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Endocrinology Vol. 149, No. 11 5872-5887
Copyright © 2008 by The Endocrine Society

Systematic Expression Analysis and Antibody Screening Do Not Support the Existence of Naturally Occurring Progesterone Receptor (PR)-C, PR-M, or Other Truncated PR Isoforms

Annemarie Samalecos and Birgit Gellersen

Endokrinologikum Hamburg, 20251 Hamburg, Germany

Address all correspondence and requests for reprints to: Birgit Gellersen, Ph.D., Endokrinologikum Hamburg, Falkenried 88, 20251 Hamburg, Germany. E-mail: gellersen{at}endokrinologikum.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Functional progesterone withdrawal associated with human parturition has been ascribed to various mechanisms modulating the function of the classical progesterone receptors (PRs), B and A, in utero. These include up-regulation of the inhibitory PR-C isoform, described as a 60-kDa protein occurring from translation initiation at codon 595. Our initial attempts to detect PR-C yielded uninterpretable results. To systematically validate antibodies for immunodetection of PR isoforms, we generated expression vectors for PR variants originating from putative start codons AUG-289, -301, -595, -632, and -692 in addition to those for PR-B and PR-A, and for alternative splice variants PR-T, PR-S, and PR-M. All constructs were subjected to in vitro and in vivo translation and immunoblotting with a panel of 13 PR antibodies. Antibodies raised against full-length PR were generally not capable of detecting N-terminally truncated forms, whereas C-terminal antibodies did not or only weakly reacted with PR-B and PR-A but produced prominent nonspecific signals. Thus, immunodetection of N-terminally truncated PR isoforms is prone to artifacts. Proteins of about 64 kDa were expressed from PR-289 and -301, but no corresponding endogenous forms were observed. PR-T, PR-S, and PR-M cDNAs yielded no detectable translation products. No protein was translated from AUG-595 in our PR-C expression vector unless a Kozak sequence was introduced, and the product was not 60 but 38 kDa in size. Thus, the 60-kDa protein called PR-C does not originate from AUG-595 and is not a naturally occurring PR isoform.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROGESTERONE (P4) plays a pivotal role in the control of reproductive functions (1), mainly mediated by the classical P4 receptors (PRs), members of the superfamily of ligand-activated transcription factors (2, 3). The PR has two major isoforms, PR-B and PR-A, that are identical in the central DNA binding domain (DBD) and the C-terminal ligand binding domain (LBD) but differ in their transcriptional activation potential. PR-B is a protein of 933 amino acids (aa); PR-A lacks the N-terminal 164 aa of PR-B, termed the B-upstream segment (BUS), is generally a weaker transactivator than PR-B, and antagonizes other steroid hormone receptors (4, 5). Selective gene ablation studies in mice revealed that the actions of PR isoforms are highly tissue specific, with PR-A being indispensable for ovarian and uterine functions, and PR-B for mammary gland development (6). Although PR-A might arise from alternative translation initiation at the Met-165 codon, overwhelming evidence suggests the existence of a separate promoter generating specific PR-A transcripts (7, 8, 9). Moreover, the extensive 5'-heterogeneity in human PR transcripts observed by Northern blotting led to the assumption that at least one additional N-terminally truncated isoform, PR-C, must be independently transcribed (9). The translational start site of PR-C was proposed to be Met-595, which is located within the DBD. Therefore, PR-C would lack the first of two zinc fingers and be defective in DNA binding. The development of an antibody directed against the very C terminus of the PR allowed Western blot analysis of such truncated isoforms, and PR-C was described as a 60-kDa protein that, upon hormone binding, translocated from the cytoplasm to the nucleus in T47D breast cancer cells (10). An expression vector with Met-595 as the start codon produced a product that did not bind DNA but interfered with PR-B function by forming heterodimers (11, 12).

The development of new region-specific PR antibodies led to the detection of additional isoforms in breast cancer cells like a 78-kDa PR isoform that migrates slightly faster than PR-A in gel electrophoresis. This isoform, which does not appear to be a proteolytic fragment of PR-B or PR-A, also seems to lack part of the N terminus and binds hormone (13).

A further level of complexity to PR expression was added by the identification of splice variants and new leader exons used in breast cancer cells, endometrial tissue, and testis. Within the originally reported genomic structure of the PR gene, encompassing eight exons, deletions of exons 2, 4, 6, 4 + 6, 5 + 6, part of exon 4, or the 5'-most 52 bases of exon 6 have been reported (14, 15, 16). The PR splice variants identified in MCF-7 cells were cloned into a PR-A background and subjected to functional studies in transfected cells (14). Although wild-type PR-B and PR-A bind DNA only upon progestin treatment, the {Delta}6 variant was found to bind DNA constitutively, whereas {Delta}4 or {Delta}5,6 failed to bind regardless of hormone.

PR variants carrying an alternative insertion of two exons between exons 4 and 5 (i45a, i45b) have been identified in endometrium (17). Upstream of exon 1, two novel noncoding leader exons (T, S) were identified that are spliced to exons 4–8 in testis and endometrium (18, 19). Translation of these mRNAs can only, if at all, be initiated at Met-692 in exon 4 to contribute to a short PR isoform termed PR-S. Finally, exon M was described to be located in intron 3 and to be spliced to exons 4–8. Interestingly, this leader exon contains an in-frame initiator codon such that 16 novel hydrophobic aa, representing a potential signal peptide, are added to the sequence encoded by exons 4–8. PR-M expressed in insect cells migrated at an apparent molecular mass of 38 kDa (20).

Although many of the aforementioned variants have not been analyzed in depth regarding their functionality and/or relative level of expression, a physiological role has been proposed for PR-C in the initiation of human parturition. High P4 levels are required throughout pregnancy to maintain a quiescent myometrium. However, in contrast to most other species, circulating P4 levels do not decline before onset of labor in humans (21). A number of underlying mechanisms have been proposed, including an increased myometrial PR-A to PR-B ratio (22), a decline in nuclear coactivators of steroid hormone receptors at term (23), and up-regulation of the unrelated membrane progestin receptors that antagonize nuclear PR function (24). Yet, the latter hypothesis is still under debate (25). Furthermore, a massive up-regulation of PR-C has been reported in the myometrium upon the onset of labor, which in turn could account for the "functional P4 withdrawal" by inhibiting full-length PR (26). PR-C, when in the cytoplasm, was proposed to sequester locally available P4 or, when located into the nucleus, to suppress PR-B transcriptional activity on PR responsive elements. A different study suggested that PR-C is the major isoform in amnion and decidua, and that expression levels sharply decrease with the onset of contractions (27). Higher PR-C levels have also been reported in endometriomas when compared with eutopic and normal endometrium (28).

The expression of variant PRs is likely to have important functional implications in P4 target tissues. Therefore, their accurate identification and quantification are of fundamental interest. For example, an important diagnostic measure for the hormone-responsiveness of breast tumors is the immunohistological determination of PR status. Numerous antibodies are on the market for visualization of total PR. In addition, antibodies have been generated that selectively recognize PR-B or PR-A in formaldehyde-fixed tissues, based on the differential N-terminal conformations of these isoforms (29). However, the identification of other PR variants by immunohistochemistry is not possible. Although RT-PCR amplification has been widely used to examine the expression of PR isoforms, this approach poses methodological problems. Primers designed to amplify transcripts for PR-C invariably coamplify those for PR-B, -A, -M, or -S. Consequently, Western blot analysis is the method of choice to identify specific variants, but there is still room for interpretation. For instance, immunoreactive bands migrating around 60 kDa in SDS-PAGE have been designated PR-C (10, 26), although the theoretical molecular mass for a PR variant comprising aa 595–933 would be 38 kDa. Yet, products of this size have been interpreted as nonspecific bands (11).

When we set out to analyze the expression of PR variants in human myometrial cells by Western blotting, we encountered unforeseen difficulties that cast doubt on the specificity of certain PR antibodies. To assess reliably the expression pattern of PR variants, we generated a series of expression vectors for N-terminally truncated isoforms, including PR-C, PR-M, and PR-S, and used these to validate systematically a panel of 13 commercially available PR antibodies. We found that antibodies raised against the C terminus of PR tend to be unreliable in Western blot applications, confounding unambiguous identification of truncated PR variants. Our expression analysis revealed that PR-M, PR-T, or PR-S mRNAs yield no translation products, and that PR-C is not a naturally occurring isoform.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning of PR isoforms
Generation of pcDNA/hPR-B and pcDNA/hPR-A in eukaryotic expression vector pcDNA3.1(+) (Invitrogen, Karlsruhe, Germany) has been described (30). The hPR-B expression vector hPR0 in pSG5 was kindly provided by P. Chambon (Université Louis Pasteur, Illkirch, France) (7). All of the following N-terminally truncated or alternatively spliced cDNA variants were generated such that they carry the same stretch of about 850 bp 3'-untranslated region (UTR) as the aforementioned PR expression vectors. For construction of pcDNA/PR-595, containing 363 bp 5'-UTR to Met-595, the C-terminal Bsp120I-XbaI fragment was retrieved from pcDNA/hPR-A and inserted into the same sites of pcDNA3.1(–). To generate pcDNA/PR-301, carrying a PR cDNA with 27 bp 5'-UTR to Met-301, the N-terminal NheI-SrfI fragment was removed from pcDNA/hPR-A, the ends polished with mung bean nuclease, and religated. Vector pcDNA/PR-289, with 219 bp 5'-UTR to Met-289, was constructed accordingly, but using the NheI-BsmBI sites in pcDNA/hPR-A. The cDNAs for splice variants PR-T, PR-S, or PR-M, which contain the alternative upstream exons T, S, or M, respectively (18, 20), spliced to exon 4 of the PR gene, were amplified by RT-PCR from human testis mRNA (PR-T, -S) or from T47D cell mRNA (PR-M). Sense primers were located in the respective upstream exons (PR-T-s, 5'-TTATACTGGGCTCTGCAGGTCATC-3'; PR-S-s, 5'-CTGGAGATCTGCTTTGGCATGGAA-3'; PR-M-s, 5'-ACCTCACCCACCCAGTGATTGTT-3'), the antisense primers in exon 7 downstream of the unique HindIII site in the PR-LBD: PR-T-s and PR-S-s were paired with PR-Ex7-as (5'-AAACGCTGTGAGCTCGACACAA-3'), and PR-M-s with PR-M-as (5'-TGACTTCGTAGCCCTTCCAAAGGAAT-3'). PCR products were cloned into pCR-Blunt II TOPO (Invitrogen), verified by sequencing, and excised with EcoRV (in the polylinker) and HindIII (in the PR LBD). These fragments were inserted into pcDNA/hPR-A from which the N-terminal NheI-HindIII fragment had been removed and the NheI overhang had been polished. Resultant constructs were termed pcDNA/PR-T, pcDNA/PR-S, and pcDNA/PR-M.

Furthermore, cDNAs were generated carrying an optimized Kozak sequence (31) embedding the putative start codons Met-595, Met-632, or Met-692. Fragments were generated by PCR on template pcDNA/hPR-A with the sense primers PR-595-ATG (same as that used in Ref. 26) (5'- caccATGGAAGGGCAGCACAACT-3'; optimized Kozak sequence is underlined; lower case letters represent bases not homologous to human PR sequence), PR-632-ATG (5'-tcacCATGGTCCTTGGAGGTCGAAAA-3') or PR-692-ATG (5'-tcaccATGgGCATTGAACCAGATGTG-3'), and an antisense primer anchored in the PR LBD 3' to the unique HindIII site (PR-C-as; 5'-GTGAGCTCGACACAACTCCTTT-3'). After restriction with HindIII, PCR products were ligated into pcDNA/hPR-A from which the N-terminal NheI-HindIII fragment had been removed and the NheI overhang had been polished. Resultant constructs were designated pcDNA/PR-595-ATG, pcDNA/PR-632-ATG, and pcDNA/PR-692-ATG.

A myc-epitope was introduced downstream of Ile-920 in PR-595-ATG by insertion of a double-stranded oligonucleotide (sense, 5'-GAACAAAAACTCATCTCAGAAGAGGATCTgata-3'; antisense, 5'-AGATCCTCTTCTGAGATGAGTTTTTGTTCtatc-3'; lower case letters represent BstXI-compatible overhangs) into the unique BstXI site of pcDNA/PR-595-ATG. This results in a duplication of the Ile residue such that the myc sequence is then followed by the sequence coding for Ile-920 to Lys-933. In the resultant plasmid pcDNA/PR-595-ATG/myc, the N-terminal MluI-HindIII fragment was swapped for the corresponding fragment from pcDNA/PR-595 to yield pcDNA/PR-595/myc.

Cell culture
The human uterine myosarcoma cell line SKUT-1B (HTB 115; American Type Culture Collection, Rockville, MD), T47D human breast cancers cells, and COS-7 African green monkey kidney cells were maintained in DMEM/Ham’s F-12 (1 + 1; Sigma, Deisenhofen, Germany) supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. For progestin treatments, cells were plated in media containing 10% dialyzed steroid-depleted fetal calf serum.

Primary cultures of human myometrial smooth muscle cells were prepared from tissues obtained from cycling women at hysterectomy for uterine prolapse or leiomyomata as described previously (30). Informed consent was obtained, and the study was approved by the local ethics committee.

Transient transfections
COS-7 cells were plated at 5 x 105 cells per six-well and transfected the following day using Polyfect (QIAGEN, Hilden, Germany). A mixture of 1.5 µg plasmid DNA in 100 µl Opti-MEM reduced serum medium (Invitrogen) with 10 µl Polyfect was added to the cells in a total of 2 ml culture medium. SKUT-1B cells were plated at 6 x 105 cells per well in poly-L-lysine coated six-well plates. The next day, medium was changed to 1.5 ml antibiotic-free culture medium, and 4 µg plasmid DNA and 10 µl Lipofectamine 2000 (Invitrogen) diluted in 0.5 ml Opti-MEM were added to the cells. For transfections in 24-well plates, numbers were scaled down by a factor of five. Proteins were harvested 24 h after transfection for both cell lines.

Indirect immunofluorescence
COS-7 cells, plated at 0.4 x 105 cells per well in eight-well chamber slides (BD Biosciences, Heidelberg, Germany), were transfected the following day with PR expression constructs using 0.2 µg DNA and 0.9 µl Polyfect. After 24 h, cells were fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.2% Triton X-100 for 10 min. After blocking in normal goat serum, primary antibody was added in PBS for 1 h at room temperature. After several washes in PBS, secondary antibody diluted in PBS/2% normal goat serum was added for 1 h. The following PR antibodies were used: PGR-312 (1:200), SP2 (1:100), or 10A9 (1:2 from ready-to-use solution) (for suppliers, see Table 1Go). Secondary antibodies were Alexa Fluor 568-conjugated goat antirabbit or antimouse IgG (Invitrogen), used at 1:500 or 1:250, respectively. Nuclei were counterstained with 0.1% 4,6-diamidino-2'-phenylindole, and cells observed in a CKX41 microscope equipped with a CC-12 digital camera (Olympus, Hamburg, Germany).


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  		TABLE 1. Details on PR antibodies as used in this study

 
Western blot analysis
Whole cells extracts were prepared in radioimmunoprecipitation buffer [10 mM Tris-HCl (pH 7.4), 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA] containing protease inhibitor cocktail (Complete; Roche Applied Science, Mannheim, Germany). Treatment of T47D cells with 100 nM R5020 (promegestone; Sigma-Aldrich, Munich, Germany) or ethanol vehicle was done for 1 h before cytoplasmic and nuclear proteins were extracted as described previously (32). Protein concentrations were determined with an assay kit from Bio-Rad Laboratories (Munich, Germany). Quick protein extracts were prepared by harvesting cells in the well of a 24-well plate directly into 30 µl heated (85 C) Laemmli gel loading buffer [100 mM Tris-HCl (pH 6.8), 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, and 20% glycerol].

PR isoforms in eukaryotic expression vectors (pcDNA3.1 or pSG5) were expressed in vitro with the TNT T7 Quick Coupled Transcription/Translation System (Promega Corp., Madison, WI) using 500 ng plasmid DNA in a final volume of 25 µl. For Western blot analysis, 5 µl of these reactions was loaded per lane.

Proteins were electrophoresed in 10% SDS-polyacrylamide gels (NuPage Bis-Tris; Invitrogen) and transferred by tank blotting onto polyvinylidene fluoride Immobilon membranes (Millipore, Eschborn, Germany). Prestained protein size markers were SeeBlue Plus 2 (Invitrogen). Antibodies directed against PR are listed in Table 1Go. The monoclonal antibody against the c-myc epitope was from Covance (Hiss Diagnostics, Freiburg, Germany) and used at a dilution of 1:1000. Tumor suppressor protein p53 was detected with monoclonal antibody DO-1 (Calbiochem; EMD, San Diego, CA; 1:1000), and lamin A/C with monoclonal antibody clone 14 (Upstate; Millipore, Schwalbach, Germany; 1:1000). Secondary horseradish peroxidase-conjugated goat antimouse or antirabbit antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA) and used at 1:10,000. Immunodetection was performed with the enhanced chemiluminescence system (SuperSignal; Pierce, Bonn, Germany).

RT-PCR, Southern blot hybridization
Total RNA was isolated from cultured cells using peqGOLD RNAPure (Peqlab, Erlangen, Germany) following the manufacturer’s instructions. Reverse transcription was done on 1 µg RNA with the ImProm-II Reverse Transcription System (Promega) in a total volume of 20 µl. PCR was performed with primer pairs PR-Ex3-s (5'-TTATGTGCTGGAAGAAATGACTGC-3') and PR-Ex8-as (5'-GTGCCCGGGACTGGATAAAT-3'), or PR-M-s and PR-M-as (see above), using 5Prime Taq polymerase (Eppendorf, Hamburg, Germany) and 0.5 µl cDNA per 20 µl reaction. PCR products were resolved in 1.5% agarose gels, stained with SYBR Gold (Invitrogen), and visualized in a Typhoon 8600 Imager (Amersham Biosciences, Freiburg, Germany). After denaturation and neutralization, DNA was transferred to positively charged nylon membrane (Roche Applied Science). Southern hybridization was performed with internal oligonucleotide probes labeled with terminal deoxynucleotidyl transferase and digoxygenin-11-deoxyuridine 5-triphosphate, and detected with the DIG Luminescent Detection Kit (Roche Applied Science). Probes were: PR-LBD-5 in exon 4 (5'-GGACATGACAACACAAAACCTGAC-3'); PR-Ex4b-as (antisense to exon 4b; 5'-CTTCCTACTTTCCCACGGA-3'); and PR-Ex7-as (see above).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vitro expression of PR variants
The N-terminally truncated isoform PR-C was first described in the PR-positive T47D human breast cancer cell line and found to be absent in PR-negative MDA-MB-231 breast cancer cells (10). For the detection of PR-C in T47D cells and myometrial samples, the C-19 antibody has been used (26). This antibody was raised against a C-terminal peptide of PR and, thus, should allow detection of PR isoforms lacking the N-terminal half of the receptor protein (Table 1Go). Nuclear extracts from human myometrial smooth muscle cells treated with P4 or vehicle for 1 h and whole cell extracts from T47D and MDA-MB-231 cells, serving as positive and negative controls, respectively, were immunoprobed with this antibody (Fig. 1Go). Surprisingly, C-19 failed to detect PR-B or PR-A in T47D cell lysates but yielded a prominent band of 96 kDa in myometrial extracts. Probing of a parallel blot with a different C-terminal antibody, MAB462 (Table 1Go), also did not show PR-B or PR-A. Instead, strong nonspecific bands were apparent in lysates from PR-negative MDA-MB-231 cells. The N-terminal antibody PGR-312 specifically detected PR-B and PR-A in T47D extracts. Antibody SP2, raised against an epitope N terminal to the DBD (Table 1Go), displayed higher sensitivity and additionally revealed, albeit faintly, nuclear accumulation of PR-B and PR-A in P4 treated myometrial cells. However, this antibody also detected a ladder of lower molecular mass proteins in T47D cells. PR-B is widely described as a 116- to 120-kDa protein (12, 33). In our electrophoresis system, PR-B comigrated with the 97-kDa size marker, consistent with its calculated mass of 99 kDa (Fig. 2Go). PR-A is reportedly a 94-kDa protein (33, 34). However, its theoretical mass of 82 kDa fits well with our observations. The strikingly discrepant immunoreactivities of the different PR antibodies did not allow unambiguous identification of PR isoforms, and no conclusion could be drawn as to which of the multiple bands detected by the C-19 or MAB462 antibodies might represent PR-C.


Figure 1
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  		FIG. 1. Differing reactivities of PR antibodies with myometrial and breast cancer cell extracts. Nuclear extracts (15 µg/lane) from myometrial cells (Myo) that had been treated with 250 nM P4 (+) or with vehicle control (–) for 1 h, and whole cell extracts from T47D (T) and MDA-MB-231 (M) cells (20 µg/lane) were analyzed by Western blotting with PR antibodies C-19, MAB462, PGR-312, or SP2. The arrow indicates stripping and reprobing of a membrane. Positions of PR-B and PR-A are labeled; migration of marker proteins is shown in kDa on the left.

 

Figure 2
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  		FIG. 2. Schematic representation of the exon structure of PR and of the PR expression vectors used in this study. Top panel, The eight exons comprising wild-type PR are shown (not drawn to scale) with the protein coding region (shaded) and 5'- and 3'-UTRs (open boxes). Recently described alternative exons T, S, M, 4a, and 4b are shown below according to their genomic position. Exons T, S, and M are spliced to exons 4–8 (dotted lines). The putative hydrophobic leader signal encoded by exon M is highlighted (black box). All methionine codons are labeled by numbers, and start codons for published isoforms PR-B, PR-A, PR-C, PR-M, and PR-S are indicated by broken arrows. Bottom panel, Compositions of PR expression vectors are depicted. All cDNAs are in pcDNA3.1 with the exception of hPR0, which is in pSG5. Initiator codons carry broken arrows, and those embedded in an artificial Kozak sequence are indicated by asterisks; the corresponding cDNAs are signified by the suffix -ATG. Theoretical masses of the encoded proteins are given in kDa.

 
Therefore, we set out to investigate systematically the specificities of commercially available PR antibodies with a view to use them to assess the expression pattern of shorter PR isoforms. Theoretically, shorter receptor variants could occur from alternative translation initiation at downstream methionine start codons or be the products of alternatively spliced transcripts. In addition to Met-1 and Met-165, which initiate PR-B and PR-A, respectively, Met-595 has been postulated to be start codon in PR-C (9). However, there are additional in-frame AUG triplets, coding for Met-289, -301, -632, -692, -756, -759, and -775, the role of which has not yet been investigated. Furthermore, we wished to address the protein expression from alternatively spliced transcripts PR-T, PR-S, and PR-M. Because the alternative upstream exons T and S are noncoding, it has been suggested that in these mRNAs, translation from AUG-692 in exon 4 generates the short PR-S isoform (Fig. 2Go) (18, 19). In contrast, the alternative leader exon M includes an in-frame start codon reported to produce PR-M, a protein of about 38 kDa (20).

We generated a panel of expression vectors with cDNAs for PR-T, PR-S, and PR-M, and for truncated PR isoforms possibly starting at Met-289, -301 and -595. These cDNAs all include the natural 5'-UTRs to the respective putative start codons. Moreover, we generated expression vectors with start codons embedded in an optimized Kozak sequence to enforce translation initiation at AUG-595, -632, and -692 (constructs PR-595-ATG, PR-632-ATG, PR-692-ATG), to be used as size references. The original PR expression vector hPR0 encodes PR-B with natural 5'-UTR, whereas we derived pcDNA/PR-B from hPR1, which carries a Kozak sequence at the Met-1 codon (8). In contrast, pcDNA/PR-A includes the Met-165 codon in its natural context with 5'-UTR and was derived from hPR2 (Fig. 2Go). The identical stretch of about 850 bp 3'-UTR was maintained in all cDNAs.

Protein expression from the aforementioned vectors was tested by in vitro transcription/translation and immunoblotting. PR-B and PR-A were effectively generated from pcDNA/PR-B and pcDNA/PR-A, respectively, whereas PR-B production from hPR0 was low (Fig. 3Go). A product migrating around 63 kDa was generated from pcDNA/PR-301, and a fainter band migrating slightly more slowly was produced from pcDNA/PR-289, indicating that Met-301 and Met-289 codons can be used for translation initiation in the reticulocyte lysate system. The aforementioned pattern detected with the SP2 antibody was largely reproduced with antibody PgR636 (epitope between BUS and DBD), whereas PGR-312, raised against the N terminus of PR-A, detected PR-B and PR-A only. All three antibodies visualized an extensive laddering pattern for PR-B and PR-A, most likely due to capturing growing peptide chains. PGR-312, having the most 5' epitope, thus detected even shorter peptides than do SP2 and PgR636. Consistent with previous reports (35, 36), antibody hPRa6 reacted with PR-B only but was very inefficient.


Figure 3
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  		FIG. 3. Western blot analysis of in vitro-translated PR isoforms. The PR expression vectors shown in Fig. 2Go were subjected to in vitro transcription/translation (lanes 1–5, 7–13). Control reactions were primed with empty expression vector (lane 6, pcDNA) or received no DNA (lane 14, mock). Immunodetection was performed with the indicated PR antibodies, and positions of isoforms are indicated by their suffices (PR-B, PR-A, PR-289, PR-301, PR-595, PR-632, and PR-692). Lanes 2–3 of the blot probed with SP2 are also shown at a shorter exposure. An arrow indicates stripping and reprobing of a membrane.

 
C-terminal antibody MAB462 was used to visualize N-terminally truncated isoforms (Fig. 3Go). A product representing PR-595 (the presumed PR-C) was only generated when a Kozak sequence was present (as in construct pcDNA/PR-595-ATG), but not from AUG-595 in its natural context (pcDNA/PR-595). Furthermore, this product was not 60 but 38 kDa in size, consistent with its predicted molecular mass. Vectors pcDNA/PR-632-ATG and pcDNA/PR-692-ATG yielded proteins of the predicted sizes (34 and 27 kDa), the latter corresponding to the PR-S protein isoform. No translation products could be detected for the PR-M, PR-T, or PR-S cDNAs, indicating that neither the putative start codon in exon M nor AUG-692 in its natural context or any of the downstream methionine codons (AUG-756, -759, -775) are used in vitro. The C-19 antibody visualized PR-595 and PR-632 (products of pcDNA/PR-595-ATG and pcDNA/PR-632-ATG), but not PR-692. Both the MAB462 and the C-19 antibody detected several nonspecific bands as apparent with mock-primed transcription/translation reaction (Fig. 3Go).

We next used the in vitro translation products PR-B, PR-A, PR-289, PR-301, PR-595-ATG, PR-632-ATG, and PR-692-ATG to validate a panel of PR antibodies (Fig. 4Go). Clone 1A6 reportedly detects PR in formaldehyde-fixed paraffin sections (37, 38), but its precise epitope is not known (Table 1Go). In our hands, this antibody reacted with PR-B, PR-A, and PR-301, but no shorter isoform, localizing its epitope to the region aa 301–595. PR-289 was not detected, likely because it is so inefficiently expressed in vitro (Fig. 3Go). The H-190 antibody reacted nonspecifically with a 51-kDa band but hardly detected PR-B, PR-A, or PR-301, even though its epitope is a PR peptide spanning aa 375–564. Monoclonal antibody 10A9, to the very C terminus of PR (aa 922–933), faintly detected PR-B, PR-A, and PR-301 but very strongly the shorter variants PR-595, PR-632, and PR-692. Again, as with the MAB462 antibody (Fig. 3Go), no PR-595 product was obtained unless AUG-595 was embedded in a Kozak context (Fig. 4Go, compare lanes 5 and 6). Another C-terminal antibody, Ab-13, failed to detect larger isoforms, and barely reacted with PR-595 and PR-632. Finally, antibody C-20, raised against an internal peptide of PR, completely failed to recognize specific products.


Figure 4
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  		FIG. 4. Validation of PR antibodies on in vitro-translated PR isoforms. A selection of PR isoforms produced by in vitro transcription/translation (lanes 1–8), and control reaction primed with empty vector (lane 9) was subjected to successive immunodetection with the indicated PR antibodies. Lanes 1–2 of the blot probed with 1A6 are also shown at a shorter exposure. An arrow indicates stripping and reprobing of a membrane.

 
In vivo expression of PR variants
Next, the PR variants were expressed in vivo in transiently transfected COS-7 cells and compared with endogenous proteins in T47D cells. MDA-MB-231 cell extract was included as a negative control (Fig. 5Go). Antibodies AB-52 and PGR-312 detected PR-B and PR-A, and antibodies 1A6, SP2, and PgR636 additionally recognized PR-289 and PR-301. In contrast to in vitro translation, all isoforms were produced at comparable levels. However, no endogenous proteins corresponding to PR-289 or PR-301 were present in T47D extracts, nor were these proteins produced as by-products from PR-B or PR-A transcripts in transfected cells. Antibodies AB-52, PGR-312, 1A6, SP2, and PgR636 produced progressively more extensive banding patterns from transfected PR-B cDNA. Because the antibodies with the epitopes closest to the N terminus, AB-52 and PGR-312, display the cleanest results for full-length PR-B, the lower molecular mass products are likely proteolytic fragments lacking the N-terminal portion recognized by these antibodies. The increasing degree of laddering using antibodies with a more centrally located epitope is also reflected in T74D lysates. However, interpretation is complicated by the potential presence of alternatively spliced variants involving exons 4–6 (see below).


Figure 5
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  		FIG. 5. Validation of PR antibodies on longer PR isoforms overexpressed in COS-7 cells. There were 11 Western blots produced in parallel carrying whole cell extracts (40 µg) from COS-7 cells transfected with pcDNA/PR-B, pcDNA/PR-A, pcDNA/PR-289, pcDNA/PR-301, or empty vector (lanes 1–5), or whole cell extracts from T47D and MDA-MB-231 cells (lanes 6–7). The indicated antibodies were used for immunodetection. Please note that the blot reacted with AB-52 (top left) required 30 min exposure to film, whereas for all other antibodies, usually seconds were sufficient. Prominent nonspecific bands are marked by an arrowhead, migration of clearly identified isoforms is indicated; it is not certain if the lowest band in T47D extract detected by 10A9 represents PR-692 (bottom right).

 
The H-190 antibody weakly reacted with PR-B and PR-A but much more strongly with nonspecific bands at 60 and 30 kDa in all three cell types. Antibody C-20, as before, completely lacked specificity but decorated a band of 30 kDa in COS-7 and MDA-MB-231 cell extracts, and an additional 38-kDa band in T47D cell lysate. Among the C-terminal antibodies, Ab-13 and C-19 were not capable of detecting the longer PR isoforms; Ab-13 strongly visualized a 97-kDa band that might be mistaken for PR-B but was most prominent in PR-negative COS-7 and MDA-MB-231 cells, and C-19 decorated a doublet in MDA-MB-231 cells that misleadingly comigrated with PR-B and PR-A (Fig. 5Go, labeled with asterisks). The most prominent signal obtained with the C-19 antibody was a 45-kDa protein of unknown nature in T47D cells. Antibodies MAB462 and 10A9, both to the same C-terminal peptide, gave very similar patterns; they weakly decorated PR-B and PR-A, and additional nonspecific bands between 42 and 45 kDa in all cell types. A faint band slightly less than 28 kDa, only visible in T47D cell extracts, might represent PR-692.

Transfection of expression vectors for shorter PR isoforms, including those for PR-C, into COS-7 cells did not result in detectable levels of expression by Western blot analysis (data not shown). Therefore, we used indirect immunofluorescence (Fig. 6Go). Expression vectors for PR-B, PR-A, PR-301, PR-595-ATG, PR-632-ATG, and PR-692-ATG were transfected into COS-7 cells. PR-B and PR-A were detected equally well by antibodies PGR-312, SP2, and 10A9. The latter two antibodies also detected PR-301. All three isoforms displayed almost exclusive nuclear localization. However, expression of the PR-595, PR-632, or PR-692 isoforms could not be demonstrated using the C-terminal antibodies MAB462 or 10A9 (data not shown). Given the excellent sensitivity of 10A9 for the immunofluorescent detection of longer PR isoforms, we concluded that COS-7 cells are not a suitable cell type for expression of short PR variants.


Figure 6
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  		FIG. 6. Immunofluorescent detection of PR isoforms in transfected COS-7 cells. Expression vectors pcDNA/PR-B (A–C), pcDNA/PR-A (D–F), or pcDNA/PR-301 (G and H) were transfected into COS-7 cells and visualized by indirect immunofluorescence with PR antibodies PGR-312 (A and D), SP2 (B, E, and G), or 10A9 (C, F, and H). Red immunofluorescence for PR is shown in the upper images, blue nuclear counterstain with 4,6-diamidino-2'-phenylindole in the lower images of each panel. No staining with any of the antibodies was obtained for transfected pcDNA/PR-595-ATG, pcDNA/PR-632-ATG, or pcDNA/PR-692-ATG (data not shown).

 
As an alternative cell model, we used the uterine myosarcoma cell line SKUT-1B for expression of shorter PR isoforms. Translation products from PR-595-ATG and PR-632-ATG cDNAs, but not from PR-692-ATG cDNA, could be demonstrated upon extended exposure with the 10A9 antibody (Fig. 7AGo). However, the resultant high background did not allow verification if a 38-kDa protein present in T47D cell lysates, run in parallel, corresponded to PR-595 (PR-C). To address this issue further, SKUT-1B cells transfected with pcDNA/PR-595 or pcDNA/PR-595-ATG, and T47D cells were harvested by direct lysis in loading buffer and immediately subjected to SDS-PAGE (Fig. 7BGo). A faint product occurring from PR-595-ATG was visualized by both MAB462 and C-19 antibodies, but there clearly was no corresponding endogenous product in T47D cells. Probing these samples with the PgR636 antibody demonstrated that SKUT-1B cells are negative for PR-B and PR-A (Fig. 7BGo).


Figure 7
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  		FIG. 7. Detection of shorter PR isoforms overexpressed in SKUT-1B cells. A, A Western blot carrying whole cell extract (30 µg) from SKUT-1B cells transfected with vectors encoding shorter PR isoforms (lanes 1–7) or empty vector (lane 8), or from T47D cells (lane 9) was detected with PR antibody 10A9. Please note that an extended exposure time of 20 min was required to obtain faint signals representing PR-595 and PR-632. All other bands in lanes 1–7 are nonspecific because they also came up in PR-negative mock-transfected SKUT-1B cells (lane 8). B, SKUT-1B cells were transfected with empty vector (lane 1), pcDNA/PR-595 (lane 2), or pcDNA/PR-595-ATG (lane 3). Quick protein extracts were prepared from transfected cells and from T47D cells (lane 4) and 5 µl loaded per lane. The indicated antibodies were used for immunodetection. The arrow denotes stripping and reprobing of the membrane; arrowheads label prominent nonspecific bands. Note that very faint signals for PR-595 are visible in lane 3 when reacted with MAB462 or C-19.

 
The specificities of the PR antibodies used in this study for Western blot detection are summarized in Table 2Go. It is immediately apparent that antibodies with N-terminal epitopes are highly specific (AB-52, hPRa6, PGR-312, 1A6, PgR636), whereas those raised against C-terminal peptides are not (C-19, 10A9, MAB462, Ab-13). C-terminal antibody C-262, listed in Table 1Go, did not produce any signal in our hands. Among the antibodies with epitopes in the central region of PR, C-20 and H-190 largely failed to detect PR but reacted nonspecifically. The exception in this group is SP2, which was not only highly specific but also proved to have the highest sensitivity of all antibodies for the detection of PR-B, PR-A, or PR-301. No antibody could be identified that was capable of detecting the full range of PR isoforms with sufficient specificity, sensitivity, and reproducibility in Western blot application.


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  		TABLE 2. Reactivity of PR antibodies with PR isoforms and unidentified proteins

 
An epitope map for the 13 PR antibodies used in this study was compiled by combining our experimental data with published information (Fig. 8Go).


Figure 8
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  		FIG. 8. Depiction of antibody epitopes along the PR protein. Solid lines represent known epitopes, dotted lines signify the region within which the epitope must be located based on published data (see Table 1Go) and our present study, and dotted lines with a question mark indicate that detailed information is missing and could not be experimentally deduced; C-20 was raised against an internal peptide, C-19 and Ab-13 against a C-terminal peptide. H, Hinge region.

 
Expression of PR-C with or without Kozak sequence
To support further our notion that no protein is translated from the Met-595 codon in its natural context, we wished to render the PR-595 isoform more readily detectable. An internal myc epitope was incorporated near the C terminus at Ile-920 such that the epitope for PR antibodies MAB462 and 10A9 (aa 922–933) was preserved (Fig. 9Go). The resultant tagged constructs pcDNA/PR-595/myc and pcDNA/PR-595-ATG/myc were transfected into SKUT-1B cells alongside their untagged counterparts. Immunoblotting with a monoclonal myc antibody revealed synthesis of PR-595/myc exclusively from the construct with a Kozak sequence (pcDNA/PR-595-ATG/myc) (Fig. 9Go). Detection with PR antibody 10A9 revealed very faint expression from PR-595-ATG, but not from PR-595 cDNA, confirming our previous observations. A dramatically stronger signal was obtained in cells transfected with myc-tagged PR-595-ATG cDNA, compared with the untagged counterpart, whereas hardly any product was demonstrable with the PR-595/myc cDNA lacking the Kozak sequence. It remains to be clarified whether the drastically stronger signal intensity of myc-tagged PR-595 compared with untagged PR-595 is due to a more favorable display of the 10A9 epitope upon insertion of the myc sequence, or if the stability of the protein is enhanced by the inserted myc sequence.


Figure 9
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  		FIG. 9. Analysis of PR-595 expression in transfected SKUT-1B cells. Left panel, Expression vectors pcDNA/PR-595 (1 ) and pcDNA/PR-595-ATG (2 ) were modified to include a c-myc epitope (black box) near the C terminus of PR, between duplicated Ile-920 residues. The epitope is followed by Ile-920 to Lys-933 of PR, which contains the antigenic sequence for monoclonal antibody 10A9. Resultant constructs are pcDNA/PR-595/myc (3 ) and pcDNA/PR-595-ATG/myc (4 ); calculated masses of encoded proteins are given in parentheses (kDa). Open boxes are UTRs; the protein-coding region is shaded. Exons are numbered. Artificial Kozak sequence around the Met-595 start codon is marked with an asterisk. The empty vector pcDNA3.1 was used as negative control (5 ). Right panel, SKUT-1B cells were transfected with the constructs shown on the left (lanes 1–4) or with empty vector (lane 5), and 30 µg whole cell extract analyzed by Western blotting with a mouse monoclonal anti-myc antibody. The same samples run on a parallel blot were detected with PR antibody 10A9. Bands labeled with an arrowhead are nonspecific. Note that a very faint signal for PR-595 is visible in lane 2.

 
In conclusion, an artificial Kozak sequence is required to render AUG-595 an initiator codon, and PR-595 (PR-C) is not produced endogenously.

Analysis of PR-S
We had repeatedly observed a 27-kDa peptide in T47D cells that comigrated with PR-692 and was detected by the C-terminal antibody 10A9. To obtain further information on this peptide, we prepared whole cell, cytosolic, and nuclear extracts from T47D cells that had been treated with R5020 or vehicle for 1 h. Detection with N-terminal PR antibody SP2 demonstrated a shift of PR-B and PR-A from the cytosolic to the nuclear fraction in response to R5020 (Fig. 10Go). Even loading and separation of cytoplasmic from nuclear proteins were confirmed by reprobing with antibodies to tumor suppressor protein p53 or the nuclear membrane protein lamin A/C, respectively.


Figure 10
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  		FIG. 10. Subcellular localization of PR isoforms in T47D cells. T47D cells were treated with vehicle (Co) or R5020 for 1 h before preparation of whole cell (W), nuclear (N), and cytoplasmic (C) extracts, and 30 µg protein was loaded per lane. In vitro translated PR-692 protein (IVT) was loaded as a size reference. Parallel blots were immunodetected with PR antibodies SP2 or 10A9. Sequential stripping and reprobing (indicated by arrows) with antibodies against p53 and Lamin A/C were done to control for even loading and for purity of the cytoplasmic vs. nuclear fractions, respectively.

 
Immunodetection of the same samples with C-terminal antibody 10A9 clearly revealed the progestin-dependent nuclear translocation and reduced mobility of PR-B and PR-A indicative of ligand-induced phosphorylation. Furthermore, 10A9 decorated additional bands of higher mobility, including a band at 27 kDa that comigrated with in vitro-translated PR-692 and underwent nuclear accumulation upon R5020 stimulation (Fig. 10Go). The nuclear localization signal of PR, located at the 3'-end of the DBD (39, 40), is lacking in PR-692; if the 27-kDa band represented PR-692, it might be hijacked to the nucleus as a heterodimer through receptor dimerization sequences that are contained in the LBD (6).

Analysis of splice variants
The numerous proteins migrating below PR-B and PR-A in T47D cell extracts, as detected by the highly specific N-terminal antibodies PgR636, 1A6, and SP2 (Fig. 5Go), might represent products from alternatively spliced PR transcripts. Deletions involving exons 2, 4, 5, and 6, or insertion of exons 4a+4b, have been reported (14, 15, 16, 17). Deletion of exon 2 results in a frame shift and early translation termination in exon 3, and would reduce the size of PR-B or PR-A by 52 kDa. However, we did not observe such C-terminally truncated forms in T47D cells using the N-terminal antibody PGR-312 (Fig. 5Go). The structure of splice variants concerning exons 4–6 is depicted in Fig. 11AGo. These are either out-of-frame variants leading to premature termination (4a/b, {Delta}6, {Delta}6,2), or the reading frame is maintained and allows read through to the C terminus of PR ({Delta}4, {Delta}5,6). Resultant proteins would have a predicted mass of 11–21 kDa less than the corresponding wild-type receptor. To assess the abundance of such splice variants by RT-PCR, we amplified the region between exons 3 and 8. We also included PR-M in our analysis, using primers specific to exons M and 7 (Fig. 11AGo), and compared the expression profile in T47D cells to that in primary myometrial cells. The major product amplified between exons 3 and 8 is 898 bp in size and, thus, represents wild-type PR (visualized by in-gel staining) (Fig. 11BGo). Southern blot hybridization with probes to exons 4 or 7 confirmed the vast abundance of wild-type transcripts. Results are similar for T47D and myometrial cells with the exception of a faint signal of 592 bp only seen in T47D. Because this amplicon was only detectable with the exon 7 probe, it is likely to represent the {Delta}4 variant. No {Delta}6 or {Delta}5,6 variants were observed. Furthermore, no product of 1130 bp was detected with a probe to exon 4b (data not shown), indicating that the 4a/b variant is not relevantly expressed in the cell types examined here. The PR-M variant was of very low abundance and required extended exposure for detection.


Figure 11
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  		FIG. 11. Expression analysis of C-terminal PR splice variants at the transcript level. A, Splice variants in the region between exons 3 and 8, as reported in the literature, are depicted. The arrows indicate PCR primers used to amplify wild-type (wt) transcripts or alternatively spliced transcripts 4a/b, {Delta}4, {Delta}6, {Delta}5,6, and {Delta}6,2. The functional domains truncated or deleted in these variants are listed; out-of-frame mutations (OOF) result in premature stop codons that are indicated by arrowheads below the amplicons. The approximate reduction in molecular mass of the resultant proteins compared with wild-type PR is given in parentheses ({Delta}). Sizes of exons or amplified portions thereof are shown in italics in base pairs, and sizes of expected amplicons are given on the right. In addition to the aforementioned splice variants, a portion of PR-M cDNA was amplified using a forward primer anchored in exon M, and a reverse primer spanning the exon 6/7 boundary. The black bars (Ex4, Ex4b, Ex7) shown on top represent exon-specific probes used for Southern hybridization. B, Primer pairs PR-Ex3-s/PR-Ex8-as or PR-M-s/PR-Ex7-as were used for PCR amplification of cDNAs prepared from T47D cells (T) or primary human myometrial cells (M). Top panel, DNA was visualized with SYBR Gold. Sizes of markers (left) and major PCR products are given in base pairs. Bottom panel, Southern blots prepared from the gels shown previously were hybridized with probes to PR exons 4 or 7. Hybridization with a probe to exon 4b did not result in detection of a 1130-bp product (see A) and is not shown.

 
In conclusion, in T47D and myometrial cells, alternatively spliced transcripts are so underrepresented relative to wild-type PR mRNAs that they are unlikely to account for additional bands in Western blots detected with N-terminal antibodies.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Specificities of PR antibodies
The first generation of monoclonal antibodies to human PR was produced against PR preparations from T47D cells or human uterine extracts that had been immunoaffinity purified using antibodies against chicken or rabbit PR (41, 42). Immunoaffinity purification with the antirabbit PR antibody yielded PR-B, PR-A, and an additional 65-kDa product. Monoclonal antibodies were raised against this preparation and their epitopes mapped. A monoclonal antibody reactive to aa 1–121 in PR recognized the B-isoform, another to aa 121–208 visualized both PR-B and PR-A, and a third monoclonal antibody with an epitope spanning aa 208–296 reacted with PR-B, PR-A as well as the 65-kDa band. The latter was interpreted as a proteolytic degradation product lacking the N terminus of PR (42). In that study a total of 39 monoclonal antibodies was obtained. Remarkably, the epitopes all mapped to various regions in the N-terminal half of the receptor. The striking lack of experimental antigenicity of the C-terminal part of PR, including the DBD and LBD, despite the prediction of antigenic epitopes, was explained by the extremely high conservation in this region of the PR between species (42).

In agreement, immunization with full-length PR-B or PR-A generally produces antibodies directed against the N-terminal part of PR. For example, the epitopes for PgR636 and PgR1294, produced against formalin-fixed PR-A, locate between aa 165 and 534 (43). Raised against native PR-B, the B30 antibody recognizes the BUS and, thus, PR-B only, and the epitope for AB-52, which detects PR-B and PR-A, spans aa 221–237 (41, 44). Immunization of mice with partially purified PR from human endometrial carcinoma generated several monoclonal antibodies, such as hPRa7, which recognizes PR-B and PR-A, and hPRa6, which is specific for PR-B in Western blot applications (35).

As a consequence, generation of antibodies against the C-terminal portion of PR required immunization with C-terminal peptides. Antibodies targeting the LBD of PR include C-262 (33), C-19, Ab-13, MAB462, and 10A9 (Table 1Go). The poor antigenicity of the LBD likely accounts for the poor specificity of several of these commercially available antibodies. For instance, the C-262 antibody reacts with an entirely unrelated 60-kDa membrane protein in cell types that are devoid of nuclear PR, such as human sperm and rat granulosa cells (45, 46). Purification studies have identified this 60-kDa protein as serpine 1 mRNA binding protein 1 (also termed RDA288) (47).

The specificity of another LBD targeting antibody, C-19, is also highly questionable. For instance, this antibody immuno-stains the plasma membrane of live human aortic endothelial cells (HAECs), which has been interpreted as evidence for the existence of a membrane-associated PR isoform (48). Similarly, C-19 also strongly immunoreacts with nonpermeabilized wild-type T47D cells and PR-deficient T47D-Y cells, whereas the antibody 6A1, to an epitope around Ser-190, specifically only stains nuclei in T47D cells upon permeabilization (49).

In Western blot applications, the C-19 antibody reportedly recognizes bands of 116 (PR-B), 94 (PR-A), and, less prominently, of 100 and 60 kDa in T47D cells. The 100-kDa band is also present in cytosolic preparations of HAECs, whereas the 60-kDa band is strongly enriched in the membrane fraction of these cells (20). In a different study, expression of PR-B and PR-A was demonstrated both in T47D cells and in myometrium using antibody PgR1294, whereas C-19 recognized the same bands in T47D cells but failed to do so in myometrial samples. Instead, two bands of about 100 and 60 kDa appeared (22). In our hands, C-19 failed to specifically detect PR-B or PR-A in T47D cells or myocytes but also, as reported by others, generated a prominent band in the 100-kDa range in myocytes. Strong evidence exists to indicate that the 100-kDa signal in myometrial preparations results from cross-reactivity of the C-19 antibody with the cytoskeletal protein {alpha}-actinin (50). Together, the C-19 antibody is prone to artifacts, and results obtained with this antibody should be interpreted with great caution.

As alternatives to C-19 for the detection of N-terminally truncated PR forms, we tested the monoclonal antibodies MAB462 and 10A9, both directed against the very C terminus of PR (aa 922–933). Although these antibodies visualized shorter PR isoforms, they were very inefficient for the detection of full-length PRs. They also generated numerous nonspecific bands and can, therefore, not be recommended for the analysis of the relative levels of endogenous PR variants by Western blotting. However, 10A9 displayed excellent sensitivity for immunofluorescent detection of full-length PRs. Finally, the C-terminal antibodies Ab-13 and C-262 largely failed to yield informative results in our immunoblotting experiments.

Identity of PR-C
Wei et al. (9) were the first to report on the existence of an N-terminally truncated PR-C receptor. Although no protein data were presented, they proposed Met-595 as the start codon for this isoform based on an earlier report demonstrating that in vitro translation of cDNA hPR54, which contains the full coding sequence of PR-A, contributes not only to PR-A but also to a 42-kDa peptide (34). These products were visualized in the in vitro translation reaction after immunoprecipitation with the PR antibody AB-52. A corresponding band was also shown in immunoprecipitated T47D lysate. However, the epitope of AB-52 was subsequently mapped to aa 221–237 (44), and, consequently, this antibody simply cannot recognize PR-C. Our own studies here confirm that AB-52 specifically reacts with PR-B and PR-A but not with downstream translation products.

The notion that PR-C is a truncated PR isoform, comprising aa 595–933, has been perpetuated in the literature (3). This conjecture appears to be supported by various reports showing protein expression from cDNAs encoding these aa. However, close scrutiny of the published expression vectors for PR-C reveals that the natural context of the Met-595 codon is not maintained. Instead, the cloning procedures incorporated restriction sites resulting in modifications of the 5'-end. Although the natural context is 5'-agagggcaATGG-3' (Met-595 codon underlined), this was changed to 5'-caccATGG-3' (26), or to 5'-gaattcggATGG-3' (11, 12), and no PR-specific 5'-UTR was included. The optimal consensus sequence for translation initiation, by the rules of Kozak, is cRccAUGG, where R is a purine (G or A). According to the scanning model of translation initiation, a weak initiation codon is only used if no stronger one is present in the transcript further upstream (31). In the PR-C cDNAs mentioned previously, no 5'-sequence, except for the restriction enzyme recognition site, was included (11, 12, 26). Furthermore, the cloning procedure used by Condon et al. (26) even created a perfect Kozak sequence. Therefore, it is not surprising that transcripts generated from these cDNAs are translated in transfected cells, but this does not reflect the natural situation. Overexpression of PR-C in Sf9 insect cells yielded a protein that comigrated with a fainter band in T47D cells, as detected with the C-terminal PR antibody C-262 (11). Molecular mass markers were not shown, but this protein was described as having an apparent molecular mass of 60 kDa. Expression of this PR-C vector by in vitro transcription and translation, or by transfection into HeLa cells, yielded a protein that binds the synthetic progestin R5020 (12). Transfection into T47D cells stably expressing the PR-responsive mouse mammary tumor virus (MMTV) long terminal repeat coupled to a CAT reporter gene showed that PR-C enhances MMTV-CAT activation through endogenous PR, both in the absence and presence of R5020 (12). These observations contrast to another report indicating that overexpression of PR-C in an immortalized human myometrial cell line suppresses transactivation of the MMTV reporter by endogenous PR-B, even in the absence of added ligand (26). The protein product of the PR-C expression vector and, thus, the size of PR-C were not shown in this study.

Our data do not support the conjecture that PR-C is a naturally occurring 60-kDa protein or that this variant encompasses aa 595–933. Our expression vector pcDNA/PR-595, containing a cDNA with the natural 5' region flanking the triplet encoding Met-595, was not translated in vitro or in vivo. When the context of the Met-595 codon was changed to an optimal Kozak sequence, identical to the one introduced by Condon et al. (26), a protein of 38 kDa was produced, consistent with its theoretical mass of 39 kDa, and distinctly smaller than 60 kDa commonly assigned to PR-C. No endogenous protein comigrating with PR-595 could be detected in myocytes or T47D cell lysates.

We reasoned that a 60-kDa PR isoform could be synthesized from the upstream codons for Met-289 or Met-301, perhaps representing the "true" PR-C. In transfected cells we could demonstrate that AUG-301 and AUG-289 are used to initiate protein translation, contributing to PR variants of about 64 kDa in size. However, utilization of these residues as start methionines required deletion of the stronger upstream initiators AUG-1 and AUG-165 from the cDNAs. Therefore, it is predicted that such truncated variants are not generated in vivo to a physiologically relevant extent. In fact, we did not observe immunoreactive bands comigrating with PR-289 or PR-301 in breast cancer extracts.

Myometrial PR isoforms and parturition
A shift in the ratio of PR isoforms in pregnant human myometrium has been put forward as an attractive hypothesis to explain "functional progesterone withdrawal" associated with the onset of parturition. Both an increase in PR-A levels relative to PR-B (22) and a dramatic up-regulation of the PR-C isoform have been reported (26). The relative level of PR-C expression before and after the onset of labor has been assessed by quantitative RT-PCR and Western blot analysis (26). The abundance of PR-C transcripts was deduced by measuring three PCR amplicon pools: one obtained with primers specific for PR-B mRNA alone; one with primers amplifying both PR-B and PR-A transcripts; and one with primers to the C terminus, presumably detecting transcripts for PR-B, PR-A and PR-C simultaneously. Using this approach the onset of labor was calculated to be associated with a more than 200-fold increase in PR-C mRNA in fundal myometrium. The C-19 antibody was used to examine expression at the protein level. Western blot analysis demonstrated an increased intensity of an immunoreactive band, designated PR-C, in cytoplasmic fractions of laboring tissue samples. A more slowly migrating band, interpreted as PR-B, also increased under these conditions, especially in the nuclear compartment (26). However, in the absence of appropriate size controls and in view of our findings and others (22), it cannot be excluded that the presumed PR-B and PR-C bands may in fact be the unrelated 100 and 60-kDa proteins frequently observed with the C-19 antibody. Consistent with this conjecture, Tyson-Capper et al. (51) also noted considerable variability in the specificity of PR antibodies raised against internal or C-terminal epitopes and concluded that they should not be used to characterize PR isoform expression in human myometrium. In contrast, four different antibodies recognizing the N terminus of PR consistently demonstrated a decrease in myometrial PR-B levels associated with term labor (51).

Expression of PR-M
PR-M is a potentially interesting variant with a putative membrane-targeting signal sequence. In the original report (20), a V5-tagged PR-M fusion protein expressed in Sf9 cells migrated around 40 kDa. In T47D cells and HAEC membranes, a minor 38-kDa band was detected upon probing with the C-19 antibody and designated as the endogenous PR-M. This conclusion was based on three pieces of evidence: 1) the apparent molecular mass in SDS-PAGE; 2) this 38-kDa band was not detectable by the C-20 antibody directed to a region absent in PR-M; and 3) the 38-kDa band, along with the other five more slowly migrating bands detected by the C-19 antibody in T47D extracts, disappeared upon peptide neutralization of the antibody. However, formal proof that the 38-kDa protein is PR-M is lacking. Notably, antibody C-19 detected the 38-kDa band in the PR-B/PR-A negative variant T74D-Y cell line (20). Yet, T47D-Y cells do not bind P4 (52), and, thus, if the prominent 38-kDa band in T47D-Y cells is indeed PR-M, this receptor variant must have lost its ligand-binding capacity despite containing the full-length LBD. Consequently, the proposed physiological role of PR-M as a mediator of nongenomic P4 actions is most questionable.

We did not observe protein expression from pcDNA/PR-M by in vitro transcription/translation nor by transfection of cells. This indicates that the in-frame AUG codon in the leader exon M does not support translation initiation, or, alternatively, that the PR-M transcript and/or protein is subject to extremely rapid turnover. Moreover, we consistently observed that the C-19 antibody strongly cross-reacts with a 45-kDa protein and, to a lesser extent, with a 35-kDa protein in T47D cell lysates. The nature of these bands is as yet unclear, but our RT-PCR analysis unequivocally demonstrated that PR-M transcripts are expressed at extremely low abundance in T47D and in myometrial cells.

PR-S and other variants
We were also not able to detect protein expression from the PR-T or PR-S cDNAs. Both transcripts carry a noncoding leader exon (T or S, respectively) and the first AUG codon that would be encountered by scanning ribosomes encodes for Met-692 (18, 19). No PR-692 protein ("PR-S") was produced from this codon in its natural context. Combined with the fact that PR-T and PR-S mRNAs, based on EST database searches, are extremely rare in the human transcriptome, this observation casts doubt on the physiological relevance of these splice variants. Although we detected a faint 27-kDa band in T47D that underwent progestin-induced nuclear translocation, we could not unambiguously identify this as PR-692 ("PR-S").

Not only PRs but also other steroid hormone receptors exist in multiple forms, the generation of which, however, differs among receptors. PR-B and PR-A result from alternative promoter usage in the same gene (7), whereas the estrogen receptor (ER) isoforms ER{alpha} and ERβ are products of separate genes (53). The glucocorticoid receptor (GR) isoforms GR{alpha} and GRβ are the result of alternative splicing at the 3'-region of the pre-mRNA, whereas their isoforms A and B occur from alternative translation initiation at either Met-1 or Met-27 (54). As for the androgen receptor (AR), the full-length protein on immunoblots can be accompanied by a minor shorter variant termed AR-A. There is evidence to suggest that such shorter AR species occur from in vitro proteolytic cleavage after repeated freeze-thaw cycles (55). We tested the possibility that this is also the reason for the appearance of the shorter PR immunoreactive bands seen in T47D cell extracts. However, no differences in the banding pattern were observed between freshly loaded lysates and rethawed preparations (data not shown).

Our analysis of C-terminal splice variants involving exons 4–6 highlights that transcripts with the wild-type PR C terminus are vastly more abundant than any splice variant in myometrial or T47D cells. Therefore, the prominent lower molecular mass bands detected with highly specific antibodies, such as PgR636, 1A6, or SP2, likely do not represent products of alternatively spliced transcripts but are proteolytic fragments. It is well established that full-length PR in vivo is subject to rapid proteasomal turnover, both in a ligand-dependent and -independent fashion (56, 57, 58). These degradation products are not visualized by PR antibodies recognizing N-terminally located epitopes, such as hPRa6, AB-52, or PGR-312.

Conclusions
Together, the currently available antibodies do not allow unambiguous identification of N-terminally truncated PR isoforms. Furthermore, our assessment of the translatability of PR-C, PR-M, or PR-S transcripts renders it very unlikely that the putatively encoded receptor isoforms are expressed in vivo to any relevant extent. Moreover, the widely studied PR-C isoform is not a naturally occurring peptide composed of aa 595–933. Because reports on the expression of this receptor variant in uterine tissues relied on antibodies with poor specificity, its proposed role in human parturition must be seriously questioned.


   Acknowledgments
 
We thank J. J. Brosens for insightful discussions and critical reading of the manuscript, and P. Chambon for the hPR0, hPR1, and hPR2 expression vectors.


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online July 10, 2008

Abbreviations: aa, Amino acid(s); AR, androgen receptor; BUS, B-upstream segment; DBD, DNA binding domain; ER, estrogen receptor; GR, glucocorticoid receptor; HAEC, human aortic endothelial cell; LBD, ligand binding domain; MMTV, mouse mammary tumor virus; P4, progesterone; PR, progesterone receptor; SDS, sodium dodecyl sulfate; UTR, untranslated region.

Received April 28, 2008.

Accepted for publication July 2, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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