Hormone-dependent regulation of intercellular adhesion molecule-1 gene expression: Cloning and analysis of 5 '-regulatory region of rat intercellularadhesion molecule-1 gene in FRTL-5 rat thyroid cells

Intercellular adhesion molecule-1 (ICAM-1) has been suggested to play an im portant role in the perpetuation of autoimmune thyroid disease. To clarify the regulation of ICAM-1 gene in thyroid cells, we investigated ICAM-I expr ession 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 h ydrocortisone, insulin, and thyrotropin (TSH) express the highest levels of ICAM-1 RNA. TSH/forskolin downregulate ICAM-I RNA levels independent of th e presence or absence of hydrocortisone or insulin. Moreover, TSH/forskolin decrease ICAM-1 RNA levels that are maximally induced by two cytokines: 10 0 ng/mL tumor necrosis factor-alpha (TNF-alpha or 100 U/ml interferon-gamma (IFN-gamma). The effect of TSH/forskolin, as well as TNF-alpha and IFN-gamm a, on ICAM-1 RNA levels is transcriptional. Thus, we cloned a 1.8-kb fragme nt of the 5'-flanking region of the rat ICAM-1 gene, upstream of the transl ational start site, and showed that TNF-alpha or IFN-gamma caused a 3.5- an d greater than 12-fold increase respectively, in its promoter activity, whe n linked to a luciferase reporter gene and stably transfected into FRTL-5 c ells. TSH or forskolin, in contrast, halved the activity of the full length chimera within 24 hours and significantly suppressed the TNF-alpha and IFN -gamma-induced increase (>50%; p < 0.02). Using 5'-deletion mutants, we loc ated the element important for the TNF-alpha effect between -431 and -175 b p; we additionally show that deletion of a NF-KB core element within this r egion, TTGGAAATTC (-240 to -230 bp), causes the loss of TNF-alpha inducibil ity. The effect of IFN-gamma could be localized between -175 bp and -97 bp from the start of translation. This region contains 2 regulatory elements k nown to be involved in IFN-gamma action in other eukaryotic cells, an IFN-g amma activated site (GAS), -138 to -128 bp, and Spl site, -112 to -108 bp. Deletion of the 10 bp GAS sequence resulted in the complete loss of IFN-gam ma induction of pCAM-175 promoter activity. TSH and forskolin action was al so mapped between -175 bp and -97 bp from the start of translation. The mut ant 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 cytokin es (TNF-alpha and IFN-gamma) to increase ICAM-1 gene expression in FRTL-5 t hyroid cells. We also localized elements on the 5'-flanking region of ICAM- I important for these actions. We propose that this TSH/cyclic adenosine mo nophosphate (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.