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Department of Surgery, University of Texas Medical Branch, Galveston, Texas, USA
1Correspondence: Department of Surgery, University of Texas Medical Branch, 301 University Blvd., Galveston, Texas 77555, USA. E-mail: ggreeley{at}utmb.edu
SPECIFIC AIMS
Apelin is the endogenous ligand for the APJ receptor. It exerts a broad range of activity including effects on heart contractility, blood pressure, appetite, pituitary, gut hormone, and gastric acid secretion.
To elucidate a molecular mechanism underlying increased breast apelin expression during pregnancy and lactation the 5'-upstream regions of the rat and human apelin genes were cloned and characterized. Putative binding sites for upstream stimulatory factor (USF) were identified. Our aim was to investigate the role of USF in the activation of breast apelin expression during pregnancy and lactation in rats.
PRINCIPAL FINDINGS
1. Cloning and characterization of the 5'-flanking regions of the rat and human apelin genes and apelin expression in cell lines
We cloned and verified sequences of the 
3000 and 4000 bp 5'-upstream fragments of the rat and human apelin genes. The rat and human apelin core promoters are –207/–1 and –100/+74 bp. Putative binding sites for the transcription factors AP1, AP2, STAT, SP-1, USF, and GKLF and AP1, USF, NF1, and AP4 were identified in the core promoters of the rat and human apelin genes. A CAAT-like, but not TATA, motif was identified.
Apelin expression levels in cultured cell lines derived from tissues that express apelin in the body were measured by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. The highest apelin mRNA levels were measured in human stomach (KATO III), human breast (Hs578T), and mouse hypothalamic (GT1–7) cells. Moderate apelin mRNA levels were measured in human colon (Caco-2) and in rat heart (H9C2) and lung (RFL6) cells. Low-apelin mRNA levels were measured in human placenta (BeWo) and liver (HepG2) cells. Rat promoter sequences showed the highest transcriptional activity in breast, liver, and colon cells, and human promoter sequences showed the highest activity in breast and liver cells.
2. Identification of putative USF-1/-2 binding sites in apelin core promoters; influence of USF overexpression and site-directed mutagenesis on promoter activity
A putative USF binding site is located at –114/–109 bp and at –84/–79 bp in the rat and human apelin promoters. The extents to which USF-1/-2 bind to these sites were examined by EMSAs using a breast cell nuclear extract. In the rat and human, a single DNA-protein complex was detected that was competed by an oligonucleotide containing the putative USF site and by a consensus USF oligonucleotide, but not by a mutated consensus USF oligonucleotide. Incubation with USF-1/-2 antibodies resulted in a supershift of the DNA-protein complexes.
In transient transfection experiments, USF-1 overexpression increased rat and human apelin core promoter activities 10- and 20-fold, USF-2 over-expression increased rat and human apelin core promoters activities 8- and 15-fold.
The extents to which putative USF sites influence basal apelin core promoter activity were assessed in transient transfection experiments using wild-type (WT) constructs (rat, –207/–1 bp; human, –100/+74 bp) or constructs with mutated USF sites. Mutation of USF sites decreased basal rat and human core promoter activities by 87 and 63%.
3. Evidence that endogenous USF is recruited to the putative USF site in the human apelin promoter in breast cells
ChIP assays were performed using breast cell (Hs578T) chromatin to investigate the extent to which endogenous USF binds to the putative USF binding site within the human apelin promoter. The 5'-upstream region containing the putative USF binding site was amplified by PCR of DNA precipitated by USF-1/-2 antibodies. Results indicate that USF-1/-2 are recruited to the human apelin promoter.
4. USF up-regulates breast apelin expression in vivo in the rat
Rat breast apelin expression increases dramatically during pregnancy and lactation (Fig. 1
A). Breast apelin expression was low in cyclic rats (Fig. 1A
, lanes 1, 6, 11) and increased 3.3- and 2.4-fold during early and late pregnancy, and 6.3- and 3.1-fold during early and late lactation (Fig. 1B
). EMSAs using nuclear extracts harvested from cyclic, timed-pregnant, or lactating rats showed that USF binding activity to a consensus USF oligonucleotide was highest during early and late pregnancy and early lactation (Fig. 1C
). USF binding activity decreased at late lactation, in concert with the reduction in breast apelin expression. Western blotting analysis (Fig. 1C
, lower part) confirmed consistent histone H3 levels in nuclear protein extracts indicating that changes in USF binding are not due to extraction differences.
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ChIP assays showed that breast DNA of 3-day lactating rats (Fig. 2
B, lanes 6–11) but not cyclic rats (Fig. 2B
, lanes 1–5) was amplified using primers specific for the putative USF binding site within the rat apelin proximal promoter. This agrees with and extends EMSA findings (Fig. 1)
and indicates that USF is involved in the up-regulation of breast apelin transcription. Control immunoprecipitations were done with a nonspecific IgG (Fig. 2D
). For a positive input control, cross-linked, and sheared genomic DNA from breast tissue served as a template (Fig. 2C
).
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CONCLUSIONS AND SIGNIFICANCE
Molecular regulation of apelin expression appears complex since stomach apelin expression is activated prenatally and declines after weaning, whereas lung apelin expression is marginal at birth and increases postnatally in rats. Breast apelin expression is negligible in the cyclic rat but increases dramatically during pregnancy and lactation.
To elucidate molecular mechanisms underlying regulation of apelin gene expression, we cloned the 5'-upstream regions of the rat and human apelin genes. Regulation of the apelin gene occurs partly at the transcriptional level since transcriptional activities of 5'-upstream fragments were highest in stomach, breast, heart, and lung cells, a finding that agrees with either apelin mRNA levels in tested cell lines, in the respective tissues in vivo, or both. The core promoters are –207/+1 and –100/+74 bp, for the rat and human apelin genes. In contrast to other gastrointestinal peptides [gastrin, ghrelin, cholecystokinin (CCK), glucagon, peptide YY, neurotensin] a TATA-box was not identified in the apelin promoters. The rat and human apelin core promoters are GC rich, many GC-rich promoters lack a typical TATA box. In some TATA-less promoters, GC-rich promoter regions contain SP1 binding sites. Putative SP1 binding sites were identified in the apelin core promoters. We also identified putative E-box DNA-binding sites in the apelin core promoters. USF-1/-2 are implicated in transcriptional activation of metabolic and breast tissue genes including the estrogen receptor 
(ER ) and the IGF2 receptor, and USF is important for lactation.
Evidence for functionality for the putative USF sites in regulation of apelin core promoter activity is given in overexpression and site-directed mutagenesis experiments. Furthermore, ChIP assays indicate that endogenous USF binds to the apelin promoter in breast cells in vitro. EMSAs show that binding of a consensus USF oligonucleotide to a breast nuclear extract is higher in pregnant and lactating rats compared with cyclic rats. This increase in binding activity correlates with increased breast apelin expression during pregnancy and lactation. Furthermore, in vivo ChIP assays indicate involvement of USF in regulation of breast apelin expression during lactation. Breast DNA from lactating but not cyclic rats is amplified by primers specific for the USF binding site in the proximal promoter region of the rat apelin gene. These findings and the USF overexpression and mutation data indicate that USF exerts a stimulatory role over breast apelin expression.
The core promoters show a uniformly high transcriptional activity in all cell lines examined. With the exception of breast (Hs578T) and colon (Caco-2) cells, the magnitudes of transcriptional activities for the rat core promoters and the larger 5'-upstream constructs are nearly equivalent, which suggests that cis-regulatory elements that control basal transcription are located primarily in the core promoter. In contrast, transcriptional activities of the larger human 5'-upstream constructs are greater than core promoter activity. Repressive elements are present in the distal, upstream region of the human apelin promoter since fragments upstream of –581/+74 bp showed a 50–60% reduced activity in liver, heart, lung, placental, and hypothalamic cells. Inhibitory elements in the distal upstream region for the rat apelin promoter were not evident.
In human liver (HepG2) and murine hypothalamic (GT1–7) cells, transcriptional activities of the rat and human 5'-upstream constructs did not correlate with expression of the endogenous gene in cell lineages or in vivo. One explanation for the low apelin promoter activity in hypothalamic cells is that the rat and human apelin promoter fragments are less active in murine-derived cells.
In summary, we have characterized the 5'-upstream regulatory regions of the rat and human apelin genes and demonstrated by transient transfection, mutagenesis, EMSA, and ChIP assays that USF is involved in up-regulation of breast apelin gene expression. More importantly, in vivo ChIP assays demonstrate that endogenous USF exerts a stimulatory role in the up-regulation of breast apelin expression during lactation and possibly pregnancy in the rat.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6315fje
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  S. Han, G. Wang, X. Qi, H. M. Lee, E. W. Englander, and G. H. Greeley Jr. A possible role for hypoxia-induced apelin expression in enteric cell proliferation Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2008; 294(6): R1832 - R1839. [Abstract] [Full Text] [PDF]
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P < 0.05 vs. 3 d lac; 
= P < 0.05 vs. 6 d preg, 3 d lac, and 16 d lac.
