A human homolog of mouse Lbh gene, hLBH , expresses in heart and activates SRE and AP1 mediated MAPK signaling pathway

A human homolog of mouse Lbh gene, hLBH, expresses in heartand activates SRE and AP-1 mediated MAPK signaling pathway

Jianping Ai Æ Yuequn Wang Æ Kunrong Tan Æ Yun Deng Æ Na Luo ÆWuzhou Yuan Æ Zequn Wang Æ Yongqing Li Æ Ying Wang ÆXiaoyang Mo Æ Chuanbing Zhu Æ Zhaochu Yin Æ Mingyao Liu ÆXiushan Wu

Received: 20 November 2006 / Accepted: 26 February 2007 / Published online: 28 March 2007

� Springer Science+Business Media B.V. 2007

Abstract It has been reported that mouse Lbh (limb-bud

and heart) can regulate cardiac gene expression by modu-

lating the combinatorial activities of key cardiac tran-

scription factors, as well as their individual functions in

cardiogenesis. Here we report the cloning and character-

ization of the human homolog of mouse Lbh gene, hLBH,

from a human embryonic heart cDNA library. The cDNA

of hLBH is 2927 bp long, encoding a protein product of 105

amino acids. The protein is highly conserved in evolution

across different species from zebra fish, to mouse, to hu-

man. Northern blot analysis indicates that a 2.9 kb tran-

script specific for hLBH is most abundantly expressed in

both embryonic and adult heart tissue. In COS-7 cells,

hLBH proteins are localized to both the nucleus and the

cytoplasm. hLBH is a transcription activator when fused to

Gal-4 DNA-binding domain. Deletion analysis indicates

that both the N-terminal containing proline-dependent

serine/threonine kinase group and the C-terminal contain-

ing ERK D-domain motif are required for transcriptional

activation. Overexpression of hLBH in COS-7 cells acti-

vates the transcriptional activities of activator protein-1

(AP-1) and serum response element (SRE). These results

suggest that hLBH proteins may act as a transcriptional

activator in mitogen-activated protein kinase signaling

pathway to mediate cellular functions.

Keywords hLBH � Transcription factor �Gene regulation � SRE � AP-1

Abbreviations

DMEM Dulbecco’s Modified Eagle

Medium

DAPI 4¢,6¢-diamidino-2-

phenylindole hydrochloride

MAPK Mitogen-activated protein

kinase

MAPKK, MKK or MEK MAPK kinase

MAPKKK or MEKK A MAPKK kinase or MEK

kinase

SRE Serum response element

AP-1 Activation protein 1

Much progress has been made toward a physiological and

clinical understanding of cardiac development and heart

disease. However, the molecular mechanisms underlying

these processes are still not completely understood [1].

Cardiogenesis requires precise regulation of gene expression

in a temporal or tissue specific manner. Many transcriptional

factors play a key role in these complex processes [1–3].

Mouse Lbh (limb-bud and heart) is a member of a highly

conserved family of small acidic proteins in vertebrates,

which displays a unique spatiotemporal gene expression

pattern during early mouse heart development. Lbh has

transcriptional activation activity in mammalian cells and

could act as a transcriptional coactivator involved in

The authors Jianping Ai and Yuequn Wang have contributed

equally to the work.

J. Ai � Y. Wang � K. Tan � Y. Deng � N. Luo �W. Yuan � Z. Wang � Y. Li � Y. Wang � X. Mo �C. Zhu � Z. Yin � M. Liu (&) � X. Wu (&)

The Center For Heart Development, Key Lab of MOE for

Development Biology and Protein Chemistry, College of Life

Sciences, Hunan Normal University, Changsha, Hunan 410081,

Peoples’ Republic of China

e-mail: [email protected]

X. Wu

email:[email protected]

123

Mol Biol Rep (2008) 35:179–187

DOI 10.1007/s11033-007-9068-4

molecular pathways that pattern the limb and the heart [4].

Lbh deregulation interferes with normal cardiac develop-

ment, in part through the attenuation of Nkx2.5 and Tbx5

transcription factor function and the Lbh transgene also

interfers with Gata4-dependent pathways. Lbh might play

an important role in transcriptional control during normal

cardiogenesis [5].

Partial trisomy 2p syndrome includes a spectrum of con-

genital heart disease (CHD) that is characterized by complex

malformations of the outflow and inflow tracts, defects in

cardiac septation, heart position, as well as abnormal ven-

tricular development. The human LBH (hLBH) maps to

chromosome 2p23, a genomic region related to CHD in

partial trisomy 2p syndrome. Previous studies have impli-

cated hLBH as a candidate gene for CHD associated with

partial trisomy 2p syndrome [5]. To specifically study the

function of the human homolog of mouse Lbh, hLBH, in

heart development and disease, we have cloned the hLBH

gene from a human embryonic heart cDNA library. Northern

blot analysis demonstrated that hLBH is most abundantly

expressed in heart at both adult and embryonic stages, which

implies that hLBH might play a role in heart development.

hLBH has a predicted 105 amino acid open reading frame

(ORF), encoding a putative protein that has a molecular

weight of approximately 16.2 kDa. The N-terminal region of

hLBH protein contains a proline-dependent serine/threonine

kinase group, and its C-terminal contains an ERK D-domain

motif (a binding site of ERK), suggesting that hLBH may

have a potential role in cell signaling.

Mitogen-activated protein kinases (MAPK) are major

components of pathways controlling embryogenesis, cell

differentiation, cell proliferation, and cell death. One of the

most explored functions of MAPK signaling is the regu-

lation of gene expression by direct or indirect phosphory-

lation and subsequent activation of transcription factors [6].

Recent studies suggest that MAPK pathways are critical

not only to the response of cardiovascular cells to extra-

cellular stress but also to developmental cues that regulate

cardiovascular development [7, 8]. In mammals, MAPK

pathways are involved in multiple cellular processes

through phosphorylation of specific endpoint targets, such

as ELK-1 and SRE, which together with SRF compose a

ternary complex that induces expression of c-fos and other

early growth response genes controlling the transition from

quiescence to proliferation [7]. The c-fos products hetero-

dimerize with c-Jun proteins to form AP-1 complexes [8].

Activation of AP-1 involves the direct phosphorylation/

dephosphorylation of AP-1 components as well as the

phosphorylation and activation of transcription factors that

induce elevated expression of c-Jun or c-fos. hLBH is a

transcription activator when fused to Gal-4 DNA-binding

domain. Overexpression of hLBH in COS-7 cells activates

the transcriptional activities of activator protein-1 (AP-1)

and serum response element (SRE), suggesting that hLBH

proteins may act as a transcriptional activator in mitogen-

activated protein kinase signaling pathway to mediate

cellular functions.

Materials and methods

Construction of cDNA library of human embryonic

heart

The 20-week human embryonic heart cDNA library was

constructed as reported previously [9]. Briefly, 5 lg

mRNA was purified from 500 lg total human embryonic

heart RNA using Rapid mRNATM purification Kit (Am-

resco). Reverse transcription reactions were performed

with the purified embryonic heart mRNA and Oligo dT-RA

primer according to cDNA Synthesis kit protocol (Takara).

After Cassette Adaptor Ligation reactions using cDNA

PCR Library Kit, cDNA amplification reactions were per-

formed with RA primer (5¢-CTGATCTAGACCTGCA

GGCTC-3), CA primer (5¢-CGTGGTACCATGGT CTA

GAGT-3), and Ex Taq (Takara).

Full-length hLBH cDNA cloning and bioinformatics

analysis

The nucleic acid sequence of mouse Lbh was obtained

from NCBI (http://www.ncbi.nlm.nih.gov) and used to

search human EST database with the BLAST searching

program (http://www.ncbi.nlm.nih.gov/blast). Through a

combined BLAST search as previously described [10], a

number of ESTs presenting the same gene (hLBH) were

identified in our database search. The first forward primer

in BM543364 and the reverse primer in BF718797

(POUT1 and POUT2, Table 1), and the second forward

primer in BM543364 and the reverse primer in BF718797

(PIN3 and PIN4, Table 1) were designed using Primer

Premier 5.0 to perform standard PCR. hLBH sequences of

ORF were confirmed by PCR amplification with a pair of

primers (PORF1 and PORF2, Table 1). The 5¢-gene spe-

cific primers (PS and PAS, Table 1) were designed for 5¢-rapid amplification of cDNA ends (RACE) according to

standard PCR methods [9, 11]. 5¢-RACE was performed

using SMARTTM RACE cDNA Amplification Kit

(Clonetech). All the PCR products were then cloned into

pMD18-T-vector (Takara) and sequenced with 377 DNA

Sequencer (ABI PRISM). Sequence analysis was per-

formed using the DNAstar program and BLAST program

from NCBI. Blastn program was used to identify the locus

of genes and to look for exons and introns. BLASTn and

Pfam 9.0 were used to analyze genomic structure and the

protein domain, respectively. The homologues of hLBH

were found with BLASTp, and the sequence alignment and

180 Mol Biol Rep (2008) 35:179–187

123

phylogenetic tree analysis were performed with MegAlign

program (DNAstar).

RNA isolation and Northern blot hybridization

Human tissues from therapeutically aborted fetuses were

obtained under the approval of Changsha Women and

Children’s Hospital, People’s Republic of China, with the

consent of the patients and in accordance with university

policy. Total RNA was isolated from various tissues

(muscle, cerebellum, cerebrum, tongue, lung, adrenal,

heart, intestine, liver from 17-week stage, and lung, liver,

muscle, kidney, brain, heart, intestine, pancreas from 25-

week stage) using standard methods [9]. 20 lg samples of

each tissue were separated by electrophoresis through

formaldehyde-agarose gel. The embryo multiple tissues

Northern blots were prepared as described in previous

studies [9]. A commercially available Northern blot con-

taining mRNA from eight adult tissues was purchased

from Clontech. The adult human Multiple Tissue North-

ern blot and the two multiple embryonic tissue blots were

hybridized with cDNA probe of hLBH. The hLBH cDNA

was labeled with [a-32p] dCTP using a Random Primer

Labeling Kit (Takara). The blots were then exposed to

X-ray films at –80�C for several days and the films were

developed. The blots were then stripped and probed with

radiolabeled b-actin cDNA (Clontech) as indicator of

mRNA loading.

Plasmid construction

The following plasmids were constructed and used for

mammalian cell transfection. (i) Construction of pEGFP-

N1-hLBH: to generate a fusion protein of hLBH with

enhanced green fluorescent protein (EGFP), the coding

region of hLBH was subcloned into the XhoI and HindIII

sites of pEGFP-N1 vector in-frame with the AAG instead

of the TAG stop codon at the end of hLBH coding se-

quence. (ii) Construction of pCMV-BD-hLBH-AS1

(pGAL4-hLBH, amino acids 1–105), and two deletion

mutations: pCMV-BD-hLBH-AS2 (amino acids 1–58 with

proline-dependent serine/threonine kinase group) and

pCMV-BD-hLBH-AS3 (amino acids 54–105 with ERK

D-domain motif): the DNA fragment containing the

whole coding region of hLBH was amplified with primers

P1 and P2 containing EcoRI and SalI sites, respectively

(Table 1). The amplified DNA fragment was cloned into

pMD18-T-vector, and then the EcoRI and SalI fragment

of hLBH was subcloned into a pCMV-BD expression

vector that contained the GAL4 DNA-binding domain

(DBD) to create a fusion protein between GAL4 DBD

and hLBH ORF. The fragments of AS2 and AS3 were

amplified separately by PCR from the pMD18T-hLBH

plasmid with two pairs of primers P1 and P4, P3 and P2.

Subsequently, EcoRI and SalI fragments were subcloned

in-frame into expression vector pCMV-BD to construct

pCMV-BD-hLBH-AS2, pCMV-BD-hLBH-AS3. (iii)

Table 1 PCR primers,

programs and cyclesPrimers Nucleotide sequences Programs Cycles

POUT1 5¢-GAGTTGTGTCCACCTTGCCGAC-3¢ 94�C, 30 s; 32

POUT2 5¢-AGGAGGAGGCCAGATAGGAGCT-3¢ 54�C, 30 s;

72�C, 2 min 30 s

PIN3 5¢-GGGACGCAGGGACCGTTTTTAA-3¢ 94�C, 30 s; 30

PIN4 5¢-GGCCAGATAGGAGCTAAGCCAT-3¢ 56�C, 30 s;

72�C, 2 min

PORF1 5¢-CCTCGAGCTTCATGTCTATATATTTCCC-3¢ 94�C, 30 s; 30

PORF2 5¢-GGA AGCTTCTGCTCTTTATTTTCTTTCG-3¢ 56.5�C, 30 s;

72�C, 1 min 30 s

PS 5¢-AGATACCCCACCAAACCCA-3¢ 94�C, 30 s; 32

PAS 5¢-GGGGCAGTGAATGGGGAAA-3¢ 51�C, 30 s;

72�C, 1 min 30 s

P1 5¢-CCCGAATTCTTCATGTCTATATATTTCC-3¢ 94�C, 30 s; 30

P2 5¢-GGAAGCTTCTGCTCTTTATTTTCTTTCG-3¢ 58�C, 30 s;

72�C, 1 min

P1 5¢-CCCGAATTCTTCATGTCTATATATTTCC-3¢ 94�C, 30 s; 30

P4 5¢-CCTAAGCTTAGCAGCAGCGGTCAAAATC-3¢ 58�C, 30 s;

72�C, 1 min

P3 5¢-GATGAATTCTCCTACCAGATCTTCCCAG-3¢ 94�C, 30 s; 30

P2 5¢-GGAAGCTTCTGCTCTTTATTTTCTTTCG-3¢ 58�C, 30 s;

72�C, 45 s

Mol Biol Rep (2008) 35:179–187 181

123

Construction of pCMV-Tag2B-hLBH: the expression

plasmid for FLAG epitopetagged hLBH, pCMV-Tag2B-

hLBH, was constructed by inserting hLBH ORF down-

stream of the FLAG epitope sequence (MDYKDDDDK)

in a pCMV-Tag2B expression vector.

Cell culture, transient transfection, and subcellular

localization analysis

COS-7 cells were maintained and passaged according to

standard methods in Dulbecco’s Modified Eagle Medium

(DMEM, Gibco BRL) supplemented with 10% fetal calf

serum (FCS) in an humidified atmosphere of 95% air and

5% CO2. Cells were transfected with pEGFP-N1-hLBH

using LipofectAMINE (Invitrogen) according to the

method described previously [12]. Forty-eight hours after

transfection, cells were fixed with 4% paraformaldehyde

for 15 min and washed with PBS three times and nuclei

were stained with 4¢, 6¢-diamidino-2-phenylindole hydro-

chloride (DAPI). Subcellular localization of the EGFP-

hLBH fusion protein was detected using an inverted fluo-

rescence microscopy (Nikon, E400).

Fig. 1 Nucleotide sequence

and deduced protein sequence

of the hLBH gene. hLBHencodes a polypeptide of 105

amino acids. Both nucleotides

and amino acids are numbered

at the left and right side of each

line, respectively. The initiation

ATG and TGA stop codons are

boxed. The putative

polyadenylation signal sequence

aataaa is underlined and in gray.

A proline-dependent serine/

threonine kinase group (PMEE

IGLSPRKDGLS) and an ERK

D-domain (LKDR

LPSIVVEPTEG) were

underlined

182 Mol Biol Rep (2008) 35:179–187

123

Transient expression reporter gene assay and deletion

analysis

To understand potential transcriptional activity of hLBH,

the pCMV-BD-hLBH-AS1, pCMV-BD-hLBH-AS2,

pCMV-BD-hLBH-AS3 and pCMV-BD were transiently

co-transfected into COS-7 cells along with the pL8G5-Luc

reporter and pLexA-VP16 using LipofectAMINE as de-

scribed above [12]. Forty-eight hours later, the luciferase

activity assay was performed according to the protocols of

Stratagene [12]. The luciferase activity was normalized for

transfection efficiency by co-transfection with pCMV-lacZ

and spectrophotometry analysis. The data are the mean of

three repeats in a single transfection experiment after

normalization for b-galactosidease activity. Each experi-

ment was repeated at least three times.

Luciferase was assayed in COS-7 cells by cotransfecting

pCMV-Tag2B-hLBH, and the luciferase reporter plasmid

pAP-1-Luc (or pSRE-Luc). Forty-eight hours later, lucif-

erase activity was measured [12]. Each experiment was

performed in triplicate and each assay was repeated at least

three times. The means of the data from three individual

transfected wells are presented after normalization for b-

galactosidase activity.

Results and discussion

Molecular characterization and evolutionary

conservation of the hLBH gene

The mouse Lbh regulates cardiac gene expression by

modulating the combinatorial activities of key cardiac

transcription factors, as well as their individual functions in

cardiogenesis [5]. To specifically study the function of the

human homolog of mouse Lbh, hLBH, in heart develop-

ment and cardiogenesis, we cloned the hLBH gene through

combined BLAST search and PCRs analysis as previously

Table 2 Genomic structure of the hLBH gene

Extron number Extron size (bp) Splice donor site Intron size (bp) Splice acceptor site

I 211 gtgagtaccctgcg 1340 gggtttcttggcag

II 103 gtgagtaagtcctg 22925 tgcttttctttcag

III 2591 ttccactctggtct

Summary of the size of the extrons and introns of the hLBH gene based on comparison of the cDNA and the genomic sequence. Invariant

nucleotides (gt/ag) are in boldface type

Fig. 2 hLBH is conserved

during evolution. (A) The

phylogenetic tree analysis of

hLBH proteins and its

homologs. The amino acid

sequences of H. sapiens hLBH

and its homologues in Pongopygmaeus (CAH90344), Musmusculus (NP_084275.3),

Gallus gallus (XP-419367.1),

Tetraodon nigroviridis(CAG10379), Danio rerio(NP_956814.1) are used in

analysis. (B) Comparison of the

amino acid sequences of hLBH,

mouse LBH and Danio reriovXcl2. Residues that are highly

conserved among these

sequences are indicated within

the top majority sequence. The

identical amino acid residues

are boxed and shaded in dark

Mol Biol Rep (2008) 35:179–187 183

123

described [10]. We screened the human EST database with

the nucleotide sequence of the mouse LBH gene. To confirm

the cDNA sequences identified from the database, two pairs

of primers (POUT1/POUT2 and PIN3/PIN4, Table 1) based

on the sequences of four overlapping ESTs (AL530914,

BM472620, BU191949 and BF718797) were used to carry

out standard PCR using the human embryonic heart cDNA

library as template. A PCR product fragment of 2608 bp was

obtained and confirmed to be the cDNA sequences of hLBH.

To obtain the full-length of cDNA, 5¢-RACE were performed

using 5¢-gene specific primers (PS and PAS) as described

previously [9, 11]. The procedure yielded a 216 bp DNA

fragment for 5¢-RACE. The products were then cloned and

sequenced. A sequence of 2927 bp constituting the full-

length novel gene was assembled, which was named hLBH

as approved by the HUGO Nomenclature Committee. The

nucleotide sequence reported here is available in GenBank

with accession number EF025586.

The hLBH gene consists of an ORF of 318 bp long

extending from the first ATG codon at nucleotide 186 to a

TGA stop codon at 503, a 185 bp 5¢-untranslated region

(UTR), and a 2424 bp 3¢-UTR with a consensus poly-

adenylation signal (aataaa) (Fig. 1). The deduced hLBH

protein has 105 amino acids (Fig. 1) with a calculated

molecular mass of approximately 16.2 kDa. Comparison of

the hLBH sequence with the genomic sequence shows that

hLBH is mapped to chromosome 2p23 and spans approx-

imately 28.5 kb on the genome. Interestingly, chromo-

somal band 2p23 is frequently triplicated in partial trisomy

2p syndrome and has been related to CHD associated with

this syndrome [5].

The hLBH gene consists of three exons and two introns.

The exon–intron boundaries conform to the consensus

splicing signals, where there are a gt and an ag dinucleo-

tide at the 5¢-donor and 3¢-acceptor site, respectively (Ta-

ble 2).

We then identified the homologues of Homo sapiens

hLBH in Pongo pygmaeus (CAH90344), Mus musculus

(NP_084275.3), Gallus gallus (XP-419367.1), Tetraodon

nigroviridis (CAG10379), Danio rerio (NP_956814.1).

The amino acid sequence of hLBH was aligned with its

homologues and an evolutionary relationship among these

proteins was examined using phylogenetic tree analysis.

hLBH is one of the conserved proteins during evolution

across different species (Fig. 2A) and hLBH protein shares

more than 95% amino acid sequence identity with mouse

Lbh protein (Fig. 2B).

Analysis of hLBH protein using the Motif Scan program

(http://scansite.mit.edu) indicates that the protein product

of hLBH contains a proline-dependent serine/threonine

kinase group (amino acids 27–41, PMEE IGLSPRKDGLS)

and an ERK D-domain (amino acids 57–71, LKDR

LPSIVVEPTE G) (Fig. 1).

hLBH is strongly expressed in heart at embryonic and

adult stages

To characterize the transcript size and expression patterns

of hLBH, adult and fetal multiple tissue northern blot were

performed using hLBH cDNA as the probe. A 2.9 kb

transcript of hLBH was most abundantly detected in human

adult heart with varying low level of expression in pla-

centa, lung, skeletal muscle, kidney and liver (Fig. 3A).

hLBH was detected mainly in heart tissues of both 25 and

17 weeks human embryo (Fig. 3B and C). The results

suggest that hLBH protein plays a role in human embryo

heart development.

Subcellular localization of hLBH protein

We examined the subcellular localization of hLBH to

determine whether it could function as a transcriptional

regulator. pEGFP-N1-hLBH was transfected into COS-7

Fig. 3 Expression of hLBH in human adult and embryonic tissues

analyzed using Northern blot. A commercially available Northern blot

membrane containing multiple human adult tissues and two

membranes containing multiple human embryonic tissues were

hybridized with a-32P random-labeled hLBH cDNA probe. b-actin

was used as a control for the amount of loaded mRNA (total RNA). A

transcript of ~2.9 kb specific for hLBH was detected in adult (A) and

embryo membranes of 25 weeks (B) and 17 weeks (C)

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cells, and 48 h after the transfection, the cells were visu-

alized with epifluorence microscope after being labeled

with DAPI to stain nuclei. EGFP-hLBH protein expression

was detected in both the nucleus and the cytoplasm

(Fig. 4A). The combined image (Fig. 4C) shows a nuclear

and cytoplasmic localization of hLBH protein in the COS-7

cells.

hLBH acts as an active transcription regulator

We studied hLBH transcriptional activity using a Gal4

reporter assay [12]. The pCMV-BD-hLBH encoding a fu-

sion protein of hLBH with the DNA binding domain (BD)

of yeast transcription factor GAL4 under the control of a

CMV promoter was constructed to examine a potential

function for hLBH in transcriptional activation. We used a

system involving co-transfection of the COS-7 cells with

pCMV-BD-hLBH and pL8G5-Luc, the latter contains eight

copies of the LexA DNA binding sites and five copies of

the Gal4 DNA binding sites linked to the luciferase re-

porter gene. GAL4-hLBH fusion protein strongly enhanced

the LexA-VP16-luciferase activity by 16.7-fold (Fig. 5A),

suggesting that hLBH is a potent transcriptional activator.

To further identify potential transcriptional regulatory

domains in hLBH, we then constructed two truncated

hLBH plasmids, pCMV-BD-hLBH-AS2 (amino acids 1–58

with proline-dependent serine/threonine kinase group) and

pCMV-BD-hLBH-AS3 (amino acids 54–105 with an ERK

D-domain region) to carry out deletion assay. As shown in

Fig. 5B, pCMV-BD-hLBH-AS2 increased the luciferase

activity of reporter gene by 5.4-fold and pCMV-BD-

hLBH-AS3 by 6.0-fold. These results indicate that both the

Fig. 4 Cellular localization of

hLBH proteins in COS-7 cells.

(A) EGFP-N1-hLBH is

localized in both the nucleus

and the cytoplasm when

transfected into the COS-7 cells.

(B) The nucleus of cells stained

with DAPI. (C) The combined

image of (A) and (B)

Fig. 5 hLBH is an active

regulator in transcription. (A)

pCMV-BD-hLBH is transiently

transfected into COS-7 cells

along with the pL8G5-Luc

reporter and pLexA-VP16. (B)

pCMV-BD-hLBH-AS1, pCMV-

BD-hLBH-AS2, and pCMV-

BD-hLBH-AS3 are transiently

transfected into COS-7 cells

along with the pL8G5-Luc

reporter and pLexA-VP16. The

data are the mean of three

repeats in a single transfection

experiment after normalization

for b-galactosidease activity.

Each experiment was repeated

at least three times

Mol Biol Rep (2008) 35:179–187 185

123

proline-dependent serine/threonine kinase group at N-ter-

minal and the ERK D-domain region at C-terminal con-

tribute to the transcriptional activation of hLBH.

hLBH activates SRE and AP-1-mediated transcriptional

activation

To investigate the role of hLBH proteins, we examined

whether hLBH was directly or indirectly involved in the

regulation of transcription factors. MAPK signal trans-

duction pathways are the most widespread mechanisms of

eukaryotic cell regulation [13]. To examine the effect of

hLBH on this specific cell-signaling pathway, we per-

formed pathway-specific reporter gene assays to measure

the transcription of AP-1 and SRE by hLBH in the COS-7

cells. Firstly, using pAP-1-Luc designed for monitoring

induction of AP-1, we tested the effect of hLBH on the

transcriptional activity of AP-1, a member of the ternary

complex. Expression of hLBH significantly increased AP-1

transcriptional activity by approximately 5.6-fold

(Fig. 6A). Furthermore, using SRE-luciferase reporter de-

signed for monitoring the induction of SRE, we demon-

strated that hLBH strongly increased SRE transcriptional

activity by 6.5-fold (Fig. 6B).

Using transient transfection and reporter assays, we

have shown that hLBH has a transcriptional activation

function and activates transcription of SRE and AP-1. One

possible scenario is that hLBH proteins act in synergy

with other transcription factors to integrate information

from multiple extracellular signals and, in turn, to induce

the necessary cellular changes required for tissue specifi-

cation and morphogenesis. MAPK pathways are critical

not only to the response of cardiovascular cells to extra-

cellular stress but also to developmental cues that regulate

cardiovascular development. MAPK superfamily is com-

posed of several subfamilies including the extracellular

signal-regulated kinase (ERK), JNK/SAPK, p38 and

ERK5 [14–18]. ERK are important signaling proteins that

phosphorylate serine or threonine followed by proline (S/

T) P sites in many different protein substrates [19]. hLBH

proteins contain consensus ERK D-domain motifs in its C-

terminal part, representing a core-binding site. ERK

binding to substrate proteins is mediated by docking sites

including the FXFP motif and the D-domain [20]. In

general, the typical D domain sequences contain a (L/I) ·(L/I) motif located 3–5 amino acids downstream from a

region containing several basic residues. The central core

(L/I) · (L/I) motif clearly plays a major role in kinase

targeting to transcription factors [21]. The effects of D-

domain combine with the effects of the local environment

of (S/T) P sites to generate a preference hierarchy.

Therefore D-domain of proteins mediates phosphorylation

by ERK and thus regulates MAPK signaling [22]. hLBH

proteins contain consensus ERK D-domain motifs in its C-

terminal part, representing a core-binding site. Our results

show that hLBH has a transcriptional activation function

and activates transcriptional activities of SRE and AP-1,

suggesting the C-terminal containing ERK D-domain

motif of hLBH may play an important role in transcrip-

tional activation. Therefore, hLBH proteins might play a

role in MAPK signaling pathway through the consensus

D-domain motifs.

In summary, we have cloned and characterized the

hLBH gene/protein. hLBH is most abundantly expressed in

embryonic and adult heart. Overexpression of hLBH acti-

vates the transcriptional activities of AP-1 and SRE, which

are potential targets of ERK, JNK, and p38 in cellular

signaling and functions. These results suggest that hLBH

may have a potential role in regulating heart development

processes and heart disease. Further functional studies will

Fig. 6 Overexpression of hLBH promotes transcriptional activities of

SRE and AP-1. (A) Activation of AP-1-Luc transcriptional activity by

the overexpressing of hLBH. (B) Activation of SRE-Luc transcrip-

tional activity by the overexpressing of hLBH. COS-7 cells

transfected with individual reporter plasmid and the corresponding

plasmids shown in the figures. Forty-eight hours later, the luciferase

activity assay was performed. The data are the mean of three repeats

in a single transfection experiment after normalization for b-

galactosidease activity. Each experiment was performed at least three

times

186 Mol Biol Rep (2008) 35:179–187

123

elucidate the roles of hLBH proteins in development and

identify the signals to which they respond.

Acknowledgments We are grateful to all members of the Center for

Heart Development, College of Life Sciences in Hunan Normal

University for their excellent technical assistance and encouragement.

This study was supported in part by the National Natural Science

Foundation of China (No. 90508004, 30470867, 30570934,

30571048), PCSIRT of Education Ministry of China (IRT0445),

National Basic Research Program of China (2005CB522505), the

Foundation of Hunan Province (No. 05FJ2007, 06JJ4120), and Sci-

entific and Research Fund of Hunan Provincial Education Department

(04C327).

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