTR of}

MKP-1mRNA. The predicted AREs were rectangled and indicated as ARE1, ARE2, and ARE3. (C) RNA pull-down assay. The biotinylated full-length MKP-1 ARE or control 18S RNA was incubated with cytoplasmic extracts from 3T3-L1 cells treated (+) or non-treated () with MDI and FBS. The RNA-proteins complexes were brought-down by Streptavidin sepharose and subjected to SDS–PAGE for Western blotting. Anti-TTP and anti-HuR antibody were used. The protein amount in the lanes of direct loading was 15% of the pull-down assay used.

plexes (data not shown). On the other hand, HuR antibody

could precipitate all our demonstrated IEG mRNAs

(

Fig. 2

A and data not shown). COX-2 mRNA has been

reported to be TTP-associated and it also belongs to class

II ARE

[32,33]

. MKP-1 mRNA is a novel target of TTP,

which has not been identified before. There are three AREs

scattered

in

633

base

of

MKP-1

mRNA

3

_UTR

(NM_013642), and one belongs to class II ARE and contains

two overlapping copies of AUUUA motifs (

Fig. 2

B).

More-over, to verify the interaction between MKP-1 mRNA and

TTP proteins, the RNA pull-down analysis was performed.

The 3

_{UTR of MKP-1 mRNA was biotin-labeled and then}

incubated with 3T3-L1 cell lysates. The pulled down

RNA–protein complexes could be detected by anti-TTP

and anti-HuR antibodies (

Fig. 2

C).

ARE is a very heterogeneous element. In a previous

report, immobilized TTP protein was used to select its

opti-mal binding site by RNA SELEX and revealed a strong

pref-erence for the extended sequence UUAUUUAUU, rather

than UAUUUAU and a simple AUUUA motif

[34]

. In

the analysis of IEG AREs, we found that UUAUUUAUU

motif was present in our identified TTP interacting mRNAs

including that of TTP itself, COX-2 and MKP-1 (

Table 2

).

Although most IEG mRNAs could not be detected in

anti-TTP antibody precipitation complexes, they also were

short-lived. It may be possible that other ARE-binding

pro-teins such as KSRP and AUF1 involve in the stability

regu-lation of other IEG mRNAs

[7,35]

.

Analysis of interaction of TTP with three MKP-1 AREs

To demonstrate TTP-binding affinity on each of three

MKP-1 AREs, RNA EMSA was performed by using

ARE1, ARE2, and ARE3 probes and recombinant

GST-TTP.

Fig. 3

A showed that two of MKP-1 AREs could be

bound with TTP proteins, and the anti-TTP antibody

could block the RNA–protein complex formation (Lane

4). Moreover, the competition experiments represented

that

their

binding

affinity

to

TTP

seemed

to

be

ARE1=ARE2>ARE3 (

Fig. 3

B). ARE1 contains two

overlap-ping AUUUA pentamers and forms one UUAUUUAUU.

ARE2 has one UUAUUUAUU, and ARE3 contains one

UAUUUAU. This result reflects the above description that

TTP prefers to recognize the UUAUUUAUU sequence.

In conclusion, we showed that differentiation inducer

could stimulate the transient expression of a panel of IEGs

in 3T3-L1 cells. These IEG mRNAs were short-lived and

belonged to different ARE classes. The RNA destabilizing

protein,

TTP,

could

interact

with

class

II

and

UUAUUUAUU-containing mRNAs. The expression and

functional activity of both MKP-1 and TTP was controlled

by MAPKs

[36–40]

, and MKP-1 was a negative regulator

of MAPK pathways

[41,42]

. We first identified that

MKP-1 mRNA was one of TTP targets. This finding

sug-gested that TTP may involve in the complex net work of

MAPKs to control the signal duration and strength

through regulation of MKP-1 mRNA stability.

Acknowledgments

This work was supported by Academia Sinica and

National Taiwan University (Grant 95R0066-BM06-04).

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