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Androgen-dependent nuclear proteins in rat ventral prostate are glycoproteins associated with the nuclear matrix
Cell Biology
International
Reports,
Vol. 12, No. 8, August
1988
RAT ANDROGEN-DEPENDENT NUCLEAR PROTEINS IN PROSTATE ARE GLYCOPROTEINS ASSOCIATED WITH THE MATRIX
VENTRAL NUCLEAR
!I . Carmo-Fonseca Department Science,
0f 2781
Cell Oeiras,
Biology, Portugal
Gulbenkian
Institute
of
ABSTRACT ventral prostate androgen-dependent nuclear The major rat were studied using isolated nuclei, nuclear proteins and nuclear envelope fractions. Nuclear and matrix fractions were subnuclear obtained characterized by microscopy electron and SDS-polyacrylamide gel A group of approximately 20 kDa electrophoresis. peptides matrices is demonstrated to be present in nuclei, nuclear and nuclear envelopes from normal prostate. course experiments indicate that Time the 20 kDa become peptides drastically reduced after 7 or 10 days following castration and are incompletely restored after 3 daily testosterone injections. binding studies demonstrate that the Lectin 20 kDa both peptides bind Wheat Germ to Concanavalin A and Th e s e Agglutinin. peptides represent the major nuclear Concanavalin A bindin:;-glycoproteins from normal prostate nuclei and nuclear matrices.
INTRODUCTION It is generally accepted that most, if not a 1 1, actions of hormones are mediated by interaction of steroid hormonecomplexes with chromatin acceptor receptor sites, which results in the activation of chromatin leads to and transcription of specific genes. enhanced their Despite im;>ortance, very little is known about the nuclear acceptor molecules for steroid receptors. Acidic and basic proteins, D?,;A a n d i?I"lrl have nuclear been implicated 1934) and more recently, (Anderson, the nuclear matrix has & been proposed as accepting receptor site (Barrack 19i;0, COLfCJ]i, Although steroid 1962). hormone receptors were identified as DI:A binding proteins soon after their w e r e demonstrated discovery, only recently specific bindin; sites on the genonc DNA (fayvar ct al, 1981). liowrver, t il e !>:!A-receptor binding alone is not enougil for and additional hormone action, involvement of nuclear (IZ:ri>:lr 8 ;I i c 1:c!rna II , 1925). protcilk.5 is re:iuirc(: 0309-1651/88/080607-14/$03.00/O
@ 1988 Academic
Press Ltd.
608
Cell Biology
International
Reports,
Vol. 12, No. 8, August
1988
Several studies report cirangcs in nuclear proteins and from rat ventral prostate induced by castration (Chung ;2 Coffey, 1971; Couch & treatment androgen 1973; Xadohama G Anderson, 1976; Ahmed cot al, Anderson, Of particular importance may be t 11e androgen1981). approximately dependent peptide (or group of peptides) of in rat ventral prostate by 18 400 - 20 000 Da, identified Kishimoto et al (19G2) and Venkatraman et al (1984). characterize androgenThis study aims to further dependent nuclear proteins from rat ventral prostate. With normal, that purpose, prostate nuclei were isolated from animals, their testosterone treated castrated and for profiles compared and tested lectin electrophoretic Additionally, normal prostate nuclei were binding. buffers extracted with non-ionic detergent, nucleases and of low and high Ionic strength in order to isolate nuclear envelope and nuclear matrix fractions. MATERIALS
AND METHODS
rats (250-3008) were Wistar used. male Adult young performed via scrotal route under ether Castration was 10 days anesthesia, the animals killed 3, 5, 7 and and daily Androgen treatment consisted of a later. injection of 2.5 mg testosterone propionate subcutaneous ethyl (Sigma Chemicals, St. Louis, USA) in 0.25 ml of starting 7 days after orchidectomy. The animals oleate, were sacrificed 18 hours after the last injection. All experiments were repeated twice or three times to assess for reproductibility. Nuclear
isolation
The ventral prostates were removed and washed in ice-cold 0.25M sucrose, 10 mM Tris pH 7.4, 1 mK bC12 (0.25 M in TM buffer). Nuclei were isolated according to sucrose the procedure of Anderson et al (1970). The prostates were with minced and homogenized 10 strokes in a Potter After infiltration trough gauze the nuclei homogenizer. sedimented for 10 min at 800g. This crude were nuclear pellet was resuspended and further purified through 2.2 M sucrose in TM buffer. Liver nuclei were isolated by centrifugation through 50 mM Tris pI1 7.4, 5 mM MgC12, as described 2.2 M sucrose, by Berezney and Coffey (1977). All buffers contained 1 mM PMSF freshly prepared from a 0.1 M stock solution in anhydrous n-propyl-alcohol.
Cell Biology
Nuclear
International
matrix
Reports,
and nuclear
Vol. 72, No. 8, August
envelope
1988
609
isolation
Prostate nuclei were treated with the following extraction as described by Kaufmann et al (1951). Freshly procedures, nuclei were resuspended in STM buffer (0.25 M isolated ) and sequentially 50 mF1 Tris pH 7.4, 5mM MgSO4 sucrose, min; extracted with 1% Triton-X-100 in STM buffer, for 10 DNase I DNase I (RNase-free) or 250 pg/ml 250 ug/ml (Sigma) plus 250 pg/ml RNase A (Sigma) in STM buffer for 60 min; LS buffer (10 mM Tris pH 7.4, 0.2 m?l MgS04 >, for and twice with HS buffer (211 NaCl added to LS 15 min; DNase RNase-free was buffer) plus 1% DTT for 15 min. USA) obtained from Worthington Biochem. Corp. (Freehold, and further purified according to Long et al (1979). 1 ml1 PMSF was added to all buffers immediately before use.
SDS-PAGE and Blotting SDS-polyacrylamide gel electrophoresis was performed using 1.5 mm thick slab gels with either 7.5% or 12% separating gels as described by Laemmli (1970). Purified nuclei were washed in 0.25 M sucrose in T?.i buffer containing 1 m?J P;4SF and directly dissolved in sample buffer. Nuclear matrix and nuclear envelope fractions were washed two or three times in 10 mlY Tris pH 7.4, 1.5 mPJ EGTA, 1 mY PFISF before dilution in the sample buffer. To prepare fractions cytoplasmic the first supernatant precipitated was with (3008) 10% trichloroacetic acid, washed in acetone and diluted into sample buffer. Samples diluted in sample buffer (containing 60 rn)I Tris pH G.S, 1.25% SDS and 0.1 !J DTT) were boiled for 3-5 min, cooled and stored at -20" C. were Gel-s stained with Coomassie blue. A11 electrophoresis from reagents were BioRad (Richmond, USA). The proteins SDS-PAGE separated were electrotransferred to 0.45 urn nitroF:llulose (Towbin et a1,1979). For lectin binding studies tile blot units were saturated with 0.3% Tween 20 (BioRad) in either phosphate buffered saline (PL:IS) or TS buffer (50 1rnI.l Tris p!i 7.4, 2OC; ,mN NaCl) for 30 min at 37" C and three times r 0 0 1~1 at temperature. The blots were then incubated wit;1 50 pgjmi. Concanavalin A (Con A) (Sigma) in TS buffer PlUS 0.05% Tween 20 for 30 nin, washed in TS buffer plus 0.05% T we e n and further incubated with 50 uz:/1:!1 horseradish perosidase (Sigma) in the same buffer. Alternatively the blots Ye r e wi t Ii incubated 50 )12/1111 wheat germ ( L?G!, ) agglutinin ;JEs conjugated to horseradish peroxidasc (Sigma) in 0.05% Tween for 1 hour. ;\fter extensive washing the b o :I II (:
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1988
peroxidase was detected by a solution of 0.06% 4-chloro-lnaphtol (Sigma) and 0.01% hydrogen peroxide in TS buffer Controls were pre-incubated in (Hawkes, 1982). 0.5 methyl-a-D-mannopyranoside or 0.4 M N-acetyl-D-glucosamine min and then incubated in, respectively, the Con for 15 or VGA solutions in the presence of the sugar. Electron
M A
microscopy
of nuclei in 0.25 M sucrose, TM buffer, Suspensions were with an equal volume of 4% glutaraldehyde in 0.1 M mixed pH 7.4 and fixed for 30 min at 4' C. cacodylate buffer, Suspensions of nuclear matrices and nuclear envelopes in HS buffer fixed for 30 min at 4" C with were to a final concentration of glutaraldehyde which was added 1% (v/v), from a freshly prepared 4% stock solution in 50 mF1 cacoclylate buffer pH 7.4, 5 mM MgSO4 (Kaufmann et al, The fixed structures were sedimented, post-fixed in 1981). and processed for Epon embedding. Sections were 1% OS04 with uranyl acetate and lead citrate and studied stained in a Jeol 100 CXII electron microscope. RESULTS establish the purity and morphological composition of To various samples of material the subnuclear fractions, both obtained at each step were examined by phase contrast light microscopy transmission and electron Distinct non-nuclear contamination is absent microscopy. purified nuclear preparations (fig. 1). from Nuclei are relatively well preserved, with inner and outer membranes clearly seen. After Triton-X-100 extraction, internal morphology remains unaltered but both nuclear membranes are absent. Digestion of nuclei with DNase (RNase-free) followed extraction with low and high salt buffers bY matrix yields a residual nuclear composed of the peripheral lamina or nuclear envelope (whether nuclei were previously treated with Triton or not), residual nucleoli an internal fibrogranular network (fig. 2). Addition and RNasc to DNase in the presence of the of reducing agent ?jTi" yields nuclear a fraction of envelopes completely dcvoitled of intranuclear material (fig. 3). SDS-PAGL profiles of prostate nuclear The proteins with s h 0 I? a m a j 0 r protein band approximate molecular of 20 1:Da. This peptide (or group of peptides) is weight detected in prostate cytoplasmic fractions or in not rat liver isolated nuclei (fig. 4).
Cell Biology
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1988
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Fig.1 Electron micrograph of purified prostate nuclei. Distinct non-nuclear contamination is absent,general morphology is well preserved,the nucleolus is clearly recognized and nuclear membranes are present. x 6 600. Electron micrograph of nuclear matrix prepared Fig. 2 the in absence of Triton X-100 and Ridase. A residual nucleolus is identified (nu), together with abundant intranuclear fibrogranular material. x 14 400. Fig. 3 Electron micrograph of nuclear envelopes uniformely devoided of intranuclear material as prepared by addition of llTT to the high salt extraction buffer. 11 400.
x
612
Cell Biology International liver nuclei
prostate cyto. nuclei 94L blk
43k
3ok
-
20.1 k
Reports, Vol. 72, No. 8, August 1988
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Fig. 4 SDS-Page acrylamide gel. Lane 1: weight markers (phosphor albumin; ovalbumin; anhydrase; trypsin inhi lactalbumin). Lane 2: prostate cytoplasmic 1iane 3: nor!:lal prostate nuclei. Lane 1:: isolnt nuclei. .4r r 0 w points t Da prostate nuclear prot
on 12 molecular ylase b; carbonic bitor;a normal fraction. isolated ed liver o the 2@!c eins.
!I!!
14.4k
4 -castratednorm
3d
5d
7d
lad
AestosteroneId 2d
3d
prostate nuclear ventral rat 5 SDS-PAGE of weight Lane 1: molecular gel. pP:FLins on 12% acrylamide prostate isolated nuclei. Lanes 3 markers. Lane 2: normal nuclei isolated from castrated rats sacrificed 3, to G: nuclei Lanes 7 to 9: 7 and 10 days after operation. 5, 2 and 3 isolated from castrated rats sacrificed after 1, Arrow points to the 20 daily injections of testosterone. kDa proteins.
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1988
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the 20 kDa androgen dependence of for To test from castrated and were nuclei isolated peptides, electrophoretic their treated animals and testosterone The first significant decrease profiles compared (fig. 5). detected 7 relative intensity of the 20 kDa band is in daily testosterone After 3 castration. days after relative a clear increase in the injections there is although it does not attain normal amount of that protein, levels. Of matrix Electrophoretic analysis nuclear and reveals three major envelope fractions nuclear polypeptides within the molecular weight range of 60 70 corresponding to the nuclear lamins (fig. 6a). The 20 kDa, are present both in nuclear matrix kDa peptides and appearing as two nuclear envelope fractions, separated close bands (fig.6b). A binding liver Con glycoproteins in blots of and proteins were detected prostate nuclear essentially as described by Hawkes (1982). However, a considerable lower background staining was achieved by, the use of Tween 20 nitrocellulose for saturation of paper. Binding was i‘f inhibited by methyl-a-Dthe presence of 0.5 mannopyranoside. As expected, from the standard protein 3, mixture (myosin, galactosidase, phosphorylase bovine serum albumin and ovalbumin ) only ovalbumin was detected. The 20 IcDa peptides are shown to represent the major Con A binding proteins from normal prostate nuclei an d nuclear matrices cyto~~lasm~fig. 7). They are absent from ;;;;I: prostate castrated prostate nuclei and liver nuclei. The major Con A binding ilrotein fro,.: liver nuclei has an approximate molecular weight of lcITIa 200 (fig. 7). WGA binds intensely to several nuclear proteins including the 20 kDa androgen dependent peptides from normal prostate nuclei and nuclear matrices (fig. 8). DISCUSSION The presence of a 21 000 Da an d r 0 g c n- d c I1)e n d en t nuclear protein from rat ventral prostate was first reported by Kishimoto et al (1982). This peptide was not extractable w i t h 5% perchloric acid and only partially extractable with 0.35 1-1 idaC1. Later, Venkatranan et al (1934) resolved this protein into a group of 18 400 - 20 Oi)O Da p C [J t i c! E S and obtained electrophoretic profiles quite si:iiilnr to those presented in this paper. Ils rioscribed by those authors, the 18 400 - 20 003 pcptides resist triton-S-100 extraction and arc associated Twiti the nuclear c ri v c 1 0 7 E . The results presented here demonstrate, in addition, &at the peljtides associate also with thcx nuclear matrix.
614
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1988
94k 2ook
116.25
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14.4k
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b
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SDS-PAGE on 7.5% acrylamide gel. Fig. Lane 1: 6 (A) molecular weight markers (myosin, galactosidase, phosphorilase b, bovine serum albumin and ovalbumin). Lane 2: isolated prostate prostate nuclei. Lane 3: nuclear matrix fraction. Lane 4: prostate nuclear envelope fraction. Arrowheads point to lamina peptides. (B) The same fractions run on 12% acrylamide gel. Arrow points to the 20 kDa proteins.
Cell Biology International
Reports, Vol. 72, No. 8, August 1988
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WGA v cyto.
*
prostatenuclei
matrix
liver nuclei
con
t rol
“N”
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7 Proteins electrophoresed on 12% gels were Fig. transfered to nitrocellulose and tested for Concanavalin A binding. Lane I: prostate cytoplasm. Lane 2: normal prostate isolated nuclei. Lane 3: isolated nuclei from 10 day castrated animals. Lane 4: normal prostate nuclear isolated liver nuclei. matrix. Lane 5: Lanes 6 and 7: isolated nuclei from prostate (6) and liver (7) premethyl-a -D-mannopyranoside. incubated with Arrowheads point to the 200 kDa peptide presumely associated with pore Arrow nuclear complexes. points to the 20 kDa prostate nuclear proteins. 8 Proteins electrophoresed on 12% gels Fig. were transfered to nitrocellulose and tested for Wh e a t germ aglutinin binding. Lane 1: prostate cytoplasm. Lane 2: normal prostate isolated nuclei, Lane 3: normal prostate nuclear matrix. Lane 4: isolated liver nuclei. Lane 5: isolated liver nuclei pre-incubated with N-acetyl-Dglucosamine. Arrow points to the 20 kDa prostate nuclear proteins,
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1988
The specific nuclear location of the 20 kDa pcptiilcs their is demonstrated :lbstnce rat prostate i. n in by cytoplasmic fractions. Although it is not possible to exclude the presence of similar proteins in other androgen this study shows that they are absent from target organs, liver nuclei. the results of time-course experiments it From may are practically be seen that the 20 kDa peptides become unaffected until 5 days after orchidectomy, but drastically reduced after 7 and 10 days. Injection Of first testosterone to castrated induces rats the daily protein increase after 3 significant only Considering that the mitotic activity in treatments. the after presents a peak the ventral prostate 2 days testosterone treatment (Tuohirnaa & Yiemi, beginning of results indicate that the 20 k Da protein these 1974), of epithelial cell number. recovery follows restoration immobilized nitrocellulose Glycoproteins on together membranes can be detected using Con A as a probe peroxidase substrate with peroxidase and a chromogenic liver (Hawkes, 1982; Rohringer et al, 1985). Isolated rat have a major high molecular weight (approx. 200 nuclei which resists Triton treatment kDa) Con A binding protein, and most probably corresponds to the 190 kDa glycoprotein by Gerace et al (1982) as a constituent of described the The same polypeptide is identified nuclear pore complex. in prostate nuclei and nuclear matrices, together with the nuclear prostate specific major kDa band of 20 glycoproteins. that It is currently considered carbohydrate lumen of are added to glycoproteins inside the residues organelles such as endoplasmic reticulum, Golgi apparatus These glycoproteins then nuclear envelope. are and on the exoplasmic face of cellular membranes, integrated external cell lumenal face of organelles or I.e., the surface (Hanover & Lennarz, 1978, 1980; Snider & Robins, 1982; Snider & Rogers, 1984; Torres & Hart, 1984). However proteins, there is evidence suggesting that glycosilated a class of glycoconjugates characterized by particularly residue bound Othe presence of a single GlcNAc occur in the polypeptide chain, also glycosidically to With cytoplasm and nucleoplasm (Holt & Hart, 1986). the tumor exception of the Simian Virus 40 large possible has suggested to be a O-linked which been antigen, (Jarvis other Butel, 1985), no glycoprotein & nucleoplasmic glycoproteins have been characterized. major The results presented here indicate that the from ventral peptides rat nuclear androgen-dependent glycoproteins containing both mannose or prostate are N-acetyl-glucosamine (Nicolson, residues glucose and unrecognized previously The association of these 1974). matrix glycoproteins with nuclear is also the
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Vol. 12, No. 8, August
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1988
association of the interpreting the d e c:on s t r a t e d . In matrix with nuclear peptides the andro~en-dependent particular attention should be taken, however, considering cross-links to that inadvertent formation of the fact the proteins during isolation procedure other nuclear might render some polypeptides nucleasc and salt-insoluble (Kauf;:lann et al, 1986). whether Eurther studies are necessary to determine these androgen-dependent nuclear proteins are involved in merely Of horlIlone action or if they mechanism the of androgen stimulation, represent a product
ACKNOWLEDGMENTS The author is grateful to Galego for the kind supply to Ii. 2. AlpiarGa and J. assistance Prof. and to helpful discussion of the
Drs. C. Rodrigues-Pousada of a DNase (RNase-free) C. Lemos for skillful Dr. J. F. David-Ferreira manuscript.
and L. sample, technical for
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Ottavianno, of a major Journal of Cell L.,
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Lennarz, W. J. (1978) The N-diacetylchitobiosylpyrophosphoryland natural membranes. 254, 9237-9246.
Lennarz, W. J. A. and assembly. Evidence that within the lumen of occurs Journal of Biological Chemistry
Identification IIawkes, R. (1982) proteins after sodium dodecyl Analytical protein blotting. and 146. IIolt, G. D. distribution Localization linked GlciJac. 8057. Jarvis, Simian Virology Radohama, histone undergoing Research
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of Concanavalin A binding electrophoresis sulfate-gel Biochemistry 123, 143-
G. W. (1986) The X-acetyl-glucosamine protein-saccharide Biological Chemistry
L. and Butel, J. D. 40 large tumor Virus 141, 173-189.
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J. s. II., K a u f ma n n , Coffey, 3. S. and Shaper, i n the isolation of rat liver considerations envelope and mat 1-i:: , nuclear complex pore Experimental Cell Research 132, 105-123.
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Kaufmann, S. H., Fields, A. P. and Shaper, J. H. (1986) concepts unanswered nuclear matrix: current and The Experimental Methods and Achievements in questions. Pathology 12, 141-171. Izaike, X., Gomi, T., H. (1982) A novel nuclear Androgcn-dependent and et Biophysics Acta 718,
Kishimoto, Nakagawa, prostate. Biochimica
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Long, B. H., Huang, C.-Y. and Pogo, A. 0. (1979) Isolation characterization of matrix in Friend and the nuclear hnRNA chromatin and interactions erytroleukemia cells: with the nuclear matrix. Cell 18, 1079-1090. Nicolson, animal 89-190.
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Payvar, F., Wrange, O., Carlstedt-Duke, Gustafsson, J.-A. and Yamamoto, K. R. glucocorticoid receptors bind selectively DNA fragment whose transcription cloned glucocorticoids in Proceedings vivo. Academy of Sciences USA 78, 6628-6632. Rohringcr, R. detection of glycoproteins enzyme probes.
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Snider and Robbins, P. W. (1982) Transnembrane orpanilatF;n "* of :,ia t u r e protein glycosilation. ol~gosaccharide-lipid is located on the luininal side of microssomes frown Chinese hamster ovary cells. Journal of Biological Chemistry 257, 6796-6801. Snider, 14. 1). and oligosaccharide-lipids of Cell 36, 753-761.
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G. (1984) Topography and terminal N-acetylglucosamine intact lymphocytes. Evidence of Biological Chemistry 259,
Towbin, Ii., Staehlin, T. and Electrophoretic transfer of proteins gels to nitrocellulose sheets: applications. Proceedings of the Sciences USA 76, 4350-4354.
Gordon, J. (1979) from polyacrylamide procedure and some National Academy of
Tuohimaa, P. and Niemi, M. (1974) Cell renewal and activity of testosterone in male sex accessory mitogenic (ed.) Male accessory sex organs, pp glands. In: D. Brandes 329-343. Academic Press, New York. Venkatraman, J. T., Howell, G. M. and (1984) Androgen-dependent peptides of nuclear Biochemical prostate envelope. Research Communications 125, 469-474.
Received:
3.2.88
Accepted:
22.6.88
Lefebvre, Y. A. the rat ventral Biophysical and
International
Reports,
Vol. 12, No. 8, August
1988
RAT ANDROGEN-DEPENDENT NUCLEAR PROTEINS IN PROSTATE ARE GLYCOPROTEINS ASSOCIATED WITH THE MATRIX
VENTRAL NUCLEAR
!I . Carmo-Fonseca Department Science,
0f 2781
Cell Oeiras,
Biology, Portugal
Gulbenkian
Institute
of
ABSTRACT ventral prostate androgen-dependent nuclear The major rat were studied using isolated nuclei, nuclear proteins and nuclear envelope fractions. Nuclear and matrix fractions were subnuclear obtained characterized by microscopy electron and SDS-polyacrylamide gel A group of approximately 20 kDa electrophoresis. peptides matrices is demonstrated to be present in nuclei, nuclear and nuclear envelopes from normal prostate. course experiments indicate that Time the 20 kDa become peptides drastically reduced after 7 or 10 days following castration and are incompletely restored after 3 daily testosterone injections. binding studies demonstrate that the Lectin 20 kDa both peptides bind Wheat Germ to Concanavalin A and Th e s e Agglutinin. peptides represent the major nuclear Concanavalin A bindin:;-glycoproteins from normal prostate nuclei and nuclear matrices.
INTRODUCTION It is generally accepted that most, if not a 1 1, actions of hormones are mediated by interaction of steroid hormonecomplexes with chromatin acceptor receptor sites, which results in the activation of chromatin leads to and transcription of specific genes. enhanced their Despite im;>ortance, very little is known about the nuclear acceptor molecules for steroid receptors. Acidic and basic proteins, D?,;A a n d i?I"lrl have nuclear been implicated 1934) and more recently, (Anderson, the nuclear matrix has & been proposed as accepting receptor site (Barrack 19i;0, COLfCJ]i, Although steroid 1962). hormone receptors were identified as DI:A binding proteins soon after their w e r e demonstrated discovery, only recently specific bindin; sites on the genonc DNA (fayvar ct al, 1981). liowrver, t il e !>:!A-receptor binding alone is not enougil for and additional hormone action, involvement of nuclear (IZ:ri>:lr 8 ;I i c 1:c!rna II , 1925). protcilk.5 is re:iuirc(: 0309-1651/88/080607-14/$03.00/O
@ 1988 Academic
Press Ltd.
608
Cell Biology
International
Reports,
Vol. 12, No. 8, August
1988
Several studies report cirangcs in nuclear proteins and from rat ventral prostate induced by castration (Chung ;2 Coffey, 1971; Couch & treatment androgen 1973; Xadohama G Anderson, 1976; Ahmed cot al, Anderson, Of particular importance may be t 11e androgen1981). approximately dependent peptide (or group of peptides) of in rat ventral prostate by 18 400 - 20 000 Da, identified Kishimoto et al (19G2) and Venkatraman et al (1984). characterize androgenThis study aims to further dependent nuclear proteins from rat ventral prostate. With normal, that purpose, prostate nuclei were isolated from animals, their testosterone treated castrated and for profiles compared and tested lectin electrophoretic Additionally, normal prostate nuclei were binding. buffers extracted with non-ionic detergent, nucleases and of low and high Ionic strength in order to isolate nuclear envelope and nuclear matrix fractions. MATERIALS
AND METHODS
rats (250-3008) were Wistar used. male Adult young performed via scrotal route under ether Castration was 10 days anesthesia, the animals killed 3, 5, 7 and and daily Androgen treatment consisted of a later. injection of 2.5 mg testosterone propionate subcutaneous ethyl (Sigma Chemicals, St. Louis, USA) in 0.25 ml of starting 7 days after orchidectomy. The animals oleate, were sacrificed 18 hours after the last injection. All experiments were repeated twice or three times to assess for reproductibility. Nuclear
isolation
The ventral prostates were removed and washed in ice-cold 0.25M sucrose, 10 mM Tris pH 7.4, 1 mK bC12 (0.25 M in TM buffer). Nuclei were isolated according to sucrose the procedure of Anderson et al (1970). The prostates were with minced and homogenized 10 strokes in a Potter After infiltration trough gauze the nuclei homogenizer. sedimented for 10 min at 800g. This crude were nuclear pellet was resuspended and further purified through 2.2 M sucrose in TM buffer. Liver nuclei were isolated by centrifugation through 50 mM Tris pI1 7.4, 5 mM MgC12, as described 2.2 M sucrose, by Berezney and Coffey (1977). All buffers contained 1 mM PMSF freshly prepared from a 0.1 M stock solution in anhydrous n-propyl-alcohol.
Cell Biology
Nuclear
International
matrix
Reports,
and nuclear
Vol. 72, No. 8, August
envelope
1988
609
isolation
Prostate nuclei were treated with the following extraction as described by Kaufmann et al (1951). Freshly procedures, nuclei were resuspended in STM buffer (0.25 M isolated ) and sequentially 50 mF1 Tris pH 7.4, 5mM MgSO4 sucrose, min; extracted with 1% Triton-X-100 in STM buffer, for 10 DNase I DNase I (RNase-free) or 250 pg/ml 250 ug/ml (Sigma) plus 250 pg/ml RNase A (Sigma) in STM buffer for 60 min; LS buffer (10 mM Tris pH 7.4, 0.2 m?l MgS04 >, for and twice with HS buffer (211 NaCl added to LS 15 min; DNase RNase-free was buffer) plus 1% DTT for 15 min. USA) obtained from Worthington Biochem. Corp. (Freehold, and further purified according to Long et al (1979). 1 ml1 PMSF was added to all buffers immediately before use.
SDS-PAGE and Blotting SDS-polyacrylamide gel electrophoresis was performed using 1.5 mm thick slab gels with either 7.5% or 12% separating gels as described by Laemmli (1970). Purified nuclei were washed in 0.25 M sucrose in T?.i buffer containing 1 m?J P;4SF and directly dissolved in sample buffer. Nuclear matrix and nuclear envelope fractions were washed two or three times in 10 mlY Tris pH 7.4, 1.5 mPJ EGTA, 1 mY PFISF before dilution in the sample buffer. To prepare fractions cytoplasmic the first supernatant precipitated was with (3008) 10% trichloroacetic acid, washed in acetone and diluted into sample buffer. Samples diluted in sample buffer (containing 60 rn)I Tris pH G.S, 1.25% SDS and 0.1 !J DTT) were boiled for 3-5 min, cooled and stored at -20" C. were Gel-s stained with Coomassie blue. A11 electrophoresis from reagents were BioRad (Richmond, USA). The proteins SDS-PAGE separated were electrotransferred to 0.45 urn nitroF:llulose (Towbin et a1,1979). For lectin binding studies tile blot units were saturated with 0.3% Tween 20 (BioRad) in either phosphate buffered saline (PL:IS) or TS buffer (50 1rnI.l Tris p!i 7.4, 2OC; ,mN NaCl) for 30 min at 37" C and three times r 0 0 1~1 at temperature. The blots were then incubated wit;1 50 pgjmi. Concanavalin A (Con A) (Sigma) in TS buffer PlUS 0.05% Tween 20 for 30 nin, washed in TS buffer plus 0.05% T we e n and further incubated with 50 uz:/1:!1 horseradish perosidase (Sigma) in the same buffer. Alternatively the blots Ye r e wi t Ii incubated 50 )12/1111 wheat germ ( L?G!, ) agglutinin ;JEs conjugated to horseradish peroxidasc (Sigma) in 0.05% Tween for 1 hour. ;\fter extensive washing the b o :I II (:
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peroxidase was detected by a solution of 0.06% 4-chloro-lnaphtol (Sigma) and 0.01% hydrogen peroxide in TS buffer Controls were pre-incubated in (Hawkes, 1982). 0.5 methyl-a-D-mannopyranoside or 0.4 M N-acetyl-D-glucosamine min and then incubated in, respectively, the Con for 15 or VGA solutions in the presence of the sugar. Electron
M A
microscopy
of nuclei in 0.25 M sucrose, TM buffer, Suspensions were with an equal volume of 4% glutaraldehyde in 0.1 M mixed pH 7.4 and fixed for 30 min at 4' C. cacodylate buffer, Suspensions of nuclear matrices and nuclear envelopes in HS buffer fixed for 30 min at 4" C with were to a final concentration of glutaraldehyde which was added 1% (v/v), from a freshly prepared 4% stock solution in 50 mF1 cacoclylate buffer pH 7.4, 5 mM MgSO4 (Kaufmann et al, The fixed structures were sedimented, post-fixed in 1981). and processed for Epon embedding. Sections were 1% OS04 with uranyl acetate and lead citrate and studied stained in a Jeol 100 CXII electron microscope. RESULTS establish the purity and morphological composition of To various samples of material the subnuclear fractions, both obtained at each step were examined by phase contrast light microscopy transmission and electron Distinct non-nuclear contamination is absent microscopy. purified nuclear preparations (fig. 1). from Nuclei are relatively well preserved, with inner and outer membranes clearly seen. After Triton-X-100 extraction, internal morphology remains unaltered but both nuclear membranes are absent. Digestion of nuclei with DNase (RNase-free) followed extraction with low and high salt buffers bY matrix yields a residual nuclear composed of the peripheral lamina or nuclear envelope (whether nuclei were previously treated with Triton or not), residual nucleoli an internal fibrogranular network (fig. 2). Addition and RNasc to DNase in the presence of the of reducing agent ?jTi" yields nuclear a fraction of envelopes completely dcvoitled of intranuclear material (fig. 3). SDS-PAGL profiles of prostate nuclear The proteins with s h 0 I? a m a j 0 r protein band approximate molecular of 20 1:Da. This peptide (or group of peptides) is weight detected in prostate cytoplasmic fractions or in not rat liver isolated nuclei (fig. 4).
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Fig.1 Electron micrograph of purified prostate nuclei. Distinct non-nuclear contamination is absent,general morphology is well preserved,the nucleolus is clearly recognized and nuclear membranes are present. x 6 600. Electron micrograph of nuclear matrix prepared Fig. 2 the in absence of Triton X-100 and Ridase. A residual nucleolus is identified (nu), together with abundant intranuclear fibrogranular material. x 14 400. Fig. 3 Electron micrograph of nuclear envelopes uniformely devoided of intranuclear material as prepared by addition of llTT to the high salt extraction buffer. 11 400.
x
612
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prostate cyto. nuclei 94L blk
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3ok
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20.1 k
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.
Fig. 4 SDS-Page acrylamide gel. Lane 1: weight markers (phosphor albumin; ovalbumin; anhydrase; trypsin inhi lactalbumin). Lane 2: prostate cytoplasmic 1iane 3: nor!:lal prostate nuclei. Lane 1:: isolnt nuclei. .4r r 0 w points t Da prostate nuclear prot
on 12 molecular ylase b; carbonic bitor;a normal fraction. isolated ed liver o the 2@!c eins.
!I!!
14.4k
4 -castratednorm
3d
5d
7d
lad
AestosteroneId 2d
3d
prostate nuclear ventral rat 5 SDS-PAGE of weight Lane 1: molecular gel. pP:FLins on 12% acrylamide prostate isolated nuclei. Lanes 3 markers. Lane 2: normal nuclei isolated from castrated rats sacrificed 3, to G: nuclei Lanes 7 to 9: 7 and 10 days after operation. 5, 2 and 3 isolated from castrated rats sacrificed after 1, Arrow points to the 20 daily injections of testosterone. kDa proteins.
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the 20 kDa androgen dependence of for To test from castrated and were nuclei isolated peptides, electrophoretic their treated animals and testosterone The first significant decrease profiles compared (fig. 5). detected 7 relative intensity of the 20 kDa band is in daily testosterone After 3 castration. days after relative a clear increase in the injections there is although it does not attain normal amount of that protein, levels. Of matrix Electrophoretic analysis nuclear and reveals three major envelope fractions nuclear polypeptides within the molecular weight range of 60 70 corresponding to the nuclear lamins (fig. 6a). The 20 kDa, are present both in nuclear matrix kDa peptides and appearing as two nuclear envelope fractions, separated close bands (fig.6b). A binding liver Con glycoproteins in blots of and proteins were detected prostate nuclear essentially as described by Hawkes (1982). However, a considerable lower background staining was achieved by, the use of Tween 20 nitrocellulose for saturation of paper. Binding was i‘f inhibited by methyl-a-Dthe presence of 0.5 mannopyranoside. As expected, from the standard protein 3, mixture (myosin, galactosidase, phosphorylase bovine serum albumin and ovalbumin ) only ovalbumin was detected. The 20 IcDa peptides are shown to represent the major Con A binding proteins from normal prostate nuclei an d nuclear matrices cyto~~lasm~fig. 7). They are absent from ;;;;I: prostate castrated prostate nuclei and liver nuclei. The major Con A binding ilrotein fro,.: liver nuclei has an approximate molecular weight of lcITIa 200 (fig. 7). WGA binds intensely to several nuclear proteins including the 20 kDa androgen dependent peptides from normal prostate nuclei and nuclear matrices (fig. 8). DISCUSSION The presence of a 21 000 Da an d r 0 g c n- d c I1)e n d en t nuclear protein from rat ventral prostate was first reported by Kishimoto et al (1982). This peptide was not extractable w i t h 5% perchloric acid and only partially extractable with 0.35 1-1 idaC1. Later, Venkatranan et al (1934) resolved this protein into a group of 18 400 - 20 Oi)O Da p C [J t i c! E S and obtained electrophoretic profiles quite si:iiilnr to those presented in this paper. Ils rioscribed by those authors, the 18 400 - 20 003 pcptides resist triton-S-100 extraction and arc associated Twiti the nuclear c ri v c 1 0 7 E . The results presented here demonstrate, in addition, &at the peljtides associate also with thcx nuclear matrix.
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94k 2ook
116.25
67k
k
43k
92.sk 3ok bb.?k
20.1 k
14.4k
a
b
4
3
2
ConA
12
7
3
4
5
6
7
SDS-PAGE on 7.5% acrylamide gel. Fig. Lane 1: 6 (A) molecular weight markers (myosin, galactosidase, phosphorilase b, bovine serum albumin and ovalbumin). Lane 2: isolated prostate prostate nuclei. Lane 3: nuclear matrix fraction. Lane 4: prostate nuclear envelope fraction. Arrowheads point to lamina peptides. (B) The same fractions run on 12% acrylamide gel. Arrow points to the 20 kDa proteins.
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WGA v cyto.
*
prostatenuclei
matrix
liver nuclei
con
t rol
“N”
a.. 8
,’
1234
5
7 Proteins electrophoresed on 12% gels were Fig. transfered to nitrocellulose and tested for Concanavalin A binding. Lane I: prostate cytoplasm. Lane 2: normal prostate isolated nuclei. Lane 3: isolated nuclei from 10 day castrated animals. Lane 4: normal prostate nuclear isolated liver nuclei. matrix. Lane 5: Lanes 6 and 7: isolated nuclei from prostate (6) and liver (7) premethyl-a -D-mannopyranoside. incubated with Arrowheads point to the 200 kDa peptide presumely associated with pore Arrow nuclear complexes. points to the 20 kDa prostate nuclear proteins. 8 Proteins electrophoresed on 12% gels Fig. were transfered to nitrocellulose and tested for Wh e a t germ aglutinin binding. Lane 1: prostate cytoplasm. Lane 2: normal prostate isolated nuclei, Lane 3: normal prostate nuclear matrix. Lane 4: isolated liver nuclei. Lane 5: isolated liver nuclei pre-incubated with N-acetyl-Dglucosamine. Arrow points to the 20 kDa prostate nuclear proteins,
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The specific nuclear location of the 20 kDa pcptiilcs their is demonstrated :lbstnce rat prostate i. n in by cytoplasmic fractions. Although it is not possible to exclude the presence of similar proteins in other androgen this study shows that they are absent from target organs, liver nuclei. the results of time-course experiments it From may are practically be seen that the 20 kDa peptides become unaffected until 5 days after orchidectomy, but drastically reduced after 7 and 10 days. Injection Of first testosterone to castrated induces rats the daily protein increase after 3 significant only Considering that the mitotic activity in treatments. the after presents a peak the ventral prostate 2 days testosterone treatment (Tuohirnaa & Yiemi, beginning of results indicate that the 20 k Da protein these 1974), of epithelial cell number. recovery follows restoration immobilized nitrocellulose Glycoproteins on together membranes can be detected using Con A as a probe peroxidase substrate with peroxidase and a chromogenic liver (Hawkes, 1982; Rohringer et al, 1985). Isolated rat have a major high molecular weight (approx. 200 nuclei which resists Triton treatment kDa) Con A binding protein, and most probably corresponds to the 190 kDa glycoprotein by Gerace et al (1982) as a constituent of described the The same polypeptide is identified nuclear pore complex. in prostate nuclei and nuclear matrices, together with the nuclear prostate specific major kDa band of 20 glycoproteins. that It is currently considered carbohydrate lumen of are added to glycoproteins inside the residues organelles such as endoplasmic reticulum, Golgi apparatus These glycoproteins then nuclear envelope. are and on the exoplasmic face of cellular membranes, integrated external cell lumenal face of organelles or I.e., the surface (Hanover & Lennarz, 1978, 1980; Snider & Robins, 1982; Snider & Rogers, 1984; Torres & Hart, 1984). However proteins, there is evidence suggesting that glycosilated a class of glycoconjugates characterized by particularly residue bound Othe presence of a single GlcNAc occur in the polypeptide chain, also glycosidically to With cytoplasm and nucleoplasm (Holt & Hart, 1986). the tumor exception of the Simian Virus 40 large possible has suggested to be a O-linked which been antigen, (Jarvis other Butel, 1985), no glycoprotein & nucleoplasmic glycoproteins have been characterized. major The results presented here indicate that the from ventral peptides rat nuclear androgen-dependent glycoproteins containing both mannose or prostate are N-acetyl-glucosamine (Nicolson, residues glucose and unrecognized previously The association of these 1974). matrix glycoproteins with nuclear is also the
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association of the interpreting the d e c:on s t r a t e d . In matrix with nuclear peptides the andro~en-dependent particular attention should be taken, however, considering cross-links to that inadvertent formation of the fact the proteins during isolation procedure other nuclear might render some polypeptides nucleasc and salt-insoluble (Kauf;:lann et al, 1986). whether Eurther studies are necessary to determine these androgen-dependent nuclear proteins are involved in merely Of horlIlone action or if they mechanism the of androgen stimulation, represent a product
ACKNOWLEDGMENTS The author is grateful to Galego for the kind supply to Ii. 2. AlpiarGa and J. assistance Prof. and to helpful discussion of the
Drs. C. Rodrigues-Pousada of a DNase (RNase-free) C. Lemos for skillful Dr. J. F. David-Ferreira manuscript.
and L. sample, technical for
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Received:
3.2.88
Accepted:
22.6.88
Lefebvre, Y. A. the rat ventral Biophysical and