The ERK1/2-hepatocyte nuclear factor 4alpha axis regulates human ABCC6 gene expression in hepatocytes.

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 4alpha (HNF4alpha) binding site. We demonstrated that HNF4alpha regulates the expression of ABCC6, acts through the putative binding site, and determines its cell type-specific expression. We also showed that HNF4alpha 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-HNF4alpha axis has an important role in regulation of the gene.

Mithramycin A suppresses expression of the human melanoma-associated gene ABCB8.

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.

ABCC6 expression is regulated by CCAAT/enhancer-binding protein activating a primate-specific sequence located in the first intron 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 (HSs) specific to cell lines expressing ABCC6. These HSs are located in the proximal promoter and in the first intron of the gene. We further characterized the role of the HSs 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 hepatocyte nuclear factor 4α (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.

Application of cellular biosensors for analysis of bioactivity associated with airborne particulate matter.

Exposure to airborne particulate matter (PM) is a known risk factor for adverse health effects observed in many environmental and occupational settings. The pathological mechanisms involved in PM-mediated toxicity depend on the size and contents of particles that vary depending on the source of emission. Chemical compositions of PM show multiple components with different bioavailabilities that are capable of acting on multiple molecular and cellular targets, making it difficult to predict PM-associated toxicity based solely on chemical analysis. The aim of the study was to develop robust, sensitive and economical assays for environmental pollutants based on genetically modified mammalian cells. We tested the suitability of two biosensor assays, Fluorescent Cell Chip and Oxibios, developed in part in our laboratories, for assessment of the potential toxicity of airborne PM. Reference PM and PM obtained by sampling of diesel exhaust and indoor air in aluminum and copper facilities in Poland were tested with the two bioassays using unified experimental protocols. Resultant data showed complex patterns of stimulatory and inhibitory activities that were consistent with the origin of PM and might be correlated with their chemical composition. The analysis was informative with regard to type and extent of possible toxicity associated with specific PM and allowed for detection of significant differences between PM from different industrial sites and particular locations within the same industrial sites as well as overall ranking of toxicity risk based on chemical analysis.