Lack of evidence for estrogen and progesterone receptors in human adipose tissue

ft. Steroid Bwchem. Molec. Bwl. Vol. 51, No. 5/6, pp. 275-281, 1994

Pergamon

0960-0760(94)00139-1

Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0960-0760/94 $7 00 + 0.00

Lack of Evidence for Estrogen and Progesterone Receptors in H u m a n Adipose Tissue M . Br6nneg

rd, 1. M . O t t o s s o n , 2 J. B 6 6 s , 1 C. M a r c u s I a n d P . B j 6 r n t o r p z

~Department of Pediatrics, Endocrine Research Unit, Karolinska Institute, Huddinge University Hospital, 141 86 Huddinge and 2Wallenberg Laboratory, Department of Heart and Lung Disease, Sahlgrenska Hospital, University of Gothenburg, Sweden We have previously presented data indicating the absence of estrogen and progesterone receptors f r o m h u m a n a d i p o s e t i s s u e b y t h e use o f specific a n t i b o d i e s ( A b b o t t ) as well as specific l i g a n d s . In a d d i t i o n , s p e c i f i c e s t r o g e n a n d p r o g e s t e r o n e c R N A p r o b e s d i d n o t h y b r i d i z e to a n y m R N A s p e c i e s in e i t h e r a b d o m i n a l o r g l u t e a l / f e m o r a l a d i p o s e t i s s u e as d e m o n s t r a t e d b y s o l u t i o n h y b r i d i z a t i o n a n d N o r t h e r n blot. In o r d e r to d e m o n s t r a t e e v e n e x t r e m e l y s m a l l q u a n t i t i e s o f g e n e p r o d u c t s we h a v e n o w u s e d t h e P o l y m e r a s e c h a i n r e a c t i o n - t e c h n i q u e to s t u d y e s t r o g e n - a n d p r o g e s t e r o n e r e c e p t o r g e n e e x p r e s s i o n . S e q u e n c e s c o r r e s p o n d i n g to e a c h specific c D N A w e r e d e m o n s t r a t e d i n d i c a t i n g small amounts of estrogen- and progesterone receptor mRNA not detected by RNA/RNA or R N A / T N A ( t o t a l n u c l e i c a c i d s ) h y b r i d i z a t i o n assays. T h e e s t r o g e n r e c e p t o r - r e g u l a t e d g e n e pS2, h o w e v e r , was n o t i n d u c e d b y e s t r o g e n s in h u m a n a d i p o s e t i s s u e in c o n t r a s t to a s i g n i f i c a n t i n c r e a s e in pS2 m R N A levels a f t e r e s t r o g e n e x p o s u r e to t h e e s t r o g e n r e c e p t o r ( + ) cell line M C F 7 . F r o m t h e s e r e s u l t s we c o n c l u d e t h a t e s t r o g e n - a n d p r o g e s t e r o n e r e c e p t o r s a r e a b s e n t f r o m h u m a n a d i p o s e t i s s u e a n d t h a t t h e e x t r e m e l y low level o f t r a n s c r i p t i o n o f t h e c o r r e s p o n d i n g g e n e s is n o t s u f f i c i e n t to a l l o w translation of the message into functional proteins.

ft. Steroid Biochem. Molec. Biol., Vol. 51, No. 5/6, pp. 275-281, 1994

tween non-genomic and genomic actions of steroids meaning that a specific tissue or cell type may actually lack intracellular receptor proteins or alternatively express extremely low levels of receptors, but be highly "membrane-responsive" [6]. T h u s , cell- and tissue specificity of response to a whole-body signal is determined by local pre-receptor, receptor and genomic differences. Adipose tissue is one of the most abundant pools of estrogen [7]. However, in spite of the remarkably high concentrations of estrogen and progesterone in comparison to other human tissues, the actual effect of these steroids in human adipose tissue has been debated [7]. Previous studies have indicated a high level of glucocorticoid receptors (GR) as well as androgen receptors in adipose tissue [8, 9]. On the contrary, the expression of estrogen (ER)- and progesterone (PR) receptors both at protein and m R N A level has been difficult to demonstrate [8]. In this study, we have applied the polymerase chain reduction (PCR)-technique in order to evaluate the expression of corresponding m R N A s for the ER and

INTRODUCTION One of the most controversial issues in endocrinology today is the n u m b e r of steroid hormone receptors necessary to obtain a biological response. In spite of the classical three step model in the mode of action of steroid hormones (ligand > receptor > biological response) [1, 2] there is substantial evidence that several other factors such as heat shock proteins contribute to determine target tissue responsiveness[3]. Therefore, this model should be referred to with caution when trying to explain specific biological effects in terms of ligand concentration and receptor structure and/or number. Furthermore, the actions of steroids appear to involve the cell membrance as well as the genome, and (although it has not been demonstrated) it is even conceivable that actions of steroids at the cell surface might be able to trigger changes in gene expression indirectly [4, 5]. During the last 10 years evidence has accumulated that synergistic interactions occur be*Correspondance to M. Br6nneg~ird. Received 19 May 1994; accepted 8 Aug. 1994. 275

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PR in human adipose tissue. We have also determined the expression levels of the glucocorticoid- and progesterone-regulated metallothionein ( M T I I A ) gene and the estrogen-regulated gene pS2 which, if induced, would indicate a receptor-mediated process [10].

MATERIALS AND METHODS

Subjects and cell cultures Subcutaneous, abdominal adipose tissue was obtained from three pre-menopausal women, 20, 41 and 47 years of age, undergoing abdominal surgery for non-malignant diseases. Body mass index was 23.8, 28.5 and 25.8 kg/m 2, respectively. None were taking oral contraceptives and none were diabetic. T h e patients had been fasting overnight and surgery was performed under general anesthesia. About 2 0 g of adipose tissue was excised during the initial phase of the operation. T h e study was approved by the Ethics Committee of the University of Gothenburg. Cells from the human breast cancer cell line M C F 7 were grown in R P M I medium 1640 (GIBCO) supplemented with 10% fetal calf serum. All experiments were performed after four cell passages with M C F 7 cells growing in monolayer culture. Cells were maintained in serum-free medium for 8 h and then treated with cortisol, progesterone and fl-estradiol at the concentrations indicated in the legends to the figures, for 6h. Before R N A or T N A (total nucleic acids) preparation, the cells were washed twice with phosphate-buffered saline, p H 7.4. Immediately after excision, the adipose tissue was placed in sterile Parker medium 199 (SBL, Stockholm, Sweden) supplemented with 1 0 m M H E P E S [N-(2hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid; Sigma, St Louis, U.S.A.], at room temperature and p H 7.4. Pieces of adipose tissue (5-20 g) were prepared under sterile conditions and used for incubations in plastic tubes (600 mg tissue/20 ml medium). Connective tissue and blood vessels were removed. T h e adipose tissue was pre-incubated in control medium (Parker medium 199 supplemented with 1 2 . S m M NaHCO3, 10 m M H E P E S , 1 °o human serum albumin; I m m u n o AG, Vienna, Austria] and 0.1 mg cephalothin (Keflin, Lilly France S.A., France) and 1000/~U insulin per ml (Actrapid, Novo Nordisk A/S, Denmark), p H adjusted to 7.4) for 3-5 days. T h e medium was changed daily. After pre-incubation, at the time for steroid hormone exposure (t = 0), about 500mg of adipose tissue was frozen in liquid nitrogen as a control. T h e rest of the tissue was divided into three groups and was given fresh medium. One third was incubated in medium with cortisol (control medium supplemented with cortisol, 1 0 - 6 M ) , o n e third in medium with estradiol (control medium supplemented with fl-estradiol, 1 0 - 6 M ) and one third was continuously incubated in the control medium. After t = 1, 2, 4, 6,

9, 12 and 24 h of incubation 500 mg of tissue from each medium was frozen in liquid nitrogen. In one incubation experiment, the 9 h samples were excluded. In selected experiments, adipose tissue was also incubated with progesterone (control medium supplemented with progesterone, 10 6 M) and 500 mg of tissue from the medium were frozen in liquid nitrogen at indicated time intervals. T h e samples were stored at - 7 0 ° C until preparation of total nucleic acids. At each time point, 50-60 mg of adipose tissue in duplicate from each medium was used for determination of heparin-releasable lipoprotein lipase ( L P L ) activity [11]. This was made to check the conditions under which the tissue was incubated. Under optimal conditions cortisol induces L P L activity within 2 4 h (after pre-incubation) in human adipose tissue indicated by a 2.6-, 3.3- and 1.3-fold induction, respectively, as compared with the initial L P L activity in the three patients investigated (data not shown).

Preparation of R N A and T N A Total RNA from adipose tissue (200mg) and cultured M C F 7 cells (approx. 10 7 cells) was prepared by the single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction according to the protocol of Chomczynski [12]. For preparation of T N A , adipose tissue or M C F 7 cells were homogenized in a sodium dodecyl sulfate (SDS) buffer (1 o.o SDS; 10 mmol/1 of Tris-HC1, p H 7.5, and 5 mmol/1 of E D T A ) , digested with 100 mg of proteinase K for 45 min at 45~C, and subsequently extracted with phenol-chloroform after the addition of isoamylic alcohol (24: 1, v/v) [13]. RNA and T N A concentrations were determined spectrophotometrically. T h e integrity of RNA samples was verified by gel electrophoresis and ethidium bromide staining.

Hybridization analysis of R N A Oligonucleotide probes (50 base pairs) were synthesized and cloned into the PstI/HindIII sites in p G E M T M - I (Promega Biotechnology, Madison, WI). T h e sequence of these inserts in p G E M T M - I was confirmed by D N A sequencing using the dideoxy chain-termination method [14]. These plasmids were used for in vitro synthesis of cRNA using SP6 RNA polymerase and the opposite m R N A strand using T 7 R N A polymerase as described below. T h e sequence of the M T I I A oligonucleotides used has been reported previously [15]. For the pS2 gene the sequence of the oligonucleotides used corresponded to nucleotides 55-106 [10]. RNA hybridization was analyzed using a solution hybridization protocol [16]. T N A samples were hybridized to approx. 20,000cpm/sample of [35S]UTP labeled RNA probes and hybrids were allowed to form in 40#1 of a buffer consisting of 0.6 mol/1 of NaC1, 30 mmol/1 of dithiothreitol ( D T T / ,

Steroid Receptors in Adipose Tissue

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Table 1. Primers used for the reverse transcription and PCR reactions Sequence 1. 2. 3. 4. 5. 6.

5'-GCC 5'-GAG 5'-GCA 5'-TTG 5'-CTC 5'-GCA

cDNA site GGC GGC GCC GGG GGG GGA

CTC GCC GCT CCA GAA TAT

GCG GCC CGC CCG TTC GAT

CAC TAC GCC CCC AAT AGC

CGH GAG CGG CCG ACT TCT

GTA TTC CGC CTG CAT GTT

GCC AAC CTT CCG GGT CCA

G-3' 643-667(as) G-3' 398-442(s) G-3' 1441-1464(as) C-3' 1255-1278(s) C-3' 236~2385(as) GAC-3' 1768 1794(s)

Sequences used for PCR reactions are from pubhshed cDNA sequences of respectwe steroid receptor. 1-2, estrogen receptor [28]; 3~4, progesterone receptor [29]; 5~6, glucocorticoid receptor [30]. Antisense (as) and sense (s) sequences are indicated. and 25°o formamide at 68'~C. After an overnight incubation, samples were digested with RNase by adding 1 ml of a solution containing 40 p g RNase A and 2/~g RNase T1 (Boehringer-Mannheim, Mannheim, Germany). Digestion was performed for 45 min at 45°C after which RNase-resistant R N A was precipitated by the addition of 0.1 ml of 6tool/1 of trichloroacetic acid. Precipitates were collected for scintillation counting by filtration on glass fiber filters (Whatman GF/Clifton, NJ). T h e amount of pS2 and M T I I A m R N A of a sample was determined in duplicate and calculated from a linear standard curve constructed from incubations with known amounts of in vitro synthesized m R N A complementary to the 35S-labeled probe as described previously [15, 16]. Results are expressed as amol m R N A / # g T N A . Means and SD were calculated for the various experimental groups. Means were compared by the M a n n - W h i t n e y U test, when appropriate and differences between groups considered significant for P values of 0.05 or less.

RESULTS

Expression of p S 2 m R N A In M C F 7 cells, ~-estradiol caused a dose-related and significant increase of pS2 m R N A . Figure 1 shows a typical dose-response curve with a maximal inductive effect on pS2 at a concentration of 1 0 - 6 M . N o effect on pS2 m R N A expression levels was observed after incubation of M C F 7 cells with progesterone or cortisol (Fig. 1). T h e time course of//-estradiol action on pS2 m R N A levels in M C F 7 cells showed a maximal effect 6 h after onset of treatment (data not shown) and based on these data M C F 7 cells were treated with each specific steroid for 6 h before T N A preparation. In h u m a n adipose tissue, however, no induction of pS2 m R N A by/~-estradiol was observed (Fig. 2). It should be noted that the basal level of expression of pS2 m R N A in both M C F 7 and adipose tissue is very low and did not indicate any differences between the two cell types. T h e fact that pS2 m R N A was not induced in adipose tissue indicate that ER is not present whereas expression of a functional ER is a requirement for the induction of ER-regulated genes.

Synthesis of c D N A and P C R c D N A was synthesized from total cellular R N A using oligo (dT) primers and M - M L V reverse transcriptase (BRL). A mix of 1 x M - M L V R T buffer ( 5 0 m M Tris-HC1, p H 8 . 3 , 7 5 m M KC1, 3 m M MgC12), 0.01 M D T T , 0.5 m M each of d A T P , d C T P , d G T P , and d T T P , 0.2/~g oligo (dT) and 200 U M M L V R T in 20/~1 were incubated at 37°C for 60 min. T h e P C R reaction was carried out in a reaction mix containing 1 × amplification buffer [ 1 . 5 m M MgC12, 0.2/~M each of d A T P , d C T P , d G T P , and d T T P , 0.2/~m of each primer, 1/~g c D N A and 2.5 U taq polymerase (Promega)], in a total volume of 100#1. T h e following P C R program was used for amplification of D N A ; 1 cycle of denaturation at 95°C for 2 min; annealing at 55°C for l m i n ; extension at 72°C for 2 min, followed by 29 cycles of denaturation at 95°C for 1 min; annealing at 55°C for 2 min; extension at 72°C for 2 min, and a final cycle of extension at 72°C for 7 m i n . For each P C R a control reaction with no addition of c D N A was carried out, excluding any contamination of foreign D N A in the samples. T h e sequence of each specific primer is shown in Table 1.

MCF7 (pS2)

Progesterone

---tb- Cortisol. < Z [-

501

Z

20 10 0

,

,

Molar Fig. 1. D o s e - r e s p o n s e c u r v e for pS2 m R N A in M C F 7 cells. MCF7 cells w e r e i n c u b a t e d with i n c r e a s i n g c o n c e n t r a t i o n s of p r o g e s t e r o n e , cortisol or p - e s t r a d i o l for 6 h , T N A was p r e p a r e d and solution h y b r i d i z a t i o n c a r r i e d out with a c R N A p r o b e for pS2 as described in M a t e r i a l s and M e t h o d s .

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300" <

250-

.~

200"

< Z

150"

Adipose tissue (MTIIA)

--It-- Progesterone --0-- Cortisol +

B-Estradiol

+

Control

---*-- B-Estmdiol

100" Adipose tissue (pS2)

50" 0

Oh

1

2h

4h

6h

1 h

2 h

Hours of incubation Fig. 2. T i m e c o u r s e c u r v e s o f p r o g e s t e r o n e , c o r t i s o l a n d f l - e s t r a d i o l a c t i o n o n M T I I A m R N A a n d pS2 m R N A l e v e l s in h u m a n a d i p o s e t i s s u e . H u m a n a d i p o s e t i s s u e w a s i n c u b a t e d w i t h 10 -6 M o f r e s p e c t i v e s t e r o i d , T N A prepared at indicated time intervals and solution hybridization carried out with cRNA probes for MTIIA and pS2, r e s p e c t i v e l y , as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s .

(10 5-10 6 M ) caused a 3-fold increase in M T I I A m R N A levels whereas only a 1.8-fold increase was M T I I A m R N A is known to be positively regulated observed in adipose tissue (P < 0.01) (Figs 2 and 3). No by both glucocorticoids, heavy metals and progesterone differences in basal expression of M T I I A m R N A were [17]. In M C F 7 cells, cortisol as well as progesterone observed between the two cell types. T h e reason for caused a dose-related increase of M T I I A m R N A . this cell-specific difference in M T I I A induction is not Figure 3 shows a typical dose-response curve where understood and has to be further evaluated. both cortisol and progesterone caused a maximal As shown in Fig. 2, cortisol at a concentration of inductive effect on M T I I A at a concentration of 1 0 6 M clearly induced the expression of M T I I A 10 5 - 1 0 - 6 M . T h e time course of cortisol and m R N A , whereas no effect was obtained with either progesterone action on M T I I A m R N A levels showed fl-estradiol or progesterone at the same concentration. a maximal effect 6-7 h after onset of treatment (data not It is known that due to differences in receptor affinity shown) and based on these data, cells were treated with for different steroids progesterone has to be added in each specific steroid for 6 h before T N A preparation. significantly higher concentrations as compared to No induction of M T I I A was observed after treatment glucocorticoids in order to obtain an effect on gene of M C F 7 cells with fl-estradiol (Fig. 3). Treatment of MCF7 cells with cortisol transcription via G R [17]. W h e n adipose tissue was incubated with progesterone, however, at a concentration of 10 3 - 1 0 - 4 M (data not shown), the m R N A levels for M T I I A increased 1.2-1.3-fold. This concenMCF 7 tration of progesterone is significantly higher than that Progesterone (MTIIA) needed for the induction of M T I I A m R N A in M C F 7 + Cortisol. < cells and probably reflects the fact that high concenZ trations of progesterone bind, with poor affinity, to the iG R to stimulate transcription.

Expression of M T I I A mRNA

:1

X

Amplification of ER, PR, and Gr cDNA in M C F 7 and adipose tissue

200 1

Molar Fig. 3. D o s e - r e s p o n s e c u r v e s f o r M T I I A m R N A in M C F 7 cells. M C F 7 cells w e r e i n c u b a t e d w i t h i n c r e a s i n g c o n c e n t r a t r i o n s o f p r o g e s t e r o n e , c o r t i s o l a n d 18-estradiol f o r 6 h, T N A p r e pared and solution hybridization carried out with a cRNA p r o b e f o r M T I I A as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s .

As indicated in Fig. 4, P C R products of ER and PR in M C F 7 cells, were easily detected after 30 cycles of amplification. All P C R products were of expected sizes corresponding to sequences indicated by selected primers in T a b l e 1. In adipose tissue amplification of an equal amount of c D N A ( m R N A ) resulted in small, barely detected amounts of P C R products for both ER and PR (Fig. 4). N o P C R products were seen in the control samples ( = n o c D N A added) (Fig. 4). G R c D N A , however, was easily amplified in both adipose tissue and M C F 7 cells (Fig. 5). All P C R reactions were

Steroid Receptors in Adipose Tissue PCR ER; PR

MCF 7 Std

ER

C

a significant M T I I A - i n d u c t i o n in M C F 7 cells were observed, did not increase M T I I A m R N A expression levels, thus indirectly indicating the lack of PR gene expression. However, at supraphysiological concentrations of progesterone a minor increase in M T I I A m R N A was observed. This effect on gene transcription by progesterone is postulated to be mediated via binding of progesterone to G R and subsequently to glucocorticoid and/or progesterone response elements [20]. Consequently, the specificity of hormone response in a

Ad PR

C

ER

C

279

PR

t~

200bp

245 210

PCR GR

~

<

I / Fig. 4. P C R p r o d u c t s o f e s t r o g e n (ER) and p r o g e s t e r o n e (PR) r e c e p t o r s f r o m adipose tissue (Ad) a n d MCF7 cells. The e t h i d i u m b r o m i d e - s t a i n e d 1% agarose gel shows t h a t P C R a m p l i f i e d ER a n d P R cDNA using p r i m e r s p r e s e n t e d in Table 1, yielded the e x p e c t e d 245 and 210bp f r a g m e n t s , respectively. C o n t r o l P C R r e a c t i o n s with no t e m p l a t e a d d e d (C) and a DNA m o l e c u l a r weight m a r k e r , phiX174 (Std), are indicated. 600bp

optimized according to the highest annealing temperature generating a P C R product. DISCUSSION In these studies we examined the relationship between the cellular ER, PR and G R m R N A content and the steroid response. T h e study was prompted by evidence that estrogens and progesterone have documented effects in human adipose tissue in spite of undetectable receptor levels [8, 18] and that there are substantial data supporting important roles for these steroids in the regulation of human adipose tissue metabolism, morphology and distribution [19]. In the ER- and PR-positive cell line M C F 7 in contrast to human adipose tissue, the ER-regulated gene pS2 displayed a classical dose-response curve when the estradiol responses were compared with increasing concentrations of fl-estradiol. T o our knowledge, there is no gene specifically regulated by progesterone. T o indirectly address the question of PR expression in human adipose tissue we therefore investigated the induction of the M T I I A gene, which is known to be positively regulated by both progesterone and glucocorticoids via PR and GR, respectively [17]. In adipose tissue, progesterone at concentrations where SBMB 51/5 6--D

Fig. 5. P C R p r o d u c t s of the GR f r o m h u m a n adipose tissue (Ad) and MCF7 cells. The e t h i d i u m b r o m i d e - s t a i n e d 1% agarose gel show t h a t P C R a m p l i f i e d GR cDNA using p r i m e r s 5 a n d 6 (Table 1), yielded the e x p e c t e d 600bp f r a g m e n t . C o n t r o l P C R r e a c t i o n s with no t e m p l a t e a d d e d (C) and a DNA m o l e c u l a r weight m a r k e r , phil74 (Std), are indicated.

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given tissue is dependent, in part, upon the expression of a particular receptor in that tissue. Previous studies have shown that the degree of glucocorticoid response of a defined target gene is linearly related to the number of ligand-bound GRs [21]. It has also been shown that receptor action at low receptor titers is limited by binding of receptor to its response element and that estrogen response element is probably markedly undersaturated by receptor at physiological conditions [22]. Similar studies have not been carried out for PR. Therefore, extremely low ER expression levels would not generate any cellular response in terms of increased transcription of ERregulated genes. The question is then why ER and PR are not expressed in human adipose tissue. Our results indicate a critical level of m R N A for translation to take place and raise the question of the biological significance of very low and by generally used techniques undetectable m R N A levels. Price and O'Brien [23] recently demonstrated ER m R N A levels in human adipocytes as well as in adipose stromal cells by PCR. Adipose stromal cells as well as adipocytes contain significant concentrations of estrogens and it is possible that the ER-gene is down-regulated. If this is the case, it contrasts well established actions of estrogens in breast tissue where female sex steroids up-regulate both ER and PR [24]. The second question is then how and if there are any effects of estrogens and/or progesterone in human adipose tissue. It is possible that steroid (e.g. progesterone) metabolites may act by way of "orphan" receptors, which are members of the steroid receptor superfamily [25]. Such a possibility has been suspected for a long time but not until recently been considered again to explain high affinity binding of 5-dihydroprogesterone in some tissues [26]. It is also established that female sex steroids per se evoke biological responses by way of non-genomic processes [4, 5]. These effects are often mediated through the release of C a 2 + from intracellular stores and probably triggered by inositol 1,4,5triphosphate generated by a steroid receptor-induced hydrolysis of membrane phosphatidyl-inositol 4,5bisphosphate[ 27]. In summary, we have presented additional evidence indicating the absence of ER and PR from human adipose tissue based on the following findings; (i) failure to induce the ER-regulated gene pS2 by fl-estradiol and (ii) failure to induce the Gr and PR-regulated gene M T I I A by progesterone at physiological concentrations. Further studies of the effects on human adipose tissue metabolism by steroid hormones are needed in order to clarify and explain these issues. Acknowledgements--This study was supported by grants from the Swedish Medical Research Councd (19X-9941, 09522 and 09743), Karolinska Institute, Harald and Greta Jeansson, and Ake Wibergs foundations. M.B. is a research fellow of the Swedish Medical Research Council. T h e authors wish to thank M s Birgitta Oden for excellent technical assistance.

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