Cloning of porcine chemerin, ChemR23 and GPR1 and their involvement in regulation of lipogenesis.

Abstract

Chemerin is a novel adipokine which is abundant in adipose tissue to promote adipocyte differentiation and with significant relativity to BMI and insulin sensitivity. We report here the molecular characterization of porcine chemerin and its receptors ChemR23 and GPR1, as well as their transcriptional regulation during lipogenesis. Chemerin was mainly expressed in liver, intestine, kidney and adipose tissue, consistent with the expression pattern of GPR1, but not ChemR23, which was predominantly present in spleen and temperately in adipose tissue. We further investigated the lipogenesis-related transcriptional activation of PPAR/ and KLF15 on chemerin and its receptors. The data showed that KLF15, but not PPAR/, can up-regulate the mRNA level of chemerin, ChemR23 and GPR1, which was consistent with the results of luciferase assay that confirmed the effect of KLF15 on ChemR23 promoter. Taken together, our data provide basic molecular information for the further investigation on the function of chemerin in lipogenesis.

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*Corresponding author. Tel: 86-27-87282669; Fax: 86-27-87287376;
E-mail: yangzq@mail.hzau.edu.cn
This paper is an article on the field of obesity research.
Received 22 March 2010, Accepted 14 June 2010
Keywords: Chemerin, Chemerin receptors, Lipogenesis, Porcine,
Transcriptional regulation
Cloning of porcine chemerin, ChemR23 and GPR1 and their
involvement in regulation of lipogenesis
Jianfeng Huang, Jian Zhang, Ting Lei, Xiaodong Chen, Yan Zhang, Lulu Zhou, An Yu, Zhilong Chen, Ronghua Zhou &
Zaiqing Yang*
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Life Science and
Technology, Huazhong Agricultural University, Wuhan, 430070, P. R. China
Chemerin is a novel adipokine which is abundant in adipose
tissue to promote adipocyte differentiation and with significant
relativity to BMI and insulin sensitivity. We report here the
molecular characterization of porcine chemerin and its re-
ceptors ChemR23 and GPR1, as well as their transcriptional
regulation during lipogenesis. Chemerin was mainly expressed
in liver, intestine, kidney and adipose tissue, consistent with
the expression pattern of GPR1, but not ChemR23, which was
predominantly present in spleen and temperately in adipose
tissue. We further investigated the lipogenesis-related tran-
scriptional activation of PPARγ γ and KLF15 on chemerin and its
receptors. The data showed that KLF15, but not PPARγ γ, can
up-regulate the mRNA level of chemerin, ChemR23 and
GPR1, which was consistent with the results of luciferase assay
that confirmed the effect of KLF15 on ChemR23 promoter.
Taken together, our data provide basic molecular information
for the further investigation on the function of chemerin in
lipogenesis. [BMB reports 2010; 43(7): 491-498]
INTRODUCTION
Obesity is a new emerging threat to human health, leading to
the increasing morbility and mortality of numbers of health
problems which contribute to the development of insulin re-
sistance, type 2 diabetes, atherosclerosis, hypertension and fat-
ty liver disease (1). Although normal fat mass is crucial for the
maintenance of systemic glucose and lipid homeostasis, ex-
cessive expansion of adipose tissue and increasing of mature
adipocytes will lead to obesity. Adipose tissue is also regarded
as an active endocrine organ and secrets various bioactive cy-
tokines termed adipokine. These factors including leptin and
adiponectin often have multiple functions and take part in di-
verse fields such as glucose/lipid metabolism, insulin action,
inflammation and appetite (2).
Chemerin was originally named as TIG2 or RARRES2 in skin
rafts (3) and then well known as a chemoattractant capable of
recruiting the immune cells which express its specific receptor
ChemR23 (i.e. NK cells, macrophages, pDCs) to the in-
flammatory sites (4, 5). Chemerin is secreted into the blood
circulation as prochemerin with little biological activity. Under
certain conditions such as coagulation, fibrinolytic, inflamma-
tory and complement cascades, prochemerin could be con-
verted into active form by serine proteases existing in the sur-
rounding microenvironment (6, 7). Prochemerin and active
chemerin may be disabled by other diverse serine proteases (8)
or processed by cysteine proteases into an anti-inflammatory
form that inhibit the expression of proinflammatory cytokines
stimulated by LPS in a ChemR23-dependent manner (9).
Several recent studies have highlighted the involvement of
chemerin in obesity, inflammation and metabolic syndromes
(10-12). Chemerin and ChemR23 are up-regulated during adi-
pocyte differentiation (13) and chemerin deficiency impairs
this process and reduces accumulation of lipid-droplet (14).
Chemerin can either activate insulin signaling through enhanc-
ing glucose uptake in 3T3-L1 adipocytes (15), or induce in-
sulin resistance in human skeletal muscle cells (16). Chemerin
may also be a potential link between steatosis and infla-
mmatory reaction in adipose tissue. Therefore growing re-
search interests are focused on the function of chemerin in glu-
cose/lipid metabolism in insulin target organs such as liver,
muscle and adipose tissue.
Besides ChemR23, orphan receptor GPR1 and CCRL2 have
been identified by in vitro assays as spare receptors for chem-
erin (17, 18). However, only ChemR23 and GPR1 are capable
to support chemerin-derived signal transduction. Because pigs
are regarded as an ideal model for obesity research and chem-
erin as a novel adipokine, we analyzed porcine chemerin and
its receptors using bioinformatics and investigated the sim-
ilarity between ChemR23 and GPR1 as well as the regulation
of their expression in lipogenesis.

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Fig. 1. Amino acid sequence alignment of ChemR23 and GPR1.
(A) Sequences from human, pig, mouse and cow were aligned by
ClustalW. CY/FNNF conservative sequences and G protein inter-
action motifs were boxed and labeled. Putative transmembrane
domains were shaded in grey. (B) Percentage of putative CY/FNNF
motif in vertebrate ChemR23 and GPR1 (accession No. are listed
in Supplement Table 4). The abbreviated species names are: Homo
sapiens (Hs); Mus musculus (Mm); Bos taurus (Bt); Sus scrofa (Ss).
RESULTS
Cloning and characterization of porcine chemerin, ChemR23
and GPR1
The porcine chemerin cDNA (Genbank acc. No. EU660865,
592 bp) contained a 35 bp 5’-UTR, a 65 bp 3'-UTR and 492
bp of CDS encoding 163 amino acids with a calculated molec-
ular weight of 18,698 Da. The coding sequence of porcine
chemerin shared 87.7% and 72.3% similarity with its human
and mouse homologs, respectively, across their entire length,
with a even higher similarity (95.2% and 81.0% to human and
mouse, respectively) in the last 21 amino acids (R140-L160)
which forms the C-terminus functional region. Chemerin com-
prises of a disordered C-terminus tail, an antiparallel β-pleated
sheet wrapping an N-terminus α-helix, is previously described
to fold as “cystatin” but in reverse orientation compared to
chemokine (19). Similar to chemokine, the basic amino acid
region (H38-I119) between the N-terminus α-helix and C-termi-
nus tail in porcine chemerin, which pI value is 9.99, is thought
to interact with sulphated glycosaminoglycan (GAG) as a che-
mokine (20) and the N-terminus of ChemR23 with pI 3.66.
However, the disordered C-terminus is diphase with pI 4.35
for G142-F157, and pI 8.75 for the last six (K158-S163) which
seems to partially explain the inhibitory action of these six
amino acids.
The uninterrupted CDS of ChemR23 (EU660866.2) covered
1092 bp and encoded 363 amino acid, showing 90.8% and
87.1% similarity with their respective human and mouse
homologs. Porcine GPR1 (FJ234899) spanned 1068 bp and en-
coded 355 amino acids, which shares 93.5% and 87.6% sim-
ilarity with human and mouse GPR1. The similarity between
ChemR23 and GPR1 was further explored because of the high
capability of GPR1 to capture the ligand chemerin. High sim-
ilarity between ChemR23 and GPR1 at amino acid level was
found in six typical vertebrates (Supplement Table 1). Bioinfor-
matic analysis also showed that ChemR23 and GPR1 have the
corresponding transmembrane domains (Fig. 1) and hydro-
philicity/hydrophobicity distribution in extracellular loop 2
and 3 (Fig. 2A, B). Consistent with these findings, the deduced
three dimensional structures of porcine ChemR23 and GPR1
had remarkably similar structures from TM1 to TM7 (Fig. 2C).
In the phylogenetic tree (Fig. 2D) constructed from ChemR23
and GPR1 amino acid sequences of various species, the top-
ology for the vertebrate ChemR23 and GPR1 was well re-
solved, suggesting a paralogous pattern of gene divergence,
i.e., evolution by gene duplication. Meanwhile, the horizon-
tally symmetrical phylogenetic tree suggested that ChemR23
and GPR1 have similar evolutional patterns since the emer-
gence of vertebrates.
Genomic structure and chromosomal mapping of porcine
chemerin and ChemR23
Based on the comparison of their genomic and cDNA se-
quences, the structure of porcine chemerin (Genbank acc. No.
GQ888511) and ChemR23 (EU660866.2) were deduced (Su-
pplement Table 2). Chemerin covered approximately 3 kb and

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Fig. 2. Analyses of the similarities between porcine ChemR23 and GPR1. Hydrophobicity oscillograms of extracellular region 2 (A) and ex-
tracellular region 3 (B) were depicted using Protscale with the option Hphob./Kyte & Doolittle. (C) Comparison of the three dimensional
structures of porcine ChemR23 and GPR1. On the left is ChemR23 (residues D29 to F349) and right is GPR1 (residues S23 to N342).
Both models were constructed based on template-based homology modeling. (D) The evolutionary relationship between ChemR23 and GPR1
among different species. The phylogenetic tree was constructed from the amino acid sequences by the neighbor-joining method using
MEGA4.0. Bootstrap values at each node represent the percentage of 100 replicates. The length of branch is proportional to the degree of
divergence and Poisson correction was applied for multiple substitutions.
comprised six exons, the open reading frame was encoded by
exon 2-5. ChemR23 spanned over 37Kb with four exons, and
the CDS was located within the last exon. All splicing sites of
exons/introns were conformed to the GT/AG rule.
Using Minnesota porcine Radiation Hybrid (IMpRH) panels,
porcine chemerin was localized to 18q24 at a distance of
0.26cR from the most significantly linked marker SWR169
(LOD=13.91). Porcine ChemR23 and GPR1 were assigned to
14q21.1 and 15q24.6, respectively, based on the annotations
of HTGs (CU468889 for ChemR23 and CU672272 for GPR1).
The corresponding chromosome regions of chemerin, ChemR23
and GPR1 in human were 7q36.1, 12q24.1 and 2q33.3,
respectively. The chromosomal locations of these three genes
were syntenic between pigs and human.
Tissue distribution of chemerin, ChemR23 and GPR1 in pigs
The expression level of porcine chemerin, ChemR23 and GPR1
in various tissues was quantitated using real-time PCR. Similar
to the expression pattern in rodents, chemerin was abundantly
present in liver, adipose tissue and kidney, but undetectable in
muscle. Compared to that in rodents, the expression level of
chemerin in adipose tissue is relatively lower in pigs (Fig. 3A)
(10).
ChemR23 was expressed mainly in spleen, temperately in
subcutaneous WAT and epididymal WAT, and less in stomach
and lung. Conversely, lung is an importantly resource for

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Fig. 3. Tissue distribution of porcine chemerin, ChemR23 and
GPR1. The mRNA expression levels of chemerin (A) and ChemR23
and GPR1 (B) were quantitated in various tissues by qRT-PCR
and normalized using GAPDH. Data were presented mean ±
SEM (n = 3).
ChemR23 in rodents. As to porcine GPR1, abundant expre-
ssion was detected in kidney, WAT, spleen and liver, similar
to the expression profile of chemerin (Fig. 3B). It should be
noted that chemerin and its two receptors were both expressed
substantially in WAT, which is in line with the important role
of chemerin in adipogenesis.
Transcriptional regulation of chemerin, ChemR23 and GPR1
during lipogenesis
KLF15 and PPARγ are the key transcriptional factors regulating
lipogenesis. The lipid accumulation was prompted in porcine
kidney cell line IBRS-2 cells overexpressing KLF15 (Fig. 4A).
The mRNA levels of chemerin, ChemR23 and GPR1 were also
up-regulated in IBRS-2 cells overexpressing KLF15, whereas
PPARγ overexpression had no effects on the transcription of
both chemerin and its receptors (Fig. 4B, C).
To further understand the transcriptional regulation of ChemR23,
the 5'-flanking region of porcine ChemR23 was amplified ac-
cording to the sequence available at HTGS database (Genbank
acc. No. CU468889). Transcription start site (TSS) was identi-
fied 254 bp upstream from intron1. The region from -1,269 to
+54 (relative to TSS), which was similar to the human/mouse
homologs without canonical TATA box or CAAT sequence
(21), was found to contain two putative PPAR binding motifs
and three KLF15 consensus binding motifs (CG/TCCCC) (22)
(Fig. 4D). This region was cloned into pGL3 vector and trans-
fected into NIH-3T3. Luciferase assay showed that KLF15, but
not PPARγ, enhances the proximate promoter activity by 2.8
folds (Fig. 4E). Even in the presence of 100 nM rosiglitazone,
the promoter activity of ChemR23 was not changed by PPARγ
significantly in IBRS-2 (Fig. 4F). However, neither KLF15 nor
PPARγ regulated the proximate promoter activity of mouse
GPR1 (Supplement Fig. 1).
DISCUSSION
Previous studies on chemerin and ChemR23 mainly used ro-
dent animal models and little was known about the molecular
characteristics of chemerin, ChemR23 and GPR1 in higher
mammals such as pigs. In the current study, chemerin, ChemR23
and GPR1 were characterized in pigs. The deduced amino
acid sequences of porcine chemerin, ChemR23 and GPR1
showed high similarities with their corresponding homologs in
other species. The “cystatin” protein fold pattern and two short
peptides named chemerin-9 and C15 existed within C-termi-
nus functional region were identified in porcine chemerin,
which are conserved in mammalian chemerin and speculated
to be potential for the combination and activation toward
ChemR23 (9, 23). Interestingly, porcine ChemR23 and GPR1
shared corresponding transmembrane domains, conserved
C(Y/F)NNF motif, similar hydrophilicity/hydrophobicity dis-
tribution in the extracellular loop 2 and 3 and nearly identical
three dimensional structures. Among various species ranging
from lower (fish) to higher vertebrate (human), ChemR23 and
GPR1 shared relatively high similarity and a similar unin-
terrupted coding region structure. Previous study showed that
GPR1 displays more potential affinity to chemerin than its
unique functional receptor ChemR23 (18), the similar protein
and genomic structures and high genetic relationship between
ChemR23 and GPR1 could partially explain the underlying
molecular mechanism.
Porcine chemerin was detected predominantly in liver,
moderately in WAT and kidney, which is similar to that in ro-
dents except that, compared to rodents, chemerin expression
level in porcine liver is higher. Porcine ChemR23 was ex-
pressed mainly in spleen and temperately in WAT, and at very
low level in lung, whereas rodent ChemR23 was most abun-
dantly present in lung and WAT (10, 13). Porcine GPR1 was
mainly expressed in kidney, WAT, spleen and liver. Since por-
cine chemerin and its receptors were uniformly at high level in

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Fig. 4. Regulation of chemerin, ChemR23 and GPR1 by KLF15 and PPARγ. (A) Red O oil staining was used to evaluate lipid content in
IBRS-2 (×200 magnification). qPCR was conducted to measure the relative mRNA expression of chemerin (B) and semi-qPCR to measure
ChemR23 and GPR1 (C). (D) The structure of ChemR23 proximate promoter (from −1,269 to +54). Transcription start site (TSS) was pre-
dicted by Promoter 2.0. The putative transcription factor-binding sites were underlined, and the core binding sites were shown in rec-
tangular boxes. The putative KLF15 binding motifs similar to consensus “CG/TCCCC” were showed in gray boxes. Luciferase assays of por-
cine ChemR23 in NIH3T3 (E) and IBRS-2 (F). The treated groups were cotransfected with pGL3-pChemR23 and pcDNA-PPARγ or pcDNA-
KLF15 respectively and compared to the control group. Cells were added with 100 nM rosiglitazone (RSG) after transfection for 16 h and
further incubated for 8 h. The data were represented as mean ± SEM (n = 3). The differences between groups were analyzed by un-
paired student’s t-test. *P＜0.05, **P＜0.01.
WAT, a storage depot for serum lipid, as well as the abun-
dance of chemerin and GPR1 in liver, we proposed that chem-
erin may be involved in the regulation of adipogenesis and
hepatic lipid metabolism (unpublished data).
Due to the high level of GPR1 in both epididymal and sub-
cutaneous fat, we speculated that GPR1 could play a role in
lipid accumulation as ChemR23 do. Several conserved tran-
scriptional factor binding elements associated with lipo-
genesis, such as C/EBP, CREB, KLF and PPAR have been found
within the promoters of mouse and human chemR23 and
GPR1. A recent study on KLFs found that inhibition of KLF15
blocks 3T3-L1 differentiation and ectopic expression of KLF15
in NIH 3T3 or C2C12 cells triggers both lipid accumulation
and the expression of PPARγ in the presence of inducers of
adipocyte differentiation (24). In our experiment, overexpression
of KLF15 but not PPARγ in porcine kidney cell line IBRS-2 in-
creased the intracellular lipid content. KLF15 but not PPARγ
up-regulated the mRNA level of chemerin, ChemR23 and
GPR1. Luciferase assay further confirmed the proximate pro-
moter of porcine ChemR23 can be activated by KLF15, but not
by PPARγ, which means KLF15 may directly act on the two re-
ceptors without enhancing PPARγ activity in pigs. Previous
studies found up-regulation of chemerin and ChemR23 mRNA
levels are significantly enhanceed by troglitazone in 3T3-L1
differentiation (13) and rosiglitazone treatment or forced ex-
pression of PPARγ can induce chemerin expression in mouse
BMSCs during adipogenic differentiation (25), but the other
group showed that troglitazone inhibits chemerin secretion
from primary human muscle cells (16). The apparent contra-
dictory observations may be caused by the distinct regulation
of PPARγ on chemerin in different tissues. Meanwhile, the
transcriptional activation of both ChemR23 and GPR1 by
KLF15 further confirms the similarity between the two re-
ceptors and suggests the potential function of GPR1 in lipo-
genesis in preadipocyte.
In conclusion, the characterization of porcine chemerin,
ChemR23 and GPR1 would provide a basis for functional
study of chemerin in lipid metabolism via the new receptor
GPR1, which may contribute to the establishment of a pig
model for obesity research.