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Caspase-14 expression in the human placenta
RBMOnline - Vol 11. No 2. 2005 236-243 Reproductive BioMedicine Online; www.rbmonline.com/Article/1822 on web 23 June 2005
Article Caspase-14 expression in the human placenta Adrian Charles is a paediatric and perinatal pathologist. While studying Natural Sciences at Cambridge University, UK, he enjoyed pathology, and this led him to train in medicine at Cambridge. After time in general medicine and paediatrics he trained in pathology, also at Cambridge. He then studied with Professor Berry in paediatric and perinatal pathology at Bristol, UK, and also undertook full time research in the molecular pathology of paediatric renal tumours leading to a MD at Cambridge. Since 1999 he has been in Perth, West Australia, developing research and clinical interests in stillbirths and the placenta.
Dr Adrian Charles Daniel WR Kam1, Adrian K Charles2, Arun M Dharmarajan1,3 School of Anatomy and Human Biology, Faculty of Life and Physical Sciences, University of Western Australia, 35 Stirling Highway Crawley, Perth, Western Australia 6009; 2King Edward Memorial Hospital for Women (KEMH), 374 Bagot Road, Subiaco, Western Australia 6008. 3 Correspondence: Tel: +61 8 64882981; Fax: +61 8 64881051; e-mail: [email protected]
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Abstract Caspase-14 is involved in epidermal differentiation, and previous studies demonstrated abundant expression in the skin. However, the expression of caspase-14 in the human placenta has not been reported. The aims of this study were to determine whether caspase-14 is expressed in the first trimester and term human placenta, and if it is associated with apoptosis in the placenta. Caspase-14 is expressed in the trophoblast cells, and in lower amounts in the mesenchyme. Western blot analysis demonstrated increased expression in the first trimester compared with term placentae. Immunohistochemistry for caspase-14 showed diffuse expression in the trophoblast layer, and not only in occasional cells that are identified by TUNEL staining. Using an explant model in which apoptosis was inhibited with superoxide dismutase (SOD), no significant differences in caspase-14 protein concentrations were seen with differing levels of apoptosis. Caspase-14 is present in the human placenta, primarily in the trophoblast, but its function is not clear, and appears not to be related purely to apoptosis. Keywords: apoptosis, caspase, caspase-14, cell death, human, placenta
Introduction The human placenta performs a variety of functions to ensure a protected environment for the development of the fetus inside the mother. It regulates the respiratory and nutrient exchange between the mother and fetus, as well as serving as an important endocrine organ that synthesizes various hormones and proteins. The syncytiotrophoblast, the outermost layer of the placenta, is a multinucleated cell layer that forms the principal interface between the maternal circulation and fetal/placental tissues.
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Placental apoptosis is a normal physiological process occurring throughout gestation, and occurs in all placental cell types, increasing significantly from the first to the third trimester (Nelson, 1996; Smith et al., 1997). Apoptosis has a critical role in placental development, including interstitial and endovascular invasion (Chan et al., 1999), maternal immune tolerance (Chan et al., 1999), and placental trophoblast turnover (Smith et al., 1997). One of the major apoptotic pathways is through activation of the caspases. Caspase-14 is the newest member identified in the caspase family (van de Craen et al., 1999). Ahmad et al.
(1998) suggested that caspase-14 might play a role in mouse death receptor and granzyme B-induced apoptosis; however, no human caspase-14 analogues of this function have yet been identified, though a role as a cytokine activator in humans has been suggested (Mikolajczyk et al., 2004). Caspase-14 is found abundantly in mammalian skin. In human skin, the caspase-14 protein is localized to the differentiating keratinocytes of the skin, supporting a role of caspase-14 in epidermal barrier formation (Chien et al., 2002). In addition, caspase-14 mRNA is expressed only in the uppermost layer of living epidermal cells, including the granular layer, hair follicles and sebaceous glands, suggesting a wider role than a proapoptotic gene (Eckhart et al., 2000). Caspase-14 is also expressed in the epithelium of the human choroid plexus, retinal pigment and in the thymic Hassall’s bodies. In the choroid plexus, caspase-14 protein is expressed in the cells that facilitate the transfer of molecules between the blood and the cerebrospinal fluid (CSF), thus forming part of the blood–CSF barrier (Lippens et al., 2003). Caspase-14 is also
Article - Caspase-14 in the human placenta - DWR Kam et al.
expressed in the skin of the embryonic and adult mouse (Kuechle et al., 2001). To date, a detailed study of caspase-14 in the human placenta has not been reported. This study examined the expression of caspase14 in the human placenta, and related the expression to apoptotic cells. Using an in-vitro model of apoptosis in first trimester villi, correlation of expression with apoptosis was examined.
Materials and methods Tissue collection This study was approved by the ethics committee of King Edward Memorial Hospital (KEMH). First trimester placentae (8–12 weeks of gestation) were collected from legally approved terminations of pregnancy, while term placentae (38–41 weeks of gestation) were collected from uncomplicated deliveries via Caesarean and vaginal delivery. For the first part of the study, these placental samples were obtained within 30 min of delivery or termination and were immediately snap frozen in liquid nitrogen and stored at –80°C, while a separate portion was fixed overnight in 10% neutral buffered formalin (NBF) for immunocytochemical analysis. In the explant study, fresh samples of placenta were transferred onto ice and delivered to the laboratory within 30 min. Human skin for positive control for caspase-14 was obtained from surgical material.
Tissue sectioning All human placenta and human skin tissue sections were cut uniformly at 5 μm thickness and mounted on silanated slides. Pre-embedded human skin for microscopic analysis was provided by Dr Diane Keeney (Vanderbilt University, Tennessee, USA). Sections were stained in haematoxylin and eosin using routine histological techniques to illustrate tissue morphology in the specimens.
Primary antibody The polyclonal caspase-14 antibody was provided by Dr Wim Declercq (University of Ghent, Belgium) and was used for both the immunocytochemistry and Western blot analysis. This antibody has previously been used to detect caspase-14 expression in other tissues (Lippens et al., 2003).
Immunohistochemistry Immunocytochemistry was conducted on normal placental tissue from first trimester (8–12 weeks of gestation, n = 5) and from term placentae (38–41 weeks of gestation, n = 4). Negative controls were performed on the human placenta and skin, in the absence of the caspase-14 primary antibody. All reagents were diluted in Tris-buffered saline solution (TBS; 0.1 mol/l Tris and 0.15 mol/l NaCl) made up with 0.025% of Tween-20 (TBSTT) and 10% goat serum (Hunter Antisera, Jesmond, NSW, Australia). Slides were heated to 60°C for 15 min and were de-waxed in two washes of absolute toluene for 5 min each, followed by rehydration from 100% ethanol to double distilled water (DDW). After de-paraffinization, the tissue sections were equilibrated in TBS for 5 min. The slides were then rinsed in
DDW and subjected to antigen retrieval in a sodium citrate buffer (10 mmol/l; pH 6.0) at 90°C for 20 min. Afterwards, the slides were cooled for 20 min at room temperature and rinsed three times in TBS, for 5 min each. Excess liquid was removed and non-specific binding of the antibodies was blocked by incubating the sections in 20% goat serum (Hunter Antisera) made in TBS-T for 1 h at room temperature. This was followed by three washes of TBS for 5 min each. After blocking, the caspase-14 antibody was applied overnight at 4°C at a 1:100 dilution, followed by three washes in TBS for 5 min each. Excess liquid was removed and the secondary antibody, biotinylated goat anti-rabbit, was applied at a 1:200 dilution and incubated for 1 h at room temperature. After incubation, excess secondary antibody was rinsed off in three washes of TBS for 5 min each. In the third step, streptavidin labelled with Alexa Fluor 546 dye (BD Biosciences, North Ryde, NSW, Australia) and DAPI were used together at a 1:100 dilution. The sections were incubated in these tertiary reagents for 1 h at room temperature, followed by another set of three TBS washes for 5 min each. To preserve the longevity of the fluorescent dyes, DAKO fluorescent mounting medium (Dako Cytomation, Botany, Australia) was applied to the sections. The slides were cover-slipped, sealed with nail polish and stored in aluminium foil at 4°C until viewing with fluorescence microscopy.
TUNEL For the staining of apoptotic cell nuclei, the ApopTag® Plus Peroxidase In-Situ Apoptosis Detection Kit (Intergen Company, New York, NY, USA) was used, as previously described (Berg et al., 2002; Lareu et al., 2003). The procedure was according to the protocol for formalin fixed, paraffin embedded tissue, using a 10 min incubation at room temperature for proteinase K digestion (20 μg/ml). Cellular nuclei were counter stained with methyl green. Sections from five first trimester and four term placentae were examined. A negative control was performed by omitting the terminal transferase enzyme from the labelling mixture. A 4-day post-weaning rat mammary gland was used as the positive control included in the ApopTag Plus Kit. Mature nulliparous Wistar rats were obtained from the Animal Resource Centre (Murdoch, Australia).
Microscopy and imaging Haematoxylin and eosin and TUNEL slides were viewed with a Leica DMRBE microscope. Images were captured using a Nikon DXM1200F digital camera with ACT-1 v2 (Nikon, Garden City, NY, USA) software. Immunostaining was viewed with a Leica DMRBE microscope and images were captured with a black and white digital camera (CCD Cool SNAP ES camera; Photometrics, Huntington Beach, CA, USA). These images were then pseudocoloured using the V++ v4.0 software.
Western blot analysis Western blot analysis was performed on five first trimester and four term placental tissues. Tissues were homogenized in sodium phosphate buffer (10 mmol/l, pH 7) containing sucrose (0.25 mol/ l), EDTA (1 mmol/l), PMSF (1 mmol/l) and trypsin inhibitor (100 mg/ml). Protein concentration of homogenates was measured (Bradford, 1976) and 30 μg resolved by 12% SDS-PAGE and
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Article - Caspase-14 in the human placenta - DWR Kam et al. transferred to Hybond-C nitrocellulose membrane (Amersham, Baulkham Hills, NSW, Australia). Membranes were blocked in 5% non-fat milk in Tris-buffered saline/0.1% Tween-20 (TBST) for 1 h at room temperature. Membranes were then separately probed with antibodies against caspase-14 at a dilution of 1:1000 in TBST for 1–2 h at room temperature. Following primary incubation, membranes were washed in TBST and incubated with a secondary antibody at a dilution of 1:20,000, followed by streptavidin–horseradish peroxidase (Roche Diagnostics, Castle Hill, NSW, Australia) at a dilution of 1:20,000 for 1 h at room temperature. Protein signals were detected by enhanced chemiluminescence (Supersignal West Pico ECL substrate; Pierce product from Australian Biosearch, Karrinyup, Western Australia) and exposed to autoradiographic film (Kodak XAR-5). The negative control consisted of omitting the primary antibody. A human skin sample was included on each gel to standardize chemiluminescence levels and exposure times. Equal sample loading was assessed by staining membranes with Ponceau S.
RNA extraction Snap-frozen placentae were homogenized and total RNA was isolated with the NucleoSpin Spin RNA II Kit (Clontech; BD Biosciences). RNA samples were resuspended in RNasefree water and stored at –80°C until used. RNA integrity was confirmed by examining the presence of the ribosomal 28S and 18S bands. RNA quantitation and purity were determined with a Beckman DU 640 spectrophotometer.
Reverse transcription–polymerase chain reaction (RT–PCR) Complementary DNA was generated from total RNA with reverse transcriptase (Promega, Delta House, Southampton, UK) as per manufacturer’s protocol. RT–PCR was performed using 1 μg of total RNA, with the volume made up to 13.5 μl with nuclease-free water. Random hexameric primers (0.5 μl, 500 ng/ μl; hexadeoxyribonucleotides, Promega) were added, secondary RNA structures were removed by heat denaturation at 70°C for 5 min, followed by incubation of the samples on ice for 5 min. The samples were briefly centrifuged and the reagents added; 5 μl 5× MMLV Reaction Buffer (Promega), 2.6 μl of 5 mmol/ l dNTPs, 1 μl MMLV-Reverse transcriptase enzyme RNase H Minus, Point Mutant (Promega), 1 μl RNasin (Promega), and 1.9 μl nuclease-free water. These samples were then mixed and reverse transcribed using a PTC-100 Peltier thermocycler (MJ Research Inc., Geneworks Pty Ltd., Adelaide, South Australia) at 25°C for 10 min, 55°C for 50 min, and at 70°C for 15 min. The reverse transcribed cDNA was purified through spin columns (Ultra Clean GelSpin Kit; GeneWorks) and eluted out in 50 μl 10 mmol/l Tris, pH 8.
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Caspase-14 mRNA expression was examined using the Rotor Gene RG-3000 (Corbett Research Inc., Mortlake, Sydney, NSW, Australia) with the fluorescent dye, SYBR Green I (Fisher Biotech, Subiaco, Western Australia). PCR primers that correspond to the entire open reading frame of human caspase-14 [10] generated a 728 bp fragment: forward 5′-ATGAGCAATCCGCGGTCTTT3′ and reverse 5′-CTACTGCAGATACAGCCGTT-3′. Reaction conditions were as per manufacturer’s protocol with optimization of primers and magnesium chloride (MgCl2) concentrations and annealing temperature. One-tenth volume of target cDNA and
master premix were incubated with 3 mmol/l MgCl2 and 0.4 pmol/μl of each primer. The reaction run programme was as follows: an initial denaturation step at 95°C for 10 min; 40 cycles at 95°C for 15 s, 55°C for 7 s and 72°C for 14 s, followed by a fluorescence measurement after each cycle. A melt curve was performed with continuous fluorescence measurement between 70 and 90°C. Heating rate was set at 0.1°C/s.
Explant culture Five fresh first trimester placental villi were snap-frozen (0 h) with RNAlater™ (Sigma-Aldrich, Castle Hill, NSW, Australia) for RNA extraction and without RNAlater™ for protein extraction. For the culture experiment, triplicate sets of fresh placental villi were placed in sterile 20-ml glass scintillation vials containing 2 ml minimum essential medium (MEM) with Earle’s salts (Gibco product from Invitrogen, Mulgrave Victoria, Australia), supplemented with 2 mmol/l L-glutamine (Gibco), 100 IU/ml of both penicillin and streptomycin (Gibco), and 0.1% fatty acidfree BSA (fraction V; Sigma, Poole, Dorset, UK). Incubations were performed at 37°C for 4 h under both presence and absence of superoxide dismutase (SOD, 75 IU/ml; Sigma). This SOD concentration has been shown to significantly reduce DNA fragmentation in the human placenta, a classical marker for the end stage of apoptosis (Rao et al., 2000). A 4-h incubation at 37°C was performed under gas conditions of 95% O2 and 5% CO2. After the incubation, the placentae were collected and snapfrozen. For each sample, fresh tissue prior to the experiment (time zero) was used to normalize the values. Samples were stored at –80˚C for Western blot analysis.
Statistical analysis All statistical analysis was carried out using SPSS v11.0.0 (Chicago, IL, USA), while the graphs were plotted using Microsoft Excel X for Mac. Tests of normality also demonstrated significance. Twotailed t-tests (paired and independent) were conducted to analyse the data. All error bars are based on the SEM.
Results The histological sections confirmed that the specimens contained only placental villi, and these demonstrated either the morphological characteristics of the first trimester or term placental villi. Caspase-14 was localized in the first trimester and the term placentae. It was located in the trophoblast, in the first trimester and the term placenta (Figures 1b, c). Some staining of the mesenchymal cells was also seen in the first trimester. There was autofluorescence of red blood cells (Figure 1c). Human skin was used as the positive control and showed the pattern of staining as previously described predominantly in the upper epidermis (Figure 1a) (Eckhart et al., 2000; Pistritto et al., 2002). TUNEL staining demonstrated positive staining of a few cells in the first trimester and the term placentae. These included nuclei in the trophoblast as well as the occasional mesenchymal cell, in both the first trimester and term placenta (Figure 2). The expression of caspase-14 protein was confirmed by Western blot analysis. The presence of a second band with a more mobile transcript was evident in the placental samples compared with the skin (Figure 3a). The results indicated that caspase-14 protein
Article - Caspase-14 in the human placenta - DWR Kam et al. is more abundant in the first trimester than the term placenta (Figure 3b). The second band suggests that there may be a splice variant(s), or post-translational modification. No significant amount of cleaved caspase-14 is identified in the placenta, unlike the skin (see Discussion). RT–PCR resulted in the amplification of a product of the correct size (728 bp) on the agarose gel electrophoresis (Figure 4). The product had a peak melting point of 92.5°C and was verified as
caspase-14 by DNA sequencing (data not shown). The relationship between caspase-14 and apoptosis was explored further using fresh first trimester villi in the explant SOD model. There was no correlation between caspase-14 expression and apoptosis. Uncleaved caspase-14 protein was again the predominant form, with also a similar duplex band as seen in the non-cultured placenta (Figure 5).
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Figure 1. Caspase-14 immunofluorescence. Caspase-14, antibody signal red, with 4,6diamidino-2-phenylindole counterstaining nuclei blue. (a) Human skin (×200). The caspase-14 immunostaining is localized to the superficial layers (SE) of the epidermis (EP). The dermis (DE) is contains some blood vessels. (b) First trimester placenta (×400). Caspase-14 is identified in the villi and localized to the trophoblast (TB). Little is seen in the mesenchymal tissue (M). (c) Term placentae (×400). The caspase-14 is found in the trophoblast layer (TB). Little is seen in the stromal tissues. The red blood cells show strong autofluorescence.
Figure 2. TUNEL assay. TUNEL-positive cells are stained brown, with methyl green counterstain of healthy nuclei. First trimester placenta with occasional positive nuclei in the syncytiotrophoblast layer and one in the stroma (×400).
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Article - Caspase-14 in the human placenta - DWR Kam et al.
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Figure 3. (a) Western blot analysis of caspase-14 in the human placenta. The large band (~29 kDa) represents the procaspase14 protein, while the lower band, observed in the human skin, is the activated caspase-14 protein (~20 kDa) that results from proteolytic cleavage. The presence of the dual bands at ~29 kDa is discussed in the text. (b) A comparison of caspase-14 expression in the first trimester and term placentae. An independent samples t-test demonstrated a significant difference in caspase-14 protein expression between the first trimester and term placentae (*P < 0.05).
Figure 4. Caspase-14 reverse transcriptase-polymerase chain reaction. Microgel DNA molecular weight ladder (lane 1). The amplified caspase14 product (728 bp) is shown in the keratinocyte culture (lane 2) and the human placenta (lane 3).
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Article - Caspase-14 in the human placenta - DWR Kam et al.
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Figure 5. (a) Western blot analysis of caspase-14 in the superoxide dismutase (SOD) and control treatment groups. (b) Quantification. There was no protein variation observed for caspase-14 before or after the 4-h experiment. In addition, a paired-samples t-test (two-tailed) indicated no significant difference of caspase-14 expression between the SOD and control treatments. (c) Inhibition of internucleosomal DNA fragmentation with SOD treatment in vitro (* denotes significant difference between 4-h control and 4-h SOD, P = 0.001). Upper panel: gel showing the fragmentation at time 0, 4-h control, and after 4-h incubation with SOD. Lower panel: comparison of densitometric measurements of the three groups showing a significant difference (adapted from Charles, 2005).
Discussion Caspase-14 is the most recently identified member of the caspase family (van de Craen et al., 1998). The exact function of this gene has not been discovered and it has not previously been examined in the placenta. Hence, the objective of this study was to detect caspase-14 in the human placenta, determine its expression pattern and investigate if this is related to cells undergoing apoptosis. There was more caspase-14 protein in the first trimester than the term placentae as found by Western blot analysis, and similar results were observed by immunohistochemistry. Compared with the first trimester, the ubiquitous cytotrophoblast layer found in the early placental villi covers of only 20% of term placental villi (Benirschke and Kaufmann, 1999). This antibody has been previously used to successfully detect caspase-14 in the human skin, and in the epithelium of the retinal pigment, thymic Hassall’s bodies and in the ependymal lining of the choroid plexus (Lippens et al., 2003). The presence of caspase-14 protein in the syncytiotrophoblast and cytotrophoblast in similar concentrations suggests that the protein in the syncytial layer is derived from the incorporation of cytotrophoblast cytoplasm into the syncytium. In the human skin, caspase-14 synthesis is restricted to the differentiating keratinocytes. As differentiation proceeds, the nuclei of terminally differentiating keratinocytes become degraded and the cornified layers are formed. These de-nucleated cells, now known as corneocytes, form the protective layer of the skin (stratum corneum) and these de-nucleated cells still contained caspase-14 protein (Lippens et al., 2003). In the placenta, the syncytium needs a constant supply of RNA from the cytotrophoblast cells for growth and maintenance of its metabolic and structural integrity (Benirschke and Kaufmann, 1999). It is likely that through this fusion process, the caspase-
14 protein expressed in the cytotrophoblast would be transferred to the syncytium. Caspase-14 was also detected in the mesenchyme of the first trimester placentae, but rarely in the term placentae. Mesenchymal cells are present in the connective tissue within the villi (Benirschke and Kaufmann, 1999). By term, most of the villi are developed and do not have proliferating mesenchymal cells. TUNEL staining demonstrated that all cell types in the human placenta underwent cell death, although the overall number of TUNEL-positive cells was low. These findings were similar to previous reports (Smith et al., 1997; Austgulen et al., 2002). As expected, small groups of nuclei located in the syncytiotrophoblast were TUNEL positive. It is unlikely that caspase-14 has a role in the end stage of apoptosis, as caspase14 was localized throughout the trophoblast cell layer and not confined to the few dying nuclei demonstrated by TUNEL, unless it is stored and only activated by the few nuclei in the syncytium undergoing apoptosis. Huppertz et al. (1999) suggested that early apoptotic processes occur in the cytotrophoblast, promoting its fusion into the syncytium. Assuming that caspase-14 has a role in apoptosis, the localization of caspase-14 to the cytotrophoblast as well as the syncytiotrophoblast may indicate a role for caspase-14 in the early apoptotic cascade in syncytial formation in the human placenta. The pattern of expression of caspase-14 in the human skin raises the suggestion that there is a modification of the normal apoptotic process in certain cell types such as the epidermis, where there is a loss of nuclei, but preservation of the cytoplasm. To date, human caspase-14 involvement in apoptosis has not been identified.
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Article - Caspase-14 in the human placenta - DWR Kam et al. Western blot analysis showed that the first trimester and term placenta both contained the caspase-14 protein, predominantly in the uncleaved state. An interesting finding was the presence of a second band a slightly less than the expected size compared with the human skin tissues. These appearances suggest there are differences in the post-translational modification(s) of the protein between skin and the placenta, or that there are splice variants between the two tissues.
The lack of caspase-14 variation in the first trimester and term placentae, as well as in the explant model, suggest that caspase14 has a separate, non-apoptotic role in the cytotrophoblast. A non-apoptotic role for caspase-14 has been suggested in the skin (Mikolajczyk et al., 2004). Caspase-14 may be involved in a specialized form of apoptosis, which is believed to occur when cytotrophoblast cells fuse and are incorporated into the syncytium.
Another observation is that little cleaved, activated caspase14 was detected. A reason why activated caspase-14 may not be seen is if cleavage occurs in the syncytial knots, which are normally shed into the maternal circulation. However, cleaved caspase-14 was not observed in the explant model, where any debris would be contained within the supernatant. Hence, it is unlikely that caspase-14 is cleaved in the human placenta, in either the normal physiological state or in the experimental conditions of apoptosis inducement and inhibition.
Acknowledgements
The SOD explant model has proved to be very useful for examining the molecular signalling pathways involved in placental apoptosis. Under defined experimental conditions, DNA fragmentation was significantly reduced with SOD treatment (Rao et al., 2000; Charles et al., 2005 Figure 1). This study also demonstrated a significant reduction of DNA fragmentation with SOD (data not shown) and an up-regulation of caspase-3 in the 4-h control group, but not in the 4-h SOD group. The data revealed SOD as a potent anti-apoptotic agent that inhibits early caspase-mediated apoptosis in the human placenta, with the treatment group demonstrating significant reductions in DNA fragmentation, as well as increase in the ratio of the anti-apoptotic genes compared with the proapoptotic genes (Charles et al., 2005). Using the explant model, no significant differences in caspase-14 protein expression were evident between the 4-h SOD and 4-h control treatment groups. There was no cleavage of caspase-14 in the cultured samples. This data collectively implies that caspase-14 is not activated and does not play a role in the apoptotic pathway in this model. In addition, Western blot analysis and immunocytochemistry revealed caspase-14 was expressed less in all human placental specimens compared with the human skin. Many tissues that express caspase-14 have a function involving barrier formation. The skin is the first line of defence against environmental conditions and pathogen entry. In the choroid plexus, the ependymal layer forms the epithelium lining that constitutes the blood–brain barrier. Caspase-14 expression was also observed to multiple cell lines derived from simple epithelia of breast, prostate and stomach (Pistritto et al., 2002). In the human placenta, caspase-14 was located in the syncytiotrophoblast, which forms the physical barrier between maternal and fetal tissues. The results of this study indicate that caspase-14 may have a role in the trophoblast homologous to its role in epidermis.
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This study is the first to demonstrate the expression of caspase14 in the human placenta. The present study located caspase14 protein and mRNA in the human placenta, and found that caspase-14 is not exclusively localized to cells undergoing apoptosis. Caspase-14 was also predominantly expressed in the uncleaved state, whereby caspases are usually activated by cleavage.
The caspase-14 antibody was kindly provided by Dr Wim Declercq (Department of Molecular Biology, University of Gent, Belgium). Dr Luis Filgueira (School of Anatomy and Human Biology, UWA) provided the immunofluorescence reagents, and Dr Diane Kenney (Vanderbilt University, Nashville) provided the human abdominal skin (embedded) and keratinocyte cDNA. We thank Dr Jan Dickinson (Maternal Fetal Medicine, KEMH) for assistance in obtaining the placentas, and Dr Fiona Wood (Clinical Cell Culture, Perth) for providing the human skin biopsy. This study was supported by grants from the Western Australian Institute for Medical Research, Women and Infants Research Foundation of Western Australia and University of Western Australia small grant scheme (AC and AD) and the Raine Medical Research Foundation, Perth, Western Australia. We thank Ms Sue Hisheh for technical assistance.
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Article - Caspase-14 in the human placenta - DWR Kam et al. Lareu RR, Lacher MD, Bradley CK et al. 2003 Regulated expresion of inhibitor of apoptosis protein 3 in the rat corpus luteum. Biology of Reproduction 68, 2232–2240. Lippens S, Van den Broecke C, Damme EV et al. 2003 Caspase14 is expressed in the epidermis, the choroid plexus, the retinal pigment epithelium and thymic Hassall’s bodies. Cell Death and Differentiation 7, 1218–1224. Mikolajczyk J, Scott FL, Krajewski S et al. 2004 Activation and substrate specificity of caspase-14. Biochemistry 43, 10560–10569. Nelson DM 1996 Apoptotic changes occur in syncytiotrophoblast of human placental villi where fibrin type fibrinoid is deposited at discontinuities in the villous trophoblast. Placenta 17, 387–391. Pistritto G, Jost M, Srinivasula SM et al. 2002 Expression and transcriptional regulation of caspase-14 in simple and complex epithelia. Cell Death and Differentiation 9, 995–1006. Rao RM, Dharmarajan AM, Rao AJ 2000. Cloning and
characterization of an apoptosis-associated gene in the human placenta. Apoptosis 5, 53–60. Smith SC, Baker PN, Symonds EM 1997 Placental apoptosis in normal human pregnancy. American Journal of Obstetrics and Gynaecology 177, 57–65. Van de Craen M, Loo GV, Pype S et al. 1998 Identification of a new caspase homologue: caspase-14. Cell Death and Differentiation 5, 838–846. Van de Craen M, Declercq W, Van den brande I et al. 1999 The proteolytic procaspase activation network: an in vitro analysis. Cell Death and Differentiation 6, 1117–1124. Received 27 January 2005; resubmitted and refereed 19 April 2005; accepted 20 May 2005.
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Article Caspase-14 expression in the human placenta Adrian Charles is a paediatric and perinatal pathologist. While studying Natural Sciences at Cambridge University, UK, he enjoyed pathology, and this led him to train in medicine at Cambridge. After time in general medicine and paediatrics he trained in pathology, also at Cambridge. He then studied with Professor Berry in paediatric and perinatal pathology at Bristol, UK, and also undertook full time research in the molecular pathology of paediatric renal tumours leading to a MD at Cambridge. Since 1999 he has been in Perth, West Australia, developing research and clinical interests in stillbirths and the placenta.
Dr Adrian Charles Daniel WR Kam1, Adrian K Charles2, Arun M Dharmarajan1,3 School of Anatomy and Human Biology, Faculty of Life and Physical Sciences, University of Western Australia, 35 Stirling Highway Crawley, Perth, Western Australia 6009; 2King Edward Memorial Hospital for Women (KEMH), 374 Bagot Road, Subiaco, Western Australia 6008. 3 Correspondence: Tel: +61 8 64882981; Fax: +61 8 64881051; e-mail: [email protected]
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Abstract Caspase-14 is involved in epidermal differentiation, and previous studies demonstrated abundant expression in the skin. However, the expression of caspase-14 in the human placenta has not been reported. The aims of this study were to determine whether caspase-14 is expressed in the first trimester and term human placenta, and if it is associated with apoptosis in the placenta. Caspase-14 is expressed in the trophoblast cells, and in lower amounts in the mesenchyme. Western blot analysis demonstrated increased expression in the first trimester compared with term placentae. Immunohistochemistry for caspase-14 showed diffuse expression in the trophoblast layer, and not only in occasional cells that are identified by TUNEL staining. Using an explant model in which apoptosis was inhibited with superoxide dismutase (SOD), no significant differences in caspase-14 protein concentrations were seen with differing levels of apoptosis. Caspase-14 is present in the human placenta, primarily in the trophoblast, but its function is not clear, and appears not to be related purely to apoptosis. Keywords: apoptosis, caspase, caspase-14, cell death, human, placenta
Introduction The human placenta performs a variety of functions to ensure a protected environment for the development of the fetus inside the mother. It regulates the respiratory and nutrient exchange between the mother and fetus, as well as serving as an important endocrine organ that synthesizes various hormones and proteins. The syncytiotrophoblast, the outermost layer of the placenta, is a multinucleated cell layer that forms the principal interface between the maternal circulation and fetal/placental tissues.
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Placental apoptosis is a normal physiological process occurring throughout gestation, and occurs in all placental cell types, increasing significantly from the first to the third trimester (Nelson, 1996; Smith et al., 1997). Apoptosis has a critical role in placental development, including interstitial and endovascular invasion (Chan et al., 1999), maternal immune tolerance (Chan et al., 1999), and placental trophoblast turnover (Smith et al., 1997). One of the major apoptotic pathways is through activation of the caspases. Caspase-14 is the newest member identified in the caspase family (van de Craen et al., 1999). Ahmad et al.
(1998) suggested that caspase-14 might play a role in mouse death receptor and granzyme B-induced apoptosis; however, no human caspase-14 analogues of this function have yet been identified, though a role as a cytokine activator in humans has been suggested (Mikolajczyk et al., 2004). Caspase-14 is found abundantly in mammalian skin. In human skin, the caspase-14 protein is localized to the differentiating keratinocytes of the skin, supporting a role of caspase-14 in epidermal barrier formation (Chien et al., 2002). In addition, caspase-14 mRNA is expressed only in the uppermost layer of living epidermal cells, including the granular layer, hair follicles and sebaceous glands, suggesting a wider role than a proapoptotic gene (Eckhart et al., 2000). Caspase-14 is also expressed in the epithelium of the human choroid plexus, retinal pigment and in the thymic Hassall’s bodies. In the choroid plexus, caspase-14 protein is expressed in the cells that facilitate the transfer of molecules between the blood and the cerebrospinal fluid (CSF), thus forming part of the blood–CSF barrier (Lippens et al., 2003). Caspase-14 is also
Article - Caspase-14 in the human placenta - DWR Kam et al.
expressed in the skin of the embryonic and adult mouse (Kuechle et al., 2001). To date, a detailed study of caspase-14 in the human placenta has not been reported. This study examined the expression of caspase14 in the human placenta, and related the expression to apoptotic cells. Using an in-vitro model of apoptosis in first trimester villi, correlation of expression with apoptosis was examined.
Materials and methods Tissue collection This study was approved by the ethics committee of King Edward Memorial Hospital (KEMH). First trimester placentae (8–12 weeks of gestation) were collected from legally approved terminations of pregnancy, while term placentae (38–41 weeks of gestation) were collected from uncomplicated deliveries via Caesarean and vaginal delivery. For the first part of the study, these placental samples were obtained within 30 min of delivery or termination and were immediately snap frozen in liquid nitrogen and stored at –80°C, while a separate portion was fixed overnight in 10% neutral buffered formalin (NBF) for immunocytochemical analysis. In the explant study, fresh samples of placenta were transferred onto ice and delivered to the laboratory within 30 min. Human skin for positive control for caspase-14 was obtained from surgical material.
Tissue sectioning All human placenta and human skin tissue sections were cut uniformly at 5 μm thickness and mounted on silanated slides. Pre-embedded human skin for microscopic analysis was provided by Dr Diane Keeney (Vanderbilt University, Tennessee, USA). Sections were stained in haematoxylin and eosin using routine histological techniques to illustrate tissue morphology in the specimens.
Primary antibody The polyclonal caspase-14 antibody was provided by Dr Wim Declercq (University of Ghent, Belgium) and was used for both the immunocytochemistry and Western blot analysis. This antibody has previously been used to detect caspase-14 expression in other tissues (Lippens et al., 2003).
Immunohistochemistry Immunocytochemistry was conducted on normal placental tissue from first trimester (8–12 weeks of gestation, n = 5) and from term placentae (38–41 weeks of gestation, n = 4). Negative controls were performed on the human placenta and skin, in the absence of the caspase-14 primary antibody. All reagents were diluted in Tris-buffered saline solution (TBS; 0.1 mol/l Tris and 0.15 mol/l NaCl) made up with 0.025% of Tween-20 (TBSTT) and 10% goat serum (Hunter Antisera, Jesmond, NSW, Australia). Slides were heated to 60°C for 15 min and were de-waxed in two washes of absolute toluene for 5 min each, followed by rehydration from 100% ethanol to double distilled water (DDW). After de-paraffinization, the tissue sections were equilibrated in TBS for 5 min. The slides were then rinsed in
DDW and subjected to antigen retrieval in a sodium citrate buffer (10 mmol/l; pH 6.0) at 90°C for 20 min. Afterwards, the slides were cooled for 20 min at room temperature and rinsed three times in TBS, for 5 min each. Excess liquid was removed and non-specific binding of the antibodies was blocked by incubating the sections in 20% goat serum (Hunter Antisera) made in TBS-T for 1 h at room temperature. This was followed by three washes of TBS for 5 min each. After blocking, the caspase-14 antibody was applied overnight at 4°C at a 1:100 dilution, followed by three washes in TBS for 5 min each. Excess liquid was removed and the secondary antibody, biotinylated goat anti-rabbit, was applied at a 1:200 dilution and incubated for 1 h at room temperature. After incubation, excess secondary antibody was rinsed off in three washes of TBS for 5 min each. In the third step, streptavidin labelled with Alexa Fluor 546 dye (BD Biosciences, North Ryde, NSW, Australia) and DAPI were used together at a 1:100 dilution. The sections were incubated in these tertiary reagents for 1 h at room temperature, followed by another set of three TBS washes for 5 min each. To preserve the longevity of the fluorescent dyes, DAKO fluorescent mounting medium (Dako Cytomation, Botany, Australia) was applied to the sections. The slides were cover-slipped, sealed with nail polish and stored in aluminium foil at 4°C until viewing with fluorescence microscopy.
TUNEL For the staining of apoptotic cell nuclei, the ApopTag® Plus Peroxidase In-Situ Apoptosis Detection Kit (Intergen Company, New York, NY, USA) was used, as previously described (Berg et al., 2002; Lareu et al., 2003). The procedure was according to the protocol for formalin fixed, paraffin embedded tissue, using a 10 min incubation at room temperature for proteinase K digestion (20 μg/ml). Cellular nuclei were counter stained with methyl green. Sections from five first trimester and four term placentae were examined. A negative control was performed by omitting the terminal transferase enzyme from the labelling mixture. A 4-day post-weaning rat mammary gland was used as the positive control included in the ApopTag Plus Kit. Mature nulliparous Wistar rats were obtained from the Animal Resource Centre (Murdoch, Australia).
Microscopy and imaging Haematoxylin and eosin and TUNEL slides were viewed with a Leica DMRBE microscope. Images were captured using a Nikon DXM1200F digital camera with ACT-1 v2 (Nikon, Garden City, NY, USA) software. Immunostaining was viewed with a Leica DMRBE microscope and images were captured with a black and white digital camera (CCD Cool SNAP ES camera; Photometrics, Huntington Beach, CA, USA). These images were then pseudocoloured using the V++ v4.0 software.
Western blot analysis Western blot analysis was performed on five first trimester and four term placental tissues. Tissues were homogenized in sodium phosphate buffer (10 mmol/l, pH 7) containing sucrose (0.25 mol/ l), EDTA (1 mmol/l), PMSF (1 mmol/l) and trypsin inhibitor (100 mg/ml). Protein concentration of homogenates was measured (Bradford, 1976) and 30 μg resolved by 12% SDS-PAGE and
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Article - Caspase-14 in the human placenta - DWR Kam et al. transferred to Hybond-C nitrocellulose membrane (Amersham, Baulkham Hills, NSW, Australia). Membranes were blocked in 5% non-fat milk in Tris-buffered saline/0.1% Tween-20 (TBST) for 1 h at room temperature. Membranes were then separately probed with antibodies against caspase-14 at a dilution of 1:1000 in TBST for 1–2 h at room temperature. Following primary incubation, membranes were washed in TBST and incubated with a secondary antibody at a dilution of 1:20,000, followed by streptavidin–horseradish peroxidase (Roche Diagnostics, Castle Hill, NSW, Australia) at a dilution of 1:20,000 for 1 h at room temperature. Protein signals were detected by enhanced chemiluminescence (Supersignal West Pico ECL substrate; Pierce product from Australian Biosearch, Karrinyup, Western Australia) and exposed to autoradiographic film (Kodak XAR-5). The negative control consisted of omitting the primary antibody. A human skin sample was included on each gel to standardize chemiluminescence levels and exposure times. Equal sample loading was assessed by staining membranes with Ponceau S.
RNA extraction Snap-frozen placentae were homogenized and total RNA was isolated with the NucleoSpin Spin RNA II Kit (Clontech; BD Biosciences). RNA samples were resuspended in RNasefree water and stored at –80°C until used. RNA integrity was confirmed by examining the presence of the ribosomal 28S and 18S bands. RNA quantitation and purity were determined with a Beckman DU 640 spectrophotometer.
Reverse transcription–polymerase chain reaction (RT–PCR) Complementary DNA was generated from total RNA with reverse transcriptase (Promega, Delta House, Southampton, UK) as per manufacturer’s protocol. RT–PCR was performed using 1 μg of total RNA, with the volume made up to 13.5 μl with nuclease-free water. Random hexameric primers (0.5 μl, 500 ng/ μl; hexadeoxyribonucleotides, Promega) were added, secondary RNA structures were removed by heat denaturation at 70°C for 5 min, followed by incubation of the samples on ice for 5 min. The samples were briefly centrifuged and the reagents added; 5 μl 5× MMLV Reaction Buffer (Promega), 2.6 μl of 5 mmol/ l dNTPs, 1 μl MMLV-Reverse transcriptase enzyme RNase H Minus, Point Mutant (Promega), 1 μl RNasin (Promega), and 1.9 μl nuclease-free water. These samples were then mixed and reverse transcribed using a PTC-100 Peltier thermocycler (MJ Research Inc., Geneworks Pty Ltd., Adelaide, South Australia) at 25°C for 10 min, 55°C for 50 min, and at 70°C for 15 min. The reverse transcribed cDNA was purified through spin columns (Ultra Clean GelSpin Kit; GeneWorks) and eluted out in 50 μl 10 mmol/l Tris, pH 8.
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Caspase-14 mRNA expression was examined using the Rotor Gene RG-3000 (Corbett Research Inc., Mortlake, Sydney, NSW, Australia) with the fluorescent dye, SYBR Green I (Fisher Biotech, Subiaco, Western Australia). PCR primers that correspond to the entire open reading frame of human caspase-14 [10] generated a 728 bp fragment: forward 5′-ATGAGCAATCCGCGGTCTTT3′ and reverse 5′-CTACTGCAGATACAGCCGTT-3′. Reaction conditions were as per manufacturer’s protocol with optimization of primers and magnesium chloride (MgCl2) concentrations and annealing temperature. One-tenth volume of target cDNA and
master premix were incubated with 3 mmol/l MgCl2 and 0.4 pmol/μl of each primer. The reaction run programme was as follows: an initial denaturation step at 95°C for 10 min; 40 cycles at 95°C for 15 s, 55°C for 7 s and 72°C for 14 s, followed by a fluorescence measurement after each cycle. A melt curve was performed with continuous fluorescence measurement between 70 and 90°C. Heating rate was set at 0.1°C/s.
Explant culture Five fresh first trimester placental villi were snap-frozen (0 h) with RNAlater™ (Sigma-Aldrich, Castle Hill, NSW, Australia) for RNA extraction and without RNAlater™ for protein extraction. For the culture experiment, triplicate sets of fresh placental villi were placed in sterile 20-ml glass scintillation vials containing 2 ml minimum essential medium (MEM) with Earle’s salts (Gibco product from Invitrogen, Mulgrave Victoria, Australia), supplemented with 2 mmol/l L-glutamine (Gibco), 100 IU/ml of both penicillin and streptomycin (Gibco), and 0.1% fatty acidfree BSA (fraction V; Sigma, Poole, Dorset, UK). Incubations were performed at 37°C for 4 h under both presence and absence of superoxide dismutase (SOD, 75 IU/ml; Sigma). This SOD concentration has been shown to significantly reduce DNA fragmentation in the human placenta, a classical marker for the end stage of apoptosis (Rao et al., 2000). A 4-h incubation at 37°C was performed under gas conditions of 95% O2 and 5% CO2. After the incubation, the placentae were collected and snapfrozen. For each sample, fresh tissue prior to the experiment (time zero) was used to normalize the values. Samples were stored at –80˚C for Western blot analysis.
Statistical analysis All statistical analysis was carried out using SPSS v11.0.0 (Chicago, IL, USA), while the graphs were plotted using Microsoft Excel X for Mac. Tests of normality also demonstrated significance. Twotailed t-tests (paired and independent) were conducted to analyse the data. All error bars are based on the SEM.
Results The histological sections confirmed that the specimens contained only placental villi, and these demonstrated either the morphological characteristics of the first trimester or term placental villi. Caspase-14 was localized in the first trimester and the term placentae. It was located in the trophoblast, in the first trimester and the term placenta (Figures 1b, c). Some staining of the mesenchymal cells was also seen in the first trimester. There was autofluorescence of red blood cells (Figure 1c). Human skin was used as the positive control and showed the pattern of staining as previously described predominantly in the upper epidermis (Figure 1a) (Eckhart et al., 2000; Pistritto et al., 2002). TUNEL staining demonstrated positive staining of a few cells in the first trimester and the term placentae. These included nuclei in the trophoblast as well as the occasional mesenchymal cell, in both the first trimester and term placenta (Figure 2). The expression of caspase-14 protein was confirmed by Western blot analysis. The presence of a second band with a more mobile transcript was evident in the placental samples compared with the skin (Figure 3a). The results indicated that caspase-14 protein
Article - Caspase-14 in the human placenta - DWR Kam et al. is more abundant in the first trimester than the term placenta (Figure 3b). The second band suggests that there may be a splice variant(s), or post-translational modification. No significant amount of cleaved caspase-14 is identified in the placenta, unlike the skin (see Discussion). RT–PCR resulted in the amplification of a product of the correct size (728 bp) on the agarose gel electrophoresis (Figure 4). The product had a peak melting point of 92.5°C and was verified as
caspase-14 by DNA sequencing (data not shown). The relationship between caspase-14 and apoptosis was explored further using fresh first trimester villi in the explant SOD model. There was no correlation between caspase-14 expression and apoptosis. Uncleaved caspase-14 protein was again the predominant form, with also a similar duplex band as seen in the non-cultured placenta (Figure 5).
a
b
c
Figure 1. Caspase-14 immunofluorescence. Caspase-14, antibody signal red, with 4,6diamidino-2-phenylindole counterstaining nuclei blue. (a) Human skin (×200). The caspase-14 immunostaining is localized to the superficial layers (SE) of the epidermis (EP). The dermis (DE) is contains some blood vessels. (b) First trimester placenta (×400). Caspase-14 is identified in the villi and localized to the trophoblast (TB). Little is seen in the mesenchymal tissue (M). (c) Term placentae (×400). The caspase-14 is found in the trophoblast layer (TB). Little is seen in the stromal tissues. The red blood cells show strong autofluorescence.
Figure 2. TUNEL assay. TUNEL-positive cells are stained brown, with methyl green counterstain of healthy nuclei. First trimester placenta with occasional positive nuclei in the syncytiotrophoblast layer and one in the stroma (×400).
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b
Figure 3. (a) Western blot analysis of caspase-14 in the human placenta. The large band (~29 kDa) represents the procaspase14 protein, while the lower band, observed in the human skin, is the activated caspase-14 protein (~20 kDa) that results from proteolytic cleavage. The presence of the dual bands at ~29 kDa is discussed in the text. (b) A comparison of caspase-14 expression in the first trimester and term placentae. An independent samples t-test demonstrated a significant difference in caspase-14 protein expression between the first trimester and term placentae (*P < 0.05).
Figure 4. Caspase-14 reverse transcriptase-polymerase chain reaction. Microgel DNA molecular weight ladder (lane 1). The amplified caspase14 product (728 bp) is shown in the keratinocyte culture (lane 2) and the human placenta (lane 3).
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Figure 5. (a) Western blot analysis of caspase-14 in the superoxide dismutase (SOD) and control treatment groups. (b) Quantification. There was no protein variation observed for caspase-14 before or after the 4-h experiment. In addition, a paired-samples t-test (two-tailed) indicated no significant difference of caspase-14 expression between the SOD and control treatments. (c) Inhibition of internucleosomal DNA fragmentation with SOD treatment in vitro (* denotes significant difference between 4-h control and 4-h SOD, P = 0.001). Upper panel: gel showing the fragmentation at time 0, 4-h control, and after 4-h incubation with SOD. Lower panel: comparison of densitometric measurements of the three groups showing a significant difference (adapted from Charles, 2005).
Discussion Caspase-14 is the most recently identified member of the caspase family (van de Craen et al., 1998). The exact function of this gene has not been discovered and it has not previously been examined in the placenta. Hence, the objective of this study was to detect caspase-14 in the human placenta, determine its expression pattern and investigate if this is related to cells undergoing apoptosis. There was more caspase-14 protein in the first trimester than the term placentae as found by Western blot analysis, and similar results were observed by immunohistochemistry. Compared with the first trimester, the ubiquitous cytotrophoblast layer found in the early placental villi covers of only 20% of term placental villi (Benirschke and Kaufmann, 1999). This antibody has been previously used to successfully detect caspase-14 in the human skin, and in the epithelium of the retinal pigment, thymic Hassall’s bodies and in the ependymal lining of the choroid plexus (Lippens et al., 2003). The presence of caspase-14 protein in the syncytiotrophoblast and cytotrophoblast in similar concentrations suggests that the protein in the syncytial layer is derived from the incorporation of cytotrophoblast cytoplasm into the syncytium. In the human skin, caspase-14 synthesis is restricted to the differentiating keratinocytes. As differentiation proceeds, the nuclei of terminally differentiating keratinocytes become degraded and the cornified layers are formed. These de-nucleated cells, now known as corneocytes, form the protective layer of the skin (stratum corneum) and these de-nucleated cells still contained caspase-14 protein (Lippens et al., 2003). In the placenta, the syncytium needs a constant supply of RNA from the cytotrophoblast cells for growth and maintenance of its metabolic and structural integrity (Benirschke and Kaufmann, 1999). It is likely that through this fusion process, the caspase-
14 protein expressed in the cytotrophoblast would be transferred to the syncytium. Caspase-14 was also detected in the mesenchyme of the first trimester placentae, but rarely in the term placentae. Mesenchymal cells are present in the connective tissue within the villi (Benirschke and Kaufmann, 1999). By term, most of the villi are developed and do not have proliferating mesenchymal cells. TUNEL staining demonstrated that all cell types in the human placenta underwent cell death, although the overall number of TUNEL-positive cells was low. These findings were similar to previous reports (Smith et al., 1997; Austgulen et al., 2002). As expected, small groups of nuclei located in the syncytiotrophoblast were TUNEL positive. It is unlikely that caspase-14 has a role in the end stage of apoptosis, as caspase14 was localized throughout the trophoblast cell layer and not confined to the few dying nuclei demonstrated by TUNEL, unless it is stored and only activated by the few nuclei in the syncytium undergoing apoptosis. Huppertz et al. (1999) suggested that early apoptotic processes occur in the cytotrophoblast, promoting its fusion into the syncytium. Assuming that caspase-14 has a role in apoptosis, the localization of caspase-14 to the cytotrophoblast as well as the syncytiotrophoblast may indicate a role for caspase-14 in the early apoptotic cascade in syncytial formation in the human placenta. The pattern of expression of caspase-14 in the human skin raises the suggestion that there is a modification of the normal apoptotic process in certain cell types such as the epidermis, where there is a loss of nuclei, but preservation of the cytoplasm. To date, human caspase-14 involvement in apoptosis has not been identified.
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Article - Caspase-14 in the human placenta - DWR Kam et al. Western blot analysis showed that the first trimester and term placenta both contained the caspase-14 protein, predominantly in the uncleaved state. An interesting finding was the presence of a second band a slightly less than the expected size compared with the human skin tissues. These appearances suggest there are differences in the post-translational modification(s) of the protein between skin and the placenta, or that there are splice variants between the two tissues.
The lack of caspase-14 variation in the first trimester and term placentae, as well as in the explant model, suggest that caspase14 has a separate, non-apoptotic role in the cytotrophoblast. A non-apoptotic role for caspase-14 has been suggested in the skin (Mikolajczyk et al., 2004). Caspase-14 may be involved in a specialized form of apoptosis, which is believed to occur when cytotrophoblast cells fuse and are incorporated into the syncytium.
Another observation is that little cleaved, activated caspase14 was detected. A reason why activated caspase-14 may not be seen is if cleavage occurs in the syncytial knots, which are normally shed into the maternal circulation. However, cleaved caspase-14 was not observed in the explant model, where any debris would be contained within the supernatant. Hence, it is unlikely that caspase-14 is cleaved in the human placenta, in either the normal physiological state or in the experimental conditions of apoptosis inducement and inhibition.
Acknowledgements
The SOD explant model has proved to be very useful for examining the molecular signalling pathways involved in placental apoptosis. Under defined experimental conditions, DNA fragmentation was significantly reduced with SOD treatment (Rao et al., 2000; Charles et al., 2005 Figure 1). This study also demonstrated a significant reduction of DNA fragmentation with SOD (data not shown) and an up-regulation of caspase-3 in the 4-h control group, but not in the 4-h SOD group. The data revealed SOD as a potent anti-apoptotic agent that inhibits early caspase-mediated apoptosis in the human placenta, with the treatment group demonstrating significant reductions in DNA fragmentation, as well as increase in the ratio of the anti-apoptotic genes compared with the proapoptotic genes (Charles et al., 2005). Using the explant model, no significant differences in caspase-14 protein expression were evident between the 4-h SOD and 4-h control treatment groups. There was no cleavage of caspase-14 in the cultured samples. This data collectively implies that caspase-14 is not activated and does not play a role in the apoptotic pathway in this model. In addition, Western blot analysis and immunocytochemistry revealed caspase-14 was expressed less in all human placental specimens compared with the human skin. Many tissues that express caspase-14 have a function involving barrier formation. The skin is the first line of defence against environmental conditions and pathogen entry. In the choroid plexus, the ependymal layer forms the epithelium lining that constitutes the blood–brain barrier. Caspase-14 expression was also observed to multiple cell lines derived from simple epithelia of breast, prostate and stomach (Pistritto et al., 2002). In the human placenta, caspase-14 was located in the syncytiotrophoblast, which forms the physical barrier between maternal and fetal tissues. The results of this study indicate that caspase-14 may have a role in the trophoblast homologous to its role in epidermis.
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This study is the first to demonstrate the expression of caspase14 in the human placenta. The present study located caspase14 protein and mRNA in the human placenta, and found that caspase-14 is not exclusively localized to cells undergoing apoptosis. Caspase-14 was also predominantly expressed in the uncleaved state, whereby caspases are usually activated by cleavage.
The caspase-14 antibody was kindly provided by Dr Wim Declercq (Department of Molecular Biology, University of Gent, Belgium). Dr Luis Filgueira (School of Anatomy and Human Biology, UWA) provided the immunofluorescence reagents, and Dr Diane Kenney (Vanderbilt University, Nashville) provided the human abdominal skin (embedded) and keratinocyte cDNA. We thank Dr Jan Dickinson (Maternal Fetal Medicine, KEMH) for assistance in obtaining the placentas, and Dr Fiona Wood (Clinical Cell Culture, Perth) for providing the human skin biopsy. This study was supported by grants from the Western Australian Institute for Medical Research, Women and Infants Research Foundation of Western Australia and University of Western Australia small grant scheme (AC and AD) and the Raine Medical Research Foundation, Perth, Western Australia. We thank Ms Sue Hisheh for technical assistance.
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