Accepted Manuscript Expression of serum amyloid A4 in human trophoblast-like choriocarcinoma cell lines and human first trimester/term trophoblast cells C. Rossmann , A. Hammer , C.N. Koyani , A. Kovacevic , M. Siwetz , G. Desoye , T.G. Poehlmann , U.R. Markert , B. Huppertz , W. Sattler , E. Malle PII:
S0143-4004(14)00225-2
DOI:
10.1016/j.placenta.2014.05.012
Reference:
YPLAC 3007
To appear in:
Placenta
Received Date: 19 March 2014 Revised Date:
27 May 2014
Accepted Date: 29 May 2014
Please cite this article as: Rossmann C, Hammer A, Koyani CN, Kovacevic A, Siwetz M, Desoye G, Poehlmann TG, Markert UR, Huppertz B, Sattler W, Malle E, Expression of serum amyloid A4 in human trophoblast-like choriocarcinoma cell lines and human first trimester/term trophoblast cells, Placenta (2014), doi: 10.1016/j.placenta.2014.05.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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“ Short Communication ”
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Revision: PL-14-00048
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Expression of serum amyloid A4 in human trophoblast-like choriocarcinoma
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cell lines and human first trimester/term trophoblast cells
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C. ROSSMANN , A. HAMMER , C.N. KOYANI a, A. KOVACEVIC , M. SIWETZ , G.
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DESOYE c, T.G. POEHLMANN , U.R. MARKERT , B. HUPPERTZ , W. SATTLER , and
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E. MALLE *
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Medical University of Graz, Institute of Molecular Biology and Biochemistry, Institute of Cell
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Biology, Histology and Embryology, c Department of Obstetrics and Gynecology, Graz, Austria
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and
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Placenta-Laboratory, Department of Obstetrics, University Hospital Jena, Jena, Germany.
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Running Head: SAA4 expression in trophoblast
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Word count excluding title page, abstract, figure legends and references: 1209
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Figures: 2
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References: 26 Supplement: 1
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* Corresponding author: Dr. Ernst Malle, Medical University of Graz, Institute of Molecular
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Biology and Biochemistry, A-8010 Graz, Austria, Tel:+43-316-380-4208, Fax:+43-316-380-
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9615; e-mail: [email protected]
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Key words: SAA4, placenta, invasion, cytokine, outgrowing extravillous trophoblast 1
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Abstract
31 Trophoblast invasion into uterine tissues represents a hallmark of first trimester placental
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development. As expression of serum amyloid A4 (SAA4) occurs in tumorigenic and invasive
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tissues we here investigated whether SAA4 is present in trophoblast-like human AC1-M59/Jeg-3
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cells and trophoblast preparations of human first trimester and term placenta. SAA4 mRNA was
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expressed in non-stimulated and cytokine-treated AC1-M59/Jeg-3 cells. In purified trophoblast
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cells SAA4 mRNA expression was upregulated at weeks 10 and 12 of pregnancy. Western-blot
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and immunohistochemical staining of first trimester placental tissue revealed pronounced SAA4
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expression in invasive trophoblast cells indicating a potential role of SAA4 during invasion.
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Introduction
42 The serum amyloid A (SAA) family comprises lipoprotein-associated proteins, encoded
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by different genes with a high allelic variation [1, 2]. In humans, the non-glycosylated SAA1/2
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proteins, highly induced during the acute-phase response [3], are considered as important clinical
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markers of inflammation [4]. While human SAA3 is a pseudogene, SAA4 codes for glycosylated
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SAA4 protein that represents the predominant SAA isoform under physiological conditions.
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Although the liver is the major source for SAA4 assumed to be constitutively expressed [5-7],
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some studies reported abundant expression specifically in tumorigenic tissues [8-11].
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Acute-phase SAA1/2 is expressed in human placenta, trophoblast cells and
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choriocarcinoma cell lines [12-14]. Therefore, the present study aimed at identifying SAA4 in (i)
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human Jeg-3 choriocarcinoma and AC1-M59 cells (hybrid of term trophoblast cells and AC-1
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cells derived from Jeg-3), (ii) trophoblast cells of human first trimester and term placenta and (iii)
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outgrowing human first trimester extravillous trophoblast (EVT) cells.
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Materials and methods
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Cell culture experiments
Placental tissues were obtained from born placentas of healthy pregnancies and from
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clinically normal human pregnancies, which were interrupted for psychosocial reasons (approved
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by the Ethical Committee at the Medical University of Graz [23-203 ex 10/11] and the Friedrich-
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Schiller-University Jena [1503-03/05]). Mononucleated human first trimester and term
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trophoblast cells were isolated as described [15, 16]. Outgrowing EVT cells were isolated from
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placental villi after terminations of pregnancy (7-10 weeks, n=5) and were incubated in Petri
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dishes for two days at 5% CO2 in DMEM/Ham’s F12 with 10% (v/v) human serum. Then the
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villi were transferred into collagen-coated 24-well plates (two explants/well) and incubated at
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2.5% O2, 5% CO2. After three days of culture the villi were removed from the plates and the
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already outgrown cells were cultured for another day at 5% CO2. Human hepatocellular
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carcinoma (HUH-7) cells, human Jeg-3 and human AC1-M59 cells were cultured as described
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[12, 17, 18]. AC1-M59 and Jeg-3 cells were seeded into cell culture dishes and upon reaching
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80% confluence, incubated for 24 h in medium containing 10 ng/ml IL-1α, IL-1β, IL-6, or TNFα
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(R&D Systems, Minneapolis, MN, USA).
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RNA isolation, RT-PCR and real-time RT-PCR (qPCR) analysis
RNA was isolated from (i) AC1-M59, Jeg-3, EVT, HUH-7 (using RNeasy Mini Kit from
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QIAGEN, Hilden, Germany) and (ii) first trimester (week 6-12) and term trophoblast cells (week
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35 and 40) using Trizol-reagent (Sigma-Aldrich, Saint-Louis, MO, USA), followed by DNase
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treatment, reverse transcription and PCR for SAA4 and GAPDH (used as a housekeeping gene,
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see Supplementary Table I) as described [19]. The qPCR protocol was performed using
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LightCycler 480 system (Roche Diagnostics, Vienna, Austria) [20]. Gene specific primers used
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for SAA4 and HPRT (used as a housekeeping gene) are listed in the Supplementary Table II.
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Relative gene expression levels compared to HPRT were calculated using ∆∆CT method.
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Western blot analysis
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Total cellular proteins were isolated [16] and 50 µg of protein (as assessed by the BCA
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method) was subjected to Western-blot analysis as described [19]. Rabbit polyclonal anti-human
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SAA4 antiserum (raised against amino acids 94-112 of human SAA4; dilution 1:300) not cross-
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reacting with human SAA1/2 was used as primary antibody [21].
89 Immunohistochemistry of first trimester placenta
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Placental specimens from elective terminations of pregnancy (7-10 weeks, n=5) were fixed
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in formaldehyde and embedded in paraffin. Sections of 5 µm were dewaxed and stained for
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SAA4 (polyclonal anti-SAA4 peptide antibody [21], dilution 1:500) or HLA-G (monoclonal
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antibody 4H84, 1:2000, BD Biosciences, NJ, USA) using labelled polymer (Thermo Fisher
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Scientific, MA, USA) and 3-amino-9-ethylcarbazole (Dako, Inc., Carpinteria, CA, USA) to detect
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antibody binding [22]. Control experiments were performed using rabbit or mouse non-immune
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serum or IgG.
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Results and discussion
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RT-PCR (Fig. 1A/B) and qPCR analysis (Fig. 1C) reveals that SAA4 mRNA expression
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in AC1-M59 cells as well as in its parental cell line Jeg-3 (Fig. 1A/B) is not altered by IL-1β, IL-
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6, and TNFα treatment. However, IL-1α significantly decreased SAA mRNA expression in both
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cell lines (Fig. 1C). This observation, as well as similar data obtained in non-differentiated human
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mesenchymal stem cells and human osteosarcoma cell lines (using RT-PCR [19]) does not
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confirm the constitutive character of apolipoprotein SAA4 [6, 7] in general.
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High SAA4 mRNA expression in human colon adenocarcinoma [10, 23] and ovarian
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carcinoma [11] suggests a possible role of SAA4 in tumorigenesis and invasion as previously
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reported for SAA1/2 [24, 25]. This seems likely, as the adhesion motif present in the N-terminal
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region of SAA1/2 that mediates binding to extracellular matrix components and cell invasion, is
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highly homologous to that present in SAA4. One of the hallmarks of first trimester placental
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development is invasion of uterine tissue by fetal trophoblast cells. Thus, SAA4 expression was
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studied in invasive trophoblast cells. While expression of SAA4 transcripts is low during early
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placental development (weeks 7-9), expression levels are increased at later first trimester
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pregnancy weeks but decreased at term pregnancy stages (Fig. 1D). Data obtained from qPCR
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analyses (Fig. 1E) confirmed results obtained with RT-PCR (Fig. 1D); highest SAA4 mRNA
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expression was found at week 10 and 12; however, at later pregnancy stages (week 35 and 40)
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SAA4 mRNA levels were similar as observed in early pregnancy weeks. Furthermore, SAA4
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mRNA expression was also found in preparations of isolated outgrowing first trimester EVT cells
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(Fig. 1F).
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Next, we tried to identify SAA4 protein in human placental tissue using an antibody
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(raised against the C-terminal portion [that differs from SAA1/2] of SAA4 [21]). Two
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immunoreactive bands corresponding to approximately 14 kDa (non-glycosylated) and a less
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intensive 19 kDa (glycosylated) protein, as reported for liver-derived human SAA4 [7], were
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identified by Western-blot in outgrowing EVT cells (Fig. 2A). However, no immunoreactive
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signals for SAA4 were detected in AC1-M59 or Jeg-3 cells under non-stimulated or cytokine-
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stimulated conditions (data not shown).
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Immunohistochemical staining revealed the presence of SAA4 in cell columns of first
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trimester placental tissue representing potentially invasive EVT cells (Fig. 2Ba). In the
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proliferative zone of cell columns near the basement membrane towards the villous stroma,
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SAA4 was barely detectable. Interestingly, the more pronounced the invasive character of
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trophoblast cells, the more pronounced is SAA4 expression (Fig. 2Ba). Areas within the decidua,
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which were invaded by trophoblast cells (Fig. 2Ca), show positivity for SAA4 (Fig. 2Cb). SAA4
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expression was detected in different EVT cell populations that had already invaded the decidua
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basalis, such as intramural trophoblast cells replacing the walls of spiral arteries (Fig. 2Cd and e),
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intraluminal trophoblast cells plugging the lumina of spiral arteries (Fig. 2Cd and e), and
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interstitial trophoblast cells (Fig. 2Cg and h), respectively. However, multinucleated trophoblast
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giant cells, a subpopulation representing the terminally differentiated sessile endpoint of invading
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trophoblast cells [26], showed no stained for SAA4 (Fig. 2Ci). Also glandular epithelial cells of
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the decidua were negative for SAA4 (Supplementary Figure I). To verify the trophoblast nature
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of these cells an antibody recognizing HLA-G (expressed only by extravillous trophoblast and
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choriocarcinoma cells) was used on serial sections (Fig. 2Ca, d, and g).
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Here we present first evidence of a pronounced expression of SAA4, so far considered as
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a protein of “unknown” function, in first trimester placental tissues. SAA4 expression in invasive
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trophoblast cells suggests a likely role of this apolipoprotein during invasion. The underlying
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mechanisms as reported for acute-phase SAA [25] are currently under investigation.
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Acknowledgements
Supported by the Austrian Science Fund (FWF, DK-MCD W1226 and SFB-LIPOTOX
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F3007).
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Figure legends
2 Figure 1
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RT-PCR and qPCR analysis for SAA4 mRNA expression in non-stimulated and cytokine-
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stimulated trophoblast cells:
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AC1-M59 and Jeg-3 cells were stimulated with different cytokines (10 ng/ml) for 24 h (A-
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C). RNA was isolated from both cell lines (A-C) as well as from cell preparations from first
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trimester and term trophoblast cells at indicated pregnancy weeks (D, E) as well as outgrowing
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EVT cells from placental villi from first trimester placentas (F). Then RNA was reverse
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transcribed, and cDNA was amplified using specific forward and reverse oligonucleotide primers
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(Supplementary Table I). RT-PCR products were separated on 1.5% agarose gels. GAPDH was
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used as a housekeeping gene.
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qPCR was performed from reverse-transcribed RNA isolated from non-stimulated and
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cytokine-stimulated AC1-M59 and Jeg-3 cells (C) and first trimester and term trophoblast cells
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(E) using specific primers (Supplementary Table II) for SAA4 and HPRT (used as a
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housekeeping gene).
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NS (non-stimulated); P (positive control: RNA was isolated from human hepatocellular
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carcinoma HUH-7 cells, diluted 1:20 in F); N1 (negative control, RNA template: negative
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controls were done for all samples); N2 (negative control, water template). T = EVT. One
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representative experiment out of three is shown. Values (C and E) are expressed as mean±SEM
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(n=4). (C) = *p≤0.05 vs. non-stimulated cells; (E) *p≤0.05 vs. first trimester trophoblast cells
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isolated from pregnancy week 6 (set as 1; x-fold expression of SAA4/HPRT mRNA is given on
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the Y-axis in (C) and (E)).
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Figure 2
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SAA4 protein expression in trophoblasts cells from first trimester and term placenta: (A) Outgrowing EVT cells were isolated, lysed and 50 µg of total protein was subjected
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to Western blot analysis using anti-human SAA4 as primary antibody [21]. As a positive control
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(P) human non-glycosylated SAA4 ([expressed in E. coli] containing an N-terminal His6-tag and
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an enterokinase cleavage site [21]) was detected as a monomeric (approximately 15.5 kDa) or
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dimeric (31 kDa) protein. One representative Western-blot is shown.
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(B) Paraffin sections of placental specimens from first trimester placental villi and (C)
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trophoblast-invaded decidua basalis were stained with anti-human SAA4 antiserum (Ba, Cb,e,h,i)
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or with anti-human HLA-G antibody (Ca,d,g) as a marker for trophoblast cells. Control
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experiments were performed with non-immune rabbit IgG (Bb and Cc) or non-immune mouse
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IgG (Cf). To detect antibody binding labelled polymer and 3-amino-9-ethylcarbazole was used.
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The indicated areas in (Ca) and (Cb) (demonstrating part of trophoblast-invaded spiral artery) are
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shown at higher magnification as (Cd) and (Ce). Arrows in (Cd) and (Ce) point towards
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intraluminal trophoblast cells while arrowheads point towards intramural trophoblast cells
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positive for SAA4. Arrows in (Cg) and (Cf) point towards interstitial trophoblast cells positive
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for SAA4. The arrowheads in (Ci) represent multinucleated trophoblast giant cells negative for
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SAA4. The respective bar size is indicated in (B) and (C).
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Highlights
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• SAA4 mRNA is expressed in Jeg-3 and AC1-M59 cells • SAA4 mRNA is expressed in first trimester/term trophoblast cells • SAA4 mRNA is upregulated at pregnancy week 10 and 12
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• SAA4 protein is present in interstitial, intramural and intraluminal trophoblast cells
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C. ROSSMANN, A. HAMMER, C.N. KOYANI, A. KOVACEVIC, M. SIWETZ, G. DESOYE, T.G. POEHLMANN, U.R. MARKERT, B. HUPPERTZ, W. SATTLER, and E. MALLE
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The authors have no conflict of interest
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Online Supplement
Expression of serum amyloid A4 in human trophoblast-like choriocarcinoma
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cell lines and human first trimester/term trophoblast cells
C. ROSSMANN, A. HAMMER, C.N. KOYANI, A. KOVACEVIC, M. SIWETZ, G. DESOYE,
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Supplementary Table I
and annealing temperature)
Primers (start position on + strand)
Accession Nr. F 5’ CCA GTG AAA GCT GGC GTT CG 3’
NM_006512
R 5’ GAG AAG TGT GTG GCT CAC AGC C 3’
GAPDH
F 5’ ACA GTC CAT GCC ATC ACT GCC 3’
M17851
R 5’ GCC TGC TTC ACC ACC TTC TTG 3’
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SAA4
bp
cycles
°C
References
40
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[Kovacevic et
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RT-PCR primers, expected amplicon size (bp), and unique PCR properties (cycle number
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58
[Iochmann et al., 1999]
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al., 2008]
For methods see:
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Kovacevic A, Hammer A, Stadelmeyer E, Windischhofer W, Sundl M, Ray A, Schweighofer N, Friedl G, Windhager R, Sattler W, Malle E. 2008. Expression of serum amyloid A
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transcripts in human bone tissues, differentiated osteoblast-like stem cells and human osteosarcoma cell lines. J Cell Biochem 103:994-1004.
Iochmann S, Reverdiau-Moalic P, Beaujean S, Rideau E, Lebranchu Y, Bardos P, Gruel Y. 1999. Fast detection of tissue factor and tissue factor pathway inhibitor messenger RNA in endothelial cells and monocytes by sensitive reverse transcription-polymerase chain reaction. Thromb Res 94:165-73.
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Supplementary Table II
Gene
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qPCR primers and amplicon size (bp)
Primers (strand)
bp
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Accession Nr. F 5’ TCA GCC GTT CCA GGG TCT AT 3’
NM_006512
R 5’ ATT CCT CAG CTT TCT CGT TGG 3’
HPRT
F 5’ CCT GGC GTC GTG ATT AGT GAT 3’
NM_000194
R 5’ AGA CGT TCA GTC CTG TCC ATA A 3’
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SAA4
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For methods see:
Rossmann R, Rauh A, Hammer A, Windischhofer W, Zirkl S, Sattler W, Malle E. 2011. Hypochlorite-modified high-density lipoprotein promotes induction of HO-1 in
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Supplementary Figure I
One representative Paraffin section of a specimen from trophoblast-invaded decidua basalis was
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stained for EVT cells using an antibody against HLA-G (a) or for SAA4 using an antibody raised against the C-terminal portion of human SAA4 (b).
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Arrows = Interstitial trophoblast cells stained for HLA-G and SAA4
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Arrowheads = glandular epithelial cells negative for HLA-G and SAA4
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S0143-4004(14)00225-2
DOI:
10.1016/j.placenta.2014.05.012
Reference:
YPLAC 3007
To appear in:
Placenta
Received Date: 19 March 2014 Revised Date:
27 May 2014
Accepted Date: 29 May 2014
Please cite this article as: Rossmann C, Hammer A, Koyani CN, Kovacevic A, Siwetz M, Desoye G, Poehlmann TG, Markert UR, Huppertz B, Sattler W, Malle E, Expression of serum amyloid A4 in human trophoblast-like choriocarcinoma cell lines and human first trimester/term trophoblast cells, Placenta (2014), doi: 10.1016/j.placenta.2014.05.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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“ Short Communication ”
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Revision: PL-14-00048
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Expression of serum amyloid A4 in human trophoblast-like choriocarcinoma
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cell lines and human first trimester/term trophoblast cells
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C. ROSSMANN , A. HAMMER , C.N. KOYANI a, A. KOVACEVIC , M. SIWETZ , G.
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DESOYE c, T.G. POEHLMANN , U.R. MARKERT , B. HUPPERTZ , W. SATTLER , and
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E. MALLE *
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Medical University of Graz, Institute of Molecular Biology and Biochemistry, Institute of Cell
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Biology, Histology and Embryology, c Department of Obstetrics and Gynecology, Graz, Austria
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and
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Placenta-Laboratory, Department of Obstetrics, University Hospital Jena, Jena, Germany.
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Running Head: SAA4 expression in trophoblast
Word count abstract: 98
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Word count excluding title page, abstract, figure legends and references: 1209
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Figures: 2
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References: 26 Supplement: 1
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* Corresponding author: Dr. Ernst Malle, Medical University of Graz, Institute of Molecular
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Biology and Biochemistry, A-8010 Graz, Austria, Tel:+43-316-380-4208, Fax:+43-316-380-
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9615; e-mail: [email protected]
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Key words: SAA4, placenta, invasion, cytokine, outgrowing extravillous trophoblast 1
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Abstract
31 Trophoblast invasion into uterine tissues represents a hallmark of first trimester placental
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development. As expression of serum amyloid A4 (SAA4) occurs in tumorigenic and invasive
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tissues we here investigated whether SAA4 is present in trophoblast-like human AC1-M59/Jeg-3
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cells and trophoblast preparations of human first trimester and term placenta. SAA4 mRNA was
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expressed in non-stimulated and cytokine-treated AC1-M59/Jeg-3 cells. In purified trophoblast
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cells SAA4 mRNA expression was upregulated at weeks 10 and 12 of pregnancy. Western-blot
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and immunohistochemical staining of first trimester placental tissue revealed pronounced SAA4
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expression in invasive trophoblast cells indicating a potential role of SAA4 during invasion.
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Introduction
42 The serum amyloid A (SAA) family comprises lipoprotein-associated proteins, encoded
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by different genes with a high allelic variation [1, 2]. In humans, the non-glycosylated SAA1/2
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proteins, highly induced during the acute-phase response [3], are considered as important clinical
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markers of inflammation [4]. While human SAA3 is a pseudogene, SAA4 codes for glycosylated
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SAA4 protein that represents the predominant SAA isoform under physiological conditions.
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Although the liver is the major source for SAA4 assumed to be constitutively expressed [5-7],
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some studies reported abundant expression specifically in tumorigenic tissues [8-11].
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Acute-phase SAA1/2 is expressed in human placenta, trophoblast cells and
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choriocarcinoma cell lines [12-14]. Therefore, the present study aimed at identifying SAA4 in (i)
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human Jeg-3 choriocarcinoma and AC1-M59 cells (hybrid of term trophoblast cells and AC-1
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cells derived from Jeg-3), (ii) trophoblast cells of human first trimester and term placenta and (iii)
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outgrowing human first trimester extravillous trophoblast (EVT) cells.
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Materials and methods
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Cell culture experiments
Placental tissues were obtained from born placentas of healthy pregnancies and from
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clinically normal human pregnancies, which were interrupted for psychosocial reasons (approved
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by the Ethical Committee at the Medical University of Graz [23-203 ex 10/11] and the Friedrich-
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Schiller-University Jena [1503-03/05]). Mononucleated human first trimester and term
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trophoblast cells were isolated as described [15, 16]. Outgrowing EVT cells were isolated from
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placental villi after terminations of pregnancy (7-10 weeks, n=5) and were incubated in Petri
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dishes for two days at 5% CO2 in DMEM/Ham’s F12 with 10% (v/v) human serum. Then the
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villi were transferred into collagen-coated 24-well plates (two explants/well) and incubated at
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2.5% O2, 5% CO2. After three days of culture the villi were removed from the plates and the
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already outgrown cells were cultured for another day at 5% CO2. Human hepatocellular
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carcinoma (HUH-7) cells, human Jeg-3 and human AC1-M59 cells were cultured as described
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[12, 17, 18]. AC1-M59 and Jeg-3 cells were seeded into cell culture dishes and upon reaching
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80% confluence, incubated for 24 h in medium containing 10 ng/ml IL-1α, IL-1β, IL-6, or TNFα
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(R&D Systems, Minneapolis, MN, USA).
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RNA isolation, RT-PCR and real-time RT-PCR (qPCR) analysis
RNA was isolated from (i) AC1-M59, Jeg-3, EVT, HUH-7 (using RNeasy Mini Kit from
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QIAGEN, Hilden, Germany) and (ii) first trimester (week 6-12) and term trophoblast cells (week
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35 and 40) using Trizol-reagent (Sigma-Aldrich, Saint-Louis, MO, USA), followed by DNase
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treatment, reverse transcription and PCR for SAA4 and GAPDH (used as a housekeeping gene,
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see Supplementary Table I) as described [19]. The qPCR protocol was performed using
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LightCycler 480 system (Roche Diagnostics, Vienna, Austria) [20]. Gene specific primers used
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for SAA4 and HPRT (used as a housekeeping gene) are listed in the Supplementary Table II.
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Relative gene expression levels compared to HPRT were calculated using ∆∆CT method.
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Western blot analysis
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Total cellular proteins were isolated [16] and 50 µg of protein (as assessed by the BCA
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method) was subjected to Western-blot analysis as described [19]. Rabbit polyclonal anti-human
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SAA4 antiserum (raised against amino acids 94-112 of human SAA4; dilution 1:300) not cross-
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reacting with human SAA1/2 was used as primary antibody [21].
89 Immunohistochemistry of first trimester placenta
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Placental specimens from elective terminations of pregnancy (7-10 weeks, n=5) were fixed
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in formaldehyde and embedded in paraffin. Sections of 5 µm were dewaxed and stained for
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SAA4 (polyclonal anti-SAA4 peptide antibody [21], dilution 1:500) or HLA-G (monoclonal
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antibody 4H84, 1:2000, BD Biosciences, NJ, USA) using labelled polymer (Thermo Fisher
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Scientific, MA, USA) and 3-amino-9-ethylcarbazole (Dako, Inc., Carpinteria, CA, USA) to detect
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antibody binding [22]. Control experiments were performed using rabbit or mouse non-immune
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serum or IgG.
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Results and discussion
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RT-PCR (Fig. 1A/B) and qPCR analysis (Fig. 1C) reveals that SAA4 mRNA expression
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in AC1-M59 cells as well as in its parental cell line Jeg-3 (Fig. 1A/B) is not altered by IL-1β, IL-
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6, and TNFα treatment. However, IL-1α significantly decreased SAA mRNA expression in both
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cell lines (Fig. 1C). This observation, as well as similar data obtained in non-differentiated human
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mesenchymal stem cells and human osteosarcoma cell lines (using RT-PCR [19]) does not
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confirm the constitutive character of apolipoprotein SAA4 [6, 7] in general.
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High SAA4 mRNA expression in human colon adenocarcinoma [10, 23] and ovarian
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carcinoma [11] suggests a possible role of SAA4 in tumorigenesis and invasion as previously
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reported for SAA1/2 [24, 25]. This seems likely, as the adhesion motif present in the N-terminal
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region of SAA1/2 that mediates binding to extracellular matrix components and cell invasion, is
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highly homologous to that present in SAA4. One of the hallmarks of first trimester placental
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development is invasion of uterine tissue by fetal trophoblast cells. Thus, SAA4 expression was
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studied in invasive trophoblast cells. While expression of SAA4 transcripts is low during early
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placental development (weeks 7-9), expression levels are increased at later first trimester
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pregnancy weeks but decreased at term pregnancy stages (Fig. 1D). Data obtained from qPCR
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analyses (Fig. 1E) confirmed results obtained with RT-PCR (Fig. 1D); highest SAA4 mRNA
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expression was found at week 10 and 12; however, at later pregnancy stages (week 35 and 40)
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SAA4 mRNA levels were similar as observed in early pregnancy weeks. Furthermore, SAA4
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mRNA expression was also found in preparations of isolated outgrowing first trimester EVT cells
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(Fig. 1F).
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Next, we tried to identify SAA4 protein in human placental tissue using an antibody
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(raised against the C-terminal portion [that differs from SAA1/2] of SAA4 [21]). Two
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immunoreactive bands corresponding to approximately 14 kDa (non-glycosylated) and a less
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intensive 19 kDa (glycosylated) protein, as reported for liver-derived human SAA4 [7], were
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identified by Western-blot in outgrowing EVT cells (Fig. 2A). However, no immunoreactive
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signals for SAA4 were detected in AC1-M59 or Jeg-3 cells under non-stimulated or cytokine-
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stimulated conditions (data not shown).
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Immunohistochemical staining revealed the presence of SAA4 in cell columns of first
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trimester placental tissue representing potentially invasive EVT cells (Fig. 2Ba). In the
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proliferative zone of cell columns near the basement membrane towards the villous stroma,
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SAA4 was barely detectable. Interestingly, the more pronounced the invasive character of
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trophoblast cells, the more pronounced is SAA4 expression (Fig. 2Ba). Areas within the decidua,
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which were invaded by trophoblast cells (Fig. 2Ca), show positivity for SAA4 (Fig. 2Cb). SAA4
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expression was detected in different EVT cell populations that had already invaded the decidua
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basalis, such as intramural trophoblast cells replacing the walls of spiral arteries (Fig. 2Cd and e),
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intraluminal trophoblast cells plugging the lumina of spiral arteries (Fig. 2Cd and e), and
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interstitial trophoblast cells (Fig. 2Cg and h), respectively. However, multinucleated trophoblast
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giant cells, a subpopulation representing the terminally differentiated sessile endpoint of invading
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trophoblast cells [26], showed no stained for SAA4 (Fig. 2Ci). Also glandular epithelial cells of
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the decidua were negative for SAA4 (Supplementary Figure I). To verify the trophoblast nature
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of these cells an antibody recognizing HLA-G (expressed only by extravillous trophoblast and
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choriocarcinoma cells) was used on serial sections (Fig. 2Ca, d, and g).
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Here we present first evidence of a pronounced expression of SAA4, so far considered as
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a protein of “unknown” function, in first trimester placental tissues. SAA4 expression in invasive
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trophoblast cells suggests a likely role of this apolipoprotein during invasion. The underlying
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mechanisms as reported for acute-phase SAA [25] are currently under investigation.
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Acknowledgements
Supported by the Austrian Science Fund (FWF, DK-MCD W1226 and SFB-LIPOTOX
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F3007).
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Figure legends
2 Figure 1
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RT-PCR and qPCR analysis for SAA4 mRNA expression in non-stimulated and cytokine-
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stimulated trophoblast cells:
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AC1-M59 and Jeg-3 cells were stimulated with different cytokines (10 ng/ml) for 24 h (A-
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C). RNA was isolated from both cell lines (A-C) as well as from cell preparations from first
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trimester and term trophoblast cells at indicated pregnancy weeks (D, E) as well as outgrowing
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EVT cells from placental villi from first trimester placentas (F). Then RNA was reverse
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transcribed, and cDNA was amplified using specific forward and reverse oligonucleotide primers
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(Supplementary Table I). RT-PCR products were separated on 1.5% agarose gels. GAPDH was
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used as a housekeeping gene.
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qPCR was performed from reverse-transcribed RNA isolated from non-stimulated and
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cytokine-stimulated AC1-M59 and Jeg-3 cells (C) and first trimester and term trophoblast cells
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(E) using specific primers (Supplementary Table II) for SAA4 and HPRT (used as a
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housekeeping gene).
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NS (non-stimulated); P (positive control: RNA was isolated from human hepatocellular
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carcinoma HUH-7 cells, diluted 1:20 in F); N1 (negative control, RNA template: negative
19
controls were done for all samples); N2 (negative control, water template). T = EVT. One
20
representative experiment out of three is shown. Values (C and E) are expressed as mean±SEM
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(n=4). (C) = *p≤0.05 vs. non-stimulated cells; (E) *p≤0.05 vs. first trimester trophoblast cells
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isolated from pregnancy week 6 (set as 1; x-fold expression of SAA4/HPRT mRNA is given on
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the Y-axis in (C) and (E)).
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Figure 2
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SAA4 protein expression in trophoblasts cells from first trimester and term placenta: (A) Outgrowing EVT cells were isolated, lysed and 50 µg of total protein was subjected
28
to Western blot analysis using anti-human SAA4 as primary antibody [21]. As a positive control
29
(P) human non-glycosylated SAA4 ([expressed in E. coli] containing an N-terminal His6-tag and
30
an enterokinase cleavage site [21]) was detected as a monomeric (approximately 15.5 kDa) or
31
dimeric (31 kDa) protein. One representative Western-blot is shown.
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(B) Paraffin sections of placental specimens from first trimester placental villi and (C)
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trophoblast-invaded decidua basalis were stained with anti-human SAA4 antiserum (Ba, Cb,e,h,i)
34
or with anti-human HLA-G antibody (Ca,d,g) as a marker for trophoblast cells. Control
35
experiments were performed with non-immune rabbit IgG (Bb and Cc) or non-immune mouse
36
IgG (Cf). To detect antibody binding labelled polymer and 3-amino-9-ethylcarbazole was used.
37
The indicated areas in (Ca) and (Cb) (demonstrating part of trophoblast-invaded spiral artery) are
38
shown at higher magnification as (Cd) and (Ce). Arrows in (Cd) and (Ce) point towards
39
intraluminal trophoblast cells while arrowheads point towards intramural trophoblast cells
40
positive for SAA4. Arrows in (Cg) and (Cf) point towards interstitial trophoblast cells positive
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for SAA4. The arrowheads in (Ci) represent multinucleated trophoblast giant cells negative for
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SAA4. The respective bar size is indicated in (B) and (C).
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Highlights
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• SAA4 mRNA is expressed in Jeg-3 and AC1-M59 cells • SAA4 mRNA is expressed in first trimester/term trophoblast cells • SAA4 mRNA is upregulated at pregnancy week 10 and 12
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• SAA4 protein is present in interstitial, intramural and intraluminal trophoblast cells
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C. ROSSMANN, A. HAMMER, C.N. KOYANI, A. KOVACEVIC, M. SIWETZ, G. DESOYE, T.G. POEHLMANN, U.R. MARKERT, B. HUPPERTZ, W. SATTLER, and E. MALLE
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The authors have no conflict of interest
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Online Supplement
Expression of serum amyloid A4 in human trophoblast-like choriocarcinoma
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cell lines and human first trimester/term trophoblast cells
C. ROSSMANN, A. HAMMER, C.N. KOYANI, A. KOVACEVIC, M. SIWETZ, G. DESOYE,
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Supplementary Table I
and annealing temperature)
Primers (start position on + strand)
Accession Nr. F 5’ CCA GTG AAA GCT GGC GTT CG 3’
NM_006512
R 5’ GAG AAG TGT GTG GCT CAC AGC C 3’
GAPDH
F 5’ ACA GTC CAT GCC ATC ACT GCC 3’
M17851
R 5’ GCC TGC TTC ACC ACC TTC TTG 3’
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SAA4
bp
cycles
°C
References
40
55
[Kovacevic et
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Gene
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RT-PCR primers, expected amplicon size (bp), and unique PCR properties (cycle number
30
58
[Iochmann et al., 1999]
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al., 2008]
For methods see:
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Kovacevic A, Hammer A, Stadelmeyer E, Windischhofer W, Sundl M, Ray A, Schweighofer N, Friedl G, Windhager R, Sattler W, Malle E. 2008. Expression of serum amyloid A
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transcripts in human bone tissues, differentiated osteoblast-like stem cells and human osteosarcoma cell lines. J Cell Biochem 103:994-1004.
Iochmann S, Reverdiau-Moalic P, Beaujean S, Rideau E, Lebranchu Y, Bardos P, Gruel Y. 1999. Fast detection of tissue factor and tissue factor pathway inhibitor messenger RNA in endothelial cells and monocytes by sensitive reverse transcription-polymerase chain reaction. Thromb Res 94:165-73.
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Supplementary Table II
Gene
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qPCR primers and amplicon size (bp)
Primers (strand)
bp
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Accession Nr. F 5’ TCA GCC GTT CCA GGG TCT AT 3’
NM_006512
R 5’ ATT CCT CAG CTT TCT CGT TGG 3’
HPRT
F 5’ CCT GGC GTC GTG ATT AGT GAT 3’
NM_000194
R 5’ AGA CGT TCA GTC CTG TCC ATA A 3’
M AN U
SAA4
105
131
TE D
For methods see:
Rossmann R, Rauh A, Hammer A, Windischhofer W, Zirkl S, Sattler W, Malle E. 2011. Hypochlorite-modified high-density lipoprotein promotes induction of HO-1 in
EP
endothelial cells via activation of p42/44 MAPK and zinc finger transcription factor Egr-
AC C
1. Arch Biochem Biophys 509:16-25.
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M AN U
SC
RI PT
Supplementary Figure I
One representative Paraffin section of a specimen from trophoblast-invaded decidua basalis was
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stained for EVT cells using an antibody against HLA-G (a) or for SAA4 using an antibody raised against the C-terminal portion of human SAA4 (b).
EP
Arrows = Interstitial trophoblast cells stained for HLA-G and SAA4
AC C
Arrowheads = glandular epithelial cells negative for HLA-G and SAA4
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