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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 1 317-321
Copyright © 2000 by The Endocrine Society

Original Studies

Prostate-Specific Antigen Synthesis and Secretion by Human Placenta: A Physiological Kallikrein Source during Pregnancy1

Manuela Malatesta, Ferdinando Mannello, Francesca Luchetti, Francesco Marcheggiani, Leone Condemi, Stefano Papa and Giancarlo Gazzanelli

Istituto di Istologia and Analisi di Laboratorio (M.M., F.M., F.M., G.G.) and Istituto di Scienze Morfologiche (F.L., S.P.), Facoltà di Scienze MFN, Università degli Studi di Urbino, and Divisione di Ginecologia and Ostetricia, Ospedale Civile (L.C.), 61029 Urbino, Italy

Address all correspondence and requests for reprints to: Dr. Ferdinando Mannello, Istituto di Istologia ed Analisi di Laboratorio, Università degli Studi, Facoltà di Scienze MFN, Via E. Zeppi, 61029 Urbino (PU), Italy. E-mail: mannello{at}bio.uniurb.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostate-specific antigen (PSA), a kallikrein-like serine protease until recently thought to be prostate specific, has been demonstrated in various nonprostatic tissues and body fluids. PSA has been also found in human endometrium and amniotic fluids, even if the significance of this novel expression is unclear. In this study, we have demonstrated by multiple techniques that human placental tissue, obtained at delivery from normal full-term pregnancies, synthesizes and secretes PSA. RT-PCR showed the presence of PSA messenger ribonucleic acid; biochemical, chromatographic, and immunological studies revealed the expression of both free and complexed PSA forms; immunoelectron microscopy indicated the syncytiotrophoblast as the site of PSA synthesis and secretion. Moreover, in vitro experiments demonstrated that PSA production and secretion are up-regulated by 17ß-estradiol, a pregnancy-related steroid hormone. These results suggest that human placenta is a source of the PSA present in amniotic fluid and maternal serum during pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE KALLIKREINS (KLK) are a family of serine proteases involved in the posttranslational processing of polypeptides to their bioactive or inactive forms. They are encoded by a multigene family, of which only three members have been characterized to date in the human: KLK 1, encoding the true glandular kallikrein; and KLK 2 and KLK 3, two genes primarily expressed in the prostate that encode for prostate-specific antigen (PSA) (1). PSA is a serine protease with chymotryptic-like activity (2) that until recently has been thought to be exclusively produced by epithelial cells of the prostate gland and then used as a marker for the diagnosis and management of prostate cancer (3). However, several studies have recently demonstrated the widespread distribution of PSA in a variety of human normal and tumoral tissues, cell lines, and biological fluids (4). Although the physiological role and the biological significance of extraprostatic PSA are currently unknown, it has been suggested that this serine protease can be regarded as a growth factor regulator produced by cells bearing steroid hormone receptors (5).

PSA immunoreactivity has been also revealed in normal and pathological amniotic fluids, with varying content in relation to gestational age: biologically and immunologically, PSA found in amniotic fluids was identical to the prostate KLK-like serine protease, but its physiological function and biological origin have not been yet clarified (6, 7, 8). A study has also demonstrated that the PSA gene is expressed in human normal cycling endometrium (9), suggesting the presence of a local KLK-kinin system in this hormone-responsive tissue. Moreover, preliminary results showing the presence of PSA protein in human at term placenta have been recently reported (10).

The present study provides biochemical, molecular, and immunocytochemical evidence for the synthesis and secretion of PSA by human placental tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Samples

Seven fresh human placentas were collected from women (aged 25–38 yr) undergoing normal, full-term pregnancies (40 ± 2 weeks) immediately after delivery. After the membranes were stripped, each placenta was immediately processed for the different analyses. Blood was also drawn from healthy control women (n = 15) and pregnant women (n = 7), and after blood clotting, the samples were centrifuged at 500 x g for 10 min, and sera were stored at -30 C until assay (<2 weeks). The subjects gave informed consent to the study, which was performed in accordance with the ethical standards of Helsinki Declaration of 1975, as revised in 1983.

PSA and protein measurements

Fragments of placenta were homogenized and then sonified on ice according to the procedure described previously (10). The lysates were centrifuged at 9150 x g at 4 C for 30 min, after which the supernatants were immediately stored at -80 C until analysis (<2 weeks). The total protein content was determined with the bicinchoninic method, using a commercially available kit (Bio-Rad Laboratories, Inc., Hercules, CA). Free and total PSA concentrations were determined in serum and cytosolic extracts of placenta with the AxSYM PSA assay (Abbott Laboratories, Abbott Park, IL) (11, 12). PSA immunoreactivity, determined for a minimum of three concentrations at least in triplicate, was expressed as micrograms per L. The detection limits of the test were 0.02 and 0.01 µg/L for total and free PSA, respectively. Placental extracts were serially diluted in PSA-negative female serum and reanalyzed for the response linearity to exclude the possibility that the detection of nonprostatic PSA was due to a matrix effect. Analytical recovery of two concentrations (6.5 and 13 µg/L) of purified PSA (Sigma, St. Louis, MO) was performed as previously detailed (13).

Immunogram and Western blotting

Sample components were separated on a 600 x 9-mm column of Sephacryl S-300 (Pharmacia Biotech, Uppsala, Sweden). The samples were applied to the column and eluted with 0.05 mol/L Tris-HCl buffer, pH 7.5. Fractions of 0.5 mL each were collected and analyzed for PSA content. Our Western blotting protocol was followed throughout, using an antihuman PSA monoclonal mouse antibody (DAKO Corp., Milan, Italy) (11, 12). PSA from culture supernatant of LNCaP, a human prostate carcinoma cell line that constitutively secretes PSA (1), was used as a positive control.

Immunoelectron microscopy

Immediately after labor, fragments of placental tissue were fixed by immersion in a mixture of 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 mol/L Sörensen phosphate buffer, pH 7.4, at 4 C for 2 h and then dehydrated and embedded in LRWhite resin (10). Ultrathin sections were processed for immunocytochemistry using a rabbit polyclonal antihuman PSA antibody (Biomeda, Foster City, CA) and a secondary gold-conjugated antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) following our previously reported protocol (12). As controls, some sections were treated in the absence of anti-PSA antibody.

Extraction of ribonucleic acid (RNA) and RT-PCR

Total RNA from placental tissue, collected immediately after labor, and from LNCaP cells was extracted using a commercial reagent, RNA-Fast (Promega Corp., Madison, WI), according to the manufacturer’s recommendations. Total RNA (5 µg) underwent RT for synthesis of the first strand of complementary DNA (cDNA), using 1 µmol/L deoxynucleoside triphosphates, 10 mmol/L dithiothreitol, and 200 U SuperScript II reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD). The reaction was performed at 42 C for 1 h, followed by a denaturation step for 5 min at 95 C. Amplification of the cDNA was performed as previously described (12). An initial denaturation step (95 C for 2 min) was followed by 40 cycles (94 C for 50 s, 61 C for 50 s, and 72 C for 90 s) and a final extension for 10 min. The PSA was amplified in 45 µL of a PCR mixture containing 1 x PCR buffer, 1.5 mmol/L magnesium chloride, 200 nmol/L of each primer, 200 µmol/L deoxynucleoside triphosphates, and 2.5 U AmpliTaq DNA polymerase (Promega Corp.). Ten microliters of each PCR reaction were electrophoresed on 1.5% agarose gels and visualized by ethidium bromide staining under a UV light source. The new PSA primer sequences, designed on the basis of sequence data obtained from the European Molecular Biology Gene Bank and used to avoid amplification of the highly homologous human glandular kallikrein gene (14), were as follows: PSA E-S, 5'-CTCTCGTGGCAGGGCAGT-3' (exon 2); and PSA AE-S, 5'-CCCCTGTCCAGCGTCCAG-3' (exon 4). The predicted PSA primer-amplified product was 485 bp in size. For placental tissue, LNCaP cells, and negative control samples, messenger RNA extraction and cDNA amplification were carried out one sample at a time to avoid cross-contamination.

Culture of explants

Placental tissue was dissected, rinsed with Earle’s Balanced Salt Solution, cultured in 25-cm2 tissue culture flasks, and maintained up to 7 days at 37 C in a 5% CO2 incubator with RPMI 1640 phenol red-free medium containing 20 mmol/L HEPES, 10% charcoal-stripped FBS, 2 mmol/L L-glutamine, nonessential amino acids, 1% antibiotic-antimycotic solution, and 0.075% NaHCO3 (Sigma). The phenol red-free media were used, because phenol red has weak estrogenic activity (15), whereas charcoal-stripped FBS is devoid of any steroid hormones. The cells were also grown in serum-free medium in the presence of 17ß-estradiol (Schering AG, Berlin, Germany). Stimulation was initiated by adding 10-7 mol/L steroidal compound dissolved in absolute ethanol and incubating the explants for up to 7 days. Tissue culture supernatants were removed for PSA analysis on days 1, 3, 5, and 7. The secretion index, defined as the secreted PSA divided by the cell-associated PSA, was expressed as a percentage. The media removed at the end of each day were centrifuged at 3000 x g for 15 min at 4 C and stored at -30 C until assay. At the end of the culture period, tissue explants were homogenized in lysis buffer, as previously described (11). LNCaP (American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 containing 10% FCS, 2 U/L penicillin, 200 µg/L streptomycin, and 5 mmol/L glutamine.

Statistical analyses

Statistical analysis of results, reported as the mean ± SE of at least three independent experiments, was performed with the StatView 4 package (Abacus Concepts, Berkeley, CA), using a Macintosh PB (Apple Computer, Cupertino, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The average serum PSA content of the healthy control women examined (n = 15) was 0.03 ± 0.01, vs. 0.15 ± 0.05 µg/L in serum from pregnant women (n = 7; P < 0.0008). The mean concentration of total PSA in placental tissues (n = 7) was 57 ± 9 µg/L, with about 30% in the free noncomplexed form (17.31 ± 2.64 µg/L). The dilution studies revealed a good linearity (n = 7; r2 = 0.98), demonstrating that placental matrix (i.e. lipids, hemoglobin, hormones, and proteins) did not affect the performance of PSA assay specific for serum samples. The immunoenzymometric tests revealed that more than 70% of the total PSA in placenta was in a bound form, and the remainder was free uncomplexed protease; these data were also confirmed by the elution chromatographic profile, revealing the highest immunoreactivity in the fraction where the PSA complex with {alpha}1-antichymotrypsin (~100 kDa) was expected (Fig. 1Go). The immunoreactivity of the free PSA form was also found in fractions 56–67 (molecular mass, 35–40 kDa); these data were confirmed by Western blotting analysis performed with a specific monoclonal antibody (Fig. 1Go).



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  		Figure 1. Elution profile of placental PSA immunogram from the Sephacryl S-300 column. The positions of the molecular mass markers are indicated at the top: IgG (158 kDa), BSA (66.2 kDa), and bovine carbonic anhydrase (31 kDa). Western blot analysis of PSA in placenta (8 ng) is reported in the inset.

 
The electron microscopic examination of immunolabeled samples of placenta revealed that most PSA labeling occurred in epithelial cells (syncytiotrophoblast) coating the villous surface (Fig. 2Go). These cells showed a diffuse cytoplasmic signal, frequently occurring on rough endoplasmic reticulum cisternae (Fig. 2aGo); moreover, the labeling appeared concentrated in the apical region, in particular in the microvilli and the cytoplasmic layer just beneath them (Fig. 2bGo). Some labeling was also observed in the basal region of these cells, in the numerous cytoplasmic protrusions spreading out in the connective matrix (Fig. 2cGo). This matrix as well as the fibroblasts distributed therein showed a weak signal (not shown). No significant labeling was observed in cell nuclei. Control samples were virtually unlabeled.



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  		Figure 2. Immunocytochemical localization of PSA in the syncytiotrophoblast. a, PSA labeling is located in the RER cisternae (arrowheads) as well as free in the cytoplasm (arrows). The nucleus (N) is almost devoid of labeling. b, In the apical region of the syncytiotrophoblast, strong labeling is present in microvilli (arrows). c, The basal region of the syncytiotrophoblast shows specific labeling in the cytoplasmic protrusions (arrows) spreading out in the connective tissue (C). The connective matrix displays a weak signal (arrowhead). The bars represent 0.25 µm.

 
As shown in Fig. 3Go, ethidium bromide-stained agarose gel electrophoresis demonstrated that placental tissue as well as LNCaP cells, which are both positive for PSA immunoreactivity, produced the expected 485-bp transcript.



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  		Figure 3. Ethidium bromide-stained agarose gel of RT-PCR products of PSA messenger RNA isolated from normal human placental tissue and LNCaP cells. MW, Molecular weight markers expressed in base pairs. Lane 1, LNCaP cells (as positive control); lane 2, placental tissue; lane 3, negative control.

 
The cellular extracts of placental explants cultured in vitro showed a total PSA content of 5.47 ± 0.07 µg/mg protein, 1.53 ± 0.06 µg of which was in the free PSA form. The explants secreted in culture medium an immunoreactive PSA protein with a steady increase in rate; the average daily secretion in medium was 0.10 ± 0.01 µg/L, and the total amount of PSA released over the 7-day period represented about 15% of the amount in the tissue explant (Table 1Go). Addition of 17ß-estradiol to the cultured placental explants resulted in a significant stimulation of PSA production (P < 0.001; Fig. 4Go).


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  		Table 1. PSA in culture media and decidua

 


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  		Figure 4. Time course of PSA release by placental explants into culture medium after 17ß-estradiol stimulation (•). The negative control ({circ}) was treated only with absolute ethanol.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PSA, until recently considered a prostate-specific serine protease (1), has been demonstrated to be a widespread biochemical marker, regulated by several steroidal compounds (5). In particular, the PSA gene may be up-regulated in physiological conditions associated with a steroid hormone overproduction (e.g. during pregnancy and in the endometrial cycle), suggesting new biological roles of PSA both in fetal growth/development (6, 7, 8) and as a potential regulator of uterine function (9). However, the source of PSA found in amniotic fluids and maternal serum has not yet been clarified.

In a preliminary study, we detected PSA in human normal placental tissue (10). In this report we demonstrated that human normal placenta synthesizes and secretes PSA. The molecular approach showed that the PSA gene is present and functionally active, the biochemical analyses showed that placental PSA occurs as both free and complexed molecules, and immunocytochemistry revealed the syncytiotrophoblast as the main responsible for placental PSA biosynthesis and secretion. In particular, the ultrastructural results suggest that the syncytiotrophoblast is a bipolar structure. PSA is synthesized in the rough endoplasmic reticulum cisternae, then transported and secreted as free/complexed molecules mainly in the apical region and in a much lower amount in the basal region. Moreover, our in vitro experiments not only confirmed that placental cells actively produce and secrete PSA, but also demonstrated that 17ß-estradiol, a steroid hormone related to pregnancy, is able to up-regulate PSA secretion. A similar phenomenon has been observed in breast cancer cell lines, when treated with several steroid compounds (15).

The expression of PSA in nonprostatic sources and, in particular, in female tissues and fluids suggests new important biological roles of this serine protease, i.e. as a potential sensitive biochemical/molecular marker of hormone responsiveness (5, 11, 15). The concomitant presence of the steroid hormones and receptors in human placenta (16) and the significant increase in PSA production under hormone stimulation (present study) suggest the possibility of placental PSA modulation by steroid hormones (17).

Our results strongly support the hypothesis that placental tissue represents a source of the PSA found in both maternal serum and amniotic fluid during pregnancy (6, 7, 8). PSA in these body fluids may play a role as a growth factor modulator and/or as a translational/posttranscriptional protein regulator. In fact, PSA hydrolyzes the insulin chains and interleukin-2 (2), enzymatically digests insulin-like growth factor-binding proteins (18), activates latent transforming growth factor (19), inactivates protein C inhibitors (20, 21), and regulates the hormonal bioactivity of PTH-related protein (22, 23). The proteolytic activity of PSA on these different biological substrates, all detected in placenta (24), could explain in part the novel potential role of PSA in this tissue, not only as a sensitive molecular marker implicated in hormone responsiveness but also as an initiator of the protease cascade, an important biological mechanism for tissue remodeling in the uterus (25, 26).


   Footnotes
 
1 This work was supported in part by a grant from the Assessorato alla Sanità, Regione Marche, Italy. Back

Received January 6, 1999.

Revised July 6, 1999.

Revised September 3, 1999.

Accepted September 14, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. McCormack RT, Wang TJ, Rittenhouse HG, Wolfert RL, Finlay JA, Sokoloff RL. 1995 Molecular forms of prostate-specific antigen and the human kallikrein gene family: a new era. Urology. 45:729–744.[CrossRef][Medline]
  2. Watt KWK, Lee PJ M, ’Timkulu T, Chan WP, Loor R. 1986 Human prostate-specific antigen: structural and functional similarity with serine proteases. Proc Natl Acad Sci USA. 83:3166–3170.[Abstract/Free Full Text]
  3. Chu MT. 1997 Prostate-specific antigen and early detection of prostate cancer. Tumor Biol. 18:123–134.
  4. Diamandis EP, Yu H. 1997 Nonprostatic sources of prostate-specific antigen. Urol Clin North Am. 24:275–282.[CrossRef][Medline]
  5. Diamandis EP, Yu H. 1995 New biological functions of prostate-specific antigen. J Clin Endocrinol Metab. 80:1515–1517.[Free Full Text]
  6. Yu H, Diamandis EP. 1995 Prostate-specific antigen immunoreactivity in amniotic fluid. Clin Chem. 41:204–210.[Abstract/Free Full Text]
  7. Melegos DN, Yu H, Allen LC, Diamandis EP. 1996 Prostate-specific antigen in amniotic fluid of normal and abnormal pregnancies. Clin Biochem. 29:555–562.[CrossRef][Medline]
  8. Filella X, Molina R, Alcover J, Carretero P, Ballesta AM. 1996 Detection of nonprostatic PSA in serum and nonserum samples from women. Int J Cancer. 68:424–427.[CrossRef][Medline]
  9. Clements J, Mukhtar A. 1994 Glandular kallikreins and prostate-specific antigen are expressed in the human endometrium. J Clin Endocrinol Metab. 78:1536–1539.[Abstract]
  10. Mannello F, Malatesta M, Fusco E, Bianchi G, Cardinali A, Gazzanelli G. 1998 Biochemical characterisation and immunolocalization of prostate-specific antigen in human term placenta. Clin Chem. 44:1735–1738.[Free Full Text]
  11. Mannello F, Sebastiani M, Amati S, Gazzanelli G. 1997 Prostate-specific antigen expression in a case of intracystic carcinoma of the breast: characterisation of immunoreactive protein and literature surveys. Clin Chem. 43:1448–1454.[Abstract/Free Full Text]
  12. Mannello F, Malatesta M, Luchetti F, Papa S, Battistelli S, Gazzanelli G. 1999 Immunoreactivity, ultrastructural localization, and transcript expression of prostate-specific antigen in human neuroblastoma cell lines. Clin Chem. 45:78–84.[Abstract/Free Full Text]
  13. Mannello F, Miragoli G, Bianchi G, Gazzanelli G. 1997 Immunoreactive prostate-specific antigen in pleural effusions. Clin Chem. 43:847–848.[Free Full Text]
  14. Corey E, Arfman EW, Liu AY, Vessella RL. 1997 Improved reverse transcriptase-polymerase chain reaction protocol with exogenous internal competitive control for prostate-specific antigen mRNA in blood and bone marrow. Clin Chem. 43:443–452.[Abstract/Free Full Text]
  15. Zarghami N, Grass L, Diamandis EP. 1997 Steroid hormone regulation of prostate-specific antigen gene expression in breast cancer. Br J Cancer. 75:579–588.[Medline]
  16. Strauss JF, Martinez F, Kiriakidon M. 1996 Placental steroid hormone synthesis: unique features and unanswered questions. Biol Reprod. 54:303–311.[Abstract]
  17. Petraglia F, de Micheroux AA, Florio P, et al. 1995 Steroid-protein interaction in human placenta. J Steroid Biochem Mol Biol. 53:227–231.[CrossRef][Medline]
  18. Cohen P, Graves HCB, Peehl DM, Kamarei M, Giudice LC, Rosenfeld RC. 1992 Prostate-specific antigen is an insuline-like growth factor binding protein-3 protease found in seminal plasma. J Clin Endocrinol Metab. 75:1046–1053.[Abstract]
  19. Killian CS, Corral DA, Kawinsky E, Constantine RI. 1993 Mitogenic response of osteoblast cells to prostate-specific antigen suggests an activation of latent TGF-ß and a proteolytic modulation of cell adhesion receptors. Biochem Biophys Res Commun. 92:940–947.
  20. Espana F, Gilabert J, Estelles A, Romeu A, Asmar J, Cabo A. 1991 Functionally active protein C inhibitor/plasminogen activator inhibitor-3 is secreted in seminal plasma and complexes with prostate-specific antigen. Thromb Res. 64:309–320.[CrossRef][Medline]
  21. Kise H, Nishioka J, Kawamura L, Suzuki K. 1996 Characterization of semenogelins and its molecular interaction with prostate-specific antigen and protein C inhibitor. Eur J Biochem. 238:88–96.[Medline]
  22. Iwamura M, Hellman J, Cockett AT, Lilja H, Gershagen S. 1996 Alteration of the hormonal bioactivity of parathyroid hormone-related protein as a results of limited proteolysis by prostate-specific antigen. Urology. 48:317–325.[CrossRef][Medline]
  23. Cramer SD, Chen Z, Peehl DM. 1996 Prostate-specific antigen cleaves parathyroid hormone-related protein in the PTH-like domain: inactivation of PTHrP-stimulated cAMP accumulation in mouse osteoblasts. J Urol. 156:526–531.[CrossRef][Medline]
  24. Lala PK, Hamilton GS. 1996 Growth factors, proteases and protease inhibitors in the maternal-fetal dialogue. Placenta. 17:545–555.[CrossRef][Medline]
  25. Salamonsen LA. 1994 Matrix metalloproteinases and endometrial remodelling. Cell Biol Int. 18:1139–1144.[CrossRef][Medline]
  26. Clements J, Mukhtar A, Yan S, Holland A. 1997 Kallikreins and kinins in physiologic events in the reproductive tract. Pharmacol Res. 35:537–540.[CrossRef][Medline]



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