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Original Article |
-Reductase Activity Compatible with Androgen Inactivation
Laboratories of Ontogeny and Reproduction (P.R.P., R.D., N.F., Y.T.), Centre Hospitalier Unìversitaire de Québec, Pavillon Centre Hospitalier de l’Université Laval, Québec, Canada G1V 4G2; Department of Obstetrics and Gynecology (C.H.B.), HealthPartners Regions Hospital, St. Paul, Minnesota 55101; and Department of Obstetrics and Gynecology and Centre de Recherche en Biologie de la Reproduction (Y.T.), Faculty of Medicine, Laval University, Québec, Canada G1V 4G2
Address all correspondence and requests for reprints to: Dr. Yves Tremblay, Laboratory of Ontogeny and Reproduction, Room T-1–58, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, 2705 Laurier Boulevard, Québec, Canada G1V 4G2. E-mail: . yves.tremblay{at}crchul.ulaval.ca
Abstract
Androgens delay lung maturation through their action on lung fibroblasts. Knowing that testosterone is secreted by the lung epithelial-like cell line A-549, we have studied the metabolism of androgens by several human lung diploid fibroblasts cell lines. No 17-ketosteroid reductase activity was detected. In contrast, testosterone was transformed mainly into androstenedione and androstanedione with no 5
-dihydrotestosterone formed, indicating the presence of 17ß- hydroxysteroid dehydrogenase (HSD) type 2 and 5-reductase activities. The eight cell lines analyzed had either a low or high 17ß-HSD type 2 activity level. No correlation between these levels and the sex or age stage of cells was established, but Northern blot analysis of human lung RNA samples of five adult subjects revealed very similar variations between subjects in the level of 17ß-HSD type 2 mRNA. The 5-reductase activity had a marked substrate preference for androstenedione, the product of 17ß-HSD type 2. When tritiated testosterone was used as substrate, only barely detectable levels of 5-dihydrotestosterone were observed by HPLC in the presence of the 17ß-HSD type 2 inhibitor EM-919. The use of unlabeled testosterone or of the antiandrogen hydroxyflutamide demonstrated that the tritiated testosterone substrate itself had no effect on levels of 5-reduction. In fact, in these cells, 5-reductase has no significant activity on testosterone, but it further converts the product of 17ß-HSD type 2, thus playing a role with 17ß-HSD type 2 in androgen inactivation. Because androgens delay lung maturation and surfactant synthesis by their action on lung fibroblasts, it is of particular interest to find that the steroid metabolism of these lung fibroblast cells is oriented toward androgen inactivation. Because lung fibroblasts of subjects with low 17ß-HSD type 2 expression levels are likely to be exposed to higher levels of androgens, an allelic variation of the 17ß-HSD-2 gene is suspected, which would result in familial incidence of respiratory distress. This is in line with reported cases of familial incidence of respiratory distress.
DURING LUNG DEVELOPMENT there is pressure by positive and negative regulators of fibroblast-epithelial communication in the control of epithelial type II (PTII) cell maturation that will end by the formation of surfactant synthesis by PTII cells (1). An improper regulation of the factors that govern these processes can delay surfactant production (2), thus increasing the risk for respiratory distress (RD), which is more frequent in male infants (3, 4). The risk for RD increases with the degree of prematurity of the baby, and, interestingly, this condition is more frequent in male than in female infants. Some growth factors such as epithelial growth factor or TGFß1 antagonize lung maturation processes (5, 6), whereas other factors such as the stromal-derived growth factor neuregulin-1 stimulates surfactant synthesis, as seen in the mouse cell line MLE12 (7). Steroids are known to take part in regulating the processes. For example, glucocorticoids stimulate fibroblast/PTII cells communication, which in turn will stimulate production of pulmonary surfactant by PTII cells. In vitro, exposure of fibroblasts to androgens decreased their ability to stimulate the production of surfactant by PTII cells through fibroblast/PTII cell communication (8). Current findings indicate that these steroidal effects are mediated through binding of androgens to androgen receptors (9, 10), which are present in both male and female lung tissues (11).
Studies of laboratory animals also indicate that androgens play a role of negative modulator on surfactant production. When 5-dihydrotestosterone (5-DHT) is administered to pregnant females, surfactant production in the lung of female fetuses is decreased to the levels of male fetuses from untreated pregnant females (9, 12). Moreover, male mice carrying the Tfm gene (male with testicular feminization), which have no functional androgen receptors, have surfactant levels comparable with that of the normal female at comparable developmental time point (13). All these results suggest that the sex difference in surfactant production is related to the metabolism of androgens. In addition, these results obtained in vivo validate the in vitro models in which androgens negatively modulate surfactant production.
Because testes do not secrete testosterone during late gestation, how could androgens modulate lung maturation at that time? In situ metabolism of androgens by the lung itself can answer this question. Tissue- and cell-specific expression of steroidogenic enzymes allows for local formation of sex steroids from circulating C19 steroid precursors of adrenal origin, namely, dehydroepiandrosterone (DHEA) and androstenedione (14). 17ß-Hydroxysteroid dehydrogenase (17ß-HSD) exists in multiple isoforms, each encoded by a specific gene and each having either a predominant reductase or a dehydrogenase activity in vivo. Thus, the presence of specific 17ßHSDs expressed in the cells will have as consequences to produce either active 17ß-hydroxyandrogens (types 1, 3, 5, or 7) or inactive 17-ketosteroids (types 2, 4, 6, or 8). Although testosterone can efficiently bind the androgen receptor, its 5-reduction by the 5-reductase leads to the formation of 5-DHT, the most potent active androgen.
Early studies report that 3-HSD and 17ß-HSD are the major enzymatic activities detected in lung slices, while low 5-reductase and 3ß-hydroxysteroid dehydrogenase/
5-4 isomerase (3ß-HSD) activities were also detected (15, 16, 17). Recently, we studied the steroidogenic capacity of the male lung adenocarcinoma epithelial cell line A-549. These are PTII-like cells that synthesize disaturated phosphatidylcholines like normal lung PTII cells do. We demonstrated that these cells produce active androgens from androstenedione. A-549 steroid metabolism involves 17ß-HSD type 5 and 3-HSD type 3 as predominant enzymes, and a low 5-reductase activity (Fig. 1
). Consequently, in this model the testosterone produced is stabilized, whereas 5-DHT is rapidly inactivated into androstane-3,17ß-diol. Therefore, androgens are thought to be produced by PTII cells in vivo, at least in the male lung.
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-DHT. The two enzymes that allow for this specific pattern of inactivation are 17ß-HSD type 2 for androgen inactivation and 5-reductase type 1, which has no significant activity on testosterone in these lung fibroblasts. In addition, these cell lines can be separated into two groups having high or low 17ß-HSD type 2 activity. Moreover, we also present a Northern blot study in which levels of 17ß-HSD type 2 mRNA also vary from subject to subject with either a high or a low expression profile. Thus, our results suggest the existence of an allelic variation in the 17ß-HSD type 2 gene that is likely to determine the predisposition of subjects to RD. Materials and Methods
Chemicals
The following reagents were purchased from NEN Life Science Products (Boston, MA): [1,2,6,7-3H]T (95.0 Ci/mmol), [1,2,6,7-3H]androstenedione (74.0 Ci/mmol), [1,2-3H]5-DHT (60 Ci/mmol), [1,2,6,7-3H]DHEA (100 Ci/mmol), [2,4,6,7-3H]estrone (114 Ci/mmol), and [6,7-3H]E2 17ß (51.0 Ci/mmol). HEPES, BICINE [N,N-bis(2-hydroxyethyl)glycine], nicotinamide adenine dinucleotide phosphate NAD(P) and its reduced form NAD(P)H, and BSA were obtained from Sigma (St. Louis, MO); fetal bovine serum (FBS) from Hyclone Laboratories, Inc. (Logan, UT); unlabeled steroids from Steraloids (Wilton, NH); and hydroxyflutamide and EM-919 from Drs. Fernand Labrie and Donald Poirier (Laboratoire d’Oncologie et d’Endocrinologie Moléculaire, Québec, Canada).
Cell cultures
CCD-32Lu, CCD-34Lu, CCD-34Sk, LL 24, MRC-5, and IMR-90 cells [respectively, CRL-1485, CRL-1491, CRL-1497, CCL-151, CCL-171, and CCL-186; American Type Culture Collection (ATCC), Manassas, VA] were grown in MEM with Earle’s buffered salt solution containing 1.5 g/liter NaHCO3, nonessential amino acids, 10% (vol/vol) FBS [except LL 24; without nonessential amino acids and with 15% (vol/vol) FBS]. For MRC-5 and IMR-90, 1.0 mM sodium pyruvate was added as recommended by ATCC. Hs 907.Lu, FHs 738Lu, and Hs 389(B).Lu cells (respectively, CRL-7657, HTB-157, and CRL-7266, ATCC) were cultured in DMEM containing 25 mM HEPES, 4.5 mg/liter glucose, and 3.7 g/liter NaHCO3, and supplemented with 10% (vol/vol) FBS according to ATCC specifications. Penicillin (50 IU/ml) and streptomycin (50 µg/ml) were added in all media. Serum complement was heat inactivated at 56 C for 20 min. All cell lines were cultured at 37 C in 5% CO2 atmosphere.
Steroid metabolism in intact cells
Cells (50,000 to 105) were incubated in 24-well plates with 1 ml of the appropriate culture media containing either tritiated testosterone (2 x 106 dpm; 9.6 nM) or androstenedione (2 x 106 dpm; 12.3 nM) for the indicated time periods. When indicated, FBS was treated with dextran-coated charcoal as described (18) to remove steroids and other small molecules. At the end of incubation, culture media were frozen until steroid extraction, and 100 µl methanol were added to each well to fix cells before determination of DNA amounts using 3,5-diaminobenzoic acid as described (19, 20). Steroids were extracted twice with 5 ml diethylether, evaporated, and applied to silica gel-coated plates and resolved in toluene-acetone (4:1, vol/vol). Plates were revealed using a Storm apparatus (Molecular Dynamics, Inc., Sunnyvale, CA) to localize the radioactive signals then spots were scraped and disintegrations per minute were determined by scintillation counting (model LS3801, Beckman, Mississauga, Ontario, Canada). Values in disintegrations per minute were used to determine the percentage of each steroid. Amounts of femtomoles of substrate and products were calculated using these percentage values and total amount of steroid added per well. Each time point was assayed in triplicate and values were normalized per microgram of DNA. Rates of conversion were determined from the average of three slopes (femtomoles per microgram per hour). Enzymatic activities were calculated using the following percentage values: 17ß-HSD type 2, [(androstenedione + androstanedione + androsterone (if any))/total steroid] x 100; 5-reductase, [(androstanedione + androsterone + 5-DHT (if any))/total steroid] x 100.
HPLC analysis
Samples of 5-DHT (see Fig. 8) were analyzed by HPLC. The very weak spots corresponding to the positions of 5-DHT on silica gel-coated plates were scraped and samples of triplicate were pooled to increase sensitivity. Samples were extracted twice with 5 ml diethylether, dried, and resuspended in 125 µl of mobile phase (H2O/acetonitrile/tetrahydrofurane 65:23:12, vol/vol/vol). Samples were run on an Ultrasphere ODS column (4.6 x 150 mm). Tritiated testosterone, 5-DHT, and androsterone were used as standards.
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Cells were homogenized in ice-cold buffer containing 20% (vol/vol) glycerol, 1.0 mM EDTA, and 4 mM potassium phosphate (pH 7.0). Samples were homogenized by hand in an all-glass Dounce homogenizer fitted with a B-pestle, centrifuged at 1,000 x g for 10 min to remove cell debris and again at 105,000 x g for 60 min. Supernatants were saved as cytosol. The microsome-enriched pellets were washed, subjected to a second round of ultracentrifugation, suspended in buffer, and saved as microsomes. Completeness of homogenization was assessed by light microscopy, as previously described (18). Protein was measured by the method of Markwell et al. (21) with BSA as the standard.
Enzymatic assays on cytosol and microsomes
17ß-HSD, 3ß-HSD, and 3-HSD were assayed as previously described (18, 22, 23). Briefly, a 10-µl aliquot of cytosol or microsomes was combined with 10 µl reaction mixture containing 1.0 mM nicotinamide nucleotide cofactor, 2.0 µM [3H]-labeled E2, testosterone, DHEA, or 5-DHT in 0.08 M BICINE [N,N-bis(2-hydroxyethyl)glycine] (pH 9.0), for dehydrogenase activities, or 2.0 µM [3H]-labeled E1 (estrone, 1,3,5-estratrien-3-ol-17-one), androstenedione, DHEA, or 5-DHT in 0.08 M HEPES (pH 7.2), for reductase activities. Assays were run at 37 C. Reaction mixtures were transferred in total to the preadsorbant layer of silica gel HL plates and fractionated into substrate and product by TLC with benzene:acetone (4:1, vol/vol) as the solvent. Following TLC, substrate and products were localized by a light misting with water and scraped into 10 ml Ecolume for liquid scintillation counting. Percentage conversion was calculated as the ratio of counts per minute in product/counts per minute in substrate plus product. This value was converted to picomoles per milligram protein per 30 min as previously described (24).
RNA extraction and Northern blot analyses
Total RNA was extracted from 4 x 106 cells using Tri-reagent, a mixture of phenol and guanidine thiocyanate in a monophasic solution (Molecular Research Center, Cincinnati, OH) as described (22). RNA (15 µg) was glyoxalized, resolved by 1.2% (wt/vol) agarose gel electrophoresis, and transferred to Hybond-XL membrane (Amersham/Pharmacia Biotech, Baie d’Urfée, Québec, Canada). Membranes were successively hybridized with the following cDNA probes: full-length 17ß-HSD type 2 fragment (22); 5-reductase type 1 (25); 5-reductase type 2 (26); and 
-actin 2-kb segment (27). All of the probes were labeled by random priming (28). Hybridizations were performed under high stringency conditions (50% formamide, 42 C), and membranes were washed in 1x saline sodium citrate, 0.1% SDS at 60 C (1x saline sodium citrate: 150 mM NaCl, 15 mM CH3COONa). Relative RNA amounts were determined directly from membranes using a Strom apparatus and the ImageQuant software (both from Molecular Dynamics, Inc., Sunnyvale, CA), or autoradiographs were quantified by densitometric scanning (BioImage Visage 110s, Genomic Solution; RMLuton, Jackson, MI) with the Whole Band Analysis software (WBA; RMLuton).
Results
Steroid metabolism by intact CCD-32Lu and Hs 907.Lu lung fibroblast cell lines
Two human lung fibroblast cell lines, namely CCD-32Lu and Hs 907.Lu, were first selected to initiate the study of steroid metabolism. These cell lines were established from male and female neonates, respectively. To study the capacity of these cells to produce androgens from steroid precursors present in the circulation, intact cells in culture were exposed to tritiated DHEA and androstenedione for 24 h. Barely detectable levels of androstenediol and testosterone were respectively obtained, indicating that there was no or very low 17-ketosteroid reductase activity in these cells (data not shown). In CCD-32Lu cells, low but significant levels of newly formed androstenedione were detected with tritiated DHEA, which is indicative of 3ß-HSD activity, and barely detectable amounts were observed for Hs 907.Lu. For both cell lines, more than 96% of the DHEA used as substrate was recovered after a 24-h incubation.
The capacity of fibroblast cells to metabolize testosterone was also evaluated. After a 24-h incubation, the major product was androstenedione (Fig. 2A). A small amount of androstanedione was detected and no 5-DHT was observed. These results show that the major steroidogenic activity in these lung fibroblast cells is a 17ß-HSD type 2-like activity and that 5-reductase activity is also present. Interestingly, a time course study using values normalized per microgram of DNA revealed that the oxidative 17ß-HSD activity was clearly higher in CCD-32Lu, compared with Hs 907.Lu (Fig. 2B). Using tritiated 5-DHT as substrate, 3-HSD activity was also detected in both cell lines (data not shown).
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17ß-HSD, 3ß-HSD, and 3-HSD activities in CCD-32Lu and Hs 907.Lu cytosol and microsomes
To better characterize the steroidogenic enzymes present in these lung fibroblast cells, the substrate and cofactor preferences of the 17ß-HSD and other steroidogenic activities were studied in cell extracts of CCD-32Lu and Hs 907.Lu cells. As shown in Fig. 3, 17ß-HSD specific activity levels were higher in CCD-32-Lu cells than in Hs 907.Lu cells. Dehydrogenase activity with E2 and testosterone exceeded reductase activity with E1 and androstenedione and the highest specific activities were associated with microsomes. Nicotinamide adenine dinucleotide (NAD) and its reduced form (NADH) were the preferred cofactors. With regard to their potential for DHEA metabolism, CCD-32Lu cells also had the highest level of activity (Fig. 4, A and B). The principal activity was microsomal conversion of DHEA to androstenediol with NADH as the preferred cofactor. Putative 3ß-HSD activity based on the conversion of DHEA to androstenedione was at the limit of detection. 5-DHT was converted to androstanedione and 3,17ß-diol with NAD and NADH as the preferred cofactors by both cells types (Fig. 4, C and D).
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Higher 17ß-HSD type 2 activity in CCD-32Lu, compared with Hs 907.Lu, is suggestive of sex-specific levels of expression. The effect of the sex or age stage of cells on levels of steroidogenic activities was addressed. Steroid metabolism in eight human lung fibroblast cell lines was compared. Characteristics of these cell lines are presented in Table 1. Cells were incubated in the presence of tritiated testosterone or androstenedione and levels of 17ß-HSD type 2 and 5-reductase activities were respectively determined. Although 5-reductase activity with androstenedione was very similar in all cell lines, 17ß-HSD type 2 activity showed large variations (Fig. 5). In fact, the lung fibroblast cell lines can be divided into two groups, the first having a very low 17ß-HSD type 2 activity (Hs 907.Lu, Hs 389(B).Lu, and LL-24) and the second presenting a strong 17ß-HSD type 2 activity, significantly higher than the 5-reductase activity estimated with [3H]-androstenedione (CCD-32Lu, MRC-5, IMR-90, FHs-738Lu, and CCD-34Lu). Because the relative level of these two activities may influence the fate of testosterone entering in the cell, the 17ß-HSD type 2 to 5-reductase activity ratio was calculated (Table 1). No correlation between the 17ß-HSD type 2 to 5-reductase ratio and either the sex or age stage of cells could be established.
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and Table 1). For all experiments, cells were cultured under conditions recommended by ATCC, which differ slightly between cell lines. To exclude the culture conditions as the cause of the observed variations of 17ß-HSD type 2 activity, each of the CCD-32Lu, MRC-5, Hs 907.Lu, and Hs-389(B).Lu cell lines was cultured under both the CCD-32Lu (high 17ß-HSD type 2 activity) and the Hs 907.Lu (low activity) culture conditions. The variability of the 17ß-HSD type 2 activity between cell lines was reproduced under each culture conditions (data not shown).
Substrate preference of the 5-reductase in lung fibroblast intact cells
The two substrates for 5-reductase enzyme are the substrate (testosterone) and the product (androstenedione) of the 17ß-HSD type 2 enzyme. Consequently, in the presence of 17ß-HSD type 2, the value obtained for 5-reductase activity may vary, depending on the tritiated substrate used, if the 5-reductase has a substrate preference in intact cells. To address this issue, the 5-reductase activity of the fibroblast cell lines was determined using both androstenedione and testosterone as substrates. The first observation of interest, shown in Fig. 6, is that the rate of 5-reduction is different, depending on the substrate used. This occurs in all of the analyzed cell lines. Our data indicate that the preferred substrate is androstenedione, which was converted, depending on the cell line, from 1.5- to 26.2-fold more efficiently than testosterone. The second observation is that the difference in the rate of 5-reduction for the two substrates is higher in cell lines with a low 17ß-HSD type 2 activity, compared with cells with a high 17ß-HSD type 2 activity. This suggests that the 5-reductase activity value estimated from [3H]-testosterone in cell lines with a high 17ß-HSD type 2 activity is overestimated because of conversion of testosterone into androstenedione.
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-reductase gene caused by the added tritiated testosterone, fibroblast cells were incubated with tritiated androstenedione in the presence of unlabeled testosterone and tritiated androstenedione or testosterone in the presence of the antiandrogen hydroxyflutamide. The 5-reductase activity was greater with androstenedione and was not affected by the presence of hydroxyflutamide or unlabeled testosterone (Fig. 7). To determine whether 5-DHT is produced by the 5-reductase expressed in lung fibroblast cells, CCD-32Lu cells were incubated with the 17ß-HSD type 2 inhibitor EM-919 (29). This inhibitor had no effect on the 5-reduction of [3H]-androstenedione (Fig. 8A), but it completely abolished the 17ß-HSD type 2 activity with [3H]-T (Fig. 8B). HPLC analysis clearly showed that 5-DHT was produced from [3H]-T in CCD-32Lu cells but only at very low levels. However, the nearly complete inhibition of 17ß-HSD type 2 activity clearly failed to increase the 5-reduction of [3H]-T to the levels observed with [3H]-androstenedione. In fact, the levels of 5-DHT synthesized from [3H]-T in the absence of 17ß-HSD type 2 activity was at least 65-fold lower then levels of androstanedione synthesized from [3H]-androstenedione.
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-reductase mRNA levels in lung fibroblast cell lines
Northern blot analyses were performed with RNA from fibroblast cell lines using 17ß-HSD type 2, 5-reductase type 1, 5-reductase type 2, and actin cDNA probes. RNA from the lung epithelial type II-like cell line A-549 was included for comparison purposes. The 17ß-HSD type 2 gene is expressed by the fibroblasts but not by the A-549 cells (Fig. 9A). The 5-reductase type 1 cDNA probe produced a hybridizing signal for all the analyzed fibroblast cell lines and A-549 cells at quite similar levels as estimated after normalization with actin expression levels (Fig. 9C and data not shown). The 5-reductase type 2 probe was negative for all cell lines (data not shown). A linear relationship was found between 17ß-HSD type 2 mRNA and activity levels for most of the lung fibroblast cell lines (Fig. 9B). Exceptions are the CCD-32Lu cells, which have a very high mRNA level relative to the activity, and the IMR-90, which exerted the higher activity to mRNA ratio.
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Samples of total RNA isolated from lung of five adult humans were tested for the presence of 17ß-HSD type 2 mRNA. This transcript was found in all of the lung tissues (Fig. 10). Interestingly, subjects can be divided into two groups with low (1-fold) or high (3.3- to 4.7-fold) 17ß-HSD type 2 mRNA levels (Fig. 10).
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We had recently reported that the lung type II-like epithelial cell line A-549 expresses a pattern of steroid metabolism in which 17ß-HSD type 5 and 3-HSD type 3 are the predominant enzymes. As a result, these cells converted androstenedione into testosterone, which was detected in the culture media. In contrast, 5-DHT was rapidly converted into 3,17ß-diol and did not accumulate in the medium (18). We now report that normal human lung fibroblast cell lines have an opposite pattern of steroid metabolism. Indeed, lung fibroblasts inactivate active androgens into their 17-ketosteroid counterparts through expression of the 17ß-HSD type 2 gene. Knowing that androgens modulate fibroblast/epithelial cell communication leading to cell differentiation and the beginning of surfactant production during pregnancy, our findings of an opposite steroid metabolism in the two cell types is of particular interest.
Although the study of steroid metabolism in intact cells revealed the presence of 17ß-HSD dehydrogenase activity (active androgens 
17-ketosteroids) higher in CCD-32Lu cells than in Hs 907.Lu cells, the study of steroid metabolism following cell fractionation into cytosol and microsomes has allowed to define the biochemical characteristics of the relevant enzyme. CCD-32Lu and Hs 907.Lu cells were assayed for 17ß-HSD activity with E2, E1, testosterone, and androstenedione. The observations that activity was significantly higher in CCD-32Lu cells, the highest specific activities were microsomal, and the E2 to testosterone activity ratio was approximately 2 are consistent with the presence of 17ß-HSD type 2 (24). That NAD and NADH were the preferred cofactors is also characteristic of the type 2 isoform. The microsomal conversion of DHEA to androstenediol and 5-DHT to androstanedione with NADH and NAD as the preferred cofactors, respectively, is also consistent with the presence of 17ß-HSD type 2. All these observations concur with results from Northern blot analyses showing higher levels of the 17ß-HSD type 2 mRNA in CCD-32Lu cells than in Hs 907.Lu cells. Other steroidogenic activities were also detected in the cell fractions. The conversion of DHEA to androstenedione and 5-DHT to 3,17ß-diol are indicative of the presence of 3ß-HSD and 3-HSD, respectively. These observations concur with the study of intact cells in which 3-HSD activity was observed for CCD-32Lu and Hs 907.Lu, and a very low 3ß-HSD activity was detected with only CCD-32Lu. The human lung fibroblast steroidogenic pathway is summarized in Fig. 11.
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-reductase activity also shows a substrate preference that favors the 5-reduction of androstenedione over that of testosterone. Consequently, the product of 17ß-HSD type 2 is further converted into other metabolites without significant conversion of testosterone into 5-DHT, which is the most potent androgen. The efficiency of cell lines to inactivate testosterone instead of increasing its androgenic potential is a function of the 17ß-HSD type 2 to 5-reductase activity ratio. The data in Table 1 allow a division of the cell lines into two groups having a high or low value for this ratio. However, it should be noted that the values were calculated using the 5-reductase activity determined from tritiated androstenedione. This value was selected to calculate the ratio because it is not affected by 17ß-HSD type 2 activity. However, when calculated using tritiated testosterone, the 5-reductase activity cannot be compared between cell lines because the conversion of testosterone into androstenedione results in overestimation of the 5-reductase activity for cell lines having high 17ß-HSD type 2 activity. Consequently, the 5-reductase activity values measured with tritiated androstenedione are, in fact, relative values that do not represent the actual level of testosterone 5-reduction.
We have clearly demonstrated that the 5-reductase expressed in human lung fibroblast cell lines exerts a strong substrate preference for androstenedione over testosterone. The addition of unlabeled testosterone did not alter the conversion of [3H]-androstenedione to androstanedione and the use of the antiandrogen hydroxyflutamide failed to increase the conversion of [3H]-T into 5-DHT, thus showing that the presence of [3H]-T did not modulate 5-reductase expression. Moreover, levels of [3H]-T 5-reduction were at least 65-fold lower than those of [3H]-androstenedione 5-reduction in the presence of EM-919. Thus, it is clear that the 5-reductase expressed in human lung fibroblast cells exerts a strong substrate preference for androstenedione. Positive signal was obtained by Northern blots with the 5-reductase type 1 probe. It was demonstrated that 5-reductase type 1 had an apparent Km value for androstenedione that was 6 times lower, and a Vmax that was 2 times higher, than that of testosterone in cell homogenates prepared from Chinese hamster ovary cells transfected with recombinant 5-reductase type 1 (30). This means that the catalytic efficiency of the enzyme for androstenedione as substrate was 12 times higher than that of testosterone. In our experiments, the difference in the catalytic efficiency of the enzyme between the two substrates was at least 5 times more pronounced. This could be explained by the fact that the Km and Vmax values were determined from cell homogenates, although in our experiments, the difference in the rate of conversion between the two substrates was determined in intact cells. In fact, our results are very informative about the role that plays 5-reductase type 1, at least in human lung fibroblasts. In these cells, the endogenous 5-reductase type 1 activity on testosterone is low enough, and its activity on androstenedione is high enough, to participate in testosterone inactivation (by removing the product of the 17ß-HSD type 2 enzyme) without formation of significant amount of 5-DHT. Thus, in these cells, the 5-reductase type 1 might play an active role in minimization of the androgen potential.
Knowing that results obtained from cell line models have to be considered with circumspection in regard to the actual in vivo situation, we propose a model of lung steroid metabolism (Fig. 12) that would be informative and useful for a better comprehension of the mechanisms regulating fibroblast/epithelial cell communication in which androgens play a role of negative modulator. We have to keep in mind that androgens delay lung maturation and surfactant production by their action on lung fibroblast cells and that the steroid metabolism of these cells, which is oriented toward androgen inactivation, is particularly adapted to reduce the risk of RD by decreasing the intracellular level of androgens. The androgen receptor has been detected in both lung fibroblasts (11) and PTII-like A549 cells (31). The occupancy of this receptor depends on local steroid metabolism, which differs between cell types. The age stage of cell lines used in this study enables us to suggest that the pattern of steroid metabolism presented in Fig. 11 might be present in fibroblasts at the time of stimulation of surfactant synthesis because the same enzymes are expressed in both cells isolated during the second trimester of pregnancy and in neonates. The first question is, What is the source of androgens in the fetal lung? In the human, there is no testosterone synthesis by the gonads at the time of stimulation of surfactant synthesis. The major source would be local formation of androgen from circulating steroid precursors of adrenal origin. In light of the results obtained from the study of the A-549 cell line (18), we can suggest that androstenedione entering from the circulation is converted into testosterone by the 17ß-HSD type 5 expressed in PTII cells. The low 5-reductase and the high 3-HSD type 3 activities present in these cells allow accumulation and secretion of testosterone but not that of 5-DHT, which is rapidly converted into 3,17ß-diol. As a paracrine factor, testosterone can reach fibroblasts in which it would be rapidly inactivated into 17-ketosteroids (androstenedione) by the 17ß-HSD type 2. The strong substrate preference of the 5-reductase type 1 expressed in fibroblasts allows further conversion of androstenedione in androstanedione without formation of a significant level of 5-DHT. Thus, the occupancy of the androgen receptor in fibroblast cells (11) might be a function of the rate of testosterone formation by PTII cells and the rate of testosterone inactivation by fibroblasts, both events being candidates for modulation.
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). Because it is expected for the male to produce more androgens than the female, males would be more affected than females by a low 17ß-HSD type 2 expression profile. Nagourney et al. (32) reported that "preterm infants born to women with a previous preterm infant affected by RDS are at an increased risk of RDS, which suggests an important genetic (or other familial) tendency in its origin." Our results suggest that the 17ß-HSD-2 gene could be a candidate to conciliate these observations.
We have demonstrated that human lung fibroblast cells have the potential to inactivate exogenous androgens through the expression of 17ß-HSD type 2 and 5-reductase type 1 genes. In our cell model, 5-reductase type 1 has no significant activity on testosterone, but it further converts the product of 17ß-HSD type 2, thus playing an important role in androgen inactivation. Finally, variation in 17ß-HSD type 2 activity and mRNA from cell line to cell line and mRNA from subject to subject open the way to further studies aiming to explore the possibility that subjects with the low 17ß-HSD type 2 expression profile could be predisposed to RD.
Acknowledgments
We thank Drs. Fernand Labrie and Donald Poirier for providing hydroxyflutamide and EM-919, respectively.
Footnotes
This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC 171140), Association Pulmonaire du Québec, and Respiratory Health Research Network from Fonds de la Recherche en Santé du Québec (FRSQ) (to Y.T.) and from HealthPartners Research Foundation (to C.H.B.).
Y.T. is recipient of a senior scholarship from le Fonds de la Recherche en Santé du Québec.
Abbreviations: DHEA, Dehydroepiandrosterone; 5-DHT, 5-dihydrotestosterone; E1, estrone, 1,3,5-estratrien-3-ol-17-one; FBS, fetal bovine serum; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase/5-4 isomerase; 17ß-HSD, 17ß-hydroxysteroid dehydrogenase; NAD, nicotinamide adenine dinucleotide; NADH, reduced form of nicotinamide adenine dinucleotide; PTII, epithelial type II cell; RD, respiratory distress.
Received March 7, 2002.
Accepted May 1, 2002.
References
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