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Molecular Cloning of Complementary DNAs for Two Human Endometrial Proteins and Cellular Localization of Their Messenger RNAs“ M. JULKUNEN,~M. SEPPALA,~AND 0.A. JANNE bDepartment of Obstetrics and Gynecology Univemity Central Hospital SF-00290 Helsinki, Finland ‘The Population Council and The Rockefeller University New York, New York 10021 The biological actions of the female sex steroids, estradiol and progesterone, that elicit opposite morphologic changes in the human endometrium occur via binding to the cognate receptor proteins. Estrogens stimulate proliferation and mitotic activity of endometrial cells, whereas progestins are antiproliferative and cause secretory changes in the glands and decidualization of the stromal cells. A number of estrogen- and progesterone-regulated proteins have been described in endometrial tissue or uterine secretions. Two well-characterized major proteins in this group are a human 0-lactoglobulin homologue (pLG/PP14), also known as placental protein 14 (PP14), and insulin-like growth factor-binding protein- 1 (IGFBP- 1). The function of /?LG/PP14 in the human endometrium is not known. In other species, 0-lactoglobulin is the major whey protein in the milk and is expressed in the mammary gland during pregnancy and lactation.’ It belongs to a secretory protein family that includes members such as retinol-binding protein and the major urinary protein, some of which bind small hydrophobic molecules. It has been suggested that 0-lactoglobulins may function in the transport of retinal.* PLG/PP 14 has also been proposed to have immunosuppressive functions in reprod~ction.~ Measurement of this protein has the potential of becoming a blood test for human endometrial functionlo*”because it is synthesized in the endometrium” and is present in the peripheral circulation at relatively high concentrations that fluctuate during the menstrual cyc1e.l) Plasma and other biological fluids contain several IGFBPs,I4 three of which have been cloned.’’-” IGFBP-1 in amniotic fluid and decidua was initially characterized as PP12 (placental protein 12)18and is synthesized by both the secretory and decidualized endometrium, where progesterone has been shown to increase its synthesis.I9Like other IGFBPs, IGFBP-1 is a modulator of the biological action of IGF-I and IGF-11.” To elucidate the primary structure of these two human endometrial proteins and to study their protein homologies and regulation of their gene expression, we have isolated and sequenced cDNA clones encoding both /3LG/PP14 and IGFBP-1. In situ hybridization technique was subsequently used to identify the cell types expressing these mRNAs. ‘This study was supported by grants from the Finnish Social Insurance Institution, the Sigrid Juselius Foundation, and the Academy of Finland. 284
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PLG/PP14 Primary Structure A monospecificPLG/PP 14 antiserum was used to isolate cDNA clones corresponding to this protein from a human cDNA library prepared from RNA of first-trimester decidua.21Approximately 0.6% of the plaques in this library contained PLG/PP14 inserts, which indicates that PLG/PP14 is a fairly abundant protein in decidual tissue of early pregnancy. To verify that the longest cDNA was an authentic PLG/PP14 clone and contained the entire protein coding region, capped sense and complementary RNA strands were synthesized using T7 RNA polymerase, and the synthetic RNAs were subsequently translated in vitro in a reticulocyte lysate. PLG/PP14 antibody precipitated a polypeptide, encoded by the sense RNA, that was identical in size with the translation product of polyadenylated RNA of human decidua.21 The amino acid sequence of the PLG/PP14 protein deduced from its cDNA is shown in FIGURE1. The nucleotide sequence of PLG/PP14 mRNA exhibits an approximately 60% sequence identity with the ovine and bovine P-lactoglobulin mRNA sequence^,^^.^^ and the derived protein sequence has a 53% sequence identity with that of horse P-lactoglobulin I protein.24The longest PLG/PP14 cDNA insert of 798 nucleotides in length contains the entire protein coding region, the complete 3’-noncoding sequence including the beginning of the poly(A) tail, and all but the first 21 nucleotides of the 5’-untranslated sequence. The sequence for these missing nucleotides was determined by direct sequencing of the PLG/PP 14 mRNA.21Similarly to other P-lactoglobulins, the PLG/PP 14 precursor protein is composed of a putative signal peptide (the first 18 amino acids) and a mature protein encompassing 162 amino acid residues. Underlined with a thick line in FIGURE1 are the first 22 NH,-terminal amino acid residues of the mature protein, which are identical with those previously determined by direct protein sequencing for the purified protein.25 There are four cysteinyl residues in the cDNA (boxed in FIG. l), which may be responsible for intrachain disulfide bridge f ~ r m a t i o n . ~These ~ , ~ ’ same cysteinyl residues are spatially conserved in the monomeric P-lactoglobulins, so that the folding and three-dimensional structure of these proteins may be similar. The secreted PLG/PP14 protein contains 17.5% carbohydrates2*;in accordance with this result, there are three potential glycosylation sites (Asn-X-Sernhr) in the cDNA sequence identified by a thin underlining in FIGURE1.
PLG/PPl4 mRNA in Different Tissues The size of the PLG/PP14 mRNA is approximately 900 nucleotides (FIG.2A-C). On Northern blot hybridization studies, this mRNA is detectable in endometrium, decidua, and fallopian tube, but not in the other human tissues studied (liver, kidney, adrenals, and p l a ~ e n t a ) . There ~ ~ , ~ ~is a clear cyclic variation in the concentration of PLG/PP14 mRNA in the nonpregnant endometrium: it is present in decreasing amounts in samples taken during the menstrual and early proliferative phases and is not detectable at all around the time of ovulation (FIG. 2A and B). The mRNA reappears 6 days after ovulation, and its concentration increases dramatically toward
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FIGURE 1. Amino acid sequences of PLG/PP14 protein deduced from the cDNA. The NH,terminal methionine of the mature protein is assigned number 1 in the sequence. The 22 NH,terminal amino acid residues identical with those determined by protein sequencingzsare noted by a thick underscore. The amino acid residues of the three potential glycosylation sites are printed in italics with a thin underscore. The four cysteinyl residues conserved among the monomeric /3-lactogtobulins are boxed. (Adapted from Julkunen et dZ1)
the end of the cycle (FIG.2A). The content of PLG/PP14 mRNA is much lower in the fallopian tube,29but, similar to the endometrium, it is expressed in this tissue in a cyclic fashion (FIG.2C). The cells that express PLG/PP14 mRNA were localized by in situ hybridization exclusively in the glandular epithelium of the endometrium (FIG. 3a and c) and the mucosal epithelium of the fallopian tubes (FIG.4a, c, and e).
Structure of the PLG/PPlI Gene
Southern blot analysis of human DNA using KpnI, XbuI, PstI, and EcoRI restriction enzymes (no recognition sites in the cDNA) shows the presence of multiple hybridizable bandsz1There are two possible explanations for this finding: (i) the PLG/ PP14 gene is a fairly large single-copy gene and encompasses at least 20 kb of human DNA, or (ii) there are several PLG/PPlCrelated gene sequences in the human genome. The ovine j3-lactoglobulin gene, thought to be a single-copy gene, has been isolated and its complete structure determined.'^^^ This gene contains seven exons, but spans only about 5 kb of ovine DNA, that is, its size is much smaller than that anticipated by Southern blotting for the human gene, should this latter one really be a single-copy gene. However, there are several examples of human genes that have much longer intronic sequences than their counterparts in other species, mainly due to the presence
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of repeated sequences in the introns of the human genes. To determine whether the human PLG/PP 14 gene actually exhibits the same overall exon-intron organization as the ovine /3-lactoglobulin gene, we are currently characterizing several genomic clones isolated from human DNA libraries and carrying out studies on the chromosomal loci that encompass this gene($.
IGFBP-1 Primary Structure
IGFBP-1 cDNA was isolated, by immunological screening, from the same firsttrimester decidual cDNA library as that for obtaining /3LG/PP14 cDNA c1ones.I’ In the in vitro transcription/translation studies, the sense RNA strand supported the synthesis of a 30-kDa peptide that was precipitated with a monospecific IGFBP-1 antiserum. Of interest, the complementary-strand RNA supported the synthesis of a 20-kDa peptide that was not, however, recognized by this antibody.” IGFBP-1 mRNA contains an open reading frame of 777 nucleotides that encode a 259-residue-long peptide (FIG.5). The signal peptide is 25 residues long and is composed of mainly hydrophobic amino acids. The middle part of the protein (residues 89- 114) contains a region rich in Pro, Glu, Ser and Thr residues, which has features of a typical PEST sequence3I and is underlined with a thin line in FIGURE 5. The presence of this sequence suggests that the intracellular half-life of IGFBP-1 protein is very short, which notion is supported by in vivo studies.32The PEST sequence appears to be unique to IGFBP-1 since a similar region is not present in the other IGFBPs.’~J’The mRNA encoding IGFBP-1 may also turn over rapidly, as is suggested by the presence of five copies of the AUUUA sequence in its 3‘-noncoding region.” The protein does not contain recognition sites for N-linked glycosylation, but regions rich in Ser and Thr can potentially serve as acceptors for 0-linked sugars. In addition to the signal peptide, the
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FIGURE 2. RNA gel blot analysis of pLG/PP14 mRNA expression in the endometrium (A and B) and fallopian tube (C).Each lane contains 2.5 pg of poly(A) RNA from the following sources: (A) proliferative endometria, days 3, 6, 7, 9, and 13 of the cycle (lanes 1 to 5 ) and secretory endometria on days 3, 6, 8, 10, 11, and 12 after ovulation (lanes 6 to 11). (B) Longer exposure of the film for lanes 2-6 shown in panel A. (C) Fallopian tubes from the proliferative phase, days 1, 3, 11, and 12 of the cycle (lanes 1 to 4), and the secretory phase, days 15, 20, and 22 of the cycle (lanes 5 to 7 ) .Exposure times of the films were 4 hours (A), 24 hours (B), and 4 days (C). (Adapted from Julkunen er d Z 9 )
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FIGURE 3. Identification by in situ hybridization of human endometrial cell types expressing PLG/PP14 and IGFBP-1 genes. Late secretory (a,b) and menstrual (day 3 of the cycle; c,d) endometrial samples were hybridized with PLG/PP14 cRNA (a,c) or IGFBP-1 cRNA @,a) probes. Original magnification x 180; reduced by 35%. (From Julkunen Reproduced by permission.)
protein has another hydrophobic region covering the residues 30-50(FIG.5). This domain and its adjacent regions contain a total of twelve cysteines and may be involved in ligand binding. All these cysteines, along with the spacing between them, are conserved among the three IGFBPs.I5-” It is also noteworthy that in the carboxy-terminus of the protein there is a tripeptide sequence, Arg-Gly-Asp (RGD), which is recognized by cell adhesion receptor^.^^ This tripeptide motif is embedded in a pentapeptide that is conserved between IGFBP-1 and IGFBP-2; however, neither the RGD sequence nor the flanking two residues are present in IGFBP-3.I6
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IGFBP-1 mRNA in Different Tissues RNA gel blot analysis of IGFBP-1 mRNA expression in different human tissues indicated that this RNA is present in liver and HepG2 cells, secretory endometrium and decidua, but not in proliferative endometrium, placenta, kidney, or adrenals. Is In contrast to this quite tissue-specific expression of IGFBP- 1 mRNA, the genes coding for other known IGFBPs appear to be expressed in most tissues. Further experi16317
FIGURE 4. In situ hybridization analysis of PLG/PP14 and IGFBP-I mRNAs in fallopian tubes. Tissue sections from late proliferative (a,b), late secretory (c,d) and menstrual (day 3 of the cycle) phases (e,f) of the cycle were hybridized with PLG/PP14 cRNA (a, c, e) or IGFBP-1 cRNA (b, d, f) probes. Original magnification x 400; reduced by 35%. (From Julkunen et aLZ9 Reproduced by permission.)
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FIGURE 5. Amino acid sequences of IGFBP-I protein deduced from the cDNA. The NH,terminal methionine of the mature protein is assigned number 1. The NH,-terminal amino acid residues identical with those determined by direct sequencing of the purified protein are noted by a thick underscore. The residues corresponding to the PEST region are printed in italics with a thin underscore. The twelve cysteinyl residues conserved among the IGFBP-1, IGFBP-2, and IGFBP-3 sequences are identified by solid dots. The potential membrane attachment tripptide RGD is boxed. (Adapted from Julkunen et al.”)
ments on endometrial samples collected throughout the menstrual cycle confirmed the presence of IGFBP-1 mRNA in the late secretory phase endometrium and during menstrual bleeding on the third day of the cycle (FIG. 6A).29Several fallopian tuba1 specimens were also studied by Northern blot analysis and by hybridization histochemistry; some, but not all, of them contain IGFBP-1 mRNA in very small amounts (FIG. 6B). In siru hybridization technique localizes the IGFBP-1 mRNA to the stromal cells of the late secretory phase endometrium (FIG.3b and d), but it is noteworthy that not all cells have detectable signals. The fallopian tube contains very low amounts of IGFBP-1 mRNA, and the silver grains in hybridization histochemistry are barely above the background, although they seem to be localized in the mucosal epithelial cells (FIG. 4b, d, and 0.
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Structure of the IGFBP-1 Gene Southern blot analysis indicated that there is a single IGFBP-1 gene in the human genome that is distinct from those encoding the other IGFBPs.15The gene spans 5.9 kb of human DNA and is organized in four ex on^.^' The promoter region is relatively uncomplicated and contains features characteristic of “housekeeping” genes and genes that are expressed in a tissue-specific manner.I5 We have mapped the IGFBP-1 gene to bands p12-pl3 on human chromosome 7.35There are two alleles of this gene due to the presence of a polymorphic Bgn site in the first i n t r ~ nEven . ~ ~ though all IGFBPs appear to be unique gene products, it is interesting that their genes share organizational similarities. For example, the size, location, and sequence of exons 1, 3, and 4 are conserved between the IGFBP-1 and IGFBP-3 gene^.'^^^^ The IGFBP-3 gene has, however, an extra intron located in the upstream portion of the 3’-untranslated region and thus contains five rather than four exons as is the case with the IGFBP-1 gene.36 As could have been inferred from their differential regulation, the promoter regions of the IGFBP-1 and IGFBP-3 genes are not very similar.
SUMMARY AND CONCLUSIONS Insulin-like growth factor binding protein- 1 (IGFBP- 1) and a human &lactoglobulin homologue (PLG/PP14) are two major secretory proteins of the human endometrium. The genes coding for these two proteins are expressed in separate types of the endometrial cells, with the IGFBP-1 gene being expressed in the stromal and the PLG/ PP14 gene in the glandular epithelial cells. Although the biological reasons for the presence and expression of IGFBP-1 and PLG/PP14 in human endometrial cells remain to be elucidated, the fact that these gene products are expressed in different endometrial cell types provides a unique opportunity to employ them as markers in studies on epithelial-to-stromal cell communication in the endometrium.
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FIGURE 6, RNA gel blot analysis of IGFBP-1 mRNA expression in the endometrium (A)and the fallopian tube (B). Each lane contains 2.5 pg poly(A) RNA from the following sources: (A) proliferative endometrial samples, day 3, 6, 7,9, 13, or 16 of the cycle (lanes 1 to 6), and secretory endometria on days 3, 6, 8, 10, 11, and 12 after ovulation. (B) Fallopian tube RNA from the proliferative phase [day 11 of the cycle (lane I)] and the secretory phase [days 15, 20,and 22 of the cycle (lanes 2, 3, and 4)]. Exposure times of the films were 6 hours (A) and 12 days (B). (Adapted from Julkunen ei aLZ9)
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The primary structures of these proteins have been deduced from their cloned cDNAs. pLG/PP 14 is highly homologous to all known P-lactoglobulins from various species. For example, horse P-lactoglobulin I monomer exhibits a 53% protein sequence identity with PLG/PP14; they have the same number of amino acid residues, and their three-dimensional structures are predicted to be similar. This latter conclusion is inferred from the fact that the four cysteinyl residues that are responsible for the formation of intramolecular bridges in P-lactoglobulins are spatially conserved in @LG/PP14. The human protein is encoded by a 900-base pair-long mRNA that is expressed in the glandular epithelial cells of the endometrium in a cyclic manner; in addition, it is found in the mucosal epithelial cells of the fallopian tubes. Several lines of circumstantial evidence suggest that the expression of the PLGflP14 gene is regulated by progesterone; however, whether this regulation is elicited by the progesterone receptor at the transcriptional level has not so far been demonstrated. The IGFBP-1 protein sequence contains 259 amino acid residues, with the propeptide possessing a 25-amino acid-long signal peptide. The NH,-terminal sequence of this and other IGFBPs is very cysteine-rich, suggesting the possibility that this domain is involved in the binding of IGF-I and IGF-I1 ligands. A PEST region, a sequence that is found in proteins with short intracellular half-lives, is included in the middle half of the IGFBP-1 polypeptide. Among the IGFBPs, IGFBP-1 appears to be the only one with a PEST sequence. The carboxy-terminal end of IGFBP-1 contains an Arg-Gly-Asp tripeptide that is also found in IGFBP-2 and may function as a cell attachment recognition signal in these proteins. The human IGFBP-1 gene is a singlecopy gene localized to chromosome 7, and its 1.6-kb mRNA is expressed in the liver, decidua of pregnancy, secretory endometrium, ovarian granulosa cells, and fallopian tubes. In the endometrium, the IGFBP-1 gene is very actively expressed in the stromal cells during the late secretory phase, suggesting that its activity is controlled by progesterone. This postulate remains to be proven, however, by direct experiments. In comparison to other known IGFBPs, the gene encoding IGFBP-1 is much more tissue-specific in its expression. This fact, along with its putative fast turnover rate (the presence of a PEST sequence), makes it tempting to speculate that IGFBP-1 plays an important regulatory role in selected cell types.
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R. A. C. T. NORTH,J. B. C. FINDLAY, 8. PAPIZ, M. Z., L. SAWER,E. E. ELIOPOULOS, SIVAPRASADARAO, T. A. JONES,M. E. NEWCOMER & P. J. KRAULIS.1986. The structure of P-lactoglobulin and its similarity to plasma retinol-binding protein. Nature 324 383-385. E. A. MOWLES,0. M. R. 9. BOLTON,A. E., K. J. CLOUGH,R. J. STOKER,A. G. POCKLEY, WESTWOOD& M. G. CHAPMAN.1987. Identification of placental protein 14 as an immunosuppressive factor in human reproduction. Lancet i: 593-595. A. KOSKIMIES, T. LAATIKAINEN, U.-H. STENMAN & M.-L. 10. SEPPALA,M., M. JULKUNEN, HUHTALA.1988. Proteins of the human endometrium: Basic and clinical studies toward a blood test for endometrial function. Ann. N. Y. Acad. Sci. 541:432-444. 11. JOSHI,S. G., R. RAO,E.E. HENRIQUES, R. S.RAIKAR& M. GORDON.1986. Luteal phase concentrations of a progestagen-associated endometrial protein (PEP) in the serum of cycling women with adequate or inadequate endometrium. J. Clin. Endocrinol. Metab. 6 3 1247- 1249. 12. JULKUNEN,M., R. KOISTINEN,J. SlOBERG, E.-M. RUTANEN,T. WAHLSTROM & M. SEPPALA.1986. Secretory endometrium synthesizes placental protein 14. Endocrinology 1 1 8 1782-1786. M., D. AFTER, M. SEPPALA,U.-H. STENMAN & H. BOHN.1986. Serum levels 13. JULKUNEN, of placental protein 14 reflect ovulation in non-conceptional menstrual cycles. Fertil. Steril. 45: 47-50. S., P. HOSSENLOPP, B. SEGOVIA,D. SEURIN,G. PORTOLAN, C. LASSARRE 14. HARDOUIN, & M. BINOUX.1987. Heterogeneity of insulin-like growth factor binding proteins and relationships between structure and affinity. I. Circulating forms in man. Eur. J. Biochem. 1 7 0 121-132. M., R. KOISTINEN,K. AALTO-SETALA,M. SEPPALA,0. A. JANNE & K. 15. JULKUNEN, KONTULA.1988. Primary structure of human insulin-like growth factor-binding protein/ placental protein 12 and tissue-specific expression of its mRNA. FEBS Lett. 236: 295-302. W. J. HENZEL,G. A. WINSLOW, S. A. SPENCER, R. HELL16. WOOD,W. J., G. CACHIANES, MISS,J. L. MARTIN& R. C. BAXTER.1988. Cloning and expression of the growth hormone dependent insulin-like growth factor-binding protein. Mol. Endocrinol. 2 1176- 1185. J.-L. MARY,J. SCHWANDER & G. HEINRICH.1989. Cloning, 17. BINKERT,C., J. LANDWEHR, sequence analysis and expression of a cDNA encoding a novel insulin-like growth factor binding protein (IGFBP-2). EMBO J. 8 2497-2502. R., N. KALKKINEN, M.-L. HUHTALA, M. SEPPALA,H. BOHN& E.-M. RUTA18. KOISTINEN, NEN. 1986. Placental protein 12 is a decidual protein that binds somatomedin and has an identical N-terminal amino acid sequence with somatomedin-bindingprotein from human amniotic fluid. Endocrinology 118: 1375- 1378. 19. RUTANEN,E.-M., R. KOISTINEN, J. SJOBERG,M. JULKUNEN, T. WAHLSTROM, H. BOHN 8r M. SEPPALA.1986. Synthesis of placental protein 12 by human endometrium. Endocrinology 118: 1067-1071. 20. RECHLER,M. M. & S. P. NISSLEY.1990. Insulin-like growth factors. In Peptide Growth Factors and Their Receptors I. M. B. Sporn & A. B. Roberts, Eds. Vol. 95: 263-267. Springer Verlag. Heidelberg. M., M. SEPPALA& 0. A. JANNE. 1988. Complete amino acid sequence of 21. JULKUNEN, human placental protein 14: A progesterone-regulated uterine protein homologous to P-lactoglobulins. Proc. Natl. Acad. Sci. USA 8 5 8845-8849. J.-C. MERCIER,S. SOULIER, J.-L. VILOTTE & J.-P. FURET. 22. GAYE,P., D. HUE-DELAHAIE, 1986. Ovine P-lactoglobulin mRNA: Nucleotide sequence and mRNA levels during functional differentiation of the mammary gland. Biochimie 68: 1097- 1107. Y. C. KANG,J. E. KINSELLA& C. A. BAR. 1987. 23. JAMIESON,A. C., M. A. VANDEYAR, Cloning and nucleotide sequence of the bovine P-lactoglobulin gene. Gene 61: 85-90. J. LIBERATORI & G. BRAUNITZER. 1984. Primary 24. CONTI,A., J. GODOVAC-ZIMMERMANN, structure of monomeric P-lactoglobulin I from horse colostrum (Equus cabullus, Perissodactyla). Hoppe-Seyler’s Z. Physiol. Chem. 3 6 5 1393-1399. M.-L., M. SEPPALA,A. NARVANEN, P. PALOMAKI, M. JULKUNEN & H. BOHN. 25. HUHTALA, 1987. Amino acid sequence homology between human placental protein 14 and P-lactoglobulin from various species. Endocrinology 1 2 0 2620-2622. J. 1988. The structural motif of P-lactoglobulin and retinol26. GODOVAC-ZIMMERMANN,
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ANNALS NEW YORK ACADEMY OF SCIENCES binding protein. A basic framework for binding and transport of small hydrophobic molecules? Trends Biochem. Sci. 1 3 64-66. PERVAIZ,S. & K. BREW.1985. Homology of j34actoglobulin, serum retinol-binding protein, and protein HC. Science 228: 335-337. BOHN,H., W. KRAUS& W. WINCKLER.1982. New soluble placental tissue proteins: Their isolation, characterization, localization and quantification. Placenta 4 67- 8 1. JULKUNEN, M., R. KOISTINEN,A.-M. SUIKKARI, M. SEPPALA& 0. A. JANNE. 1990. Identification by hybridization histochemistry of human endometrial cells expressing mRNAs encoding a uterine P-lactoglobulin homologue and insulin-like growth factorbinding protein-1. Mol. Endocrinol. 5: 700-707. HARRIS,S., S. ALI, S. ANDERSON,A. L. ARCHIBALD & A. J. CLARK.1988. Complete nucleotide sequence of the genomic ovine j3-lactoglobulin gene. Nucleic Acid Res. 16: 10379-10380. ROGERS,C., R. WELLS& M. RECHSTEINER.1986. Amino acid sequences common to rapidly degraded proteins: The PEST hypothesis. Science 234 364-368. RUTANEN,E.-M., M. SEPPALA,R. PIETILA& H. BOHN.1984. Placental protein 12 (PP12): Factors affecting levels in late pregnancy. Placenta 5: 243-248. RUOSLAHTI, E. & M. D. PIERSCHBACHER. 1986. Arg-Gly-Asp: A versatile cell recognition signal. Cell 44: 517-518. BRINKMAN, A., C. A. H. GROFFEN,D. J. KORTLEVE & S. L. S. DROP.1988. Organization of the gene encoding the insulin-like growth factor binding protein IBP-1. Biochem. Biophys. Res. Commun. 157: 898-907. ALITALO,T., K. KONTULA,R. KOISTINEN,K. AALTO-SETALA,M. JULKUNEN, 0. A. JANNE, M. SEPPALA& A. DE LA CHAPELLE.1989. The gene encoding the human low-molecular weight insulin-like growth factor binding protein (IGF-BP25): Regional localization to 7p12-pl3 and description of a DNA polymorphism. Hum. Genet. 83: 335-338. CUBBAGE,M. L., A. SUWANICHKUL & D. R. POWELL.1990. Insulin-like growth factor binding protein-3. Organization of the human chromosomal gene and demonstration of promoter activity. J. Biol. Chem. 265: 12642-12649.
Molecular Cloning of Complementary DNAs for Two Human Endometrial Proteins and Cellular Localization of Their Messenger RNAs“ M. JULKUNEN,~M. SEPPALA,~AND 0.A. JANNE bDepartment of Obstetrics and Gynecology Univemity Central Hospital SF-00290 Helsinki, Finland ‘The Population Council and The Rockefeller University New York, New York 10021 The biological actions of the female sex steroids, estradiol and progesterone, that elicit opposite morphologic changes in the human endometrium occur via binding to the cognate receptor proteins. Estrogens stimulate proliferation and mitotic activity of endometrial cells, whereas progestins are antiproliferative and cause secretory changes in the glands and decidualization of the stromal cells. A number of estrogen- and progesterone-regulated proteins have been described in endometrial tissue or uterine secretions. Two well-characterized major proteins in this group are a human 0-lactoglobulin homologue (pLG/PP14), also known as placental protein 14 (PP14), and insulin-like growth factor-binding protein- 1 (IGFBP- 1). The function of /?LG/PP14 in the human endometrium is not known. In other species, 0-lactoglobulin is the major whey protein in the milk and is expressed in the mammary gland during pregnancy and lactation.’ It belongs to a secretory protein family that includes members such as retinol-binding protein and the major urinary protein, some of which bind small hydrophobic molecules. It has been suggested that 0-lactoglobulins may function in the transport of retinal.* PLG/PP 14 has also been proposed to have immunosuppressive functions in reprod~ction.~ Measurement of this protein has the potential of becoming a blood test for human endometrial functionlo*”because it is synthesized in the endometrium” and is present in the peripheral circulation at relatively high concentrations that fluctuate during the menstrual cyc1e.l) Plasma and other biological fluids contain several IGFBPs,I4 three of which have been cloned.’’-” IGFBP-1 in amniotic fluid and decidua was initially characterized as PP12 (placental protein 12)18and is synthesized by both the secretory and decidualized endometrium, where progesterone has been shown to increase its synthesis.I9Like other IGFBPs, IGFBP-1 is a modulator of the biological action of IGF-I and IGF-11.” To elucidate the primary structure of these two human endometrial proteins and to study their protein homologies and regulation of their gene expression, we have isolated and sequenced cDNA clones encoding both /3LG/PP14 and IGFBP-1. In situ hybridization technique was subsequently used to identify the cell types expressing these mRNAs. ‘This study was supported by grants from the Finnish Social Insurance Institution, the Sigrid Juselius Foundation, and the Academy of Finland. 284
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PLG/PP14 Primary Structure A monospecificPLG/PP 14 antiserum was used to isolate cDNA clones corresponding to this protein from a human cDNA library prepared from RNA of first-trimester decidua.21Approximately 0.6% of the plaques in this library contained PLG/PP14 inserts, which indicates that PLG/PP14 is a fairly abundant protein in decidual tissue of early pregnancy. To verify that the longest cDNA was an authentic PLG/PP14 clone and contained the entire protein coding region, capped sense and complementary RNA strands were synthesized using T7 RNA polymerase, and the synthetic RNAs were subsequently translated in vitro in a reticulocyte lysate. PLG/PP14 antibody precipitated a polypeptide, encoded by the sense RNA, that was identical in size with the translation product of polyadenylated RNA of human decidua.21 The amino acid sequence of the PLG/PP14 protein deduced from its cDNA is shown in FIGURE1. The nucleotide sequence of PLG/PP14 mRNA exhibits an approximately 60% sequence identity with the ovine and bovine P-lactoglobulin mRNA sequence^,^^.^^ and the derived protein sequence has a 53% sequence identity with that of horse P-lactoglobulin I protein.24The longest PLG/PP14 cDNA insert of 798 nucleotides in length contains the entire protein coding region, the complete 3’-noncoding sequence including the beginning of the poly(A) tail, and all but the first 21 nucleotides of the 5’-untranslated sequence. The sequence for these missing nucleotides was determined by direct sequencing of the PLG/PP 14 mRNA.21Similarly to other P-lactoglobulins, the PLG/PP 14 precursor protein is composed of a putative signal peptide (the first 18 amino acids) and a mature protein encompassing 162 amino acid residues. Underlined with a thick line in FIGURE1 are the first 22 NH,-terminal amino acid residues of the mature protein, which are identical with those previously determined by direct protein sequencing for the purified protein.25 There are four cysteinyl residues in the cDNA (boxed in FIG. l), which may be responsible for intrachain disulfide bridge f ~ r m a t i o n . ~These ~ , ~ ’ same cysteinyl residues are spatially conserved in the monomeric P-lactoglobulins, so that the folding and three-dimensional structure of these proteins may be similar. The secreted PLG/PP14 protein contains 17.5% carbohydrates2*;in accordance with this result, there are three potential glycosylation sites (Asn-X-Sernhr) in the cDNA sequence identified by a thin underlining in FIGURE1.
PLG/PPl4 mRNA in Different Tissues The size of the PLG/PP14 mRNA is approximately 900 nucleotides (FIG.2A-C). On Northern blot hybridization studies, this mRNA is detectable in endometrium, decidua, and fallopian tube, but not in the other human tissues studied (liver, kidney, adrenals, and p l a ~ e n t a ) . There ~ ~ , ~ ~is a clear cyclic variation in the concentration of PLG/PP14 mRNA in the nonpregnant endometrium: it is present in decreasing amounts in samples taken during the menstrual and early proliferative phases and is not detectable at all around the time of ovulation (FIG. 2A and B). The mRNA reappears 6 days after ovulation, and its concentration increases dramatically toward
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FIGURE 1. Amino acid sequences of PLG/PP14 protein deduced from the cDNA. The NH,terminal methionine of the mature protein is assigned number 1 in the sequence. The 22 NH,terminal amino acid residues identical with those determined by protein sequencingzsare noted by a thick underscore. The amino acid residues of the three potential glycosylation sites are printed in italics with a thin underscore. The four cysteinyl residues conserved among the monomeric /3-lactogtobulins are boxed. (Adapted from Julkunen et dZ1)
the end of the cycle (FIG.2A). The content of PLG/PP14 mRNA is much lower in the fallopian tube,29but, similar to the endometrium, it is expressed in this tissue in a cyclic fashion (FIG.2C). The cells that express PLG/PP14 mRNA were localized by in situ hybridization exclusively in the glandular epithelium of the endometrium (FIG. 3a and c) and the mucosal epithelium of the fallopian tubes (FIG.4a, c, and e).
Structure of the PLG/PPlI Gene
Southern blot analysis of human DNA using KpnI, XbuI, PstI, and EcoRI restriction enzymes (no recognition sites in the cDNA) shows the presence of multiple hybridizable bandsz1There are two possible explanations for this finding: (i) the PLG/ PP14 gene is a fairly large single-copy gene and encompasses at least 20 kb of human DNA, or (ii) there are several PLG/PPlCrelated gene sequences in the human genome. The ovine j3-lactoglobulin gene, thought to be a single-copy gene, has been isolated and its complete structure determined.'^^^ This gene contains seven exons, but spans only about 5 kb of ovine DNA, that is, its size is much smaller than that anticipated by Southern blotting for the human gene, should this latter one really be a single-copy gene. However, there are several examples of human genes that have much longer intronic sequences than their counterparts in other species, mainly due to the presence
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of repeated sequences in the introns of the human genes. To determine whether the human PLG/PP 14 gene actually exhibits the same overall exon-intron organization as the ovine /3-lactoglobulin gene, we are currently characterizing several genomic clones isolated from human DNA libraries and carrying out studies on the chromosomal loci that encompass this gene($.
IGFBP-1 Primary Structure
IGFBP-1 cDNA was isolated, by immunological screening, from the same firsttrimester decidual cDNA library as that for obtaining /3LG/PP14 cDNA c1ones.I’ In the in vitro transcription/translation studies, the sense RNA strand supported the synthesis of a 30-kDa peptide that was precipitated with a monospecific IGFBP-1 antiserum. Of interest, the complementary-strand RNA supported the synthesis of a 20-kDa peptide that was not, however, recognized by this antibody.” IGFBP-1 mRNA contains an open reading frame of 777 nucleotides that encode a 259-residue-long peptide (FIG.5). The signal peptide is 25 residues long and is composed of mainly hydrophobic amino acids. The middle part of the protein (residues 89- 114) contains a region rich in Pro, Glu, Ser and Thr residues, which has features of a typical PEST sequence3I and is underlined with a thin line in FIGURE 5. The presence of this sequence suggests that the intracellular half-life of IGFBP-1 protein is very short, which notion is supported by in vivo studies.32The PEST sequence appears to be unique to IGFBP-1 since a similar region is not present in the other IGFBPs.’~J’The mRNA encoding IGFBP-1 may also turn over rapidly, as is suggested by the presence of five copies of the AUUUA sequence in its 3‘-noncoding region.” The protein does not contain recognition sites for N-linked glycosylation, but regions rich in Ser and Thr can potentially serve as acceptors for 0-linked sugars. In addition to the signal peptide, the
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FIGURE 2. RNA gel blot analysis of pLG/PP14 mRNA expression in the endometrium (A and B) and fallopian tube (C).Each lane contains 2.5 pg of poly(A) RNA from the following sources: (A) proliferative endometria, days 3, 6, 7, 9, and 13 of the cycle (lanes 1 to 5 ) and secretory endometria on days 3, 6, 8, 10, 11, and 12 after ovulation (lanes 6 to 11). (B) Longer exposure of the film for lanes 2-6 shown in panel A. (C) Fallopian tubes from the proliferative phase, days 1, 3, 11, and 12 of the cycle (lanes 1 to 4), and the secretory phase, days 15, 20, and 22 of the cycle (lanes 5 to 7 ) .Exposure times of the films were 4 hours (A), 24 hours (B), and 4 days (C). (Adapted from Julkunen er d Z 9 )
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FIGURE 3. Identification by in situ hybridization of human endometrial cell types expressing PLG/PP14 and IGFBP-1 genes. Late secretory (a,b) and menstrual (day 3 of the cycle; c,d) endometrial samples were hybridized with PLG/PP14 cRNA (a,c) or IGFBP-1 cRNA @,a) probes. Original magnification x 180; reduced by 35%. (From Julkunen Reproduced by permission.)
protein has another hydrophobic region covering the residues 30-50(FIG.5). This domain and its adjacent regions contain a total of twelve cysteines and may be involved in ligand binding. All these cysteines, along with the spacing between them, are conserved among the three IGFBPs.I5-” It is also noteworthy that in the carboxy-terminus of the protein there is a tripeptide sequence, Arg-Gly-Asp (RGD), which is recognized by cell adhesion receptor^.^^ This tripeptide motif is embedded in a pentapeptide that is conserved between IGFBP-1 and IGFBP-2; however, neither the RGD sequence nor the flanking two residues are present in IGFBP-3.I6
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IGFBP-1 mRNA in Different Tissues RNA gel blot analysis of IGFBP-1 mRNA expression in different human tissues indicated that this RNA is present in liver and HepG2 cells, secretory endometrium and decidua, but not in proliferative endometrium, placenta, kidney, or adrenals. Is In contrast to this quite tissue-specific expression of IGFBP- 1 mRNA, the genes coding for other known IGFBPs appear to be expressed in most tissues. Further experi16317
FIGURE 4. In situ hybridization analysis of PLG/PP14 and IGFBP-I mRNAs in fallopian tubes. Tissue sections from late proliferative (a,b), late secretory (c,d) and menstrual (day 3 of the cycle) phases (e,f) of the cycle were hybridized with PLG/PP14 cRNA (a, c, e) or IGFBP-1 cRNA (b, d, f) probes. Original magnification x 400; reduced by 35%. (From Julkunen et aLZ9 Reproduced by permission.)
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LEU VAL LEU LEU LEU LEU THR VAL GLN VAL GLY VAL THR ALA GLY 10 1 0 0 ALA PRO TRP GLN CYS ALA PRO CYS SER ALA GLU LYS LEU ALA LEU a 20 a 30 CYS PRO PRO VAL SER ALA SER C?S SER GLU VAL THR ARG SER ALA 0
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GLY CYS GLY CYS CYS PRO MET CYS ALA LEU PRO LEU GLY ALA ALA 0 50 0 60 CYS GLY VAL ALA THR ALA ARG CYS ALA ARG GLY LEU SER CYS ARG 70 ALA LEU PRO GLY GLU GLN GLN PRO LEU H I S ALA LEU THR ARG GLY 90 0 80 GLN GLY ALA CYS VAL GLN GLU SER ASP ALA SER ALA PRO H I S ALA 100 ALA GLU ALA GLY SER PRO GLU SER PRO GLU SER THR GLU I L E THR 110 120 GLLI GLU GLU LEU LEU ASP ASN PHE H I S LEU MET ALA PRO SER GLU 130 GLU ASP H I S SER I L E LEU TRP ASP ALA I L E S E R THR TYR ASP GLY 150 140 SER LYS ALA LEU H I S VAL THR ASN I L E LYS LYS TRP LYS GLU PRO 160 CYS ARG I L E GLU LEU TYR ARG VAL VAL GLU SER LEU ALA LYS ALA 180 170 GLN GLU THR SER GLY GLU GLU I L E SER LYS PHE TYR LEU PRO ASN 190 CYS ASN LYS ASN GLY PHE TYR H I S SER ARG GLN CYS GLU THR SER 210 200 MET ASP GLY GLU ALA GLY LEU CYS TRP CYS VAL TYR PRO TRP ASN 220
GLY LYS ARG I L E PRO GLY SER PRO GLU L 1E I1230 CYS GLN MET TYR PHE ASN VAL GLN ASN
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FIGURE 5. Amino acid sequences of IGFBP-I protein deduced from the cDNA. The NH,terminal methionine of the mature protein is assigned number 1. The NH,-terminal amino acid residues identical with those determined by direct sequencing of the purified protein are noted by a thick underscore. The residues corresponding to the PEST region are printed in italics with a thin underscore. The twelve cysteinyl residues conserved among the IGFBP-1, IGFBP-2, and IGFBP-3 sequences are identified by solid dots. The potential membrane attachment tripptide RGD is boxed. (Adapted from Julkunen et al.”)
ments on endometrial samples collected throughout the menstrual cycle confirmed the presence of IGFBP-1 mRNA in the late secretory phase endometrium and during menstrual bleeding on the third day of the cycle (FIG. 6A).29Several fallopian tuba1 specimens were also studied by Northern blot analysis and by hybridization histochemistry; some, but not all, of them contain IGFBP-1 mRNA in very small amounts (FIG. 6B). In siru hybridization technique localizes the IGFBP-1 mRNA to the stromal cells of the late secretory phase endometrium (FIG.3b and d), but it is noteworthy that not all cells have detectable signals. The fallopian tube contains very low amounts of IGFBP-1 mRNA, and the silver grains in hybridization histochemistry are barely above the background, although they seem to be localized in the mucosal epithelial cells (FIG. 4b, d, and 0.
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Structure of the IGFBP-1 Gene Southern blot analysis indicated that there is a single IGFBP-1 gene in the human genome that is distinct from those encoding the other IGFBPs.15The gene spans 5.9 kb of human DNA and is organized in four ex on^.^' The promoter region is relatively uncomplicated and contains features characteristic of “housekeeping” genes and genes that are expressed in a tissue-specific manner.I5 We have mapped the IGFBP-1 gene to bands p12-pl3 on human chromosome 7.35There are two alleles of this gene due to the presence of a polymorphic Bgn site in the first i n t r ~ nEven . ~ ~ though all IGFBPs appear to be unique gene products, it is interesting that their genes share organizational similarities. For example, the size, location, and sequence of exons 1, 3, and 4 are conserved between the IGFBP-1 and IGFBP-3 gene^.'^^^^ The IGFBP-3 gene has, however, an extra intron located in the upstream portion of the 3’-untranslated region and thus contains five rather than four exons as is the case with the IGFBP-1 gene.36 As could have been inferred from their differential regulation, the promoter regions of the IGFBP-1 and IGFBP-3 genes are not very similar.
SUMMARY AND CONCLUSIONS Insulin-like growth factor binding protein- 1 (IGFBP- 1) and a human &lactoglobulin homologue (PLG/PP14) are two major secretory proteins of the human endometrium. The genes coding for these two proteins are expressed in separate types of the endometrial cells, with the IGFBP-1 gene being expressed in the stromal and the PLG/ PP14 gene in the glandular epithelial cells. Although the biological reasons for the presence and expression of IGFBP-1 and PLG/PP14 in human endometrial cells remain to be elucidated, the fact that these gene products are expressed in different endometrial cell types provides a unique opportunity to employ them as markers in studies on epithelial-to-stromal cell communication in the endometrium.
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FIGURE 6, RNA gel blot analysis of IGFBP-1 mRNA expression in the endometrium (A)and the fallopian tube (B). Each lane contains 2.5 pg poly(A) RNA from the following sources: (A) proliferative endometrial samples, day 3, 6, 7,9, 13, or 16 of the cycle (lanes 1 to 6), and secretory endometria on days 3, 6, 8, 10, 11, and 12 after ovulation. (B) Fallopian tube RNA from the proliferative phase [day 11 of the cycle (lane I)] and the secretory phase [days 15, 20,and 22 of the cycle (lanes 2, 3, and 4)]. Exposure times of the films were 6 hours (A) and 12 days (B). (Adapted from Julkunen ei aLZ9)
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The primary structures of these proteins have been deduced from their cloned cDNAs. pLG/PP 14 is highly homologous to all known P-lactoglobulins from various species. For example, horse P-lactoglobulin I monomer exhibits a 53% protein sequence identity with PLG/PP14; they have the same number of amino acid residues, and their three-dimensional structures are predicted to be similar. This latter conclusion is inferred from the fact that the four cysteinyl residues that are responsible for the formation of intramolecular bridges in P-lactoglobulins are spatially conserved in @LG/PP14. The human protein is encoded by a 900-base pair-long mRNA that is expressed in the glandular epithelial cells of the endometrium in a cyclic manner; in addition, it is found in the mucosal epithelial cells of the fallopian tubes. Several lines of circumstantial evidence suggest that the expression of the PLGflP14 gene is regulated by progesterone; however, whether this regulation is elicited by the progesterone receptor at the transcriptional level has not so far been demonstrated. The IGFBP-1 protein sequence contains 259 amino acid residues, with the propeptide possessing a 25-amino acid-long signal peptide. The NH,-terminal sequence of this and other IGFBPs is very cysteine-rich, suggesting the possibility that this domain is involved in the binding of IGF-I and IGF-I1 ligands. A PEST region, a sequence that is found in proteins with short intracellular half-lives, is included in the middle half of the IGFBP-1 polypeptide. Among the IGFBPs, IGFBP-1 appears to be the only one with a PEST sequence. The carboxy-terminal end of IGFBP-1 contains an Arg-Gly-Asp tripeptide that is also found in IGFBP-2 and may function as a cell attachment recognition signal in these proteins. The human IGFBP-1 gene is a singlecopy gene localized to chromosome 7, and its 1.6-kb mRNA is expressed in the liver, decidua of pregnancy, secretory endometrium, ovarian granulosa cells, and fallopian tubes. In the endometrium, the IGFBP-1 gene is very actively expressed in the stromal cells during the late secretory phase, suggesting that its activity is controlled by progesterone. This postulate remains to be proven, however, by direct experiments. In comparison to other known IGFBPs, the gene encoding IGFBP-1 is much more tissue-specific in its expression. This fact, along with its putative fast turnover rate (the presence of a PEST sequence), makes it tempting to speculate that IGFBP-1 plays an important regulatory role in selected cell types.
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