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Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2289-2296
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Departments of Medicine, Pathology, Anesthesiology, and
Oncology, Johns Hopkins University School of Medicine, Baltimore, MD.
Gender differences in vascular thromboses are well known, and there
is evidence that platelets may be involved in these differences and
that sex hormones affect platelet function. We characterized the
expression of the estrogen receptor
Thromboembolic complications of coronary and cerebral
atherosclerotic diseases are the number 1 cause of death for both men and women in our society.1 Gender differences in the
epidemiology of thromboembolic diseases have been described in a number
of clinical settings. The incidence of cardiovascular disease is lower
in premenopausal women than in men, but is increased for women after
menopause,2 suggesting cardioprotective effects of
estrogens. Observational studies led to the generally accepted belief
that hormone replacement therapy (HRT) in postmenopausal women was
beneficial, and one of the proposed mechanisms of the beneficial
effects of HRT was the ability to lower low-density lipoprotein
cholesterol and raise high-density lipoprotein
cholesterol.3,4 However, recent data from the Heart and
Estrogen/Progestin Replacement Study (HERS) indicated that HRT in
postmenopausal women with known coronary artery disease produced no
overall benefit, despite the expected improvement in serum
cholesterol.5 These unexpected findings raise the
possibility that HRT may have a biologic effect that counteracts its
beneficial effect on lipids. Substantial evidence indicates that HRT
predisposes to venous thrombosis,6,7 but little information
exists regarding a possible prothrombotic effect of HRT on platelets in
the coronary circulation, where high shear forces exert an especially
important effect on the generation of platelet thrombi.8
There is both indirect and direct evidence that sex hormones affect
human platelet biology. For example, several physiologic properties of
platelets from women have been shown to vary with the phase of the
menstrual cycle. We have found that platelets from women bound more
fibrinogen during the luteal phase (defined as 14 or fewer days from
the onset of the next menstrual cycle, or the second half of the
menstrual cycle) of the menstrual cycle than during the follicular
phase, suggesting a hormonal regulation of glycoprotein (GP) IIb-IIIa
activation.9 The number of To explore the possibility that gender differences in platelet function
are due to sex-hormone-mediated genomic or nongenomic events, we
investigated whether the megakaryocyte/platelet lineage contains
receptors for sex hormones. We found that megakaryocytes and platelets
express the estrogen receptor (ER) Reagents
Human subjects
Enrichment of CD34+ cells from leukopheresis units or bone marrow To obtain stem cells from leukopheresis units, low-density mononuclear cells from normal donors were first separated by centrifugation over HISTOPAQUE 1077 (Sigma) according to the manufacturer's suggested protocol. Mononuclear cells were washed twice in 1% BSA in phosphate-buffered saline (PBS) (pH = 7.4, 0.137 mol/L NaCl, 4.3 mmol/L Na2HPO4, 1.4 mmol/L KH2PO4, 2.7 mmol/L KCl) without Ca+2 and Mg+2. Cells were incubated with biotinylated anti-CD34+ antibody (gift from the Johns Hopkins Oncology Center, Baltimore, MD) for 30 minutes and washed, and then CD34+ cells were isolated from the mononuclear cell fraction by passage over an avidin column (CellPro, Bothell, WA). In a second protocol, donor bone marrow for allogeneic transplantation was processed by our clinical Graft Engineering Laboratory (Johns Hopkins University, Baltimore, MD). Small mononuclear cells were separated from marrow by clinical elutriation centrifugation. This lymphoid-rich fraction was labeled with biotinylated anti-CD34 antibody and passed over a CEPRATE column (CellPro). The CD34-depleted fraction was washed, relabeled with biotinylated anti-CD34 antibody, washed again, and passed over an avidin column as previously described for leukopheresis products. Yields from the 2 preparation techniques were as follows: leukopheresis, 0.8 to 1.2 × 106 CD34+ cells from 1 × 109 mononuclear cells; CD34-depleted bone marrow fraction, 3 to 7 × 106 CD34+ cells from 1 × 109 mononuclear cells. CD34+ cells were resuspended in Stem Pro culture media without phenol red and without serum, cultured with 1 ng/mL IL-3 for 2 days, and immediately harvested (termed day-0 cells) or treated with 50 ng/mL PEG-rhMGDF for 7 days (termed day-7 cells). Approximately 25% of the day-7 cells were megakaryocytes as assessed by flow cytometry with the use of the megakaryocyte-specific marker GPIb or GPIIb.Cell lines and cell culture The human erythroleukemia (HEL) and Dami megakaryocytic cell lines were obtained from the American Type Culture Collections (ATCC; Rockville, MD). The Dami cells were obtained from the ATCC in 1989 and demonstrate greater GPIIIa (integrin 3) expression than
do our HEL cells, despite their likelihood of being a subclone of
HEL. PC3 and LNCap, both human prostate cancer cell
lines, were gifts from Dr John Isaacs, and T47D, a human breast cancer cell line19 was a gift from Dr Saraswati Sukumar (both from Johns Hopkins University). These cell lines were used as controls for
ER  , ER , the progesterone receptor (PR), and AR. HEL and Dami
cells were grown in RPMI 1640 containing 10% serum and 1% penicillin/streptomycin. T47D cells were grown in RPMI 1640 containing 10% serum, 1% penicillin/streptomycin, and 0.2 IU/mL insulin. PC3
cells were cultured in Ham's F12K media containing 10% FBS and 1%
penicillin/streptomycin with 2 mmol/L
L-glutamine, and 1.5 g/L sodium bicarbonate. Depending
on the experiment, cells were cultured in FBS (GibcoBRL) or
charcoal-stripped FBS. The FBS was stripped with the use of a standard
protocol (Sigma) containing 0.25% charcoal (vol/vol) and 0.0025%
dextran (vol/vol) in 10 mmol/L HEPES (pH 7.4).
Platelet preparation Whole blood was obtained into acid-citrate-dextrose (0.1 mol/L trisodium citrate, 0.11 mol/L dextrose, and 71 mmol/L citric acid monohydrate) anticoagulant with the use of a 19-gauge needle and platelet-rich plasma (PRP), prepared as previously described.20 The top, middle, and bottom one third (by volume) of the PRP were designated PRP-upper, PRP-middle, and PRP-lower, respectively. Total red blood cells, white blood cells, and platelets were counted. Platelets were obtained by centrifugation of PRP at 800g for 20 minutes. Gel-filtered platelets were also prepared from PRP obtained as described previously. Then, 7.5 µmol/L PGI2 in 1:50 dilution and freshly prepared apyrase (20 µL/mL) were added, and the PRP was centrifuged at 800g for 20 minutes. The platelet pellet was resuspended in 1 mL buffer (138 mmol/L NaCl, 12 mmol/L NaHCO3, 10 mmol/L KCl, 5.5 mmol/L glucose, 0.36 mmol/L Na2HPO4, 0.35% BSA, and 10 mmol/L HEPES, pH 7.4) containing apyrase and PGI2. Platelets were purified over a Sepharose CL-2B (Pharmacia, NJ) column. The eluate possessed normal adenosine-diphosphate-inducible aggregation in the presence of fibrinogen.Reverse transcription-polymerase chain reaction Total RNA was prepared from different cell samples with the use of a single-step guanidine thiocyanate/phenol kit (RNA STAT-60; Tel-Test, Friendswood, TX). One microgram of total RNA was reverse-transcribed in a 20-µL reaction mixture containing 50 mmol/L KCl, 10 mmol/L Tris-HCl; pH 8.3, 4 mmol/L MgCl2, 1 mmol/L dNTPs (Pharmacia), 100 pmol random hexanucleotides (Pharmacia), 15 units of ribonuclease inhibitor (Gibco BRL), and 220 units M-MLV reverse transcriptase (Gibco BRL) at 37°C for 1 hour. Polymerase chain reaction (PCR) conditions varied slightly according to primers, but the ranges were as follows: 30 to 35 cycles of 1 minute at 94°C, 30 to 60 seconds at 52°C to 60°C, and 30 to 60 minutes at 72°C. Then, 1× PCR buffer (20 mmol/L Tris-HCl, pH 8.0, 50 mmol/L KCl) was used in the presence of 200 mmol/L deoxynucleotide-triphosphates (dNTPs), and 2.0 mmol/L MgCl2, 10 pmol of each primer per reaction, and 2.5 units of Gibco Taq polymerase. Then, 10 µL of the PCR reactions was separated on 2% agarose gels and visualized by ethidium bromide staining. All reverse transcription (RT) - PCR analyses were confirmed at least twice in separate experiments.
Immunostaining and fluorescence microscopy Cells were washed once in PBS containing 0.05% sodium azide (Sigma), cytofuged onto glass slides, fixed in 4% paraformaldehyde for 10 minutes at 4°C, washed 3 times in PBS, and washed twice in PBS with 50 mmol/L NH4Cl. Cells were permeabilized with 0.05% saponin in PBS containing 10% normal goat serum for 30 minutes at 22°C. The first primary antibody incubation was performed in PBS containing 10% normal goat serum and 0.05% saponin for 1 hour at 37°C followed by washing in PBS containing 0.05% saponin 3 times for 5 minutes each at 22°C. Cells were then incubated with the first fluorochrome-conjugated secondary antibody for 30 minutes at 37°C followed by washing 3 times in PBS containing 0.05% saponin for 5 minutes each at 22°C. The nucleic acid staining dye Dapi (10 mg/mL) was diluted in the preparatory solution A of Slowfade Antifade kit (Molecular Probes, Eugene, OR) in 1:100 dilution. After an initial primary and secondary antibody staining, the procedure was repeated for the second primary and secondary antibody staining, and the slides were then mounted in the preparatory solution A containing Dapi. Each fluorochrome was analyzed individually by means of an inverted confocal laser scanning Fluorescence Microscope (LSM; Zeiss, Germany).
Western immunoblotting Immunoblotting was performed as previously described.26 Cells were lysed in 15 mmol/L Hepes, pH 7.0, 145 mmol/L NaCl, 0.1 mmol/L MgCl2, 10 mmol/L ethylene glycol-bis-N,N,N',N'-tetraacetic acid (EGTA), 1% Triton X-100, 1 mmol/L NaVO4, 250 µg/mL 4-2-aminoethyl-benzene sulfonylfluoride, 15 µg/mL of protease inhibitors (chymostatin, antipain, and pepstatin), and 55 µg/mL of the protease inhibitor leupeptin. Protein concentration was determined by the Bradford technique. For AR analysis, the lysing detergent was 2% sodium dodecyl sulfate (SDS). Lysates were separated by 8% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Amersham, Buckinghamshire, England). A series of polyclonal antisera were used to probe for ER: L-20 and
N-19 (Santa Cruz Biotechnology, Inc), anti-ER (Upstate
Biotechnology, Lake Placid, NY), and Ab-1 (Oncogene
Research Products, Cambridge, MA). A series of polyclonal antisera were
used to probe for AR: PG-21 (Upstate Biotechnology), C19 and
N-20 (Santa Cruz Biotechnology), and NCL-ARp (Vector
Laboratories, Burlingame, CA). Numerous experiments indicated that the
anti-ER from Upstate Biotechnology, and PG21 yielded the best
results. Blocking peptides from the amino termini of ER and AR
were purchased from Upstate Biotechnology, and Santa Cruz
Biotechnology, respectively. The specificity of fragments detected by
immunoblot was assessed by preincubating the primary antibody with the
various peptides. For 10 µg/mL of antibody, 35 to 70 µmol/L peptide was used.
Radioimmunoassay Total testosterone was measured with the use of the Coat-A-Count Kit (Diagnostic Products, Los Angeles, CA) according to the manufacturer's protocol. Testosterone levels in charcoal-stripped FBS were undetectable (lower limit of detection is 0.14 nmol/L) and in FBS was 3.5 nmol/L.
Identification of RNA transcripts for sex hormone receptors Specific nuclear and cytoplasmic receptors mediate the biologic effects of sex hormones. We surveyed the megakaryocyte/platelet lineage for the expression of ER, ER , PR, and AR. Using total RNA from
various cell lines and oligonucleotide primers specific for each
receptor cDNA, we probed for the respective transcripts by RT-PCR.
T47D, a breast cancer cell line, served as positive control for all 4 receptors (Figure 1, lane 7) and
demonstrated the ability of our primers to amplify the expected
targets. ER and AR transcripts were identified in day-0
CD34+ cells not treated with PEG-rhMGDF (lane 1) and
appeared to increase after CD34+ cells were treated with
PEG-rhMGDF for 7 days (lane 2). The ER and AR RT-PCR products from
the day-7 cells were cloned, and nucleotide sequencing confirmed them
to be authentic ER and AR transcripts (data not shown).
Approximately 25% of the day-7 cells expressed the
megakaryocyte-specific marker GPIIb, and many showed an increased cell
size and a nucleus with several lobes, morphologic features typical of
megakaryocytes (Figures 2 and 3). ER and PR messenger RNAs (mRNAs)
were not observed in the day-0 or day-7 cells (Figure 1, lanes 1 and
2), or after longer exposure to PEG-rhMGDF (not shown). The absence of
ER and PR transcripts in CD34+ or
CD34+-derived cells was not due to poor quality RNA, since
actin (Figure 1, bottom panel), ER , and AR transcripts were
readily amplified in all RNA preparations.
Localization and distribution of the estrogen receptor Expression of the estrogen receptor in platelets of either sex by this technique (not
shown). The anti-ER antisera used in the immunofluorescence analyses do not work on Western blotting (according to the
manufacturer), and perhaps they were less efficient in detecting their
target under the conditions of our platelet immunofluorescence
experiments. However, Western blot analysis showed both ER and AR
protein in human platelets (Figure 4).
Specificity for both platelet ER and AR was demonstrated by the
ability of the appropriate peptide to block antibody binding (Figure 4,
lanes 5-8), while an irrelevant peptide did not block antibody binding
(Figure 4, lanes 9-12). LNCap and HEL cells contained the expected
approximately 65-kd ER and approximately
110-kd AR proteins. Figure 4A suggests that ER in
platelets may be slightly larger than in HEL and LNCap cells. A second
preparation of anti-ER antisera also suggested platelet ER was larger in platelets (not shown). To some extent, these studies were limited by reagents (see "Discussion"), but perhaps platelet ER has alternately spliced mRNAs or
posttranslation modifications. In data not shown, a different anti-AR
antibody bound to the same 110-kd band on
Western blot, providing further evidence that this polypeptide in
platelet and HEL cells is the authentic AR. We suspect the signal at
approximately 62 kd may represent a proteolytic fragment
of AR, which has been repeatedly observed with AR from prostatic
cells,30-32 since we observed almost exclusively the
approximately 62-kd fragment until lysing cells in an SDS
buffer.
Ex vivo effect of sex hormones Sex hormones can autoregulate their corresponding receptors,33,34 often in a complex fashion. In these next studies, we wanted to determine whether sex hormones affected receptor expression in HEL cells. Using estradiol, we observed no changes in ER expression (data not shown). However, as shown in Figure
5, HEL-cell AR expression varied according
to the testosterone exposure, with an increase in AR at 1, 5, and 10 nmol/L testosterone (compare Figure 5B-5D with Figure 5A,
the no-testosterone control). However, AR expression was reduced when
cells were treated with 100 nmol/L testosterone (Figure
5E). The lack of AR induction in FBS containing 100 nmol/L testosterone (Figure 5E) was a consistent finding, suggesting that regulation of AR expression is not a linear function of
testosterone concentration. A similar dose-response was seen when cells
were cultured in charcoal-stripped FBS (data not shown). The reason for
the punctate appearance of the AR seen in HEL cell nuclei is unknown,
but in light of the numerous alternately spliced forms of
the AR mRNA that have been reported, perhaps this represents the
"speckle" or "coiled body" nuclear structures known
to be rich in splicing factors.35,36 We further pursued
these hormonal effects on AR expression using ex vivo-generated
megakaryocytes. As with HEL cells, we observed prominent AR expression
in megakaryocytes treated with 10 nmol/L testosterone
(Figure 6G) and a reduction in AR
expression with 100 nmol/L testosterone (Figure 6K compared with
Figures 6C and 6G). The difference in AR expression between HEL cells
and megakaryocytes in response to no testosterone (Figure 5A versus
Figure 6C) is most likely due to the persistent inhibitory effects of
ethanol on AR expression37 at the 48-hour time point (Figure 5) versus loss of ethanol via evaporation by 13 days (Figure 6).
Although sex hormones were first reported to affect platelet
function more than 25 years ago, little mechanistic data exist and no
previous information has been available on either ER
Submitted April 6, 1999; accepted November 30, 1999.
Supported in part by grants HL58564 and HL03454 from the National Institutes of Health.
Reprints: Paul F. Bray, Smith Tower 1295, Baylor College of Medicine, 6550 Fannin, Houston, TX 77030; e-mail: pbray{at}bcm.tmc.edu
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
1. National Heart, Lung, and Blood Institute. Morbidity and Mortality: 1996 Chartbook on Cardiovascular, Lung, and Blood Diseases. Bethesda, MD: National Institutes of Health; 1996. 2. Colditz GA, Willett WC, Stampfer MJ, Rosner B, Speizer FE, Hennekens CH. Menopause and the risk of coronary heart disease in women. N Engl J Med. 1987;316:1105[Abstract]. 3. Walsh BW, Schiff I, Rosner B, Greenberg L, Ravnikar V, Sacks FM. Effects of postmenopausal estrogen replacement on the concentrations and metabolism of plasma lipoproteins. N Engl J Med. 1991;325:1196[Abstract]. 4. Sullivan JM, Fowlkes LP. Estrogens, menopause, and coronary artery disease. Cardiol Clin. 1996;14:105[Medline] [Order article via Infotrieve].
5.
Hulley S, Grady D, Bush T, et al.
Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women: Heart and Estrogen/progestin Replacement Study (HERS) Research Group.
JAMA.
1998;280:605 6. Daly E, Vessey MP, Hawkins MM, Carson JL, Gough P, Marsh S. Risk of venous thromboembolism in users of hormone replacement therapy. Lancet. 1996;348:977[Medline] [Order article via Infotrieve]. 7. Castellsague J, Perez Gutthann S, Garcia Rodriguez LA. Recent epidemiological studies of the association between hormone replacement therapy and venous thromboembolism: a review. Drug Saf. 1998;18:117[Medline] [Order article via Infotrieve]. 8. Ruggeri ZM. Mechanisms initiating platelet thrombus formation. Thromb Haemost. 1997;78:611[Medline] [Order article via Infotrieve]. 9. Faraday N, Goldschmidt-Clermont PJ, Bray PF. Gender differences in platelet GPIIb-IIIa activation. Thromb Haemost. 1997;77:748[Medline] [Order article via Infotrieve].
10.
Jones SB, Bylund DB, Rieser CA, Shekim WO, Byer JA, Carr GW.
11. Tarantino MD, Kunicki TJ, Nugent DJ. The estrogen receptor is present in human megakaryocytes. Ann N Y Acad Sci. 1994;714:293[Medline] [Order article via Infotrieve]. 12. Chen LY, Mehta JL. Further evidence of the presence of constitutive and inducible nitric oxide synthase isoforms in human platelets. J Cardiovasc Pharmacol. 1996;27:154[Medline] [Order article via Infotrieve]. 13. Marcus AJ, Silk ST, Safier LB, Ullman HL. Superoxide production and reducing activity in human platelets. J Clin Invest. 1977;59:149.
14.
Broudy VC, Lin NL, Fox N, Taga T, Saito M, Kaushansky K.
Thrombopoietin stimulates colony-forming unit-megakaryocyte proliferation and megakaryocyte maturation independently of cytokines that signal through the gp130 receptor subunit.
Blood.
1996;88:2026
15.
Ajayi AA, Mathur R, Halushka PV.
Testosterone increases human platelet thromboxane A2 receptor density and aggregation responses.
Circulation.
1995;91:2742 16. Johnson M, Ramey E, Ramwell PW. Sex and age differences in human platelet aggregation. Nature. 1975;253:355[Medline] [Order article via Infotrieve]. 17. Wattel E, Cambier N, Caulier MT, Sautiere D, Bauters F, Fenaux P. Androgen therapy in myelodysplastic syndromes with thrombocytopenia: a report on 20 cases. Br J Haematol. 1994;87:205[Medline] [Order article via Infotrieve]. 18. Sullivan PS, Jackson CW, McDonald TP. Castration decreases thrombocytopoiesis and testosterone restores platelet production in castrated BALB/c mice: evidence that testosterone acts on a bipotential hematopoietic precursor cell. J Lab Clin Med. 1995;125:326[Medline] [Order article via Infotrieve]. 19. Westley B, Rochefort H. A secreted glycoprotein induced by estrogen in human breast cancer cell line. Cell. 1980;20:253. 20. Faraday N, Goldschmidt-Clermont P, Dise K, Bray PF. Quantitation of soluble fibrinogen binding to platelets by fluorescence-activated flow cytometry. J Lab Clin Med. 1994;123:728[Medline] [Order article via Infotrieve]. 21. Green S, Walter P, Kumar V, et al. Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature. 1986;320:134 |
and androgen receptor (AR): testosterone regulates AR expression
Top
Abstract
Introduction
(ER 
cells derived from CD34+ cells and
in HEL cells. Western immunoblotting confirmed the presence of ER 
× 174 DNA digested with HaeIII endonuclease).
Sizes of PCR products are shown in "Materials and methods."
