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a Department of Molecular and Structural Biology, University of Aarhus, 8000 Aarhus C, Denmark
b Department of Clinical Biochemistry, Statens Serum Institut, 2300 Copenhagen S, Denmark
c Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota 55905
d Department of Immunology and Medicine, Mayo Clinic, Rochester, Minnesota 55905
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ProMBP is composed of a strongly acidic 90-residue propart [6] and a highly basic mature portion of 117 residues [7]. It is an extensively and very heterogeneously glycosylated proteoglycan, migrating in SDS-PAGE as a smear corresponding to 50–90 kDa, not visible using Coomassie Brilliant Blue staining [2, 8]. ProMBP has been isolated from the blood of pregnant women in complex only with either PAPP-A, angiotensinogen, or complement C3dg [2, 9]. However, proMBP has recently been studied in cultures of interleukin-5-stimulated umbilical cord stem cells, and it is processed in the maturing eosinophil granule to 14-kDa MBP and localized to the granule core [10]. The mature MBP is a cytotoxic protein, constituting more than 50% of the protein content of the granules in eosinophil leukocytes [11, 12]. It is released from the eosinophil leukocyte by degranulation, and plays multiple roles in the effector functions of these cells [13].
PAPP-A not complexed with proMBP cannot be isolated from pregnancy serum [3], but it has recently been detected in conditioned media from human fibroblasts. Further, it was established that PAPP-A cleaves insulin-like growth factor (IGF) binding protein-4 in an IGF-dependent manner [14]. The function of proMBP in pregnancy is unknown. It has been reported that the PAPP-A/proMBP complex is absent from maternal serum in pregnancies in which the mother is carrying a fetus with Cornelia de Lange syndrome [15].
Recently, PAPP-A and proMBP in conjunction with SP1 have been shown to be effective markers for detecting fetuses affected with Down syndrome in Weeks 7–12 of gestation [16–19]. In addition, proMBP has been suggested as a serum and histologic marker for the malignant potential in trophoblastic neoplasia [20, 21].
In term placenta, PAPP-A mRNA is synthesized by the syncytiotrophoblast and by the trophoblast-derived septal X cells, as determined by in situ hybridization [22]. In the same study, PAPP-A was colocalized, using proMBP-adsorbed polyclonal anti(PAPP-A/proMBP), to the septal X cells and the syncytial lining. Both proMBP and proMBP mRNA have been localized to the septal X cells by immunofluorescence and in situ hybridization, respectively [23, 24]. In nonpregnant individuals, synthesis of PAPP-A has been reported in a number of tissues, e.g., the corpus luteum [25], endometrium [26–28], prostate [29], testis [30], liver, pancreas, myocardium, spleen, bone marrow [31], trophoblastic tumors [32, 33], and breast carcinoma [34, 35]. These investigations were based on techniques using polyclonal antibodies, which are now known to recognize several other proteins, including eosinophil MBP, recombinant proMBP, SP1, and haptoglobin [2, 36–39].
We measured the levels of PAPP-A and proMBP mRNA in a number of reproductive and nonreproductive tissues using a sensitive semiquantitative reverse transcription (RT) polymerase chain reaction (PCR) assay. The method is based on coamplification of the cDNA and a deletion variant thereof that is used as internal standard (IS). The amounts of PAPP-A and proMBP mRNA are normalized against the total amount of mRNA in the sample, determined as the amount of ß-actin mRNA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Term placental tissue (outer maternal side) from cesarean sections was provided by the Department of Gynecology and Obstetrics, Aarhus University Hospital, Aarhus, Denmark. First-trimester trophoblast tissue was from the Danish Cancer Society, Aarhus. Prostate tissue from hyperplasias and adenocarcinomas was provided by the Department of Experimental Clinical Oncology, Aarhus University Hospital. Mononuclear cells from bone marrow, prepared as described [40], were obtained from the Department of Hematology, Aarhus County Hospital, Denmark. Normal breast tissue, and samples from lobular and ductal breast carcinomas were provided by the Department of Pathology, Aarhus County Hospital. Samples from ascending colon and kidney cortex were from Mayo Clinic, Rochester, MN. Samples from ovary, endometrium, myometrium, and tuba uterina, provided by the Department of Gynecology and Obstetrics, Aarhus University Hospital, were from hysterectomies from normal postmenopausal women (age < 50 yr). A blood sample was drawn from a pregnant woman (first trimester). All tissue samples were stored in liquid nitrogen.
Extraction of mRNA and cDNA Synthesis
Frozen tissue samples were pulverized using a mortar embedded in dry ice. Approximately 20 mg tissue powder or 106 cells were then lysed in 1 ml lysis/binding buffer (0.5 M LiCl, 10 mM EDTA, 5 mM dithiothreitol, 1% SDS, 100 mM Tris-HCl, pH 8.0) using a glass homogenizer (Wheaton, Millville, NJ). Poly(A)+ RNA was isolated using the Dynabeads mRNA DIRECT kit (Dynal A/S, Oslo, Norway), according to the manufacturer's instructions. Poly(A)+ RNA was eluted from the oligo(dT) Dynabeads by incubation in 20 µl 2 mM EDTA for 2 min at 65°C. First-strand cDNA was synthesized immediately thereafter by incubating 50% of the eluted poly(A)+-RNA for 60 min at 42°C with 4 units avian myeloblastosis virus reverse transcriptase, 10 pmol oligo(dT)24, 1 pmol 5'-AAACCCATTTTATTGCAGGGAGG-3' (MBP-specific primer [nt 840–818 in the proMBP cDNA sequence]), 1 pmol 5'-CTGTGGTTGTGTGACAAATGGC-3' (PAPP-A-specific primer [nt 4936–4915 in the PAPP-A cDNA sequence]), 40 units RNasin (Promega, Leiden, Holland), 1 mM dNTP, and 5 mM Mg2+, in 20 µl of the supplied buffer. All reagents, except primers, were from Promega (Madison, WI). The remaining poly(A)+ RNA was processed in parallel without addition of reverse transcriptase. The resulting cDNA was diluted (1:4n, n = 1 to 10) in dideoxy H2O and used directly as template for competitive PCR, or stored at -20°C until use.
Preparation of IS Templates
The IS is a deletion variant of the respective cDNA PCR product (Fig. 1) that can be amplified with the same primers as the cDNA. The PAPP-A IS was constructed by primer-mediated deletion as previously described [41]. Briefly, the 5'-CAGTCAGCTGCTCAACGGAAGGACTCACATTGG-3' (nt 4712–4731 and 4789–4805 in the PAPP-A cDNA sequence) was used with 5'-GGAGGCTCTGGGACTGCAC-3' (nt 4904–4886) as primers in a PCR, using first-strand cDNA from placenta as template, to make a 62-basepair (bp) deletion variant of the PAPP-A cDNA PCR product with the same primer binding sequences as the PAPP-A cDNA. An MBP IS was constructed by excision of a HinP1I-MspI fragment (nt 447–522 in the proMBP cDNA sequence), resulting in a 76-bp deletion variant of the cDNA PCR product. The ß-actin IS was constructed by excision of a HinP1I-MspI fragment (nt 1045–1136 in the ß-actin cDNA sequence), resulting in a 92-bp deletion variant. For construction of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) IS, the primer 5'-AACGGGAAGCTCACTGGCATGATGACATCAAGAAGGTGGTG-3' (nt 674–694 and 765–787 in the GAPDH cDNA sequence) was used with 5'-CCACCACCCTGATGTCGTAGC-3' (nt 977–957) in a PCR using first-strand cDNA as template to make a 73-bp deletion variant of the GAPDH PCR product with the same primer binding sequences as the cDNA PCR product. The IS's were purified from agarose gels and verified by sequence analysis. The fixed amount of IS added to each PCR was taken from the same batch stored in ready-to-use aliquots at -20°C.
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Primers for Competitive PCR
Primers for competitive PCR were as follows: ß-Actin: 5'-CACCCAGCACAATGAAGATCAAG-3' (nt 1003–1025) and 5'-GTCAAGAAAGGGTGTAACGCAAC-3' (nt 1207–1185); PAPP-A: 5'-CAGTCAGCTGCTCAACGGAA-3' (nt 4712–4731) and 5'-GGAGGCTCTGGGACTGCAC-3' (nt 4904–4886); MBP: 5'-TTAGTCAAGCTTGGTTTACTTGC-3' (nt 423–445) and 5'-GGAAGTCTTCTGAGGCAGTGG-3' (nt 720–700); and GAPDH: 5'-AACGGGAAGCTCACTGGCATG-3' (nt 674–694) and 5'-CCACCACCCTGTTGCTGTAGC-3' (nt 977–957). Numbers in parentheses refer to the positions in the corresponding cDNA sequences. Gene and cDNA sequences were obtained from GenBank (accession numbers: GAPDH, J02642 and J04038; ß-actin, X00351 and M10277; MBP, X14088 and M34462; PAPP-A, X68280). All primers were from DNA Technology (Aarhus, Denmark).
Competitive PCR
All PCRs were performed in a total volume of 50 µl with 1.5-unit SuperTaq (HT Biotechnology, Cambridge, UK), 0.25 nM dNTP (Pharmacia, Upsala, Sweden), 80 pmol of each primer, SuperTaq buffer, and 1 µl internal standard template (except blank control) in glass tubes using an Abacus thermal cycler (Denzyme, Aarhus, Denmark) with a ramp rate of 4°C/sec. Diluted aliquots of all reagents (stored at -20°C) were used to prepare a reaction mixture of which 49 µl was pipetted to each tube in a series of PCR experiments. A series included reactions with a dilution series of first-strand cDNA from one tissue, a dilution series from another tissue, one control with IS as the only template, and one blank control (which was taken from the master PCR mixture before addition of the IS). After addition of 1 µl diluted cDNA template, 37 cycles of PCR were performed using the following parameters: 94°C for 30 sec (90 sec in the first cycle), annealing for 30 sec (see below), and 72°C for 40 sec (400 sec in the last cycle). Annealing temperatures were 62°C for ß-actin and GAPDH, 60°C for PAPP-A, and 58°C for MBP. The amount of IS added to each PCR was determined empirically so that the dilution used for each IS template was in a linear region of the double logarithmic plot of the PCR product as a function of the dilution factor (not shown). This ensured that the amplifications were in the exponential phase throughout the 37 cycles. The PCR primers in each primer pair were positioned on different exons, enabling an easy detection of possible genomic DNA contamination (Fig. 1 and Table 1). No genomic DNA contamination of the cDNA preparations were observed in any of the tissues examined.
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Quantification
For each dilution series, the two samples, with about equal amounts of cDNA and IS PCR products, as judged by gel-electrophoresis in a 2.5% agarose gel (Fig. 2), were separated on a Hewlett-Packard (Palo Alto, CA) 1084 HPLC instrument equipped with a Waters (Milford, MA) Gen-Pak FAX nonporous ion-exchange column using a 20-min linear gradient from 0.3 to 0.7 M NaCl in TE buffer, pH 7.5 (10 mM Tris-HCL, 5mM EDTA) at 60°C (Fig. 3). The dilution, DIeq, that would have resulted in equimolar amounts of cDNA and IS PCR products, was calculated from equation 1,
where CP is the amount of cDNA PCR product, determined as the total A260 absorption; IP is the amount of IS PCR product, determined as the total A260 absorption corrected for the difference in size from the cDNA product; D is the actual dilution of the cDNA preparation; and DIeq(x) is the dilution that would result in equal molar amounts of IS and cDNA PCR product (x is either PAPP-A, proMBP, ß-actin, or GAPDH).
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A DIeq value was determined for each of the gene products as the mean value obtained from PCR of two independent dilution series and from two cDNA dilutions in each series. Finally, the specific abundance of PAPP-A or proMBP mRNA, A(x), was determined (equation 2):
where A(x) is the specific abundance of individual mRNA species. Thus, the specific abundance is a measure of the mRNA level of the gene of interest, normalized against a measure of the total mRNA in the sample. Given a constant amount of ß-actin mRNA molecules per cell, which is a reasonable assumption, the specific abundance, A, is independent of the amount of tissue used.
RNA Dot Blot Analysis
A 32P-labeled PAPP-A cDNA fragment pPA-1 [4] and a 32P-labeled MBP PCR product (see above) were hybridized to a human RNA master blot (Clontech, Palo Alto, CA) according to the manufacturer's instructions. After two washes with 0.15 M NaCl, 15 mM sodium citrate, 0.1% SDS, pH 7.0, at 65°C for 30 min, autoradiography was performed for 24 h using a phosphorimager (Molecular Dynamics). The human RNA master blot contains samples from 50 different tissues spotted on the membrane (see Fig. 5 legend). RNA amounts from all tissues were normalized against eight different housekeeping gene transcripts on the master blot.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Messenger RNA was extracted from frozen, homogenized tissue samples using oligo(dT)-coupled magnetic beads. This is a fast and easy protocol ensuring minimal degradation. To increase the sensitivity, proMBP-, PAPP-A-, and oligo(dT)-specific primers were used in the first-strand cDNA synthesis. Serial dilutions were made with the pool of cDNA obtained from the RT reaction. These cDNA dilutions were used as templates in competitive PCRs, with fixed amounts of gene-specific IS template added. From measurements of the ß-actin mRNA levels, the specific abundance was calculated for each tissue as detailed in the Materials and Methods section. We also measured the levels of GAPDH mRNA. As expected, the specific abundance of GAPDH mRNA showed minimal variation (128 ± 58 [SD]). This validates normalization against ß-actin mRNA.
PAPP-A and ProMBP mRNA Levels
We measured the specific abundance of PAPP-A and proMBP mRNA in a total of 43 samples from 13 different tissues, using the semiquantitative RT-PCR method described above. The results are summarized in Figure 4, in which the mean specific mRNA abundance for each tissue is shown relative to the specific abundance in term placenta, which contained the highest level measured for both PAPP-A and proMBP. The specific abundance of PAPP-A and proMBP mRNA was dramatically lower in first-trimester placenta than in term placenta (75- and 17-fold, respectively). It is also evident that all tissues examined contained measurable amounts of both mRNA species (Fig. 4). In endometrium from postmenopausal women, the PAPP-A mRNA level was 250-fold lower than in term placenta. Most other tissues examined had a specific PAPP-A mRNA abundance 500- to 3000-fold lower than in term placenta. In bone marrow cells, in which proMBP mRNA is expected at a relatively high level, the specific proMBP mRNA abundance was 230-fold lower than in term placenta. In breast tissue it was 800-fold lower than in term placenta, whereas the proMBP mRNA abundance was more than 1300-fold lower than in term placenta in all other tissues tested.
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Analysis of the mRNA in 1.5 ml whole blood drawn from a pregnant woman showed a very low ß-actin mRNA level and no detectable PAPP-A or proMBP mRNA. Thus, blood present in tissue samples cannot interfere with the measurements of the specific abundances of mRNA species.
Dot Blot Experiments
In addition to the tissue samples analyzed by semiquantitative RT-PCR, a rapid screen for tissues producing high amounts of PAPP-A or proMBP mRNA was carried out. This was done by hybridizing a specific 32P-labeled PAPP-A or proMBP cDNA probe to a membrane containing RNA from 50 different human tissues. As expected, placenta showed a very high signal for both mRNA species. The only other tissue with a PAPP-A signal above background was kidney (Fig. 5A). With this method, proMBP mRNA was detected in placenta and bone marrow, and at very low levels in kidney (Fig. 5B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Both PAPP-A and proMBP are among the most highly expressed genes in placenta, representing 1% (PAPP-A) and 5% (proMBP) of the total number of clones in two placental cDNA libraries (UniGene at http://inhouse.ncbi.nlm.nih.gov/UniGene/Hs.Home.html, library 398 and 399, respectively). In these libraries, PAPP-A and proMBP clones were among the five most abundant. Therefore, the mRNA specific abundances calculated here for a number of tissues are low compared to the levels in placenta (Fig. 4). In placenta, both PAPP-A and proMBP mRNA are readily detected by in situ hybridization [23–25]. In bone marrow, proMBP mRNA could not be detected by this technique (unpublished results), even though the mature MBP constitutes more than 50% of the total granule protein of the eosinophil leukocyte [12]. This highlights the relevance of an RT-PCR-based assay for the determination of PAPP-A and proMBP mRNA levels.
We found that the ratio between the specific abundance of proMBP and PAPP-A mRNA in placenta is not constant during pregnancy: levels of both mRNA species are lower in first-trimester placenta than in term placenta, but the level of PAPP-A mRNA increases relatively more than the level of proMBP mRNA. This is in good agreement with the change in the molar ratio of proMBP and PAPP-A serum levels, which goes from a 10-fold excess of proMBP in the first trimester to a 4-fold excess in the third trimester [9].
The finding that PAPP-A mRNA is synthesized in all the examined tissues, reproductive as well as nonreproductive, is surprising, and indicates that PAPP-A functions outside pregnancy. The recent demonstration that PAPP-A in conditioned media from human fibroblasts specifically cleaves IGF binding protein (BP)-4 [14], which is an inhibitor of IGF action, makes it likely that PAPP-A plays a localized role in the IGF/IGFBP-4 system. Because none of the tissues analyzed transcribe only one of the two mRNA species, it is tempting to hypothesize that proMBP plays a role in regulation of PAPP-A activity. Specifically, we speculate that proMBP is an inhibitor of PAPP-A proteolytic activity. The inhibitory effect of proMBP may not be complete, since the PAPP-A/proMBP complex isolated from pregnancy serum did show proteolytic activity [14]. In the majority of tissues, the mRNA abundance relative to term placenta is higher for PAPP-A than proMBP. However, the molar concentration of PAPP-A in the tissue may not necessarily exceed that of proMBP. All mRNA levels are expressed relative to the level in term placenta (Fig. 4), where the proMBP mRNA abundance is higher than that of PAPP-A [22]. Interestingly, in the tissues in which proMBP or MBP are known to be present in excess of PAPP-A, i.e., bone marrow cells (eosinophil leukocytes) and placenta, the specific abundance of proMBP mRNA is higher than that of PAPP-A relative to term placenta.
Earlier reports on localization of PAPP-A in tissues have resulted in contradicting results, and the question of nonplacental PAPP-A synthesis has been a subject of controversy (see [42, 43] for recent reviews). All previous investigations have been based on polyclonal antisera, and a number of reports have appeared describing the polyspecificity and heterogeneity of different preparations of these antisera [2, 36–38]. Some investigators have further purified the antisera preparations to minimize the polyspecificity, but only one [22] has taken into account that polyclonal antisera raised against PAPP-A, now known to be PAPP-A/proMBP, invariably will recognize the proMBP part of the PAPP-A/proMBP complex, as well as the mature eosinophil MBP [2]. To address the question of PAPP-A synthesis, and to detect and discriminate between PAPP-A and proMBP antigens in tissues, mRNA assays and monoclonal antibodies, respectively, must be used.
Testing different tissues for the presence of specific mRNAs is routinely done by RNA blotting techniques such as Northern or dot blotting. But Northern blotting of large mRNA species such as the PAPP-A mRNA, is technically difficult, and the sensitivity is relatively low. We attempted to detect PAPP-A and proMBP mRNA in a range of tissues by screening a commercial RNA dot blot containing normalized amounts of RNA from 50 different human tissues. A positive response above background was seen for placenta, kidney (very low), and bone marrow (only proMBP). Hence neither PAPP-A nor proMBP is synthesized in nonplacental tissues in quantities comparable to those in the placenta. We thus developed the semiquantitative RT-PCR assay described above. RT-PCR has been shown to be 1000- to 10 000-fold more sensitive than traditional RNA blotting techniques [44, 45], and we were able to detect and quantitate both PAPP-A and proMBP mRNA in all the tissues tested. In a number of these, such as colon, prostate, and uterus (endometrium and myometrium), neither PAPP-A nor proMBP mRNA was detectable when the commercial RNA dot blot was screened with PAPP-A or proMBP specific probes (Fig. 5), clearly demonstrating the higher sensitivity and the usefulness of the RT-PCR assay. The actual quantification of the products from the competitive PCR is done by ion exchange chromatography on an HPLC system, an accurate method that involves a minimum of post-PCR handling [46, 47].
An alternative way of detecting specific mRNA synthesis is by in situ hybridization. This technique has the advantage that it locates the cells that synthesize the mRNA but the disadvantage of being less sensitive, as mentioned above. The fact that the mRNA levels detected in this study in several of the tissues tested are relatively low reflects that the synthesis of PAPP-A and proMBP mRNA is limited to a few specific cells in the tissue. Immunohistochemical investigations with monoclonal antibodies are in progress. These studies confirm localization of the antigens in and around a very limited number of cells within each tissue (unpublished results).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Lars Sottrup-Jensen, Department of Molecular and Structural Biology, University of Aarhus, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark. FAX: 45 8612 3178; lsj{at}mbio.aau.dk
3 Current address: M&E Biotech A/S, Kogle Allé 6, 2970 Hørsholm, Denmark.
Accepted: May 25, 1999.
Received: March 11, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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