Regulation of intercellular adhesion molecule-1 expression by retinoic acid: analysis of the 5' regulatory region of the gene.

Human intercellular adhesion molecule-1 (ICAM-1), a specific ligand for the lymphocyte-function-associated antigen-1, plays an important role in immune responses. ICAM-1 expression is regulated by various proinflammatory cytokines, by PMA, and by retinoic acid. In this study, we have investigated the mechanisms of transcriptional control involved in the stimulation of ICAM-1 gene expression by retinoic acid in SK-N-SH cells. Northern-blot analysis demonstrated that ICAM-1 mRNA is maximally induced at 24 hr, suggesting that it is not an early-response gene with respect to retinoic-acid responsiveness, whereas the retinoic acid receptor-beta mRNA level was maximal 12 hr following retinoic acid treatment. To analyze the 5'-regulatory region of the ICAM-1 gene, an EcoRI/SaII fragment spanning the first 1.3 kb upstream of the translational start site was used to direct the expression of a linked luciferase reporter gene in transient transfection assays in SK-N-SH cells. A 24-hr treatment of transfected cells with 10 microM retinoic acid resulted in a 10- to 13-fold increase in luciferase activity compared with untreated cells. Deletion mutant analysis revealed that a region located between -393 and -176 bp from the translational start site is critical for retinoic acid stimulation of luciferase activity. This region harbors a consensus sequence for a retinoic-acid-responsive element (RARE) homologous to the element found upstream of the alcohol dehydrogenase-3 gene. Co-transfection of expression vectors encoding the retinoic acid receptor-alpha, -beta, or -gamma, with reporter plasmids harboring the putative RARE, confirmed that the ICAM-1 gene is regulated by retinoic acid in a retinoic acid receptor-dependent fashion.

Cloning of the human gene for intercellular adhesion molecule 1 and analysis of its 5'-regulatory region. Induction by cytokines and phorbol ester.

Human intercellular adhesion molecule-1 (ICAM-1), a specific ligand for the lymphocyte function-associated Ag-1 (LFA-1), plays an important role in leukocyte-endothelial cell interactions. It is induced by proinflammatory cytokines such as IL-1, TNF-alpha, or IFN-gamma. However, little is known concerning the intracellular regulatory mechanisms which trigger ICAM-1 up-regulation. In order to study potential regulatory elements involved in ICAM-1 induction we have cloned the human ICAM-1 gene and 5 kb of its 5'-regulatory region. The sequence of the cDNA was found to be distributed over seven exons separated by six introns, whereby each of the five extracellular Ig-like domains of ICAM-1 is encoded by its own exon. The upstream sequence harbors a number of sequence motifs implicated in the regulation and expression of eukaryotic genes, including binding sites for the transcription factors SP-1, AP-1, and NF-kB. Primer extension and S1 nuclease analysis revealed two transcription initiation sites 319 bp and 41 bp upstream of the translation start site. Consensus TATA boxes were found at the expected positions about 25 bp upstream of both start sites. Reverse transcriptase polymerase chain reaction showed differential use of the two TATA boxes in A549 and HS913T cells. Both RNA seem to code for the same for of ICAM-1 protein. For regulation studies a 1.3-kb EcoRI/SalI fragment of the 5'-flanking region was used to promote transcription of a linked luciferase reporter gene in transient-transfection assays in A549 and HS913T cells. Treatment of A549 cells with IL-1 or TNF-alpha resulted in a two- or fourfold increase in luciferase activity. Furthermore, a sixfold induction could be achieved after treatment with the phorbol ester PMA. In contrast, agents that increase intracellular cAMP levels did not induce luciferase activity. Northern blot analysis was used to investigate the kinetics of ICAM-1 mRNA synthesis upon induction with TNF-alpha and PMA. These data suggest that the up-regulation of ICAM-1 by cytokines occurs at least partly at the transcriptional level. Deletion analysis of the 1.3-kb fragment of the 5'-flanking region revealed sequences responsible for promotion and inhibition of transcription. In particular, two functionally distinct regions have been characterized: a short fragment containing an NF-kB binding site has been shown to function as an activator, followed immediately downstream by a sequence acting as a silencer element. Therefore, ICAM-1 gene expression seems to be modulated by multiple cis-acting elements.