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-alpha (TNF-alpha) or 100 U/ml interferon-gamma (IFN-gamma). The effect of TSH/forskolin, as well as TNF-alpha and IFN-gamma, 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-alpha or IFN-gamma 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-alpha and IFN-gamma-induced increase (>50%; p < 0.02). Using 5'-deletion mutants, we located the element important for the TNF-alpha effect between -431 and -175 bp; we additionally show that deletion of a NF-kappaB core element within this region, TTGGAAATTC (-240 to -230 bp), causes the loss of TNF-alpha inducibility. 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 known to be involved in IFN-gamma action in other eukaryotic cells, an IFN-gamma 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-gamma 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 mutl, 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-alpha and IFN-gamma) 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.
Park
E S
ES
Department of Internal Medicine, School of Medicine, Chungnam National University, Taejon, Korea.
You
S H
SH
Kim
H
H
Kwon
O Y
OY
Ro
H K
HK
Cho
B Y
BY
Taniguchi
S I
SI
Kohn
L D
LD
Shong
M
M
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D005786:Gene Expression Regulation / Q000502:physiology*; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis* / Q000235:genetics*; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011915:Rats, Inbred BUF; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology; D017384:Sequence Deletion; D020079:Terminal Repeat Sequences; D013961:Thyroid Gland / Q000378:metabolism*; D013963:Thyroid Hormones / Q000502:physiology*; D014162:Transfection
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013963:Thyroid Hormones; D018799:Intercellular Adhesion Molecule-1; D008156:Luciferases