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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 21, 2005 as doi:10.1096/fj.04-3619fje. |
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Institute of Biochemistry, Johannes Gutenberg-University Mainz, Mainz, Germany; and
* Institute of Human Genetics, Justus Liebig-University Giessen, Giessen, Germany
1Correspondence: Institute of Biochemistry, Johannes Gutenberg-University Mainz, Becherweg 30, D-55128 Mainz, Germany. E-mail: bio.chemie{at}uni-mainz.de
SPECIFIC AIMS
Increasing the expression of ADAM10 is a potential strategy to prevent Alzheimer disease. A specific aim was to identify the promoter of the ADAM10 gene and to characterize elements essential for strong promoter activity. We looked for an inducer of ADAM10 expression, analyzed SNPs located in the human ADAM10 promoter region, and describe the genomic organization of the human ADAM10 gene.
PRINCIPAL FINDINGS
1. The genomic organization and the promoter of human and mouse ADAM10 genes are highly conserved
By comparing ADAM10 cDNA sequences with genomic databases, we have delineated the genomic organization of human and mouse ADAM10 genes. Both gene loci share remarkable similarities: they comprise 
160 kbp and are composed of 16 exons. With exception of exon 16, which is 764 bp in length, all other exons of the human ADAM10 gene are relatively small, ranging from 93 to 499 bp. The intron sizes range between 482 and 35262 bp with the largest introns located primarily at the 5'-end of the human gene.
Within the first 500 bp upstream of either translation initiation site, human and mouse ADAM10 genes are evolutionarily highly conserved; each contains characteristic Sp1 sites and a CAAT box, but a TATA box is absent. The high degree of sequence identity and the presence of putative regulatory elements indicate that this region is essential for regulation of ADAM10 transcription.
2. Cloning and functional characterization of the human ADAM10 promoter
Within 2.2 kbp upstream of the human ADAM10 translation initiation site multiple putative transcription factor binding sites for Brn-2, SREBP, Oct-1, Creb1/cJun, USF, Maz, MZF-1, NF-
B, and tetinoid receptors are located. Therefore, we analyzed this region (from –2179 to –1) with regard to its participation in regulation of gene transcription. We cloned it from BAC clone RPCI 11 123C21 and placed a promoterless luciferase cDNA reporter sequence under control of this putative promoter element.
Because ADAM10 is known to be ubiquitously expressed, we examined whether this chimeric gene is translated in different cell lines. After transient transfection of HEK293, HepG2, SH-SY5Y, and IMR32 cells, which are representative for kidney, liver, and neural cells, strong luciferase gene expression was observed. Compared with the negative control, a promoterless luciferase reporter construct, a 68-fold increase in relative luciferase activity in SH-SY5Y cells, a 37- to 44-fold increase in HEK293 and IMR32, and a 20-fold increase in HepG2 cells was measured. This finding demonstrates that the region from –2179 to –1 upstream of the ADAM10 translation initiation site represents a functional promoter.
To identify the core promoter element essential for transcription of the human ADAM10 gene and identify regions involved in regulation of ADAM10 transcription, we generated different sequentially deleted 5'-flanking regions and cloned them into the promoterless firefly luciferase reporter plasmid pGL3-basic (Fig. 1
).
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After cotransfection of HEK293 cells with these deletion constructs and a renilla luciferase reporter plasmid, the induced firefly luciferase activity was determined relative to the renilla luciferase normalizing standard.
Our experiments reveal that neither strong positive nor negative regulatory elements are present in the region between –2093 and –555 and that the elements important for ADAM10 expression are downstream of –555 (Fig. 1)
.
The region essential for basal ADAM10 promoter activity was mapped by us to comprise nucleotides –508 to –300. A potential Maz binding site (–491), a CAAT-box (–480), a Sp1 binding site (–366), and an USF element (–317) are present and the removal of these putative transcription factor binding sites in the –300/–1 construct strongly decreased the promoter activity. Thus, the first 508 bp upstream of the translation initiation codon are necessary for basic ADAM10 promoter activity.
3. Sp1 sites and an USF in the 5'-UTR are essential for ADAM10 promoter function
Site-directed mutagenesis of the USF consensus sequence decreased ADAM10 promoter activity to 54%. Thus, the USF element plays a central role in ADAM10 transcription.
Site-directed mutagenesis within the CAAT box and Sp1 sites did not influence promoter activity. However, band shift assays and overexpression of human Sp1 site binding proteins in insect cells unambiguously showed that Sp1 and USp3 induce expression controlled by the ADAM10 promoter.
4. Retinoic acid increases human ADAM10 promoter-mediated gene expression
Identification of the ADAM10 promoter allowed us to generate a stable neural SH-SY5Y cell line expressing luciferase under control of the ADAM10 –2179/–1 region. This line was used for screening of luciferase expression inducers. We found that retinoic acid (RA) increases reporter gene expression by 50% (Fig. 2
A). Moreover, retinoic acid treatment of wild-type SH-SY5Y cells also increased the transcription of the endogenous ADAM10 gene to 250% (Fig. 2B
). By using EMSA we demonstrated that nuclear proteins are bound to one of two RA-responsive elements located in the ADAM10 promoter (Fig. 2C
).
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5. SNPs in the ADAM10 promoter are not necessarily associated with Alzheimer disease
We carried out genetic studies in 105 Alzheimer disease patients and 83 age-matched controls. Information about polymorphisms in the promoter region of ADAM10 was obtained from the NCBI single nucleotide polymorphisms (SNPs) database. For the present study, we selected SNPs at positions –279 and –630, which result in an A>G and in a G>T nucleotide exchange, respectively. We discovered by SSCP analysis and subsequent DNA sequencing two additional SNPs at positions –348 (C>T) and –927 (insertion of GAGA).
The ADAM10 SNP genotypes for Alzheimer disease patients and controls were in Hardy-Weinberg equilibrium. However, we observed no significant differences in genotype distribution between AD patients and control individuals.
CONCLUSIONS AND SIGNIFICANCE
Since increasing the amount of the 
-secretase ADAM10 has been shown to prevent amyloid pathology in an AD mouse model, up-regulation of this enzyme might be a valuable therapeutic approach. Therefore, strategies to increase transcription of the ADAM10 gene are the focus of our current work.
After identification and analysis of four SNPs in the ADAM10 promoter region, we observed no association between individuals suffering from AD and control subjects. Although these variations in the ADAM10 locus were not associated with AD, we cannot exclude the possibility that other SNPs in the ADAM10 locus are involved in the pathogenesis of Alzheimer disease.
We cloned a functional promoter of the human ADAM10 gene and detected the highest ADAM10 promoter activity in neural cells and reduced activities in kidney and liver cells. This finding agrees with reports showing that ADAM10 is ubiquitously expressed, but demonstrates that the ADAM10 promoter is most active in neural cells and points to a favored transcription of ADAM10 in the brain.
We identified Sp1 sites and the USF element as important for ADAM10 reporter gene expression. Some of these transcription factor binding sites are also present in promoters of other genes involved in Alzheimer disease, including APP and presenilin genes: Sp1 sites have been reported for APP, presenilin-1, and presenilin-2 promoters. In vitro transcription and cotransfection studies showed that USF activates transcription from the human APP promoter.
Identification of the ADAM10 promoter allowed us to generate a stable cell line expressing luciferase under control of that particular region. Using this cell line we identified retinoic acid as an enhancer of ADAM10 promoter-mediated gene expression (Fig. 2)
. Since we have mapped the ADAM10 core promoter region and found that this section is conserved between humans, rats, and mice, the efficiency of transcriptional activators may also be monitored in vivo in transgenic mice.
Since Alzheimer disease is associated with decreased -secretase activity and impaired retinoic acid metabolism, our novel findings directly link two cellular pathways involved in the pathogenesis of the disease and suggest that a decreased ADAM10 activity may be caused by impaired responsiveness to retinoic acid (Fig. 3
).
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3619fje;
This article has been cited by other articles:
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  N. Gavert, M. Sheffer, S. Raveh, S. Spaderna, M. Shtutman, T. Brabletz, F. Barany, P. Paty, D. Notterman, E. Domany, et al. Expression of L1-CAM and ADAM10 in Human Colon Cancer Cells Induces Metastasis Cancer Res., August 15, 2007; 67(16): 7703 - 7712. [Abstract] [Full Text] [PDF]
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Brn-2, 
SREBP, 
Oct-1, 
CREB, 
Sp1, 
CAAT box, and 
USF elements. The black line represents the 5' genomic region of the ADAM10 gene, the gray line the ADAM10 cDNA sequence, and the rectangle the firefly luciferase coding region (LUC). On the right side the activities of these deletion mutants in HEK293 cells are shown. Measured firefly luciferase activity was normalized to renilla luciferase activity of the cotransfected internal control plasmid, phRL-SV40. The highest ADAM10 promoter activity as obtained after transfection with pCP9.3 (–2179/–54) was set to 100%. All values represent the increase in firefly luciferase activity relative to pCP9.3 (–2179/–54) and are the mean ± SD of 3 independent experiments.
-secretase action, should be prevented. The membrane-bound C-terminal APP fragment remaining after