Hormone-Dependent Regulation of Intercellular Adhesion Molecule-1 Gene Expression: Cloning and Analysis of 5′-Regulatory Region of Rat Intercellular Adhesion Molecule-1 Gene in FRTL-5 Rat Thyroid Cells

Intercellular adhesion molecule-1 (ICAM-1) has been suggested to play an important role in the perpetuation of autoimmune thyroid disease. To clarify the regulation of ICAM-1 gene in thyroid cells, we investigated ICAM-1 expression in the FRTL-5 thyroid cell model and defined several elements in the 5′-regulatory region that are important for transcriptional regulation of the rat ICAM-1 gene. Cells maintained in medium with 5% serum but without hydrocortisone, insulin, and thyrotropin (TSH) express the highest levels of ICAM-1 RNA. TSH/forskolin downregulate ICAM-1 RNA levels independent of the presence or absence of hydrocortisone or insulin. Moreover, TSH/forskolin decrease ICAM-1 RNA levels that are maximally induced by two cytokines: 100 ng/mL tumor necrosis factor-α (TNF-α) or 100 U/ml interferon-γ (IFN-γ). The effect of TSH/forskolin, as well as TNF-α and IFN-γ, on ICAM-1 RNA levels is transcriptional. Thus, we cloned a 1.8-kb fragment of the 5′-flanking region of the rat ICAM-1 gene, upstream of the translational start site, and showed that TNF-α or IFN-γ caused a 3.5- and greater than 12-fold increase respectively, in its promoter activity, when linked to a luciferase reporter gene and stably transfected into FRTL-5 cells. TSH or forskolin, in contrast, halved the activity of the full length chimera within 24 hours and significantly suppressed the TNF-α and IFN-γ-induced increase (>50%; p < 0.02). Using 5′-deletion mutants, we located the element important for the TNF-α effect between -431 and - 175 bp; we additionally show that deletion of a NF-κB core element within this region, TTGGAAATTC (-240 to -230 bp), causes the loss of TNF-α inducibility. The effect of IFN-γ could be localized between -175 bp and -97 bp from the start of translation. This region contains 2 regulatory elements known to be involved in IFN-γ action in other eukaryotic cells, an IFN-γ activated site (GAS), -138 to -128 bp, and Sp1 site, -112 to -108 bp. Deletion of the 10 bp GAS sequence resulted in the complete loss of IFN-γ induction of pCAM-175 promoter activity. TSH and forskolin action was also mapped between -175 bp and -97 bp from the start of translation. The mutant construct, pCAM-175delGAS mut1, which has no GAS sequence, exhibited no TSH-mediated suppression of promoter activity. We thus show that TSH/cAMP can downregulate ICAM-1 gene expression and inhibit the activity of cytokines (TNF-α and IFN-γ) to increase ICAM-1 gene expression in FRTL-5 thyroid cells. We also localized elements on the 5′-flanking region of ICAM-1 important for these actions. We propose that this TSH/cyclic adenosine monophosphate (cAMP) action is a component of the mechanism to preserve self-tolerance of the thyroid during hormone-induced growth and function of the gland, and it may attenuate cytokine action during inflammatory reactions.