1. ¹Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
2. ²Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

Correspondence: Dr Jouni Uitto, Department of Dermatology and Cutaneous Biology, Jefferson Medical College, 233 S 10th Street, Suite 450 BLSB, Philadelphia, Pennsylvania 19107, USA. E-mail: Jouni.Uitto@jefferson.edu

³These authors contributed equally to this work

Received 15 July 2005; Revised 13 September 2005; Accepted 22 September 2005; Published online 22 December 2005.

Abstract

ABCC6, a member of the adenosine 5'-triphosphate-binding cassette family of genes, encodes multidrug resistance-associated protein 6, a putative transmembrane transporter expressed primarily in the liver and to a significantly lower extent in other tissues. Mutations in ABCC6 result in pseudoxanthoma elasticum, a multi-system heritable connective tissue disorder with variable phenotypic expression. To examine the transcriptional regulation and tissue-specific expression of this gene, we cloned 2.6 kb of human ABCC6 promoter and developed a series of 5'-deletion constructs linked to luciferase reporter gene. Transient transfections in a number of cultured cell lines of diverse origin identified a specific NF-B-like sequence (-235/-226), which conferred high level of expression in HepG2 hepatoma cells, inferring liver specificity. The functionality of the promoter fragments was confirmed in vivo by tail vein injection followed by luciferase reporter assay. Testing of selected cytokines revealed that transforming growth factor (TGF)- upregulated, while tumor necrosis factor (TNF)- and interferon (IFN)- downregulated the promoter activity in HepG2 cells. The responsiveness to TGF- was shown to reside primarily within an Sp1/Sp3 cognate-binding site at -58 to -49. The expression of the ABCC6 promoter was also shown to be markedly enhanced by Sp1 protein, as demonstrated by cotransfection of ABCC6 promoter–luciferase constructs and an Sp1 expression vector in Drosophila SL2 cells, which are devoid of endogenous Sp1. Furthermore, four additional transcription factors, with their cognate-binding sequences present in DNA, were shown to bind the 2.6-kb promoter fragment by protein/DNA array. Collectively, the results indicate that human ABCC6 displays tissue-specific gene expression, which can be modulated by proinflammatory cytokines. These findings may have implications for phenotypic expression of heritable and acquired diseases involving abnormality in the ABCC6 gene.