Progesterone Inhibits Glucocorticoid-Dependent Aromatase Induction in Human Adipose fibroblasts
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401 Progesterone inhibits glucocorticoid-dependent aromatase induction in human adipose fibroblasts M Schmidt, C Renner and GLo¨ ffler Institut fu¨ r Biochemie, Genetik und Mikrobiologie, Universita¨t Regensburg, D-93040 Regensburg, Germany (Requests for offprints should be addressed to M Schmidt, LS Biochemie III, Institut fu¨ r Biochemie, Genetik und Mikrobiologie, Universita¨t Regensburg, D-93040 Regensburg, Germany) Abstract In fibroblasts derived from human adipose tissue, aro- whereas progesterone at near-physiological concentration matase induction is observed after exposure to 1 µM (100 nM) does not inhibit aromatase activity in isolated cortisol in the presence of serum or platelet-derived microsomes. Semi-quantitative reverse transcriptase PCR growth factor (PDGF). Progesterone suppresses this induc- analysis shows reduced amounts of aromatase mRNA tion in a dose-dependent manner, 10 µM resulting in in progesterone-treated cells, indicating specific inhibition complete inhibition. A reduced cortisol concentration of the glucocorticoid-dependent pathway of aromatase (0·1 µM) concomitantly reduces the progesterone concen- induction. The inhibitory effect of progesterone is not tration required for effective inhibition (10–100 nM). This blocked by the anti-progestin ZK114043, excluding action effect of progesterone is specific, as neither the release of via progesterone receptors and indicating competition cellular enzymes nor aromatase induction by dibutyryl- for the glucocorticoid receptor. Progesterone must be con- cAMP, which acts independently from cortisol, are sidered a potential physiological inhibitor of glucocorticoid- affected. However, the inhibitory effect of progesterone dependent aromatase induction in adipose tissue. It is requires its presence throughout the induction period. proposed that it is a suppressor of aromatase induction in Kinetic studies in intact cells reveal a reduced number adipose tissue in premenopausal women. of aromatase active sites upon progesterone treatment, Journal of Endocrinology (1998) 158, 401–407 Introduction ticoid action is a prerequisite for estrogen production in adipose tissue. At present, there is little knowledge The enzyme aromatase is responsible for the production of about physiological mechanisms that may inhibit the estrogens from androgenic precursors (for review see glucocorticoid-dependent pathway of aromatase induction Simpson et al. 1989, 1994). In premenopausal women it in adipose fibroblasts. However, the increased rates of is found mainly in the ovaries and during gestation in the estrogen synthesis in adipose tissue from older (Hemsell placenta (Meyer 1955, Richards 1994). In men and et al. 1974) or obese (MacDonald et al. 1978) patients could postmenopausal women, estrogens are produced mainly in be caused by either the enhanced action of inducers or a fibroblasts from adipose tissue (Simpson et al. 1994). They loss of inhibitors in these patients. contain considerable aromatase activity, but its regulation We report here that progesterone inhibits the is only partially understood. Glucocorticoids are powerful glucocorticoid-dependent, but not the cAMP-dependent, inducers of aromatase, but their action depends on the pathway of aromatase induction in human adipose fibro- presence of serum (Simpson et al. 1981) or serum-derived blasts in vitro. We provide evidence that this inhibition growth factors like platelet-derived growth factor (PDGF) is at the transcriptional level and that it results from (Schmidt & Lo¨ffler 1994). Dibutyryl-cAMP (Bu2cAMP) the interaction of progesterone with the glucocorticoid is also able to induce aromatase, but this effect is inhibited receptor. by serum or serum-derived growth factors (Mendelson et al. 1982, 1986). Gonadotropins, which stimulate estro- Materials and Methods gen production in ovaries, have no influence on estrogen production in adipose fibroblasts (Simpson et al. 1989). Aromatase activity in vivo in adipose tissue from healthy Materials donors depends on transcription of the aromatase gene Cell culture media, fetal calf serum (FCS), antibiotics, starting from glucocorticoid-dependent promotors (Harada trypsin solution and collagenase were purchased et al. 1993, Mahendroo et al. 1993). Therefore glucocor- from Biochrom (Berlin, Germany), BSA from Biomol Journal of Endocrinology (1998) 158, 401–407 1998 Society for Endocrinology Printed in Great Britain 0022–0795/98/0158–0401 $08.00/0 Downloaded from Bioscientifica.com at 09/25/2021 03:10:29PM via free access 402 M SCHMIDT and others · Progesterone inhibits aromatase induction (Hamburg, Germany), HEPES, biotin, Norit A, dextran the kinetic experiments), was added 6 h before the in- and Serva Blue G-250 from Serva (Heidelberg, Germany), cubation was terminated. Incorporation of 3H into and cortisol, progesterone, transferrin and pantothenate H2O was measured essentially as described previously from Sigma (Deisenhofen, Germany). Recombinant (Ackerman et al. 1981). Aromatase activity is given as pmol human PDGF-BB was obtained from BTS (St Leon-Rot, testosterone used/6 h per mg protein calculated as de- Germany). [1â,2â-3H]Testosterone was from NEN/ scribed by Cole & Robinson (1990) and experimentally DuPont (Dreieich, Germany). RNeasy columns came confirmed for our assay in the laboratory. Whole cell from Qiagen (Hilden, Germany), RNasin from Promega protein was determined by an improved protocol of the (Mannheim, Germany), Moloney murine leukemia virus method of Bradford (1976), as described by Peterson (MMLV) reverse transcriptase (RT) from Amersham (1983). (Braunschweig, Germany), Taq DNA polymerase from PAN Systems (Nu¨rnberg, Germany) and nylon mem- branes from Appligene (Heidelberg, Germany). All other Aromatase assay using placental microsomes molecular biology reagents, including the DIG labeling The placental microsomal preparation and the microsomal and detection systems were from Boehringer-Mannheim assay were carried out as described by Kellis & Vickery (Mannheim, Germany). Insulin was a gift from Dr Brocks, (1987). Farbwerke Hoechst AG (Frankfurt, Germany). ZK114043 was kindly provided by Dr Parczyk, Schering AG (Berlin, Germany). All other reagents were of analytical grade and Assay of lactate dehydrogenase and alkaline phosphatase were obtained from Merck (Darmstadt, Germany). activities Lactate dehydrogenase activity was determined by the procedure of Bergmeyer & Bernt (1974), and alkaline Cells and cell culture phosphatase activity as described by Walter & Schu¨tt Human adipose tissue for the preparation of adipose (1974). After removal of the supernatant for determination fibroblasts was obtained during plastic breast surgery from of aromatase activity, the cell layers were washed with PBS healthy women. The donors gave informed consent and then lysed in 1 ml PBS by sonification. The lysates according to a protocol, which was approved by the were centrifuged (13 500 g; 5 min) and directly assayed for Ethikkommission of the University of Regensburg. Cells enzyme activity and cellular protein content. were isolated as described (Schmidt & Lo¨ffler 1997) and were subcultivated after treatment with 0·25% trypsin in PBS; the detached cells were diluted with medium and RT-PCR analysis of aromatase expression placed in 24-well plates or 100-mm dishes (for RNA Cells were grown in 100-mm dishes and treated in parallel isolation) and grown as described previously (Schmidt & to the cells used for the aromatase assay. RNA was isolated Lo¨ffler 1997). For pretreatment before aromatase induc- via RNeasy affinity columns following the manufacturer’s tion, the confluent monolayers were washed free of serum instructions. For RT-PCR, 1 µg total RNA was reverse- constituents and incubated for 48 h with serum-free transcribed (1 h/37 )C) using 200 U MMLV RT and medium (SF-medium), with replacement of the medium subsequently amplified with 2·5 U Taq DNA polymerase after 24 h. SF-medium consisted of Dulbecco’s modified for the given number of cycles (1 min/94 )C, 1 min/ Eagle’s medium and Ham’s F12 medium in a ratio of 3:1 64 )C, 30 s/74 )C) using the oligonucleotides 5*-ttatgagg (both without phenol red) supplemented with penicillin agcatgcggtacc (forward) and 5*-tagtgttccagacacctgtc (100 U/ml), streptomycin (0·1 mg/ml), transferrin (2 µg/ (reverse). After agarose gel electrophoresis and blotting by ml), pantothenate (17 µM), biotin (1 µM) and insulin standard protocols (Sambrook et al. 1989), the PCR (1 nM). Culture conditions included a humidified products were hybridized with 100 ng of a digoxigenin- atmosphere with 5% CO2 at a temperature of 37 )C. labeled probe corresponding to the amplified 432 bp cDNA fragment covering parts of exons 9 and 10 of the aromatase gene and detected using the DIG luminescent Induction of aromatase and assay of aromatase activity detection system according to the manufacturer’s instruc- After the pretreatment phase, aromatase activity in adipose tions. The identity of the cloned fragment used for the tissue stromal cells was induced by supplementation of generation of the probe was confirmed by sequencing. SF-medium with cortisol and either 10% FCS or 0·5 nM PCR products were quantified by means of a video PDGF-BB. Alternatively, Bu2cAMP (1 mM) was used as densitometer. inducer in the absence of cortisol and serum. Progesterone and other antagonists were given immediately before induction was started. The total time of induction was Statistical analysis 24 h. The substrate for the aromatase enzyme, [1â,2â- Comparative analyses of aromatase activities between