ABCC6 mutations are responsible for the development of pseudoxanthoma elasticum, a rare recessive disease characterized by calcification of elastic fibers. Although ABCC6 is mainly expressed in the liver the disease has dermatologic, ocular, and cardiovascular symptoms. We investigated the transcriptional regulation of the gene and observed that hepatocyte growth factor (HGF) inhibits its expression in HepG2 cells via the activation of ERK1/2. Similarly, other factors activating the cascade also inhibited ABCC6 expression. We identified the ERK1/2 response element in the proximal promoter by luciferase reporter gene assays. This site overlapped with a region conferring the tissue-specific expression pattern to the gene and with a putative hepatocyte nuclear factor 4␣ (HNF4␣) binding site. We demonstrated that HNF4␣ regulates the expression of ABCC6, acts through the putative binding site, and determines its cell type-specific expression. We also showed that HNF4␣ is inhibited by the activation of the ERK1/2 cascade. In conclusion we describe here the first regulatory pathway of ABCC6 expression showing that the ERK1/2-HNF4␣ axis has an important role in regulation of the gene.

Pseudoxanthoma elasticum (PXE), a rare recessive genetic disease causing skin, eye and cardiovascular lesions is characterized by the calcification of elastic fibers. The disorder is due to loss-of-function mutations of the ABCC6 gene but the pathophysiology of the disease is still not understood. Here we investigated the transcriptional regulation of the gene, using DNase I hypersensitivity assay followed by luciferase reporter gene assay. We identified three DNase I hypersensitive sites (HS) specific to cell lines expressing ABCC6. These HS are located in the proximal promoter and in the first intron of the gene. We further characterized the role of the HS by luciferase assay and demonstrated the transcriptional activity of the intronic HS. We identified the CCAAT/Enhancer binding protein ß (C/EBPß) as a factor binding the second intronic HS by chromatin immunoprecipitation and corroborated this finding by luciferase assays. We also showed that C/EBPß interacts with the proximal promoter of the gene. We propose that C/EBPß forms a complex with other regulatory proteins including the previously identified regulatory factor HNF4α. This complex would account for the tissue-specific expression of the gene and might serve as a metabolic sensor. Our results point toward a better understanding of the physiological role of ABCC6.

The role of the ABCB8 gene in human cells is poorly understood, although it has been suggested to be involved in multidrug resistance in some types of cancers (e.g., melanomas). In this study, the main mechanism of transcriptional regulation of the ABCB8 gene was characterized. EMSA and ChIP assays revealed that the transcription factor Sp1 binds to the ABCB8 core promoter region, and Sp1 consensus elements were crucial for promoter activity in a luciferase reporter gene assay. Mithramycin A, an inhibitor of Sp1 binding, downregulated the expression of ABCB8 (and other ABC genes) in a concentration-dependent manner and sensitized a melanoma cell line to doxorubicin treatment. These findings may have therapeutic applications in at least a subset of melanoma patients.