THE ANATOMICAL RECORD 229:321-333 (1991)

I mmunocytochemical Localization of Cathepsin D in Rat Ventral Prostate: Evidence for Castration-Induced Expression of Cathepsin D in Basal Cells MICHAEL J. WILSON, JOHN N. WHITAKER, AND AKHOURI A. SINHA Research Service, Minneapolis VA Medical Center (M.J.W., A.A.S.) and Departments of Laboratory Medicine and Pathology (M.J. W.) and Genetics and Cell Biology (A.A.S.), University of Minnesota, Minneapolis, Minnesota; Birmingham VA Medical Center and Departments of Neurology and Cell Biology and Anatomy (J.N.W.), University of Alabama, Birmingham, Alabama 35294

ABSTRACT Cathepsin D (EC3.4.23.5) is a n aspartyl endopeptidase involved in lysosomal proteolysis. Its functional role is uncertain. This study was undertaken to determine the cellular and subcellular distribution of cathepsin D in the normal rat ventral prostate and its possible role in the castration-induced atrophy of the gland. Cathepsin D was localized immunohistochemically to perinuclear lysosomes in secretory cells, in capillary endothelial cells, and, occasionally, in stromal cells of the untreated animal. Castration resulted in a n increased number of cathepsin D-positive cells in the stroma within 24 hr. By 48 h r after castration autophagolysosomes formed in secretory cells and apoptotic bodies appeared in the epithelium. Although apoptotic bodies generally contained immunoreactive cathepsin D, a subpopulation of larger apoptotic bodies, which commonly rested on the basement membrane and contained multiple inclusions, were more variable in cathepsin D expression. The induction of cathepsin D in dendritic cells basally oriented in the epithelium was noted a t 4 days of castration. These cells had a phagocytic phenotype, were distributed periodically along the basement membrane, and were not found in ductal epithelia. Treatment with actinomycin D or hydrocortisone to reduce the rate of regression of the ventral prostate blocked the appearance of these cathepsin D-positive, basally oriented epithelial cells. Our data indicate that this cathepsin D-positive, phagocytic cell differentiates from a cell resident in the prostatic epithelium. We suggest that it differentiates from basal cells in the secretory tubuloalveolar portion of the gland and that it is involved in the destruction of regressed secretory cells. The maintenance of the structural integrity and secretory function of the prostate gland by androgens is well established. In the rat, the ventral prostatic lobe is formed of branching tubuloalveolar structures whose epithelium of tall columnar cells is interspersed with small basal cells (Moore et al., 1930; Price and Williams-Ashman, 1964). The tubuloalveolar glands are supported by a loose connective tissue containing blood vessels, smooth muscle fibers, and stromal cells such a s mast cells, fibroblasts, and occasional leukocytes. The tubuloalveolar glands are contiguous with ductal elements which drain into the urethra. Castration results in a rapid loss of epithelial secretory cell cytoplasm within 4 days (Huttunen e t al., 1981) followed by a decline in cell number (Lesser and Bruchovsky, 1974). The reduction in secretory cell cytoplasm is marked morphologically in columnar cells with swelling of the endoplasmic reticulum and the formation of supranuclear autophagolysosomes (Helminen and Ericsson, 1972; Dahl and Kjaerheim, 1973; Kerr and Searle, 1973). Cell death is mediated a t least in part 0 1991 WILEY-LISS, INC

through a process of apoptosis in which nuclear and cytoplasmic components condense and then either are expelled into the lumen of the gland or are phagocytosed by other secretory cells or macrophage-like cells (Kerr and Searle, 1973). The latter increase in number in the epithelium following castration. Although regression is accompanied by diminished metabolic and synthetic activities, Bruchovsky et al. (1975) postulated that atrophy of the prostate is a n active process and is not merely the result of suppressed anabolic events. Such a postulate rested on the observation that the loss of prostatic cells after castration was considerably more rapid than the observed rate of cell turnover (8%in 3 days) in the intact animal.

Received May 1, 1990; accepted August 3, 1990. Address reprint requests to Michael J. Wilson, Ph.D., Research Service (151),VA Medical Center, One Veterans Drive, Minneapolis, MN 55417.

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Proteinases are a group of hydrolytic enzymes considered to have fundamental roles in the atrophic process in the prostate. However, individual proteinases such as cathepsin D (Helminen et al., 1972; Chiang et al., 1982; Tanabe et al., 19821, plasminogen activator (Rennie et al., 1984; Wilson et al., 1987), and calpain (Theis and Wilson, 1988) do not show a concordant change in the prostate after castration. As part of our studies of the role of proteinases in prostatic function, we undertook a n immunocytochemical investigation of the cellular and subcellular distribution of cathepsin D using immunocytochemistry. We report here a differential expression of cathepsin D in columnar cells and basal cells in the rat ventral prostate epithelium.

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MATERIALS AND METHODS Animals

Days of Castration Mr

0

2

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7

95.555 -

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18.4 12.4 -

Fig. 1. Immunoblot of cathepsin D in extracts of rat ventral prostate in response to castration. Extract protein (20 pg) was electrophoresed and transferred to nitrocellulose paper, and immunohistochemical detection was done as described in “Materials and Methods.” The extracts electrophoresed in individual lanes included that of intact (0, zero days of castration) and animals castrated for 2,4,and 7 days.

Increased synthesis of certain proteins and induction of specific mRNAs occur during the early phase of castration-induced regression of the prostate (Anderson et al., 1983; Montpetit et al., 1986; Lee and Sensibar, 1987; Saltzman et al., 1987). One component of the active atrophic process is a n increase in the 3ctivities of lysosomal hydrolytic enzymes such as acid ribonuclease (Engel et al., 1980) and cathepsin D (Helminen et al., 1972; Chiang et al., 1982; Tanabe et al., 1982). These enzymes can be expected to function in the autophagolysosomes formed in columnar cells and in the phagolysosomes of macrophage-like cells (Helminen and Ericsson, 1972; Dahl and Kjaerheim, 1973; Kerr and Searle, 1973). Further evidence for positive control mechanisms in prostatic regression is the apparent requirement for RNA and protein synthesis in the atrophic process. Thus, actinomycin D or cycloheximide treatment combined with castration reduced the rate of involution of the gland and concomitantly suppressed the increase in cathepsin D activity (Stanisic et al., 1978; Tanabe et al., 1982). Furthermore, hydrocortisone diminishes the rate of castration-induced regression and plasminogen activator activity of the prostate (Rennie et al., 1988).

Male Sprague-Dawley rats were purchased from BioLab (White Bear Lake, MN) and were maintained on standard laboratory chow and water ad libitum with a 12 h r light:12 h r dark photoperiod. All studies were conducted a s approved by the Animal Studies Subcommittee of the Minneapolis VA Medical Center. Sexually mature animals (70-90 days of age) were castrated via the scrota1 route while under methoxyflurane anesthesia. Animals (5 per group) were sacrificed a t 1, 2, 4,7, 10, and 15 days postcastration. One lobe of the ventral prostate was removed, spread on a piece of dental wax, fixed by immersion (24 hr) in Bouin’s fluid, and embedded in paraplast. Tissue sections of 5-6 pm were prepared for immunohistochemistry. Other lobes were fixed in cold 3.7% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) containing 2.5% sucrose, 1% CaCl,, and with or without 0.25% glutaraldehyde. Osmium tetroxide was not used for fixation. In another experimental group the effects of actino-

Fig. 2. Light micrograph of immunohistochemical localization of cathepsin D in the ventral prostate of the control (noncastrated) rat illustrates the presence of cathepsin D in granules (arrows) in the perinuclear region of the columnar secretory cells (light hematoxylin counterstain). Cathepsin D-positive granules are also observed in capillary endothelial cells (arrowhead). X 371. Fig. 3. A light micrograph of a negative control sample incubated without the primary antibody but with preimmune rabbit serum does not show localization of cathepsin D in the ventral prostate of the intact (noncastrated) rat (light hematoxylin counterstain). Basal cells are marked with arrows. x 468. Fig.4.A light micrograph of the ventral prostate (light hematoxylin counterstain) 1 day after castration. It illustrates localization of cathepsin D in some stromal cells (double arrows) and in perinuclear region of the secretory epithelial cells (arrow). x 450. Fig. 5. T w o days after castration, a light micrograph of the ventral prostate illustrates cathepsin D-positive large autophagolysosome complexes (arrows) in the supranuclear cytoplasm of columnar cells (light hematoxylin counterstain). Apoptotic bodies (double arrows) in the supranuclear cytoplasm of columnar cells show a distinct “halo” around the dense, cathepsin D-positive material, while other apoptotic bodies (inserts A, x 526 and B, x 533) contain condensed material that is cathepsin D positive (small arrowheads), stains with hematoxylin (small arrows), or demonstrates both hematoxylin staining and peroxidase reaction products for cathepsin D localization (large double arrows). Some cells in the stroma (arrowheads) are positive for cathepsin D. x 445.

FUNCTIONAL ROLE OF CATHEPSIN D IN PROSTATE

Figs. 2-5.

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Figs. 6-9

FUNCTIONAL ROLE OF CATHEPSIN D IN PROSTATE

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phoresed in 10%polyacrylamide gels with 0.01% SDS at 15°C (Laemmli, 1970). Thereafter the gels were washed for 30 min (2 changes) in 25 mM Tris (pH 8.3), 192 mM glycine, and 20% methanol (viv) and transferred electrophoretically to nitrocellulose paper using this buffer system at 30 V overnight a t room temperature in a Bio-Rad (Richmond, CA) transblot system (Sinha et al., 1989). The nitrocellulose sheet was used for immunochemical localization of cathepsin D using the polyclonal antibody described above and a Bio-Rad immunoblot assay kit with alkaline phosphatase-labeled goat antirabbit IgG. In brief, nitrocellulose sheets were incubated a t room temperature in 3%gelatin-Tris-buffered saline (TBS; pH 7.5) for 1 hr, washed for 10 min (2 changes) in 0.05% Tween-20-TBS, and incubated for 2 hr with the rabbit anti-rat cathepsin D (1:2,500 in TBS). The strips were then washed 10 min (2 changes) Antibody in 0.05% Tween-20-TBS, and incubated 1 h r with laThe primary antibody used was a polyclonal rabbit beled goat anti-rabbit globulin (1:3,000), and washed antiserum prepared against rat cathepsin D purified for 10 min (2 changes) in 0.05% Tween-20-TBS7 folfrom rat brain. The antiserum was absorbed with rat lowed by a wash with TBS alone. Subsequently, they serum proteins using solid phase immunoabsorption were incubated for 30 min in a solution containing 0.3 and with rat brain extract freed of cathepsin D by af- mg/ml nitroblue tetrazolium, 0.15 mg/ml BCIP (5finity chromatography with pepstatinyl-Sepharose bromo-4-chloro-3-indoyl phosphate toluidine salt), (Pierce Chemical Co., Rockford, IL) (Whitaker et al., 0.017% N,N-dimethylformamide, and 1 mM MgCl, in 1981; Snyder et al., 1985).The specificity of the serum 0.1 M carbonate buffer (pH 9.8) to localize alkaline was examined in Western blots (see below and Fig. 1). phosphatase. The strips were then washed and stored in distilled water. Electrophoresisand Immunoblotting The following prestained proteins (Diversified BioOne lobe of the ventral prostate of intact rats and tech, Newton Centre, MA) were electrophoresed with those castrated for 2, 4, 7, and 14 days was removed the samples and used as molecular weight markers: and frozen on solid CO,. These tissues were minced phosphorylase B (95.5 kDa), glutamate dehydrogenase with fine scissors and were homogenized separately (55.0 kDa), ovalbumin (43.0 kDa), lactic dehydroge(10% homogenate) in a medium containing 0.075 M nase (36.0 kDa), carbonic anhydrase (29.0 kDa), lactoNaC1, 2.5 mM sodium phosphate, and 0.25% Triton X- globulin (18.4 kDa), and cytochrome C (12.4 kDa). 100 (pH 7.81, using 20 strokes of a Teflon-glass tissue lmmunocytochemistry grinder. The homogenates subsequently were centrifuged at 12,OOOg for 20 min at 4°C. Aliquots of superImmunocytochemical procedures followed the protonatants were combined with a n equal volume of so- col described previously by Hsu et al. (1981) and Sinha dium dodecyl sulfate (SDS) buffer (3% SDS, 20% e t al. (1986). Briefly, deparaffnized tissue sections glycerol, 10% 2-mercaptoethanol, 62.5 mM Tris-HC1, were incubated in 0.6% methanolic hydrogen peroxide pH 6.8) and aliquots (20 pg protein) were electro- for 30 min to block endogenous peroxidase activity, rehydrated through graded alcohols and distilled water, rinsed for 10 min in 0.05 M PBS containing 2% normal rabbit serum (NRS), and incubated overnight in humidified trays a t 4°C with cathepsin D antiserum (diFig. 6. An electron micrograph illustrates localization of cathepsin D with immunogold particles in the perinuclear lysosomes (arrows) in lution 1:5,000) in PBS-1% bovine serum albumin). Opthese secretory cells from the ventral prostate of a control (noncas- timal dilution of the antibody was determined before trated) rat. The lumen (L) of the acinus is shown in the upper left undertaking the study. Slides were rinsed thoroughly hand corner. x 8,643. with PBS containing 1% NRS, covered with biotinyFig. 7. An electron micrograph of tissue from a control (noncas- lated goat anti-rabbit globulin (Vector Labs, Burlintrated) animal of a thin section was processed without the primary game, CA) for 30 min, rinsed with PBS containing 1% antibody but with preimmune rabbit serum for a negative control. NRS, and covered with avidin-biotin-peroxidase comThe electron micrography shows an absence of lysosomal localization of gold particles in the ventral prostate of the rat. The lumen (L) is plex (Vector Labs) for 30 min. They were then rinsed with 0.5 M phosphate buffer (pH 7.6) and incubated illustrated. x 8,250. with fresh filtered 3,3’-diaminobenzidine solution (0.25 Fig. 8. An electron micrograph of the ventral prostate 2 days after mg/ml; Sigma Chem. Co.) in phosphate buffer, with castration illustrates cathepsin D localization with gold particles in 0.01% hydrogen peroxide. Chromogenic development, the lysosomes (arrows) of the basal portion of cells subadjacent to the viewed through a light microscope, was no longer than basement membrane (BM). x 10,243. 10 min. Subsequently, slides were washed in running Fig. 9. An electron micrograph of the ventral prostate 2 days after tap water, dipped 3-4 times in 0.125% aqueous oscastration is positive for cathepsin D in autophagolysosomes (double mium-tetroxide solution to enhance areas of positivity, arrow) in the supranuclear cytoplasm of a secretory epithelial cell, as well as lysosomes (arrows). Note the gold particles and the lumen (L). washed again in water before counterstaining with x 8,654. Harris’ hematoxylin, dehydrated, and mounted in Per-

mycin D were examined. Eleven rats were injected S.C. a t the time of castration and then daily with 50 pg actinomycin D (Sigma Chemical Co., St. Louis, MO) in 0.20 ml saline and 11 controls received 0.20 ml saline at the same times. Five animals from each treatment group were sacrificed at 2 days and 6 were sacrificed at 4 days of treatment. In a third experiment, rats (5 per group) received one on the following treatments via i.p. injection upon castratrion and then daily for 4 days: 1 ml phosphate-buffered saline (PBS); 1 ml 5 mg/ml hydrocortisone sodium succinate (Sigma Chemical Co.) in PBS; 1m l 2 5 mg/ml hydrocortisone in PBS; or 1 ml 125 mg/ml of the proteinase inhibitor tranexamic acid (trans-4-[aminomethyI]-cyclohexanecarboxylic acid) in PBS. One lobe of the ventral prostate of these experimental animals was fixed in Bouin’s solution a s described above.

~~

~

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Figs. 10-14.

FUNCTIONAL ROLE OF CATHEPSIN D IN PROSTATE

mount. Along with the experimental tissue sections, negative control slides from all cases were incubated with rabbit preimmune serum (dilution 1:5,000) in lieu of primary antibody. Fixation and Embedding for Electron Microscopy

Selected paraformaldehyde-fixed specimens were washed several times with phosphate buffer or PBS, and embedded in LR white (LRW) or Lowicryl (K4M) resins (Polyscience, Inc., Warrington, PA) a s reported by Sinha et al. (1987). Briefly, the LRW-embedded specimens were dehydrated in 50% and 75% ethanol, infiltrated in the resin (2:1, LRW:75% ethanol for 2-3 hr; 100% LRW, 2 changes) at 4°C and at room temperature, embedded in sealed gelatin capsules, and polymerized at 4°C overnight with two 18 inch UV bulbs (15 watts, No. F15TB/BL bulbs, GTE Products Corp., Danvers, MA) at 30 cm in a n aluminum-lined polymerization chamber. For K4M embedding, specimens also were dehydrated for 10 min each in 50%, 7596, and 90% N,N-dimethylformamide (DMF; Matheson, Coleman, and Bell, Norwood, OH), infiltrated a t 4°C with K4M and DMF (ratios 1:2 K4M:DMF, 1 : l K4M:DMF, and 100% K4M; 2 changes each for 10, 20, and 30 min, respectively). Tissue was then embedded in sealed gelatin capsules and polymerized overnight with a UV light a s described above. Several thick sections from the LRW- or K4M-embedded specimens were stained with toluidine blue for light microscopy. Thin sections were collected on uncoated 200 to 300 mesh nickel or gold grids. lrnrnunoelectron Microscopy

For cathepsin D localization with protein A-gold complex, the protocol of Bendayan et al. (1986) was

Fig. 10.Four days after castration a light micrograph of the ventral prostate shows intense localization of cathepsin D in cells (arrows) resting on and parallel to the basement membrane (light hematoxylin counterstain). These cells have a distribution pattern and position in the epithelium like the basal cell. Localization of cathepsin D in lysosomes of the cuboidal secretory cells is also illustrated (arrowhead). x 492. Fig. 11. An electron micrograph of the ventral prostate 4 days after castration demonstrates cathepsin D localization in a large lysosomal complex (double arrows) found in a cell parallel to the basement membrane (BM). Localization is also observed in lysosomes in cuboidal secretory cells (arrows). The lumen (L) is shown in the upper right portion of the figure. X 8,628. Fig. 12.Seven days after castration a light micrograph of the ventral prostate shows a periodic distribution along the basement membrane of the basally localized cathepsin D-positive cells (light hematoxylin counterstain). However, in areas of ductal epithelium (arrows), relatively few of these cathepsin D-positive cells are observed. There is considerable condensation of the stroma a s compared with the intact animal (Figs. 2, 3). x 187. Fig. 13. A light micrograph of a tangential section of the ventral prostate a t 7 days following castration (light hematoxylin counterstain). The basally oriented cathepsin D-positive cells show a distinct dendritic morphology (arrows). x 518. Fig. 14.A light micrograph of the ventral prostate 14 days after castration illustrates cathepsin D-positive cells in the basal portion of the epithelium as well a s an occasional positive cell in the stroma (light hematoxylin counterstain). x 190.

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modified slightly (Sinha et al., 1987). Briefly, thin sections were hydrated in PBS, etched for 2 min in 10% H,Oz, and washed thoroughly with PBS. The grids were floated on drops of 0.1% or 1%bovine serum albumin (BSA) for 5-10 min and incubated overnight a t 4°C with cathepsin D antibody (1:3,500 in PBS and BSA). They were then washed with PBS (several changes), blotted with filter paper, and incubated for 60 min with protein A complexed with 10-15 nm gold particles ( 1 : l O in PBS; E-Y Lab. Inc., San Mateo, CA) in a moist chamber a t room temperature, washed several times with PBS, and rinsed with distilled water. The negative control grids were incubated with 1% NRS instead of primary antibody and protein A-gold, or with PBS and protein A-gold. Experimental and control grids were stained with uranyl acetate and lead citrate, and examined with a Zeiss 1OC electron microscope. Unstained sections were also examined. RESULTS Cathepsin D in the Intact Raf

The secretory epithelium of the rat ventral prostate comprises predominantly tall columnar cells oriented perpendicular to the basement membrane and occasional basal cells wedged between the secretory cells (Figs. 2, 3). The columnar cells contained basally located nuclei with prominent nucleoli and were characterized with a basophilic cytoplasm with a supranuclear clear zone corresponding to the Golgi region. Cathepsin D was found predominantly associated with granules in the perinuclear cytoplasm of the columnar cells (Fig. 2). Autophagolysosomes and apoptotic bodies were only rarely observed in the secretory epithelium. Periacinar smooth muscle cells as well as blood vessels and stroma cells are observed in the loose connective tissue of the supporting stroma. An occasional cell positive for cathepsin D localization was found in the stroma. In addition, immunoreactive cathepsin D was found in the endothelial cells of capillaries in the stroma (Fig. 2). No peroxidase reaction product was found in tissue sections incubated with preimmune rabbit serum in place of the cathepsin D antiserum (Fig. 3). Ultrastructural examination of cathepsin D localization using the immunogold technique showed gold particles specifically over perinuclear lysosomes in the ventral prostate of the intact rat (Figs. 6,7). The possible molecular species of cathepsin D in the rat ventral prostate were examined by immunoblotting following separation of extract proteins by SDS-polyacrylamide gel electrophoresis. In the intact rat, a single band of cathepsin D was detected with a molecular mass of approximately 43 kDa (Fig. 1). The Effect of Castration

The principal result a t 24 h r of castration was the appearance of cathepsin D-positive cells in the stroma (Fig. 4). An occasional autophagolysosome was observed in the supranuclear cytoplasm of secretory cells. At 2 days of castration the stroma was marked with the cathepsin D-positive cells (Fig. 51, some of which were polymorphonuclear leukocytes. In addition, the secretory cells were now characterized by the presence of large autophagolysosomes in the supranuclear cytoplasm with some cathepsin D-positive granules remaining in the basal perinuclear cytoplasm. The for-

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Figs. 15-18.

FUNCTIONAL ROLE OF CATHEPSIN D IN PROSTATE

mation of the autophagolysosome appears to occur through the fusion of lysosomes in the supranuclear cytoplasm. Apoptotic bodies were observed scattered through the secretory epithelium in 2 day castrates (Fig. 5). The autophagolysosomes and smaller apoptotic bodies in the supranuclear cytoplasm were strongly positive for cathepsin D localization. However, the larger apoptotic bodies which often had multiple masses of condensed material within the halo area demonstrated a varying degree of cathepsin D localization (Fig. 5 inserts). The condensed material in some of the large apoptotic bodies stained with hematoxylin without localization of cathepsin D, whereas others contained immunoreactive cathepsin D. In addition, there were some of these large apoptotic bodies in which one enclosed condensed mass was basophilic and another positive for cathepsin D. The immunogold technique demonstrated ultrastructurally the lysosoma1 localization of cathepsin D in the basal cytoplasm of columnar cells (Fig. 8) and in the supranuclear autophagolysosomes (Fig. 9) 2 days after castration. At 4 days of castration, cells in the epithelium in the position of basal cells, i.e., resting parallel on the basement membrane, showed intense cathepsin D localization (Fig. 10). These cells a t the ultrastructural level demonstrated large vacuoles which contained multiple components of varied electron density (Fig. 11) and showed localization of cathepsin D. These basally oriented cathepsin D-positive cells were very prominent in epithelial areas of cuboidal secretory cells at 4 and 7 days of castration and showed a distinct periodic distribution along the basement membrane (Fig. 12). They were not found along the entire tubulo-alveolar structure but were absent from areas more interior to the gland that probably were ductal in function (Fig. 12). These basally localized, cathepsin D-positive cells also had a distinct dendritic morphology which was best observed in tangential sections (Fig. 13). At 7 days of castration, the cuboidal secretory cells had fewer lysosomes, with one prominently displayed in the supranuclear cytoplasm. In these animals, there appeared to be fewer cathepsin D-positive cells in the stroma. At 14 days castration (Fig. 141, there were fewer basally oriented cathepsin D-positive cells as compared with the 7 day castrate (Fig. 12). The cuboidal cells contained a few cathepsin D-positive granules. An examination of immunoblots for cathepsin D indicated that there was a n increase in the relative amount of cathepsin D in the ventral prostate beginning a t about 4 days of castration (Fig. 1).This increase in cathepsin D is predominantly in the 43 kDa form although a t 4 and 7 days of castration, a n immunore-

Figs. 15-1 8. Light micrographs showing immunohistochemical localization of cathepsin D after 4 days of castration in which animals were treated daily with (Fig. 15) saline ( x 471), (Fig. 16) actinomycin D ( x 436), (Fig. 17) hydrocortisone ( x 4721, or (Fig. 18) tranexamic acid ( x 475). Basally oriented cathepsin D-positive cells (arrows) are observed in the saline (Fig. 15) and tranexamic acid (Fig. 18ktreated animals. They are only occasionally observed in animals treated with actinomycin D (Fig. 16) or hydrocortisone (Fig. 17). The actinomycin D (Fig. 16) and hydrocortisone (Fig. 17btreated animals demonstrate an increased number of lysosomes as compared with the salinetreated (Fig. 15)control. Light hematoxylin was used a s counterstain.

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active band of 30 kDa, was also found. The latter represents the heavy chain of the less-expressed two-chain form of rat cathepsin D (Yonezawa et al., 1988). The Effect of Actinomycin D and Hydrocortisone Treatments

The effect of agents known to retard the regression of the ventral prostate following castration were examined with respect to cathepsin D localization. The treatment of rats with actinomycin D for 4 days of castration appeared to have a t least two effects on cathepsin D localization in the prostate epithelium (Fig. 16). The secretory cells had a n increase in cathepsin D-positive granules as compared with 4 day castrates (Fig. 10) or the saline-treated controls (Fig. 15).Furthermore these cells contained fewer large supranuclear autophagolysosomes (Fig. 16). Second, the basally positioned cells on the basement membrane were only occasionally positive for cathepsin D. This was in marked contrast to the accumulation of immunoreactive cathepsin D in these cells in castrated saline-treated control animals (Fig. 15). The treatment of rats with hydrocortisone during the 4 days following castration also prevented the appearance of the cathepsin D-positive cells parallel to the basement membrane (Fig. 17). Its effect in the secretory epithelial cells was similar to that observed with actinomycin D but some autophagolysosomes were found in the supranuclear cytoplasm. Hydrocortisone treatment seemed to inhibit the fusion of lysosomes in the supranuclear cytoplasm presumably by interfering with the formation of autophagolysosomes. Occasional cathepsin D-positive basal cells were observed in the hydrocortisone-treated animals, whether the dose was 5 or 25 mg per day. The cellular localization of cathepsin D in rats treated during the 4 days of castration with the proteinase inhibitor tranexamic acid did not differ from that of the saline-treated control animals (Fig. 18). Specifically, the cytoplasmic distribution of lysosomes in the secretory cells and the appearance of the cathepsin D-positive cells parallel with the basement membrane were similar. DISCUSSION Our observations suggest that cathepsin D functions in the catabolism of cellular proteins during castrationinduced regression of the ventral prostate and that androgens exert a differential effect on the cellular localization and projected role of cathepsin D in this process. Autophagolysosomes, which degrade secretory cell cytoplasm, form through fusion of lysosomes but with little apparent change in total cathepsin D. In contrast, there is a n induction of cathepsin D in basally oriented cells which develop phagocytic features. Cathepsin D thus appears to play a role in two stages in prostatic involution: 1) the loss of secretory cell cytoplasm associated with a decrease in cell volume and 2) in the subsequent removal of regressed secretory cells by this phagocytic cell. Cathepsin D has a clear role in the degradation of secretory cell proteins through the lysosomal system in castrated animals. The formation of large autophagolysosomal bodies, positive for cathepsin D, paralleled the castration-induced involution of the supranuclear cytoplasm of columnar cells. The development of auto-

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phagolysosomes appeared to involve movement of perinuclear lysosomes to the supranuclear position in the cell and their fusion with cytoplasmic components. This conclusion is based on the relative decrease in perinuclear localization of lysosomes and the blockage of autophagolysosome formation with the concomitant accumulation of lysosomes following actinomycin D treatment. The onset of autophagolysosome formation was related temporally to the appearance of apoptotic bodies in the epithelium. Our study has shown more than one population of apoptotic body in the ventral prostate epithelium. These populations can be determined by their size and expression of cathepsin D which suggests that they are formed by different mechanisms. Most apoptotic bodies were positive for cathepsin D a s revealed by the presence of immunoperoxidase reaction product, but a subset of apoptotic bodies did not demonstrate immunoreactive cathepsin D. The latter group were among the larger apoptotic bodies which commonly rested on the basement membrane and contained multiple inclusions. Cathepsin D localization was observed in some inclusions whereas others stained only with hematoxylin. The latter may represent condensed chromatin. The absence of cathepsin D in some apoptotic bodies may indicate that this proteinase has a limited role in chromatin degradation or that cathepsin D becomes associated with chromatin material only at a specific stage of its degradation in the apoptotic body. The smaller apoptotic bodies in the supranuclear cytoplasm of secretory cells which demonstrate abundant cathepsin D localization could represent the final stages in autolysis of secretory cell cytoplasm while the larger apoptotic bodies with multiple inclusions could represent cellular material phagocytized by adjacent epithelial cells. In the stroma, cathepsin D-positive cells were first observed at day 1 of castration. Some of these cells are polymorphonuclear leukocytes; others could be macrophages or other cell types. It is possible that the influx of these cells into the stroma may accompany the increased imbibition of water (Lee et al., 1986) and resultant transitory elevation of fractional weight of interacinar tissue (Huttunen et al., 1981) that occurs by 16 h r postcastration. The most striking feature of cathepsin D localization in the regressing ventral prostate epithelium is the appearance a t 4 days of castration of cells parallel to the basement membrane that are laden with immunoreactive cathepsin D. These cells probably are the macrophage-like cells described by Helminen and Ericsson (1972) and Kerr and Searle (1973). It is likely that the induction of cathepsin D in these cells is responsible for the increase in cathepsin D activity in ventral prostate regression noted by others (Helminen et al., 1972; Chiang et al., 1982; Tanabe et al., 1982). Our findings agree with the interpretation (Ballard and Holt, 1968; Kerr and Currie, 1980) that the removal of dying cells and the ultimate digestion of apoptotic bodies result from lysosomal enzymes of the phagocytic cell rather than the dying cell. Thus, in ventral prostate atrophy, cell loss can occur through cells being sloughed into the acinar lumen or engulfed and destroyed by these phagocytic cells. Our immunocytochemical findings indicate a greater prevalence of these basally oriented

cathepsin D-positive cells, particularly at 7 days postcastration, than morphologically active macrophagelike cells (English et al., 1989). This difference in observations could be due to sampling procedures since these cathepsin D-positive cells are localized more to the periphery of the gland. In addition, many macrophage-like cells could contain cytoplasmic vacuoles of a size and/or density that would make them less evident morphologically but their presence could be detected due to their expression of cathepsin D. The origin of these basally positioned, cathepsin Dpositive cells in the epithelium is not fully established, but an increased metabolic activity of basal cells (judged to be basal cells by their morphology and position in the epithelium) as a result of castration has been described by others. These observations include a wave of mitotic activity at 3 days (Secchi and Bonne, 19731, development of acid phosphatase-positive dense bodies (Brandes, 1974), and a n increased uptake of 3Huridine (Baulieu et al., 1975). More recently Evans and Chandler (1987) have confirmed the wave of cell division a t 3 days castration and the cytologic features of basally positioned cells and postulated that the macrophage-like cells are a product of cell division of infiltrating monocytes. Although some of our observations are consistent with the cathepsin D-positive cells being macrophages, our data more strongly support a hypothesis that they differentiate from a cell originating in the epithelium (i.e., the basal cell). The temporal relationship of cathepsin D-positive cells appearing in the stroma before such cells are detected in the epithelium and the presence of large cathepsin D-positive phagolysosomal bodies are suggestive of the former. However, we did not observe any invasion of the epithelium by cathepsin D-positive cells from the stroma. Rather, the periodic spatial distribution of these cells along the basement membrane is not characteristic of a macrophage invasion but is strikingly similar to the distribution of basal cells as detected by immunohistochemistry of cytokeratin (Verhagen et al., 1988).These cells persist in the epithelium at 14 days of castration, well past the time (7 days) when wet weight, protein, and DNA content reach the basal castrate level. In addition, they become less intense in cathepsin D localization and appear to remain in the epithelium following testosterone treatment of 14 day castrated animals (unpublished results). Finally, colloidal iron-dextran complexes that are injected at the time of castration to mark macrophages are found in stromal cells a t 4 days of castration, but not in any epithelial cell (unpublished results). The possibility of the basal cell differentiating into a phagocytic cell is a new concept for hypothesized functions of this cell in the prostatic epithelium. The basal cell has been described in the prostatic epithelium of several species including the rat, dog, and human. It is a small cell (low cytoplasm to nuclear ratio) devoid of many cytoplasmic organelles but rich in free ribosomes. Because of this undifferentiated appearance it has been thought of a s a reserve or progenitor cell whose progeny differentiate into secretory cells. This hypothesis has been supported by observations of basal cell proliferation in prostatic tissues placed into organ culture (Chandler and Timms, 1977; Merchant et al.,

FUNCTIONAL ROLE OF CATHEPSIN D IN PROSTATE

1983) and their sensitivity in vitro to testosterone (Chandler and Timms, 1977) and carcinogen-directed (Sanefuji et al., 1979) differentiation. Cells with epithelial location and morphologic properties apparently intermediate between basal and secretory cells have been described in the normal prostate of the dog (Timms et al., 1976) and mouse (Sinha and Bentley, 1984) and in the normal and hyperplastic prostates of the human (Mao and Angrist, 1966; Dermer, 1978; Cleary et al., 1983; Sinha et al., 1989). However, others have not found such intermediate cell types (see Evans and Chandler, 1987). The concept of a strict progenitor cell function of basal cells is also not supported by studies which show that both secretory and basal cells of castrated mice and rats incorporate 3H-thymidine and divide following testosterone replacement (Sinha and Bentley, 1984; Evans and Chandler, 1987; English et al., 1987). The role of the basal cell as a reserve cell has also been questioned because of its distinctive pattern of cytokeratin expression (Wernert et al., 1987). Although a cytokeratin distinctive to myoepithelial cells in other organs has been demonstrated in prostatic basal cells by immunocytochemistry (Caselitz et al., 1986), prostatic basal cells do not demonstrate the ultrastructure of myoepithelial cells and hence would not appear to serve t h a t function in the prostate (Ichihara and Pelliniemi, 1975). Basal cells have also been proposed to have a role in transportation of material between secretory cells in the acinus and the extra-acinar space. Adenosine triphosphatase activity has been described on the basal cell plasma membrane (Mao and Angrist, 1966) and micropinocytotic vesicles are found on their basal surfaces (Timms e t al., 1976; Ichihara et al., 1985). The pinocytotic vesicles in the rat show strong alkaline phosphatase activity (Kimura and Ichihara, 1980). In addition, complex plasma membrane infoldings and interdigitations between basal and secretory cells have been found (Mao and Angrist, 1966). These ultrastructural features and the frequent close proximity of blood capillaries in the stroma to basal cells (Timms et al., 1976; Ichihara et al., 1985) support the concept of their transport role. Not all basal cells in the ventral prostate respond to castration by the induction of cathepsin D. The basal cells that are in the ductal epithelium have a fine structure and location in the epithelium similar to that of basal cells in the secretory epithelium of the acinus (Ichihara et al., 1978). However, cells that are in the position of basal cells in the ducts do not show cathepsin D localization. This finding is supportive of the observations of Sugimura et al. (1986) who noted that castration results in a distal to proximal regression of the ventral prostate in mice; that is, there is a loss of ductal tips and branch points of the branched tubuloalveolar structure of the prostate whereas the ducts survive albeit in a n atrophic condition. Since not all basal cells respond to castration in a similar manner, the induction of cathepsin D activity and the phagocytic phenotype in the responding basal cells would appear to be secondary to another stimulus, perhaps one from the stroma. While our data support the lysosomal role of cathepsin D catabolism of cellular proteins, there appears to be a dichotomy in androgen regulation of cathepsin D

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in the ventral prostate. Thus, while there is a clear induction of cathepsin D in basal cells upon the removal of testicular androgens, autophagolysosome formation in secretory cells appears to occur through fusion of lysosomes with no accumulation of additional lysosomes. Cathepsin D is widely distributed in mammalian tissues but its cellular content within a n organ varies widely (Whitaker et al., 1981; Whitaker and Rhodes, 1983). The uneven distribution of cathepsin D and its regulation in sensitive cells by thyroxine (Satav and Katyare, 19811, estrogen (Westley and May, 1987), and progesterone (Maudelonde et al., 1990) implicate a variety of physiologic controls. There is clear evidence for differential effects of steroid hormones on cathepsin D in the female reproductive system. In the rat uterus, the amount of cathepsin D as well as its activity is increased following administration of either estradiol or progesterone. However, the increase in cathepsin D following estrogen treatment is concomitant with a n increase in general protein synthesis whereas the increase stimulated by progesterone is specific for cathepsin D and not total protein (Elangovan and Moulton, 1980). In the human endometrium, progesterone but not estradiol increases immunoreactive cathepsin D (Maudelonde et al., 1990). However, the synthesis of a pro-cathepsin D and its mRNA are stimulated in MCF7 breast tumor cells by estrogen (Westley and May, 1987; Cavailles et al., 1988) and its concentration in primary breast tumors correlates with the estrogen receptor and not progesterone status of the tumor (Maudelonde et al., 1988). The induction of cathepsin D in prostatic basal cells appears to be a correlate of the phagocytic differentiation of these cells. These data suggest that the basal cell plays a role in regulating the number of secretory cells in the ventral prostate. The phagocytic differentiation of basal cells coincides temporally with castration-induced cell loss from the gland and occurs in the secretory epithelium where such cell loss is noted. This suggests that the basal cell has a key role in the antagonistic effect of androgen on prostatic cell death (Isaacs, 1984). On the other hand, testosterone may function in basal cells of the prostate more than just by repressing their transformation to phagocytes. Androgen-induced proliferation of basal cells may be part of the regulatory process determining the size of the secretory epithelium possibly through a mechanism independent of the basal cell’s status a s a stem cell. ACKNOWLEDGMENTS

The authors are grateful to Ms. Mary Vogel for treatment of animals, Mr. Andy Valls for tissue processing and paraffin sections, Ms. Onofrea deLeon for immunohistochemical technical assistance, and to Ms. Marie Starks for secretarial assistance. This work was supported by the Department of Veterans Affairs Medical Research funds to all three authors. LITERATURE CITED Anderson, K.M., J. Baranowski, S.G. Economous, and M. Rubenstein 1983 A qualitative analysis of acidic proteins associated with regressing, growing or dividing rat ventral prostate cells. The Prostate, 4.151-166. Ballard, K.J., and S.J. Holt 1968 Cytological and cytochemical studies of cell death and digestion in the foetal rat foot: The role of macrophages and hydrolytic enzymes. J. Cell Sci., 3.245-262.

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FUNCTIONAL ROLE OF CATHEPSIN D IN PROSTATE rate of 3H-uridine incorporation in regressing rat ventral prostate. Invest. Urol., 16t15-18. Sugimura, Y., G.R. Cunha, and A.A. Donjacour 1986 Morphological and histological study of castration-induced degeneration and androgen-induced regeneration in the mouse prostate. Biol. Reprod., 34t973-983. Tanabe, E.T., C. Lee, and J.T. Grayhack 1982 Activities of cathepsin D in rat prostate during. - castration induced involution. J. Urol., 127t826-828. Theis, J.M., and M.J. Wilson 1988 The Ca2+-dependent protease inhibitor of rat ventral prostate: Properties of the inhibitor and effects of castration on Ca2+-dependent protease and inhibitor activities. Int. J. Biochem., 20t909-916. Timms, B.G., J.A. Chandler, and F. Sinowatz 1976 The ultrastructure of basal cells of rat and dog prostate. Tissue Res., 173t543-554. Verhagen, A.P.M., T.W. Aalders, R.C.S. Ramaekers, F.M.J. Debruyne, and J.A. Schalken 1988 Differential expression of keratins in the basal and luminal compartments of rat prostatic epithelium during degeneration and regeneration. The Prostate, 13: 25-38.

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