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Circulation Research. 2001;88:438-442

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(Circulation Research. 2001;88:438.)
© 2001 American Heart Association, Inc.

Molecular Medicine

Human Platelets Contain a Glycosylated Estrogen Receptor ß

Michele L. Nealen, K. Vinod Vijayan, Everlie Bolton, Paul F. Bray

From the Department of Medicine (M.L.N., K.V.V., E.B., P.F.B.) and the Program in Cellular and Molecular Medicine (M.L.N.), Johns Hopkins University School of Medicine, Baltimore, Md.

Correspondence to Paul F. Bray, MD, Baylor College of Medicine, Thrombosis Research Section, Department of Medicine, One Baylor Plaza, BCM 286, Room N1319, Houston, TX 77030. E-mail pbray{at}bcm.tmc.edu


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—Platelets play an important role in the coronary thrombus formation that leads to myocardial ischemia and infarction. Gender differences in the development of coronary heart disease and its outcomes are partly regulated by estrogen and its receptors, but the roles of the latter in thrombogenicity are less well-defined. We previously demonstrated the presence of estrogen receptor (ER) ß in cells of the megakaryocytic lineage. In this study, we characterize human platelet ERß and its expression using biochemical and molecular biological techniques. Western immunoblotting showed that platelet ERß migrated with an apparent molecular mass {approx}3.7 kDa larger than ERß in a variety of cell lines (including those of prostate and breast origin). A rigorous investigation of platelet ERß mRNA by reverse transcriptase–polymerase chain reaction revealed normal transcripts and a single alternately spliced mRNA. However, this variant form was smaller, lacking exon 2, and could not account for the larger protein size seen in platelets. Treatment of ERß with N-glycosidase F, which removes core carbohydrate residues, caused a more rapid migration through polyacrylamide gels but had no effect on ERß from human cell lines. We conclude that the larger form of ERß in human platelets is not attributable to alternate mRNA splicing but primarily to tissue-specific glycosylation.


Key Words: platelet • estrogen receptor • glycosylation


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Platelets are largely responsible for the formation of the occlusive thrombus in coronary arteries at the site of atherosclerotic plaque rupture.1 2 3 Although this is the normal response in an injured vessel, it serves as a roadblock for normal blood flow through the coronary vessels, creating ischemic conditions that may lead to myocardial infarction. Many studies have noted that estrogen may modulate the risks and outcomes of atherosclerotic disease.4 5 6 7 8 9 10 For example, premenopausal women are less likely to experience coronary atherosclerosis than men of the same age, but after menopause, women are equally or more likely than men their age to develop coronary atherosclerosis.11 12 The results of the Heart and Estrogen/Progestin Replacement Study (HERS)6 showed that hormone-replacement therapy conferred no long-term benefits in women with established coronary artery disease. In fact, the treatment group had worse outcomes at 1 year despite the expected beneficial effects on lipid levels. Platelets are one potential target for estrogen modulation within the cascade of factors involved in ischemic atherosclerotic events. Because we had previously shown that platelets from estrogenized women had a more thrombotic phenotype than platelets from men,13 we believed that the findings of the HERS raised the question of whether hormone-replacement therapy may enhance the platelet component of the pathophysiology of coronary artery disease.

Estrogen can modulate changes nongenomically (by affecting Ca2+ flux, lipid metabolism, etc)5 7 14 15 and genomically (through the estrogen receptor [ER]),8 16 17 but relatively little is known about the effects of estrogens on platelets. There are 2 forms of the estrogen receptor, discovered 10 years apart, named ER{alpha} and ERß to designate the order in which they were identified.18 19 These two forms of the receptor share considerable sequence identity in their DNA and hormone-binding domains; they diverge considerably in their activation domains.20 ER{alpha} and ERß are coexpressed in many human tissues but have different downstream effects in response to various estrogenic ligands.21 22 23 ER{alpha} knockout mice show estrogen responsiveness, suggesting that ERß can function in the absence of ER{alpha}.24 25 Both receptors have been cloned from human tissue,18 26 but neither has been cloned from platelet material.

Previously, we identified ERß transcript and protein in megakaryocytes, platelets, and human erythroleukemia cells and noted that platelet ERß protein was larger than the protein in breast or prostate.27 In this study, we demonstrate that platelet ERß protein is glycosylated, offering an explanation for the larger size of the protein in platelets. We also identify two ERß transcripts from platelets, one of the expected size and one in which exon 2 is deleted. This is the first novel isoform of ERß described in human platelets. The implications of tissue-specific glycosylation of this receptor are discussed.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
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Reagents
The breast-cancer cell lines MCF-7 and T47D were obtained from Dr Chi Dang and Dr Saraswati Sukumar, respectively, and the LNCaP prostate-cancer cell line was obtained from Dr John Isaacs, all from Johns Hopkins University, Baltimore, Md. Unless indicated, all cell culture and reverse transcriptase–polymerase chain reaction (RT-PCR) reagents were from Life Technologies, and other chemicals were from Sigma Chemicals. Polyclonal antibodies to ERß were from Upstate Biotechnology or Santa Cruz Biotechnology. Peroxidase-conjugated secondary antibodies were from American Qualex or Santa Cruz Biotechnology.

Use of Human Subjects
Healthy human volunteers were recruited to give blood, and informed consent was obtained. Approval for use of human donors was obtained from the Institutional Review Board at Johns Hopkins School of Medicine, and procedures used were within the guidelines established by the university and the Helsinki Declaration of 1975. A total of 5 healthy women (age range 24 to 27 years) and 13 men (age range 23 to 49 years) were used.

Western Blotting
Platelet and cell lysates were prepared as previously described,27 with platelets being lysed in buffers containing 1% Triton X-100 and cell lines being lysed in buffers containing 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS. Protein concentration in the lysates was quantified using the BCA protein assay (Pierce), and polypeptides were separated by electrophoresis through 10% polyacrylamide gels. The gels were transferred to nitrocellulose, and the blots were incubated with different anti-ERß antibodies, as previously described.27

RT-PCR Characterization of ERß mRNA
MCF-7 cells were grown in DMEM-F12 supplemented with FBS (Gemini Bio-Products) and penicillin/streptomycin. T47D cells were grown in RPMI 1640 medium supplemented with 10 µg/mL insulin and penicillin/streptomycin. Human erythroleukemia cells (American Type Culture Collection) and LNCaP cells were grown in RPMI 1640 medium supplemented with FBS and penicillin/streptomycin. Cell lines and platelets were pelleted, and total RNA was isolated with RNAStat-60 (Tel-Test) according to the manufacturer’s instructions. RT-PCR was carried out as described previously.27 Amplified products were cloned into bacterial vector pCR 2.1 using the TA cloning system (Invitrogen), and regions of interest were sequenced using universal primers on an automated DNA sequencer.

Protein Predictions
PredictProtein (http://www.embl-heidelberg.de/predictprotein/predictprotein.html) was used to predict domain structure and potential modification sites of ERß. The published sequence of ERß (Genbank No. AB006590) was used; topology of membrane proteins, as well as identification of potential membrane helices, signal peptides, O-linked glycosylations, N-linked glycosylations, phosphorylation sites, and myristoylation sites, was determined.

N-Glycosidase F Digestions
LNCaP (20 µg) and platelet lysates were treated with 1 U of N-glycosidase F (Roche Molecular Biochemicals) in a final volume of 20 µL for 18 hours at 30°C and subjected to Western blotting. As a positive control for deglycosylation, an anti-integrin {alpha}v antibody (Life Technologies) was used.


*    Results
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 *Results
down arrowDiscussion
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ERß is expressed in normal breast and prostate tissue, and cell lines such as T47D and MCF-7 (of breast origin) and LNCaP (of prostate origin) are commonly used in the study of estrogen receptors.28 29 30 Compared with the ERß seen in these cell lines, ERß from platelets migrated more slowly by SDS-PAGE (Figure 1Down). Four polyclonal antibodies (Santa Cruz ERß L-20 and N-19, Upstate Biotechnology anti-ERß, and Oncogene Research ERß [Ab-1]) were used to verify the identity of ERß. All produced the same results: ERß protein from platelets was {approx}3.7 kDa larger (range from 7 different experiments, 1.9 to 6.6 kDa) than ERß protein from nonplatelet sources. In addition to the LNCaP and T47D cells, platelet ERß was larger than ERß in Dami (human megakaryocytic), Du145 (human prostate), PC-3 (human prostate), HS578T (human breast), and Chinese hamster ovary cell lines (data not shown). The size difference was consistently present in platelets from both female and male subjects.



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  		Figure 1. Human platelet ERß is larger than ERß in breast and prostate. LNCaP (20 µg) (a prostate-cancer line) and T47D (a breast-cancer line) and 20 µg (lane 3) and 40 µg (lane 4) of platelet (plt) lysates were resolved on a 10% SDS polyacrylamide gel, transferred to nitrocellulose, and probed with polyclonal antibody, L-20, to human ERß. The protein identified in platelets is {approx}3.7 kDa larger than that in T47D and LNCaP cells. These and other cells have established the size range of ERß as 52 to 63 kDa.29 30 31 Competition studies with ERß peptides confirmed the specificity of the proteins recognized (not shown).

We considered whether alternate splicing could account for the larger ERß seen in platelets. Because platelets are anucleate and have only trace amounts of RNA, we used RT-PCR to characterize ERß mRNA. The TableDown and Figure 2ADown contain the nucleotide sequence and location of the primers used in these studies. Primers from the coding region were able to amplify ERß from both platelets and nonplatelet sources (Figures 2BDown through 2D). Figure 2BDown shows that 1 of the 2 sequences amplified from platelet cDNA using primers MN5 and MN14 was a smaller product than expected. Amplified products from this region were subcloned and sequenced, and nucleotide sequences were compared with published sequences. This comparison confirmed that both products correspond to ERß. The smaller product produced from the RT-PCR of platelet cDNA (Figure 2BDown) corresponds to a splicing variation described previously in human pituitary adenomas.31 As shown in Figure 3Down, exon 2 is skipped in this transcript, which results in a frameshift and premature termination of the transcript. If this truncated protein were expressed, it would contain only 122 amino acids with a size of {approx}13.5 kDa and, therefore, could not account for the larger size of ERß seen in platelets. The larger product amplified from primers MN5 and MN14 was determined to be cDNA corresponding to the known sequence (ie, containing exon 2). No sequence divergence was observed in this part of the coding region of platelet ERß that would explain the larger size seen in Figure 1Up. Primers corresponding to the remaining regions of ERß were used to amplify ERß from platelets and cell-line sources; no size anomaly was noted in the rest of the cDNA (Figures 2CDown and 2DDown). Thus, neither alternate mRNA splicing nor other nucleotide sequence differences could account for the larger size of ERß in platelets.


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  		Table 1. List of Oligonucleotide Primers Used to Amplify ERß Sequence



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  		Figure 2. RT-PCR characterization of platelet ERß. A, Schematic of ERß primer sites. MN5 and MN14 are expected to generate a product of 745 bp; MN11 and MN12 are expected to generate a product of 624 bp; and MN13 and MN8 are expected to generate a product of 645 bp. -> indicates a primer corresponding to sense sequence; <- indicates a primer corresponding to antisense sequence. B through D, Open arrows indicate the expected product; lane 1 contains 0.5 µg of 100-bp DNA ladder. B, ERß amplification using primers MN5 and MN14. RT-PCR of RNA isolated from human platelets (lane 2) produces both a product of expected size (745 bp) and an unexpected product ({approx}600 bp). The 600-bp product is not detected in RT-PCR reactions from either MCF-7 (lane 3) or T47D (not shown). Lane 4, No template control. Filled arrow indicates the novel ERß product. C, ERß amplification using primers MN11 and MN12. Only the expected product of 624 bp is observed from platelets (lane 2) and breast-cancer cells (MCF-7, lane 3; T47D, lane 4). Lane 5, No template control. D, ERß amplification using primers MN13 and MN8. Only the expected product of 645 bp is observed from T47D (lane 2) and platelets (lane 3). Lane 4, No template control.



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  		Figure 3. Schematic of mRNA splicing of the ERß gene. Normal splicing is shown above the gene, and the deleted exon 2 variant found in platelets is shown below the gene.

Posttranslational modifications represent another means by which protein size may vary. Recent reports identifying membrane-associated forms of estrogen receptors32 33 34 35 raise the possibility that the size difference between platelet ERß and nonplatelet ERß was attributable to glycosylation. We digested both platelet and LNCaP lysates with several glycosidases to see if carbohydrate removal changed the electrophoretic mobility of ERß. Figure 4Down shows a Western blot of lysates digested with N-glycosidase F, an enzyme that removes sugar chains joined to a polypeptide at asparagine residues (N-linked). After enzyme digestion, the size of platelet ERß is much more similar to LNCaP ERß (shown in 2 different experiments in Figures 4ADown and 4BDown), suggesting that the ERß size difference we observed was largely attributable to N-linked glycosylation. This finding was consistent over several experiments. The lack of mobility shift in LNCaP ERß was not attributable to the inability of N-glycosidase F to act in these lysates, because the mobility of glycoprotein integrin {alpha}v was reduced as expected (Figure 4CDown). Digestion with enzymes to remove O-linked glycosylation yielded no change in the mobility of platelet ERß, even after the potential complex sugar chains were reduced by neuraminidase digestion (data not shown).



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  		Figure 4. Evidence for platelet ERß glycosylation. Lysates of LNCaP or platelets were incubated with or without N-glycosidase F (N Glyc F) and probed by Western immunoblotting with an anti-ERß antibody (UPS). Results of 2 separate experiments (A and B) are shown. After N-linked carbohydrates are removed with N-glycosidase F, the size difference between ERß from platelets and from LNCaP is largely eliminated. N-glycosidase F treatment was complete, as indicated by the complete loss of the higher platelet ERß form. C, Demonstration that N-glycosidase F is able to deglycosylate LNCaP lysates. Lysates were prepared and treated as described above and probed with an anti-integrin {alpha}v antibody. Arrowheads indicate {alpha}v before (lane 1) and after (lane 2) treatment with N-glycosidase F.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The explanation for the disparate responses to estrogen observed in different tissues36 37 38 is unknown, although the discovery of a second estrogen receptor in 1996 expanded the possible mechanisms that might account for these differences. The most significant findings in our present study are, first, the confirmation of ERß in human platelets; second, the demonstration that platelet ERß is {approx}3.7 kDa larger than ERß from other tissues; and, third, the finding of tissue differences in ERß glycosylation. These findings raise additional mechanistic possibilities to explain tissue-specific responses to estrogen.

We found that the platelet form of the full-length ERß protein migrates more slowly on a polyacrylamide gel than ERß from other human cell lines. This difference was consistent across many platelet samples from both genders and across a wide range of donor ages. Alternate mRNA splicing is one mechanism that could result in a larger protein product. Although a larger splicing variant has been observed in rat tissue,39 our RT-PCR data show that only the full-length transcript and the exon 2–deleted form were present in human platelets. The most likely explanation for our data is that the platelet ERß transcripts of normal size detected by RT-PCR are responsible for the larger than expected polypeptide seen by immunoblotting. The polypeptide could be posttranslationally processed, which would result in slower migration than would be predicted from its amino acid sequence.

Lu et al40 first described forms of human ERß lacking exons, finding deletions of exon 5 or of exons 5 and 6 in human breast tumors. Using RT-PCR followed by cloning and sequencing, we have identified a splicing variant of ERß in platelets that lacks the second exon. This is the first description of an exon-deleted form of ERß from human platelets; a similar splice variant of ERß has been identified in pituitary adenomas.31 Although we did not formally quantify the 2 transcripts, the relative intensities of the amplified PCR products suggested substantial quantities of the exon 2–deleted form. If expressed, the resulting polypeptide would be small (13.5 kDa) and would have limited functional capacity as an estrogen receptor, because it would lack the ligand-binding domain, including activation function 2 and all of the DNA-binding domain. However, it could serve as a sink for coregulators or other factors that may interact with the ERß N-terminal domain, decreasing the ability of wild-type ERß to function normally.

We investigated several possible posttranslational mechanisms that might account for the increase in ERß size in platelets. Phosphatase treatment of platelet lysates caused no change in ERß-apparent mobility, making phosphorylation an unlikely contributor to this size difference (data not shown). Glycosylation is another posttranslational mechanism that could potentially increase the mass of the protein. Typically, glycosylation occurs in the rough endoplasmic reticulum, and entrance into it requires a hydrophobic signal sequence, most often in the N-terminus. We used several methodologies to predict ERß hydrophobicity but found nothing suggestive of a signal peptide (data not shown). Nevertheless, there are examples of proteins translocating across membranes despite the absence of a signal peptide (eg, human plasminogen activator41 and ovalbumin42 ). In addition, rat ERß has been reported to be glycosylated on serine or threonine (O-linked glycosylation),43 but this modification is not sensitive to N-glycosidase F. We determined that deglycosylating enzymes for removing N-linked, but not O-linked, sugars resulted in a form of ERß that was closer to the predicted size of the polypeptide. This suggested that most of the size difference between ERß from platelets and cell lines is attributable to glycosylation. Analysis with the PredictProtein program noted several potential sites for N-linked glycosylation sites (asparagine residues in the appropriate amino acid context) on ERß at residues N17, N55, N61, and N407. This glycosylation may be regulated by a tissue-specific mechanism, and additional cell lineages will need to be tested to see if this is unique to platelets. Tissue-specific glycosylation has been described previously,44 45 46 often attributable to tissue-specific expression of the glycosyltransferase responsible for adding the glycosyl moiety to the protein.47 48 Addressing the functional consequences of platelet ERß glycosylation is beyond the scope of these experiments, but 3 asparagine residues lie within the region of ERß known to contain transcriptional activity, and 1 asparagine is in the ligand-binding domain. Additional studies with purified megakaryocytes are required to address this possibility.

Our findings do not speak directly to the role of ERß in platelets. ERß-deficient mice are viable, but their platelet function has not been studied.49 50 The receptor may exert its effects solely at the level of transcription regulation (in the precursor megakaryocyte), and its presence in platelets could be merely residual. It is possible that the exon-deleted isoform may heterodimerize with the form of ERß we observe in platelets. Perhaps this unusual heterodimer could modulate the effects of estrogen in a tissue-specific manner. Alternatively, ERß could have nongenomic effects on platelets and megakaryocytes. We have shown previously that platelet reactivity varies with both the phase of the menstrual cycle and levels of sex hormones,13 51 findings that could be mediated by platelet ERß. Whether platelet-specific ERß glycosylation represents a novel mechanism for regulating the effects of estrogens awaits additional studies.


*    Acknowledgments
 
This work was supported by the National Institutes of Health Heart, Lung and Blood Institute grant HL58564.


*    Footnotes
 
Original received March 28, 2000; resubmission received December 1, 2000; revised resubmission received January 4, 2001; accepted January 5, 2001.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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