ALBERT

Description: Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a member of the immunoglobulin super-family. It is expressed on the surfaces of activated monocytes, dendritic cells and macrophages. These immune cells use ALCAM through homotypic and heterotypic adhesions to control multiple stages in the inflammatory response. Indeed, anti-ALCAM antibodies and recombinant soluble ALCAM significantly inhibit monocyte transendothelial migration, stabilization of the immunological synapse and dendritic cell-mediated T-lymphocyte proliferation. Despite this significance, there is currently no understanding of how the human ALCAM gene is regulated. In this study, we identified the mechanisms for transcription, basal transcriptional activation and immunosuppressive silencing of the ALCAM gene. A common site for transcription of the ALCAM gene was identified 350 base pairs (bp) upstream from the translational start site. Multiple truncated fragments of the ALCAM promoter was cloned from genomic DNA and sub-cloned upstream of a promoterless luciferase vector. A proximal 650-bp promoter sequence conferred tissue-independent activation in hematopoietic, epithelial and endothelial cells. A canonical Sp1 binding sequence at −550 upstream of the translational start site was mapped within this proximal positive regulatory promoter region. Site-directed mutagenesis revealed this sequence was essential for optimum ALCAM promoter activity. Importantly, Sp1 occupied the cognate sequence in vivo as determined by chromatin immunoprecipitation assays. Over-expression of Sp1 significantly increased ALCAM promoter activity whereas a control expression vector had no impact. DNA sequences in the interval −600 to −800 negatively influenced promoter activity in a tissue-specific manner. This region contained a putative binding sequence for the aryl hydrocarbon receptor (Ahr), which highlighted ALCAM as a potential target of the immunosuppressing ligand dioxin. This hypothesis was tested by examination of whether ALCAM activation is blocked by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) in monocytes differentiating into macrophages and dendritic cells. Expression of ALCAM was increased 3–5-fold in HL-60 and THP-1 monocytes treated with the differentiating agent phorbol 12-myristate 13-acetate. TCDD dose dependently blocked this activation, indeed, the highest concentration of TCDD (25 nM) used in this study completely blocked ALCAM activation in both monocytic cells. In conclusion, we have unveiled for the first time, the molecular basis for transcription and basal trans-activation of the human ALCAM gene, and identified the Ahr-pathway as a powerful silencer of ALCAM gene activation. Further studies of the ALCAM promoter, may clarify how this gene is up-regulated as part of the inflammatory response, and how it is silenced by immunotoxins. Heterologous expression of ALCAM may be a potential strategy to mitigate the immunosuppressive effects of dioxins and polycyclic aromatic hydrocarbons.