Vol. 166, No. 3, 1990 February 14, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1300-1307
EXPRESSION OF RETINOIC ACID RECEPTOR GENES AND THE LIGAND-BINDING SELECTIVITY OF RETINOIC ACID RECEPTORS (RAR'S) Yuichi
Hashimoto',
Institute l-l-l
2
Faculty
Hiroyuki
Kagechika
2
,
and Koichi
of Applied Microbiology, University of Yayoi, Bunkyo-ku, Tokyo 113, Japan
Shudo 2 Tokyo
of Pharmaceutical Sciences, University of Tokyo 7-3-l Hongo, Bunkyo-ku, Tokyo 113, Japan
Received December 18, 1989
Retinoids of a structurally new type, 4-(5,6,7,8-tetrahydro-5,5,8,8-tetrathyl-2-naphthalenylcarb~oyl)benzoic acid (Am80) and (E)-4-[3-(3,5-di-tert-butylphenyl)-3-oxo-l-propenyl]benzoic acid (Ch55). were used for the investigation of retinoid in some human cell lines. receptors Previously, using these retinoids, we have established that HL-60 cells possess two retinoid receptors, RAR-a and RAR-fi . In this paper, we report coexpression of RAR-a - and RAR-fl -genes established by Northern analysis. Further, the binding selectivity of the RAR products toward all-trans-retinoic acid (RA), Am80 and Ch55 was studied by filter bindmssay. The affinity of the ligands toward RAR-a decreased in the order of Ch55 > RA > Am80. The affinity of the ligands toward RAR-6 decreased in the order of Ch55 > Am80 > RA. SUMMARY:
01990
Academic
Press,
Inc.
Retinoids play a fundamental role in normal cell growth and cell differentiation, and are defined as substances that can elicit specific biological responses by binding to and activating a specific receptor or set of receptors." 2, The elucidation of the molecular mechanism of retinoidal action requires identification of specific retinoid receptor(s). As human retinoid receptor genes, three types of receptor genes, RAR-a -, RAR-fi - and RARy -genes, have been cloned.3-7) These genes belong to the erbArelated steroid/thyroid nuclear receptor gene superfamily.*) On the other hand, synthetic retinoids which are more potent and more specific than all-trans-retinoic acid (RA) are useful tools for investigation of retinoid receptors. We have developed a structurally new type of retinoids, retinobenzoic acids.g-* *I Among the synthesized retinobenzoic acids, 4-(5,6,7,8tetrahydro-5,5,8,8-tetramethyl-2-naphthayl)benzoic acid (Am80)s) and (E)-4-[3-(3,5-di-tert-butylphenyl)-3-oxo-lpropenyllbenzoic acid (Ch55)lo1 (Fig.1) are especially superior 0006-291X/90$1.50 Copyright All rights
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
1300
Vol. 166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
COOH COOH
Am80
Fig.1.
Structures
Ch55
of RA, Am80 and Ch55.
Using these retinoids, we tools for such investigations.1z-'5' have recently established the presence of two types of retinoid receptors in human leukemia cells HL-60'2-15' : One is RAR-fi (RSBP-1),'2. 14' which can be extracted selectively from HL-60 cells with low salt (0.15 M KCl) buffer, though more than 80% of the retinoid-specific binding activity remains in the nuclear The other is RAR-a (RSBP-2),14' fraction. which requires high - 0.6 M KCl) buffer for efficient extraction. RAR-a salt (0.3 and RAR-6 can be separated by anion exchange HPLC (Mono Q).'3-'5' Coexistence of M-a and RAR-fi products in the nucleus of HL-60 cells has also been established by immunohistochemical technique by Gaub et a1.16' In this paper, we report the existence of RAR's and the expression of mRNA of RAR genes in some human cell lines, and the ligand binding selectivity of the products.
MATERIALS
AND METHODS
Chemicals: Am80 and Ch55 were synthesized as described was prepared at Amersham.'*' previously.9' lo' 3H-Am80 (65Ci/mmole) Cell Culture: HL-60 cells and suspension culture of HeLa cells were maintained in RPM11640 medium supplemented with fetal calf serum (FCS)(S% v/v) under 5% carbon dioxide at 37 "C . HeLa Sa cells were maintained in MEM medium supplemented with 5% FCS under 5% carbon dioxide at 37 "C . THP-1 cells treated with tetradecanoyl-phorbol 13-acetate (TPA) were supplied by Ajinomoto Co. RAR-expression plasmids: HeLa Si cells were Transfection of transfected with RAR-a - or RAR-4 -expression plasmid". 5' by using the calcium phosphate technique (CellPhect Transfection Kit, Pharmacia). Preparation of RAR fraction: RAR's were extracted from cultured cells as described previously.'2. 17' For the selective extraction were broken by hypotonic shock, and then homoof m-0 , cells genized in low salt (0.15 M KCl) buffer containing protease inhibitor cocktail (PIC).'p' For the extraction of RAR-a and were homogenized in high salt (0.6 M KCl) buffer MR-13 , cells containinu PIG."' The extracts were centrifuaed 11050006 x 1 hr). and then
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1300-1307
EXPRESSION OF RETINOIC ACID RECEPTOR GENES AND THE LIGAND-BINDING SELECTIVITY OF RETINOIC ACID RECEPTORS (RAR'S) Yuichi
Hashimoto',
Institute l-l-l
2
Faculty
Hiroyuki
Kagechika
2
,
and Koichi
of Applied Microbiology, University of Yayoi, Bunkyo-ku, Tokyo 113, Japan
Shudo 2 Tokyo
of Pharmaceutical Sciences, University of Tokyo 7-3-l Hongo, Bunkyo-ku, Tokyo 113, Japan
Received December 18, 1989
Retinoids of a structurally new type, 4-(5,6,7,8-tetrahydro-5,5,8,8-tetrathyl-2-naphthalenylcarb~oyl)benzoic acid (Am80) and (E)-4-[3-(3,5-di-tert-butylphenyl)-3-oxo-l-propenyl]benzoic acid (Ch55). were used for the investigation of retinoid in some human cell lines. receptors Previously, using these retinoids, we have established that HL-60 cells possess two retinoid receptors, RAR-a and RAR-fi . In this paper, we report coexpression of RAR-a - and RAR-fl -genes established by Northern analysis. Further, the binding selectivity of the RAR products toward all-trans-retinoic acid (RA), Am80 and Ch55 was studied by filter bindmssay. The affinity of the ligands toward RAR-a decreased in the order of Ch55 > RA > Am80. The affinity of the ligands toward RAR-6 decreased in the order of Ch55 > Am80 > RA. SUMMARY:
01990
Academic
Press,
Inc.
Retinoids play a fundamental role in normal cell growth and cell differentiation, and are defined as substances that can elicit specific biological responses by binding to and activating a specific receptor or set of receptors." 2, The elucidation of the molecular mechanism of retinoidal action requires identification of specific retinoid receptor(s). As human retinoid receptor genes, three types of receptor genes, RAR-a -, RAR-fi - and RARy -genes, have been cloned.3-7) These genes belong to the erbArelated steroid/thyroid nuclear receptor gene superfamily.*) On the other hand, synthetic retinoids which are more potent and more specific than all-trans-retinoic acid (RA) are useful tools for investigation of retinoid receptors. We have developed a structurally new type of retinoids, retinobenzoic acids.g-* *I Among the synthesized retinobenzoic acids, 4-(5,6,7,8tetrahydro-5,5,8,8-tetramethyl-2-naphthayl)benzoic acid (Am80)s) and (E)-4-[3-(3,5-di-tert-butylphenyl)-3-oxo-lpropenyllbenzoic acid (Ch55)lo1 (Fig.1) are especially superior 0006-291X/90$1.50 Copyright All rights
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
1300
Vol. 166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
COOH COOH
Am80
Fig.1.
Structures
Ch55
of RA, Am80 and Ch55.
Using these retinoids, we tools for such investigations.1z-'5' have recently established the presence of two types of retinoid receptors in human leukemia cells HL-60'2-15' : One is RAR-fi (RSBP-1),'2. 14' which can be extracted selectively from HL-60 cells with low salt (0.15 M KCl) buffer, though more than 80% of the retinoid-specific binding activity remains in the nuclear The other is RAR-a (RSBP-2),14' fraction. which requires high - 0.6 M KCl) buffer for efficient extraction. RAR-a salt (0.3 and RAR-6 can be separated by anion exchange HPLC (Mono Q).'3-'5' Coexistence of M-a and RAR-fi products in the nucleus of HL-60 cells has also been established by immunohistochemical technique by Gaub et a1.16' In this paper, we report the existence of RAR's and the expression of mRNA of RAR genes in some human cell lines, and the ligand binding selectivity of the products.
MATERIALS
AND METHODS
Chemicals: Am80 and Ch55 were synthesized as described was prepared at Amersham.'*' previously.9' lo' 3H-Am80 (65Ci/mmole) Cell Culture: HL-60 cells and suspension culture of HeLa cells were maintained in RPM11640 medium supplemented with fetal calf serum (FCS)(S% v/v) under 5% carbon dioxide at 37 "C . HeLa Sa cells were maintained in MEM medium supplemented with 5% FCS under 5% carbon dioxide at 37 "C . THP-1 cells treated with tetradecanoyl-phorbol 13-acetate (TPA) were supplied by Ajinomoto Co. RAR-expression plasmids: HeLa Si cells were Transfection of transfected with RAR-a - or RAR-4 -expression plasmid". 5' by using the calcium phosphate technique (CellPhect Transfection Kit, Pharmacia). Preparation of RAR fraction: RAR's were extracted from cultured cells as described previously.'2. 17' For the selective extraction were broken by hypotonic shock, and then homoof m-0 , cells genized in low salt (0.15 M KCl) buffer containing protease inhibitor cocktail (PIC).'p' For the extraction of RAR-a and were homogenized in high salt (0.6 M KCl) buffer MR-13 , cells containinu PIG."' The extracts were centrifuaed 11050006 x 1 hr). and then
