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Expression of CD44 adhesion molecules on human placentae
European Journal of Obstetrics & Gynecology and Reproductive Biology 128 (2006) 243–247 www.elsevier.com/locate/ejogrb
Expression of CD44 adhesion molecules on human placentae Chel Hun Choi a, Cheong Rae Roh a, Tae-Joong Kim a, Yoon-La Choi b, Jeong-Won Lee a, Byoung-Gie Kim a,*, Je-Ho Lee a, Duk-Soo Bae a a
Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea b Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Received 15 August 2005; received in revised form 22 December 2005; accepted 3 January 2006
Abstract Objective: This study was performed to characterize CD44 expression in human placentae. Study design: We conducted reverse transcriptase polymerase chain reaction (RT-PCR) analyses of CD44H (hematopoietic form, also called CD44s) and CD44R1 (epithelial form) in 14 normal term placentae, and immunohistochemical analysis of CD44H and CD44v6 in 31 placentae (25 term and 6 first-trimester). Results: All 14 term placentae expressed CD44H (double bands) suggesting expression of alternative isoforms on the placentae. CD44R1 was expressed on 3 of 14 (21.4%) term placentae. Immunohistochemical analysis revealed expression of CD44H in all the placentae examined. However, positive staining for CD44v6 was observed in 3 of 6 (50%) first-trimester and only 6 of 25 (24%) term placentae showing decreased expression on term placentae. Positive staining for CD44v6 was observed on the trophoblast surface of immature villi especially in the sub-chorionic area where hypoxic conditions prevail. Conclusion: These results suggest that CD44 splicing variants might play a role in the invasion of trophoblast into maternal tissue in early pregnancy. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: CD44H; CD44R1; CD44v6; Placentae
1. Introduction CD44 proteins are a ubiquitously expressed family of surface adhesion markers involved in cell–cell and cell– matrix interactions. These multiple protein isoforms are encoded by alternative splicing of a single gene and are further modified by post-translational modifications. Genomic analysis of CD44 revealed existence of 20 exons over a length of approximately 60 kilobases [1]. The five N-terminal exons plus the five C-terminal exons encode CD44H. The CD44 variant isoform that contains sequences encoded by
* Corresponding author at: Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Kangnam-Ku, Seoul 135-710, Republic of Korea. Tel.: +82 2 3410 3513; fax: +82 2 3410 0630. E-mail address: [email protected] (B.-G. Kim).
variant exon sequences 8, 9, and 10 is known as CD44R1 [2]. CD44v6 isoforms contains sequences encoded by exon 6 [3]. Extracellular matrix components have been described as ligands for the CD44H, such as hyaluronic acid (HA), collagen, fibronectin, and sulfated proteoglycan. Binding to HA is mediated via two regions encoded by exons 2s and 5s [4]. Differences in HA binding state of CD44 are cell specific and have been shown to be related to posttranslational modifications [5]. CD44H is known to mediate adhesion of lymphocytes to high endothelial venules in vitro [6,7], promotion of normal B-cell development in vitro [8], cell migration [9], and stimulation of cytokine release by macrophages [10]. The role of CD44H in tumorigenesis has been suggested by studies in which neutralizing antibodies were induced to block tumor cell invasion into extracellular matrix in vitro [11,12], correlation of CD44 density with increased metastatic potential of melanoma cell lines [13], and
0301-2115/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2006.01.017
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association between CD44 expression and survival in nonHodgkin’s lymphoma [14]. In contrast to CD44H, CD44 variants are not well characterized. Functional differences seem to exist between CD44 variant isoforms and CD44H, although the structural differences and the exact ligands recognized by these molecules have not been well defined. In a previous study, we examined CD44 transcript alternative splicing patterns in gynecologic malignancies and observed increased frequency of CD44R1 expression during ovarian and cervical carcinoma progression [15]. CD44v6 has been shown to have a crucial role in the movement and homing of antigen activated lymphocytes in lymph nodes and has been associated with tumor invasion, progression and metastasis [3,16,17]. In further elucidating the functional role of CD44 in normal and malignant tissues, the human placenta may be a useful model as its trophoblast comprises highly differentiated villous and invasive extravillous entities [18]. In this study, we used RT-PCR to examine CD44 transcript splicing patterns in normal human placentae in order to determine whether this tissue also displays altered CD44 expression. Immunohistochemical staining of CD44 protein was performed to determine which cell population might be responsible for CD44 expression.
2. Materials and methods Fourteen term placentae were obtained after spontaneous delivery, frozen immediately and stored at 80 8C until mRNA isolation. mRNA was extracted from each sample with the QuickPrep Micro mRNA Purification Kit (Pharmacia P-L Biochemicals, Milwaukee, Wisconsin) according to manufacturer’s recommendations. First-strand cDNA was synthesized with a First-Strand cDNA Kit (Pharmacia P-L Biochemicals) according to the recommendations of the supplier. About 0.1 mg mRNA (8 ml) was reverse transcribed at 37 8C for 1 h using 5 ml of bulk first-strand cDNA mixture (Cloned, FPL C purified murine reverse transcriptase, RNA guard, RNase/DNase-Free BSA, dATP, dCTP, dGTP and dTTP in aqueous buffer), 1 ml (0.002 mg) random hexadeoxynucleotide primers, and 1 ml (0.1 M) of dithiothreitol solution. CD44 cDNA was subjected to PCR using a GeneAmp PCR Core Reagent Kit (Perkin-Elmer-Cetus, Norwalk, Connecticut) for 32 cycles in triplicate samples using the following temperatures and time sequences: 94 8C for 1 min, 50 8C for 1 min, 72 8C for 1 min. Five microliters of first-strand cDNA was added to 45 ml of PCR mixture containing 5 ml 10 reaction buffer (100 mM Tris–HCl, pH 8.3, 500 mM KCl, and 1 mg/ml of gelatin), 3 ml MgCl2 (25 mM), 0.5 ml a-32P-dCTP, 1 ml (25 pM) of each primer, 4 ml of dNTP mixture (10 mM each of dATP, dCTP, dGTP, dTTP), 0.25 ml (1.25 U) Taq DNA polymerase, and 30.25 ml of sterile distilled H2O. The PCR primers employed S1 (50 -TCCCAGACGAAGACAGTCCCTGGAT-30 ) and AS1
(50 -CACTGGGGTGGAATGTGTCTTGGTC-30 ), immediately flank the region of the CD44 open-reading frame involved in the alternative splicing of several exons. We used water instead of template DNA as a negative control. PCR amplification products were labeled with [a-32P]dATP and resolved by 6% polyacrylamide gel electrophoresis (PAGE). Following autoradiography of these gels, bands corresponding to PCR products were quantitated by laser densitometry. Southern blot analysis was performed by transferring PCR products separated on 2% agarose gels to nylon membrane filters and then hybridizing these to [32P]-labeled CD44 cDNA probes. In addition to RT-PCR analysis of placental tissue, 25 samples of formalin-fixed, paraffin-embedded tissue sections of third-trimester placentae from term delivery and 6 samples of first-trimester placentae obtained from curettage due to incomplete abortion were analyzed by immunohistochemical staining. Immunohistochemical staining was performed by the microwave antigen retrieval method. Briefly, paraffin-embedded tissue sections were cut to 5 mm thick and deparaffinized in a graded series of alcohols. Endogenous peroxidase activity was quenched by incubation of sections in 1% hydrogen peroxide for 15 min at room temperature. Tissues were washed in phosphate buffered saline (1 PBS), and incubated with anti-human CD44H, CD44v6 monoclonal antibodies (R&D Systems, Minneapolis, Minnesota) diluted 1:1000 in 1 PBS for 1 h at room temperature. Slides were washed three times in 1 PBS and then incubated with biotinylated anti-mouse secondary antibody for 30 min at room temperature. Antigen–antibody complexes were detected using the avidin–biotin–peroxidase method using diaminobenzidine as a chromogen substrate (Vectastain ABC-kit, Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. Tissue sections were lightly counter-stained with hematoxylin and then examined by light microscopy. Normal uterine cervical tissues were used as a positive control and primary antibody was omitted as a negative control.
3. Results Several amplification products were obtained by RTPCR, and Southern blot analysis confirmed that two bands (84 and 482 bp) corresponded to the CD44H and CD44R1 transcripts, respectively (data not shown). All normal term placentae expressed CD44H, but CD44R1 expression was observed in only 3 of 14 (21.4%) samples examined (Fig. 1). Interestingly, double bands were observed for CD44H suggesting that alternative splicing isoforms were amplified in 12 of 14 (85.7%) placentae examined. A few additional bands of unknown significance were identified in some of the samples. Immunohistochemical analysis revealed that CD44H was expressed in all first-trimester and term placentae (Fig. 2) agreeing with results obtained by RT-PCR analysis. In first-
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Fig. 1. PCR amplification products of CD44 cDNA from 14 term placentae. CD44H (84 bp) transcript was amplified in all of the specimens, and CD44R1 (482 bp) in the specimens in lanes 7, 8 and 10 only.
trimester placentae, positive reaction was most frequently seen on the trophoblast cell surface lining the chorionic villi and in the intermediate trophoblasts of the implantation sites. In term placentae, positive reaction was noted on the cell surface of the trophoblast cells lining the chorionic villi. Staining intensity was strong and continuous along the villous outline. CD44v6 was expressed in 3 of 6 (50%) first-trimester and only 6 of 25 (24%) term placentae (Fig. 3). Staining intensity of the CD44v6 decreased when compared to first-trimester placentae. There was a distinct positive reaction on the cell surface of the syncytiotrophoblast and cytotrophoblast lining the chorionic villi and intermediate trophoblast of the implantation sites on the first-trimester placentae. Staining intensity was distinct but discontinuous and multifocally scattered throughout the placental tissue (Fig. 3a). Similar staining pattern was observed on term placentae (Fig. 3b). Positive staining on term placentae was observed exclusively on the trophoblast surface of the subchorionic area in which hypoxic conditions prevail and on the trophoblast surface of immature villi frequently associated with hypoxia in the fetus (Fig. 3b).
Fig. 3. (a) Positive immunostaining of CD44v6 in first-trimester placental tissue. Positive reaction was observed in the surface of syncytiotrophoblast and cytotrophoblast lining the chorionic villi of immature placenta (magnification 200). (b) Positive immunostaining of CD44v6 in third-trimester placental tissue. Positive staining was seen in the trophoblastic surface (with a discontinuous pattern) lining the chorionic villi. Nonspecific weak staining of fetal vessel wall and red blood cells are also noted (magnification 200).
4. Discussion
Fig. 2. Positive immunostaining of CD44H in first-trimester placental tissue. Note strong positive reaction in the trophoblast cell surface. Nonspecific weak staining of fetal vessel wall and red blood cells are also noted (magnification 200).
In humans, CD44 is present as a single copy gene on the short arm of chromosome 11 and gives rise to several mRNA transcript isoforms through a complex pattern of alternative splicing [19–21]. CD44 and its variant isoforms are expressed not only in lymphocytes but also in various other normal tissues [22–24]. The human placenta has many similarities to malignant tumors and expression of cellular proto-oncogenes that might be involved in the rapid growth and differentiation of placentae has been reported [25,26]. CD44 expression in placenta has been reported in several studies. Fox et al. [23] reported that placental cytotrophoblasts exhibited strong positive immunoreaction to CD44H and CD44 variant isoforms (CD44v3, v4/5, and v6) but not to CD44v8/9. Goshen et al. [27] reported that intermediate trophoblasts exhibited the standard form of CD44 while extravillous trophoblasts, which are responsible for the
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invasive characteristics of the placenta, were positive for the alternatively spliced form, the CD44v7/8. Marzioni et al. [18] suggested that CD44 may play an important role in tissue remodeling later in gestation. Fig. 1 shows double bands for CD44H suggesting that more than one isoforms of CD44H might be expressed on normal placentae. A CD44 variant isoform, CD44R1 was expressed in 21.4% of term placentae examined. However, we were not able to demonstrate in this study, the significance of CD44H expression and its alternative transcriptional splicing on normal placentae. Using immunohistochemistry and RT-PCR analysis of CD44, we showed that all normal first-trimester and term placentae expressed CD44H. However, whereas we observed strong positive immunostaining of CD44H and CD44v6 on the surface of syncytiotrophoblast and cytotrophoblast lining the chorionic villi and on the intermediate trophoblasts at implantation sites of the firsttrimester placentae (Figs. 2 and 3a), Marzioni et al. [18] reported that CD44 was primarily expressed on the placenta from week 16 of gestation. This discrepancy may have resulted from epitope variation as recognized by the different antibodies or the limited number of placentae used. CD44v6 immunostaining intensity and frequency decreased in term placentae, although the specimen number in each group was not enough to show statistical significance. This may suggest that expression of CD44v6 in the extravillous trophoblast may play an important role in the invasion of the trophoblast into maternal decidua during placentation, as in the aberrant expression of CD44 variant isoforms in the metastatic process in some types of epithelial neoplasms. Given that positive staining of each CD44v6 was exclusively observed on the trophoblast cell surface of sub-chorionic areas associated with hypoxic conditions in term placentae, we suggest that transcription of CD44 variant isoforms on the trophoblast may be regulated by placental hypoxia. Also, the fact that hypoxia may be a regulatory factor in CD44 expression was reported by Hasan et al. [28], who reported that expression of CD44 was downregulated after cells were exposed to hypoxia in human melanoma cell lines. The authors subsequently suggested that cell detachment after decreased adhesion appears to be a stress response, which may enable malignant cells to escape hypoxia in vivo, with the potential to form new foci of tumor growth. We suggest that further studies should be carried out to elucidate the role of hypoxia as a regulatory factor for CD44 transcription on placentae and other tissues.
5. Conclusion Our results suggest that alternative expression of CD44 variants on human placental trophoblasts may play a crucial role in the invasion of trophoblast into maternal decidua in early pregnancy. However, the biological significance of this
expression pattern is not clear. We suggest that further studies should be carried out to elucidate the expression pattern of CD44 variants on the placenta through the entire gestational period and in other tissues.
References [1] Screaton GR, Bell MV, Jackson DG, Cornelis FB, Gerth U, Bell JI. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc Natl Acad Sci USA 1992;89:12160–4. [2] Cooper DL, Dougherty G, Harn HJ, et al. The complex CD44 transcriptional unit; alternative splicing of three internal exons generates the epithelial form of CD44. Biochem Biophys Res Commun 1992;182:569–78. [3] Wielenga VJ, Heider KH, Offerhaus GJ, et al. Expression of CD44 variant proteins in human colorectal cancer is related to tumor progression. Cancer Res 1993;53:4754–6. [4] Peach RJ, Hollenbaugh D, Stamenkovic I, Aruffo A. Identification of hyaluronic acid binding sites in the extracellular domain of CD44. J Cell Biol 1993;122:257–64. [5] Lesley J, English N, Perschl A, Gregoroff J, Hyman R. Variant cell lines selected for alterations in the function of the hyaluronan receptor CD44 show differences in glycosylation. J Exp Med 1995;182:431–7. [6] Jalkanen S, Bargatze RF, de los Toyos J, Butcher EC. Lymphocyte recognition of high endothelium: antibodies to distinct epitopes of an 85–95-kDa glycoprotein antigen differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial endothelial cells. J Cell Biol 1987;105:983–90. [7] Estess P, DeGrendele HC, Pascual V, Siegelman MH. Functional activation of lymphocyte CD44 in peripheral blood is a marker of autoimmune disease activity. J Clin Invest 1998;102:1173–82. [8] Miyake K, Medina KL, Hayashi S, Ono S, Hamaoka T, Kincade PW. Monoclonal antibodies to Pgp-1/CD44 block lympho-hemopoiesis in long-term bone marrow cultures. J Exp Med 1990;171:477–88. [9] Thomas L, Byers HR, Vink J, Stamenkovic I. CD44H regulates tumor cell migration on hyaluronate-coated substrate. J Cell Biol 1992; 118:971–7. [10] Webb DS, Shimizu Y, Van Seventer GA, Shaw S, Gerrard TL. LFA-3, CD44, and CD45: physiologic triggers of human monocyte TNF and IL-1 release. Science 1990;249:1295–7. [11] Merzak A, Koocheckpour S, Pilkington GJ. CD44 mediates human glioma cell adhesion and invasion invitro. Cancer Res 1994;54:3988–92. [12] Weber GF, Bronson RT, Ilagan J, Cantor H, Schmits R, Mak TW. Absence of the CD44 gene prevents sarcoma metastasis. Cancer Res 2002;62:2281–6. [13] Birch M, Mitchell S, Hart IR. Isolation and characterization of human melanoma cell variants expressing high and low levels of CD44. Cancer Res 1991;51:6660–7. [14] Jalkanen S, Joensuu H, Soderstrom KO, Klemi P. Lymphocyte homing and clinical behavior of non-Hodgkin’s lymphoma. J Clin Invest 1991;87:1835–40. [15] Lee JH, Kang YS, Kim BG, et al. Expression of the CD44 adhesion molecule in primary and metastatic gynecologic malignancies and their cell lines. Int J Gynecol Cancer 1995;5:193–9. [16] Jungling B, Menges M, Goebel R, et al. Expression of CD44v6 has no prognostic value in patients with colorectal cancer. Z Gastroenterol 2002;40:229–33. [17] Arch R, Wirth K, Hofmann M, et al. Participation in normal immune responses of a metastasis-inducing splice variant of CD44. Science 1992;257:682–5. [18] Marzioni D, Crescimanno C, Zaccheo D, Coppari R, Underhill CB, Castellucci M. Hyaluronate and CD44 expression patterns in the human placenta throughout pregnancy. Eur J Histochem 2001;45:131–40.
C.H. Choi et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 128 (2006) 243–247 [19] Goodfellow PN, Banting G, Wiles MV, et al. The gene, MIC4, which controls expression of the antigen defined by monoclonal antibody F10.44.2, is on human chromosome 11. Eur J Immunol 1982;12:659–63. [20] Jackson DG, Buckley J, Bell JI. Multiple variants of the human lymphocyte homing receptor CD44 generated by insertions at a single site in the extracellular domain. J Biol Chem 1992;267:4732–9. [21] Konig H, Ponta H, Herrlich P. Coupling of signal transduction to alternative pre-mRNA splicing by a composite splice regulator. Embo J 1998;17:2904–13. [22] Lesley J, Hyman R, Kincade PW. CD44 and its interaction with extracellular matrix. Adv Immunol 1993;54:271–335. [23] Fox SB, Fawcett J, Jackson DG, et al. Normal human tissues, in addition to some tumors, express multiple different CD44 isoforms. Cancer Res 1994;54:4539–46.
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[24] Mackay CR, Terpe HJ, Stauder R, Marston WL, Stark H, Gunthert U. Expression and modulation of CD44 variant isoforms in humans. J Cell Biol 1994;124:71–82. [25] Adamson ED. Expression of proto-oncogenes in the placenta. Placenta 1987;8:449–66. [26] Ohlsson R. Growth factors, protooncogenes and human placental development. Cell Differ Dev 1989;28:1–15. [27] Goshen R, Ariel I, Shuster S, et al. Hyaluronan CD44 and its variant exons in human trophoblast invasion and placental angiogenesis. Mol Hum Reprod 1996;2:685–91. [28] Hasan NM, Adams GE, Joiner MC, Marshall JF, Hart IR. Hypoxia facilitates tumour cell detachment by reducing expression of surface adhesion molecules and adhesion to extracellular matrices without loss of cell viability. Br J Cancer 1998;77:1799–805.
Expression of CD44 adhesion molecules on human placentae Chel Hun Choi a, Cheong Rae Roh a, Tae-Joong Kim a, Yoon-La Choi b, Jeong-Won Lee a, Byoung-Gie Kim a,*, Je-Ho Lee a, Duk-Soo Bae a a
Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea b Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Received 15 August 2005; received in revised form 22 December 2005; accepted 3 January 2006
Abstract Objective: This study was performed to characterize CD44 expression in human placentae. Study design: We conducted reverse transcriptase polymerase chain reaction (RT-PCR) analyses of CD44H (hematopoietic form, also called CD44s) and CD44R1 (epithelial form) in 14 normal term placentae, and immunohistochemical analysis of CD44H and CD44v6 in 31 placentae (25 term and 6 first-trimester). Results: All 14 term placentae expressed CD44H (double bands) suggesting expression of alternative isoforms on the placentae. CD44R1 was expressed on 3 of 14 (21.4%) term placentae. Immunohistochemical analysis revealed expression of CD44H in all the placentae examined. However, positive staining for CD44v6 was observed in 3 of 6 (50%) first-trimester and only 6 of 25 (24%) term placentae showing decreased expression on term placentae. Positive staining for CD44v6 was observed on the trophoblast surface of immature villi especially in the sub-chorionic area where hypoxic conditions prevail. Conclusion: These results suggest that CD44 splicing variants might play a role in the invasion of trophoblast into maternal tissue in early pregnancy. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: CD44H; CD44R1; CD44v6; Placentae
1. Introduction CD44 proteins are a ubiquitously expressed family of surface adhesion markers involved in cell–cell and cell– matrix interactions. These multiple protein isoforms are encoded by alternative splicing of a single gene and are further modified by post-translational modifications. Genomic analysis of CD44 revealed existence of 20 exons over a length of approximately 60 kilobases [1]. The five N-terminal exons plus the five C-terminal exons encode CD44H. The CD44 variant isoform that contains sequences encoded by
* Corresponding author at: Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Kangnam-Ku, Seoul 135-710, Republic of Korea. Tel.: +82 2 3410 3513; fax: +82 2 3410 0630. E-mail address: [email protected] (B.-G. Kim).
variant exon sequences 8, 9, and 10 is known as CD44R1 [2]. CD44v6 isoforms contains sequences encoded by exon 6 [3]. Extracellular matrix components have been described as ligands for the CD44H, such as hyaluronic acid (HA), collagen, fibronectin, and sulfated proteoglycan. Binding to HA is mediated via two regions encoded by exons 2s and 5s [4]. Differences in HA binding state of CD44 are cell specific and have been shown to be related to posttranslational modifications [5]. CD44H is known to mediate adhesion of lymphocytes to high endothelial venules in vitro [6,7], promotion of normal B-cell development in vitro [8], cell migration [9], and stimulation of cytokine release by macrophages [10]. The role of CD44H in tumorigenesis has been suggested by studies in which neutralizing antibodies were induced to block tumor cell invasion into extracellular matrix in vitro [11,12], correlation of CD44 density with increased metastatic potential of melanoma cell lines [13], and
0301-2115/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2006.01.017
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association between CD44 expression and survival in nonHodgkin’s lymphoma [14]. In contrast to CD44H, CD44 variants are not well characterized. Functional differences seem to exist between CD44 variant isoforms and CD44H, although the structural differences and the exact ligands recognized by these molecules have not been well defined. In a previous study, we examined CD44 transcript alternative splicing patterns in gynecologic malignancies and observed increased frequency of CD44R1 expression during ovarian and cervical carcinoma progression [15]. CD44v6 has been shown to have a crucial role in the movement and homing of antigen activated lymphocytes in lymph nodes and has been associated with tumor invasion, progression and metastasis [3,16,17]. In further elucidating the functional role of CD44 in normal and malignant tissues, the human placenta may be a useful model as its trophoblast comprises highly differentiated villous and invasive extravillous entities [18]. In this study, we used RT-PCR to examine CD44 transcript splicing patterns in normal human placentae in order to determine whether this tissue also displays altered CD44 expression. Immunohistochemical staining of CD44 protein was performed to determine which cell population might be responsible for CD44 expression.
2. Materials and methods Fourteen term placentae were obtained after spontaneous delivery, frozen immediately and stored at 80 8C until mRNA isolation. mRNA was extracted from each sample with the QuickPrep Micro mRNA Purification Kit (Pharmacia P-L Biochemicals, Milwaukee, Wisconsin) according to manufacturer’s recommendations. First-strand cDNA was synthesized with a First-Strand cDNA Kit (Pharmacia P-L Biochemicals) according to the recommendations of the supplier. About 0.1 mg mRNA (8 ml) was reverse transcribed at 37 8C for 1 h using 5 ml of bulk first-strand cDNA mixture (Cloned, FPL C purified murine reverse transcriptase, RNA guard, RNase/DNase-Free BSA, dATP, dCTP, dGTP and dTTP in aqueous buffer), 1 ml (0.002 mg) random hexadeoxynucleotide primers, and 1 ml (0.1 M) of dithiothreitol solution. CD44 cDNA was subjected to PCR using a GeneAmp PCR Core Reagent Kit (Perkin-Elmer-Cetus, Norwalk, Connecticut) for 32 cycles in triplicate samples using the following temperatures and time sequences: 94 8C for 1 min, 50 8C for 1 min, 72 8C for 1 min. Five microliters of first-strand cDNA was added to 45 ml of PCR mixture containing 5 ml 10 reaction buffer (100 mM Tris–HCl, pH 8.3, 500 mM KCl, and 1 mg/ml of gelatin), 3 ml MgCl2 (25 mM), 0.5 ml a-32P-dCTP, 1 ml (25 pM) of each primer, 4 ml of dNTP mixture (10 mM each of dATP, dCTP, dGTP, dTTP), 0.25 ml (1.25 U) Taq DNA polymerase, and 30.25 ml of sterile distilled H2O. The PCR primers employed S1 (50 -TCCCAGACGAAGACAGTCCCTGGAT-30 ) and AS1
(50 -CACTGGGGTGGAATGTGTCTTGGTC-30 ), immediately flank the region of the CD44 open-reading frame involved in the alternative splicing of several exons. We used water instead of template DNA as a negative control. PCR amplification products were labeled with [a-32P]dATP and resolved by 6% polyacrylamide gel electrophoresis (PAGE). Following autoradiography of these gels, bands corresponding to PCR products were quantitated by laser densitometry. Southern blot analysis was performed by transferring PCR products separated on 2% agarose gels to nylon membrane filters and then hybridizing these to [32P]-labeled CD44 cDNA probes. In addition to RT-PCR analysis of placental tissue, 25 samples of formalin-fixed, paraffin-embedded tissue sections of third-trimester placentae from term delivery and 6 samples of first-trimester placentae obtained from curettage due to incomplete abortion were analyzed by immunohistochemical staining. Immunohistochemical staining was performed by the microwave antigen retrieval method. Briefly, paraffin-embedded tissue sections were cut to 5 mm thick and deparaffinized in a graded series of alcohols. Endogenous peroxidase activity was quenched by incubation of sections in 1% hydrogen peroxide for 15 min at room temperature. Tissues were washed in phosphate buffered saline (1 PBS), and incubated with anti-human CD44H, CD44v6 monoclonal antibodies (R&D Systems, Minneapolis, Minnesota) diluted 1:1000 in 1 PBS for 1 h at room temperature. Slides were washed three times in 1 PBS and then incubated with biotinylated anti-mouse secondary antibody for 30 min at room temperature. Antigen–antibody complexes were detected using the avidin–biotin–peroxidase method using diaminobenzidine as a chromogen substrate (Vectastain ABC-kit, Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. Tissue sections were lightly counter-stained with hematoxylin and then examined by light microscopy. Normal uterine cervical tissues were used as a positive control and primary antibody was omitted as a negative control.
3. Results Several amplification products were obtained by RTPCR, and Southern blot analysis confirmed that two bands (84 and 482 bp) corresponded to the CD44H and CD44R1 transcripts, respectively (data not shown). All normal term placentae expressed CD44H, but CD44R1 expression was observed in only 3 of 14 (21.4%) samples examined (Fig. 1). Interestingly, double bands were observed for CD44H suggesting that alternative splicing isoforms were amplified in 12 of 14 (85.7%) placentae examined. A few additional bands of unknown significance were identified in some of the samples. Immunohistochemical analysis revealed that CD44H was expressed in all first-trimester and term placentae (Fig. 2) agreeing with results obtained by RT-PCR analysis. In first-
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Fig. 1. PCR amplification products of CD44 cDNA from 14 term placentae. CD44H (84 bp) transcript was amplified in all of the specimens, and CD44R1 (482 bp) in the specimens in lanes 7, 8 and 10 only.
trimester placentae, positive reaction was most frequently seen on the trophoblast cell surface lining the chorionic villi and in the intermediate trophoblasts of the implantation sites. In term placentae, positive reaction was noted on the cell surface of the trophoblast cells lining the chorionic villi. Staining intensity was strong and continuous along the villous outline. CD44v6 was expressed in 3 of 6 (50%) first-trimester and only 6 of 25 (24%) term placentae (Fig. 3). Staining intensity of the CD44v6 decreased when compared to first-trimester placentae. There was a distinct positive reaction on the cell surface of the syncytiotrophoblast and cytotrophoblast lining the chorionic villi and intermediate trophoblast of the implantation sites on the first-trimester placentae. Staining intensity was distinct but discontinuous and multifocally scattered throughout the placental tissue (Fig. 3a). Similar staining pattern was observed on term placentae (Fig. 3b). Positive staining on term placentae was observed exclusively on the trophoblast surface of the subchorionic area in which hypoxic conditions prevail and on the trophoblast surface of immature villi frequently associated with hypoxia in the fetus (Fig. 3b).
Fig. 3. (a) Positive immunostaining of CD44v6 in first-trimester placental tissue. Positive reaction was observed in the surface of syncytiotrophoblast and cytotrophoblast lining the chorionic villi of immature placenta (magnification 200). (b) Positive immunostaining of CD44v6 in third-trimester placental tissue. Positive staining was seen in the trophoblastic surface (with a discontinuous pattern) lining the chorionic villi. Nonspecific weak staining of fetal vessel wall and red blood cells are also noted (magnification 200).
4. Discussion
Fig. 2. Positive immunostaining of CD44H in first-trimester placental tissue. Note strong positive reaction in the trophoblast cell surface. Nonspecific weak staining of fetal vessel wall and red blood cells are also noted (magnification 200).
In humans, CD44 is present as a single copy gene on the short arm of chromosome 11 and gives rise to several mRNA transcript isoforms through a complex pattern of alternative splicing [19–21]. CD44 and its variant isoforms are expressed not only in lymphocytes but also in various other normal tissues [22–24]. The human placenta has many similarities to malignant tumors and expression of cellular proto-oncogenes that might be involved in the rapid growth and differentiation of placentae has been reported [25,26]. CD44 expression in placenta has been reported in several studies. Fox et al. [23] reported that placental cytotrophoblasts exhibited strong positive immunoreaction to CD44H and CD44 variant isoforms (CD44v3, v4/5, and v6) but not to CD44v8/9. Goshen et al. [27] reported that intermediate trophoblasts exhibited the standard form of CD44 while extravillous trophoblasts, which are responsible for the
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invasive characteristics of the placenta, were positive for the alternatively spliced form, the CD44v7/8. Marzioni et al. [18] suggested that CD44 may play an important role in tissue remodeling later in gestation. Fig. 1 shows double bands for CD44H suggesting that more than one isoforms of CD44H might be expressed on normal placentae. A CD44 variant isoform, CD44R1 was expressed in 21.4% of term placentae examined. However, we were not able to demonstrate in this study, the significance of CD44H expression and its alternative transcriptional splicing on normal placentae. Using immunohistochemistry and RT-PCR analysis of CD44, we showed that all normal first-trimester and term placentae expressed CD44H. However, whereas we observed strong positive immunostaining of CD44H and CD44v6 on the surface of syncytiotrophoblast and cytotrophoblast lining the chorionic villi and on the intermediate trophoblasts at implantation sites of the firsttrimester placentae (Figs. 2 and 3a), Marzioni et al. [18] reported that CD44 was primarily expressed on the placenta from week 16 of gestation. This discrepancy may have resulted from epitope variation as recognized by the different antibodies or the limited number of placentae used. CD44v6 immunostaining intensity and frequency decreased in term placentae, although the specimen number in each group was not enough to show statistical significance. This may suggest that expression of CD44v6 in the extravillous trophoblast may play an important role in the invasion of the trophoblast into maternal decidua during placentation, as in the aberrant expression of CD44 variant isoforms in the metastatic process in some types of epithelial neoplasms. Given that positive staining of each CD44v6 was exclusively observed on the trophoblast cell surface of sub-chorionic areas associated with hypoxic conditions in term placentae, we suggest that transcription of CD44 variant isoforms on the trophoblast may be regulated by placental hypoxia. Also, the fact that hypoxia may be a regulatory factor in CD44 expression was reported by Hasan et al. [28], who reported that expression of CD44 was downregulated after cells were exposed to hypoxia in human melanoma cell lines. The authors subsequently suggested that cell detachment after decreased adhesion appears to be a stress response, which may enable malignant cells to escape hypoxia in vivo, with the potential to form new foci of tumor growth. We suggest that further studies should be carried out to elucidate the role of hypoxia as a regulatory factor for CD44 transcription on placentae and other tissues.
5. Conclusion Our results suggest that alternative expression of CD44 variants on human placental trophoblasts may play a crucial role in the invasion of trophoblast into maternal decidua in early pregnancy. However, the biological significance of this
expression pattern is not clear. We suggest that further studies should be carried out to elucidate the expression pattern of CD44 variants on the placenta through the entire gestational period and in other tissues.
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