Steroid sulfate sulfatase in human benign prostatic hyperplasia: Characterization and quantification of the enzyme in epithelium and stroma

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J. steroid Biochem.Vol. 33, No. 2, pp. 195-200,1989 Copyright 0 1989 Maxwell

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STEROID SULFATE SULFATASE IN HUMAN BENIGN PROSTATIC HYPERPLASIA: CHARACTERIZATION AND QUANTIFICATION OF THE ENZYME IN EPITHELIUM AND STROMA HARTMUTKLEIN,THOMASMOLWITZ~ and WILPRIEDBARTSCH* Dept of Clinical Chemistry, Medical Clinic, University of Hamburg, D-2000 Hamburg 20, F.R.G. (Receioed 7 October 1988) Smnmary--Characteristics and activities of e&one sulfate (EIS) and dehydr~piandrosterone sulfate (DHAS) sulfatases were studied in epithelium and stroma of benign hyperplastic tissues from human prostates. Tissues were obtained by suprapubic pros~t~tomy, and epithelium and stroma were separated mechanically by standard techniques. The assay procedure comprised homogenization in Tris-buffer, incubation of the homogenate with [3H]ElS or [)H]DHAS, separation of free steroids from nonhydrolyzed steroid sulfates by extraction with ether, and their flnal quantification by LSC. The main results were: (1) The pH-optimum of the sulfatase was found at pH 7.0. (2) The highest specific sulfatase activity was found in the epithelium and was associated with its nuclear fraction. (3) Michaelis-Menten constants Km bM) were 8.7 f 1.4 (7) and 4.3 f 0.8 (S), maximum velocity rates V_ (nmoi/h x mgDNA) were 47.4 f 8.8 (7) and 8.4 f 1.5 (5) for ElS and DHAS, respectively (means + SEM (n)). (4) The enzymatic cleavage of El-sulfate was com~tively inhibited by DHA-sulfate and vice persu with inhibition constants Ki @M) of 4.0 + 0.5 (2) for ElS and 2.7 & 0.4 (2) for DHAS. On the basis of these findings, possible roles of steroid sulfate-sulfatams in forming precursors of active androgens and estrogens from the high amounts of ElS and DHAS in blood are discussed. INTRODUCTION Steroid sulfates quantitatively dominate the circulating steroid pool in blood. In man, plasma levels of dehydroepiandrosterone sulfate (DHAS) as well as of estrone sulfate (ElS) by far exceed the concentrations of the respective free steroids. Particularly DHAS has the highest plasma concentration of all detectable steroid metabolites, thus exceeding the level of the nonconjugated DHA by a factor of about 500 (11. Similarly, ElS is found in an about 20 fold higher concentration in plasma as compared to free El [2,3J. Rather little is known about the biological importance of these steroid sulfates, except in placental estrogen biosynthesis. This tissue converts fetal DHAS to estradiol [4] using subsequently catalyzing activities of four enzymes, i.e. steroid sulfatesulfatase, the 3jI-hydroxysteroid dehydrogenasef 4,Gsomerase complex, aromatase, and the 17/Ihydroxysteroid dehydrogenase (17p-HSDH). The role of estrogens in the induction or promotion of prostatic growth, particularly of benign prostatic hyperplasia (BPH), is still open to question [5-S]. Furthermore, controversy exists concerning the question whether androgens can be aromatized directly in BPH tissue [g-13]. In addition to descriptive findings *Correspondence to Dr Wilfried Bar&h at his present address: Fraunhofer Institute for Toxicology und Aerosol Research, Niko~-F~hs-Str. 1, D-3000 Hannover 61, Federal Republic of Germany. tPart of doctoral thesis.

in human prostates [14,15], particularly animal studies support the hypothesis that estrogenic effects might be involved in the development of BPH [I@-191. Intraprostatic formation of estrogens via the aromatase, however, did not seem to us to be an important pathway in estrogen supply of the human prostate [ 131. Besides the aromatase, two alternative pathways, both of which require the cleavage of a steroid sulfate and subsequent 17/Lreduction, could form estrogenic compounds within prostatic tissue: The cleavage and 17~-reduction of ElS would result in the formation of estradiol, the most potent natural estrogen. Correspondingly, 5-androstene-3fl, 178 -dial could be formed from DHAS. This steroid has also been reported to bind to the estrogen receptor and to induce estrogen dependent processes [20-221. 17flreduction of El as well as of DHA in human BPH tissue has already been described [23]. In earlier studies, we [24] and others [6,25) noticed that the tissue levels of DHA and El in human benign hyperplastic prostates exceed those found in plasma. This tissue-plasma gradient may be explained by the presence of steroid sulfate-sulfatase activity within the prostate which in cooperation with the 17/IHSDH could form hormonal active compounds from available steroid sulfates. These findings may point to another possible biological role of steroid sulfates and of their enzymatic cleavage by sulfatase. The presented study described localization and properties of this enzyme in human BPH tissue.

195

HARTMUT KLEIN et al.

196

Tris-buffer, shred further with an Uitraturrax and filtered again with strong suction. The retained Chemicals stroma was washed with T&buffer, chilled with [6,7-“H]ElS (52 Ci/mmol), [7-3H]DHAS (23 Ci/ liquid nitrogen, pulverized, and was finally homogemmol), [4-“C]El (55 mCi/mmol), [4-“C]DHA nized with 4 vols of T&buffer in a Dounce appar(50 mci~mmol), and liquid ~inti~ation cocktail atus. (Biofluof) were obtained from New England For preparation of subcellular fractions by Nuclear (Dreieich). Nonlabelled steroid sulfates were differential centrifugation, epithelium was homopurchased from Serva (Heidelberg). All other chemi- genized in “D.&buffercontaining 0.25 M sucrose. The cals (reagent grade) were from E. Merck (Darm- 800g and 800-100,OOOgpellets (both resuspended stadt). Purity of the radiolabelled steroid sulfates in T~s-buffer) and the l~,~ g su~matant of this was checked by TLC (Silica gel G, ethyl acetate- homogenate represented the nuclear, mitochonmethanol-concentrated aqueous ammonia 75 : 25 : 2, drial + microsomal, and cytosolic fractions, respecvol~vol/vol). If less than 95% of the radioactivity was tively. Nuclei were further purified as described found in the region of the reference substance, the previously [28]. Before assay of sulfatase, the purified steroid sulfate was dissolved in Tris-buffer (10 mM nuclei were finally washed with T&-buffer in order to -I-+-HC~, 2 mM EDTA, 5 mM sodium tide, 10mM reduce the sucrose contamination. magnesium chloride, pH 7.4), and the free labelled steroid was extracted with cold ether. The nonla- Determinationof enzyme activities belled steroid sulfates were also dissolved in TrisSamples were allowed to thaw at 4°C and epithebuffer, and contaminations of free steroids were lium homogenates were further diluted with 1 or 3 correspondin~y removed by ether extraction. Steroid vols of Tris-buffer (final dilutions: I+ 9 and 1 + 19) sulfate concentrations of the remaining aqueous solu- for assay of DHAS or ElS sulfatase, respectively. tions were subsequently determined by one of the Reaction mixtures of totally 400~1 in Pyrex@ glass follo~ng procedures: DHAS was measured by RIA tubes with screw caps and Teflon@seals consisted of using a commercially available assay system (Coat- 200+1 portions of these diluted homogenates and A-Count DHEA-SO, ‘25I Kit, Diagnostic Products 100~1 of each substrate, radio-labelled and nonlaCarp, Los Angeles, Calif.) according to the recom- belled, both dissolved in T&buffer. Substrate conmendations of the manufacturer. ElS was hydro- centrations ranged 0.2-75.4 PM for ElS and lyzed, and the free El was quantified by fluorescence ,0.2-85.5 FM for DHAS with l~,~dprn of the spectroscopy using standard techniques [261. respective tritiated steroid sulfate. Reactions were started by addition of the homogenate and immediate Tissues transfer into a shaking waterbath. After incu~tion at Prostates (weighing 30-160 g, mean 52 g) were 37°C for 30 min, lo-ml portions of ether were added obtained from 11 patients (age 56-82 yrs, mean together with 1OOOdpm of either [“C]El or 70 yrs) by suprapubic pros~~tomy. After extirpa- [“CJDHA in 100~1 of T&-buffer in order to stop the tion, the tissue was immediately placed on ice. One reaction, to extract the formed free steroid by vigorpart was taken for histological examination, which ous shaking for 2 min, and to determine the recovery coniirmed the clinical diagnosis of BPH in all of the of this extraction process. Separation of the two cases. The remainder of the organ was rapidly trans- phases by freezing was performed as previously deported to the laboratory, cut into pieces of about scribed in detail [29], and the ether phases were 3 mm diameter, chilled with liquid nitrogen, and decanted into scintillation vials. After evaporation of stored frozen at -20°C until further processing. the solvent, IO-ml portions of Biofluof were added, the vials were shaken, and analyzed for 3H and “C Preparationof tissue ~#~raetions content in a Packard Tri-Garb@460C liquid scintillaAll procedures were carried out at 0_4”C, Mecha- tion counter with quench correction by external ma1 separation of ~itheli~ and stroma essentially standardization. In order to measure sulfatase activities as functions followed the method of Cowan et al. [27] as modified by Krieg et al. [14]. In brief, frozen pieces of tissue of the pH, this procedure was slightly modified. The were mixed with 4 volumes of T~s-buffer, shred in a tissue was homogen~~ in 155mM sodium chloride, Btihler rotating edge-homogenizer (two periods of 30 which was also used to dissolve the tritiated as well and 15 s, respectively, with a cooling interval of 30 s) as the nonlabelled substrates. Tris/EDTA/phosphateand the resulting tissue mash was filtered with gentle buffers (25 mM Tris, 25 mM sodium phosphate, suction through 150-pm nylon gauze. From the 5mM EDTA, 25mM magnesium chloride) were filtrate, the epithelium was pelleted by centrifugation adjusted to various pH values between 4.0 and 9.0 by at IOOOg,taken up in 4 vofs (wt/vol) of Tris-buffer, addition of either sodium hydroxide solution or and homogenized in a Dounce apparatus with a hydrochloric acid. Reaction mixtures of totally 700 ~1 tight-fitting pestle. This homogenate was frozen in were composed of 400-~1 portions of these buffers, liquid nitrogen and stored at -20°C. The material 200-~1portions of the homogenate prepared in saIine, remaining on the nylon sieve was also taken up in and another 100~1 of 155mM saline with dissolved EXPERIMENTAL

Steroid sulfate sulfatase labelled and nonlabelled substrate. All assays were run in duplicate with parallel incubation of heatdenaturated samples (95’C, 30 min) for blank correction, For substrate saturation curves, series of experiments with 10 different substrate concentrations were performed. K,,, and V_ values were read from double-reciprocal plots according to Lineweaver and Burk [30] with regression lines calculated by the method of least squares. Inhibition constants Ki for competitive inhibition of EiS hydrolysis by DHAS and vice versa were determined according to the method of Dixon [31]. Miscellaneous

Protein and DNA were quantified by the Biuret reaction and the modified Burton reaction as described previously [29, 321. Statistical comparisons were performed using the two-tailed t-test.

Fig. 2. Cleavage rate of EIS as a function of the enzyme concentration. Different portions of (1 + 19)-diluted prostatic epithelium were incubated in reaction mixtures of totally 600~1 of T&-buffer (10mM) with 1OpM of substrate for 3Omin at 37°C and pH7.4. Amount of homogenate used for standard assays indicated.

RESULTS

Evaluation of optimal incubation conditions

Cleavage of ElS and DHAS were investigated as functions of substrate and enzyme concentration, incubation time, temperature, and pH. Due to the high enzyme activity in this compartment, all optimization experiments were performed with isolated epithelium, which was diluted 1 + 9 for assays with DHAS and 1 + 19 for assays with ElS. With these amounts of tissue, acceptable conversion rates between 5% and 20% were obtained with substrate concentrations in the range 0.2-90 FM. Linearity between incubation time (Fig. 1) as well as amount of enzyme (Fig. 2) on the one hand and the velocity of the reaction on the other hand could be. demonstrate up to 60 min and 300 ~1 of the diluted epithelial homogenate, respectively. With 30 min and 200 ~1 of homogenate, standard assay conditions were chosen within these ranges of linearity. For both substrates, variation of the incubation temperature (Fig. 3) and of the buffer pH (Fig. 4) revealed maximal enzyme activities at 60°C and pH 7.0, respectively. The sulfa-

incubation time (min) Fig. 1. Cleavage rate of ElS as a function of the incubation time. ZOO-~1portions of (1 + 9)diluted prostatic epitheli~ in reaction mixtures of totally 400 ~1 of 10 mM T&-buffer were used in these experiments and incubated with 15 PM of the substrate at 37°C and pH 7.4 as outlined in the Experimental section. Incubation interval of standard assays indicated. SB 33/2-D

temperature PC) Fig. 3. Cleavage rates of ElS (O---O) and DHAS (O---O) as functions of the incubation temperature. 200~1 portions of (1 + 9)-diluted prostatic epithelium were incubated in reaction mixtures of totally 4OOfll of Trisbuffer (10 mM) with 15 FM of the respective substrate for 30 min at 37°C and pH 7.4. Temperature of standard assays indicated.

tase was found to be more resistant to an alkaline than to an acidic milieu. For the standard assay conditions, physiological values of 37°C and pH 7.4 were chosen. Q~~jt,v control

The assay procedure revealed acceptable characteristics with respect to specificity, sensitivity, and preci-

PH Fig. 4. Cleavage rates of ElS (()---a) and DHAS (O---O) as functions of the solvent pH value. 2OOql portions of (1 + 9)-dihtted prostatic ~i~e~~ in reaction mixtures of totally 700 ~1 were incubated with 8.6 MM of the respective substrate for 30 min at 37’C. Mixtures of 300-~1 portions of 155 mM sodium chloride and 400-pl portions of Tris(25 mM)/EDTA(S mM)/phosphate(25 mM)-buffer were used as matrix. pH of standard assays indicated.

198

HARTMLJT KLEINet al. Table 1. Subcellular distribution of steroid sulfatesulfatase in epithelium of human BPH tissue as measured by differential centrifugation. Sulfatase activities were measured by one-point assays with 30 gM of the respective substrate and am given as percent of the initial activity detectable in unfractionated epithelial homogenate. Means of two determinations

Whole epithelium 800 g fraction Purified nuclei

EIS

DHAS

100% 108% 89%

100% 90% 61%

1.5% 1.3%

2.4% 2.1%

Table 2. Characterization of steroid sulfate stdfatase in epithelium of human BPH tissue. Comprison of the maximal velocities (I’,,,,,) for cleavage of the two alternative substrates EIS and DHAS, of their MichaelisMenten constants (K,,), and of the inhibition constants (K.) of both steroid sulfates for mutual competitive inhibition of the& cleavage. Means f SEM (number of experiments). ‘Substrate DHAS, ‘substrate EIS F18

DHAS

47.4 + 8.8 (7)

8.4 + I .5 (5)

8.7 & 1.4 (7)

4.3 + 0.8 (5)

2.7 + 0.4’ (2)

4.0 + 0.5’ (2)

V

(nEol/h mgDNA) :mol/l)

Mitochondrial +

microsomal fraction Cytosol

sion. Recovery rates of El and DHA during the separation of free steroids from their nonhydrolyzed sulfates were about 94%, and no nonhydrolyzed steroid sulfates could be detected in the organic phase. The results were not influenced by other enzymes present in prostatic tissue: Products of secondary metabolic modification of the formed free steroids, e.g. the 17fl-reduction products 17/Iestradiol and 5-androstene-3/$17/I-dial were simultaneously extracted and were consequently included in the account of sulfatase activity. Under the standard assay conditions, blanks were found to be in the order of l-2%, and conversion rates of 3% were considered to be the detection limit. Coefficients of variation were found below 10% for intra-assay as well as inter-assay precision. Localization and characterization of the enzyme

Related to the amount of tissue, sulfatase activities measured in isolated epithelium by far exceeded activities detected in stromal preparations. In two independent experiments, enzyme activities in stroma did not significantly differ from values which had to be expected from the contamination of these preparations by residual epithelium. Therefore, our further experiments were focused on the epithelial activity. cell fractionation experiments by During

differential centrifugation, recovery of sulfatase activity from step to step exactly paralleled the recovery of DNA. The bulk of the enzyme activity initially measured in epithelial homogenate was found associated with the 800 g pellet and later with the fraction of purified nuclei, whereas only trace amounts of sulfatase activity could be detected in the mitochondrial + microsomal and cytosolic fractions (Table 1). These findings indicate a nuclear or perinuclear localization of the sulfatase. Additionally, these experiments revealed the enzyme’s sensitivity to high osmolarity: the presence of 1.0 M sucrose reduced conversion rates by 50%. Concentrations in the reaction mixture up to 0.4 M did not influence the activity (data not shown). Maximum velocities (V_) and Michaelis-Menten constants (K,,,) of the ElS as well as DHAS cleavage determined by saturation analysis (Fig. 5) in purified epithelium of several prostates are given in Table 2. In contrast to the similar K,,, values in the PM range, all samples demonstrated I-fold higher VmX values for hydrolysis of ElS as compared to DHAS. DHAS competitively inhibited the cleavage of ElS (Fig. 6) and vice oersa. The inhibition constants (Ki) of both sulfates were found in the order of their K,,, values (Table 2). DISCUBBION

Our kinetic data concerning DHAS sulfatase activity in human BPH tissue support earlier data of

Fig. 5. Estrone sulfate-sulfatase activity in epithelium of human BPH tissue as a function of the substrate concentration. 2OOql samples of (1 + 19)-diluted epithelium in reaction mixtures of totally 400~1 T&buffer (10mM) were incubated with increasing concentrations of ElS as described in the Experimental section. Means of duplicate determination. Insert: Double reciprocal plot according to Lineweaver and Burk [30]. Typical example of the experiments summarized in Table 2.

Ki=lSpM

1

2

3

4

DHAS WI)

Fig. 6. Competitive inhibition of the ElS cleavage by DHAS. 200-4 portions of (1 + 9)-diluted prostatic epithelium were incubated under standard conditions with either 0.6 or 2.3pM of ElS and increasing concentrations of DHAS. Reaction velocities were plotted according to Dixon [31]. Means of duplicate determinations.

Steroid sulfate sulfatase Farnsworth [33]. He found a K,,, of S~LM, which is very similar to the results of our experiments. This author also concluded from measnrements in predominantly epithelial and stromal tissue specimens that the enzyme in question is mainly located in the former compartment of BPH, as we do from our measurements in the separated tissue fractions. EIS sulfatase activity in whole human BPH tissue has been detected earlier by Carlstrom et al. 1341. Although no K, values were given, these investigators stated a severalfold higher activity in prostatic tissue than in muscle tissue. We assume that both activities are properties of one enzyme. Similar K,,, values, a high degree of correlation between individual V,, values, the similar subcellular localization, and the in~bition data favour this assumption. These data, along with the pH-optimum, the thermal stability, and the higher reaction rates for the substrate ElS as compared to DHAS are features of enzymes detected in other human organs, e.g. the endometrium [35], liver [36], lung [37], fetal membranes and decidua [38], but differ with respect to the substrate specificity from those detected in amnion tissue [39], which cleave only ElS. Intraprostatic cleavage of steroid sulfates yielding active estrogenic compounds may be important in regulating the intensity of estrogenic stimulation and the growth of the prostate, and finaly may also be involved in the growth control of the diseased prostate, i.e. BPH. Our study clearly demonstrates the major prerequisite of this pathway, the presence of the respective enzyme apparatus, i.e. the steroid sulfate sulfatase, The estimation of its biolo~~al significance and of its involvement in the intracellular formation of free steroids, however, requires further resolution of two problems: -From the cellular localization of steroid sulfate sulfatase, it can be concluded that the enzyme is not an important secretory protein of the prostate. This means, that the relatively polar substrates have to pass the cell mambrane before cleavage. A transport mechanism for these compounds is not known. -In view of the high metabolic activity of the steroid sulfate sulfatase, the high DHAS and ElS levels in plasma, and a plasma contamination of prostatic tissue in the range of 10% [40], care has to be taken in the interpretation of prostatic unconjugated DHA and El con~ntrations. During homogenization of the tissue, intracellular sulfatase could cleave extracellular sulfates to an unknown extent and artificially increase concentrations of unconjugated steroids, unless special precautions have been taken. Therefore, it might be difficult to discriminate between in oiuo alterations in unconjugated DHA and El levels and artefacts. Experiments from which the biological importance of the sulfatase pathway can be derived have been performed in mammary carcinoma. These tumors

199

also possess steroid sulfate sulfatase. Vignon et al. [41] have shown that the behaviour of ElS in cultured breast cancer cells is similar to that of the unconjugated steroid. Santner et al. [42] have compared the relative importance of the sulfatase and aromatase pathways in the intratissular production of estrone in mammary carcinomas, and they came to the conclusion that the sulfatase pathway might be more important in these tissues although both seem to contribute to estrone aromatase

formation. In view of low or even absent activity in the prostate [13], the sulfatase

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