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Characterisation of cholinesterase expression during murine embryonic stem cell differentiation
Chemico-Biological Interactions 175 (2008) 156–160
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Characterisation of cholinesterase expression during murine embryonic stem cell differentiation L.E. Sperling a,∗ , G. Steinert a , J. Boutter a , D. Landgraf a , J. Hescheler b , D. Pollet c , P.G. Layer a a b c
Institute of Zoology, Darmstadt University of Technology, Schnittspahnstrasse 3, D-64287 Darmstadt, Germany Institute of Neurophysiology, University of Cologne, D-50931 Cologne, Germany Institute of Chemistry and Biotechnology, University of Applied Science, D-64287 Darmstadt, Germany
a r t i c l e
i n f o
Article history: Available online 6 June 2008 Keywords: Acetylcholinesterase Butyrylcholinesterase Embryoid bodies Embryonic stem cells In vitro differentiation
a b s t r a c t It is already established that cholinesterases (ChEs) appear in every embryonic blastema at a very early stage of development, independently from innervation. Embryonic butyrylcholinesterase (BChE) is typically found in cells engaged in proliferation processes, while acetylcholinesterase (AChE) is expressed by cells undergoing morphogenetic processes. In order to better define the regulation of cholinesterases during development, we examined their expressions during in vitro differentiation of two murine embryonic stem cell lines by reverse transcription polymerase chain reaction, histochemistry and enzyme activity measurements. AChE and BChE activity and mRNA were present in the undifferentiated stem cells. To test whether the ChEs expression is regulated during differentiation, we employed the embryoid bodies (EBs) culture method, allowing the cells to differentiate, to then collect them at various stages in culture. Interestingly, phases of differentiation were accompanied by increased AChE transcripts; BChE expression was constant, decreasing at later differentiation stages. Cholinesterase activities showed corresponding patterns, with AChE activity increasing at later stages in culture and BChE slightly decreasing. Histochemistry revealed that AChE and BChE activities were mutually exclusive, being expressed by different cell subpopulations. Thus, we have demonstrated that mouse embryonic stem cells express cholinesterases, the enzymes are functional and their expression is regulated during differentiation. Therefore, it appears that their functions under these conditions are not related to synaptic transmission, but for the developmental processes. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Mammals contain two cholinesterases (ChEs): acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The two enzymes differ genetically, structurally, in their expression patterns and kinetics. AChE regulates cholinergic neurotransmission by hydrolyzing acetylcholine, whereas BChEs function remains unclear; AChE is predomi-
∗ Corresponding author. Tel.: +49 6151 166105; fax: +49 6151 166548. E-mail address: [email protected] (L.E. Sperling). 0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2008.05.034
nantly expressed in neurons and muscle, while BChE occurs in non-synaptic sites, such as liver, lung, plasma or glia. However, AChE occurs also in non-neuronal and embryonic tissues like blood cells, epithelial cells and vascular endothelial cells or during neural tube development [1]. This distribution of ChEs led to the idea that ChEs have other functions unrelated to synaptic transmission [1–3]. Various experimental data strongly support a role for ChEs in the control of cell proliferation and the onset of differentiation. AChE is involved in promoting neurite growth [4], in hematopoiesis [5,6], osteogenesis [7] and apoptosis [8] and BChE is associated with thrombopoiesis and
L.E. Sperling et al. / Chemico-Biological Interactions 175 (2008) 156–160
megakaryocytopoiesis [9], or is shown to be involved in retinal and glial proliferation [10,11]. The observation that mice live without AChE and the high levels of BChE activity in tissues of AChE-knockout mice, suggests that BChE activity facilitated their survival and that BChE has an essential function as a back-up for AChE [12]. Embryonic stem cells (ESCs) represent a unique experimental model for studying the role of a specific molecule or pathway during tissue development. Embryoid bodies are aggregates of stem cells, representing an in vitro tool to investigate developmental processes. It has been suggested that during EB formation, the tissues undergo developmental patterns similar to those during in vivo growth of the embryo [13]. Our laboratory has previously demonstrated that embryonic stem cells express most of the cholinergic markers [14]. Here, we set out to characterize the expression of cholinesterases during the in vitro differentiation of ESCs by formation of EBs and we report a regulation of the ChEs expression during ESCs differentiation. 2. Materials and methods 2.1. Cell culture Two murine embryonic stem cell lines CGR8 and D3 were used for this study. Undifferentiated CGR8 cells were grown on gelatinized-flasks in Glasgow’s buffered minimal essential medium (Gibco, Germany) supplemented with 100 units/ml leukemia inhibitory factor (LIF) (Sigma, Germany), 0.05 mM -mercaptoethanol (-ME), 2 mM l-glutamine, 100 units/ml penicillin, 0.01 mg/ml streptomycin and 10% fetal calf serum (Perbio, Germany). The D3 cells were grown on gelatinized flasks (0.2% gelatine) in DMEM supplemented with 15% fetal calf serum (BiochromAG, Germany), 2 mM l-glutamine, 50 U/ml penicillin, 50 g/ml streptomycin, 1% non-essential amino acids, 0.1 M -mercaptoethanol and 1000 U/ml leukemia inhibitory factor. To induce ESC differentiation, the cells were cultured in GMEM supplemented with 20% FCS, 1% non-essential amino acids and -ME as spheres of cells referred to as EBs using the hanging-drop method. 2.2. RNA isolation and RT-PCR Total RNA from undifferentiated CGR8 and D3 cells was prepared with RNeasy kit (Qiagen, Germany) or TriReagent (Sigma, Germany) according to the manufacturer’s instructions. For reverse transcription polymerase chain reaction (RT-PCR) analysis, 1 g total RNA previously treated with DN-ase to avoid contamination with genomic DNA was reverse-transcribed for 1 h at 42 ◦ C using an oligo dT primer and following manufacturer’s protocol (Reverse Transcription Systems, Promega, Germany). For PCR reactions, the primers used to amplify and the PCR conditions were previously described [14]. Shortly, PCR for GAPDH was run for 27 cycles and for AChE and BChE for 30 cycles to avoid entering in the saturation phase of the PCR. For detecting AChE in the D3-cell line, PCR was run for 32 cycles. Additionally, PCR products were sequenced to confirm their identity.
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2.3. Protein determination, Ellman assay and statistics Protein concentrations were routinely determined according to the method of Bradford. Bovine serum albumin was used as a standard. Cholinesterase activities were determined from whole cell extracts by the Ellman assay; the detailed procedure is described elsewhere [14]. All assays were carried out at least in triplicates. The results were statistically analyzed by one-way ANOVA followed by Tuckey’s multiple comparison test using the software GraphPad Prism 3.0. P-Values <0.5 were considered significant. 2.4. Karnovsky and Roots staining, microscopy and photography Cholinesterase activity staining was described elsewhere [15]. Shortly, sections of EBs were incubated with the staining solution for 2–3 h at 37 ◦ C for AChE and up to 6 h for BChE. Acetylthiocholine was used as substrate for the AChE staining with 10−4 M iso-OMPA to inhibit BChE and butyrylthiocholine was used as substrate for BChE staining with 10−4 M BW284c51 to inhibit AChE. Stained cells were documented using a Zeiss Axiophot microscope with DIC (Nomarski) and fluorescence optics (10× and 20× objectives). Photomicrographs were taken using an Intas camera and a computer program (Diskus 1280, CH Hilgers, Königswinter). The figures were produced using Adobe Photoshop 7. 3. Results and discussion To determine if cholinesterase expression is regulated during differentiation of murine embryonic stem cells, we used the EB model. Two murine embryonic stem cell lines, CGR8 and D3, were cultured as spheres and induced to differentiate by removal of LIF from the culture media. Mouse embryonic stem (ES) cells are maintained and self-renew (proliferation without differentiation) in media containing LIF. Pluripotency of the cells (their stemness) was confirmed by examining the expression of pluripotency markers such as Oct4, Nanog and alkaline phosphatase. EBs were collected at different days in culture and used for detection of cholinesterase transcripts and enzyme activities. The RT-PCR analysis revealed that mRNA for AChE and BChE is present in stem cells and EBs (Fig. 1). Interestingly, we found that AChE transcripts are up-regulated, starting at day 6 in culture (Fig. 1A) and BChE expression was high at the beginning and was slightly down-regulated at late stages in culture. A slight decrease of AChE transcripts was observed around day 8 in culture, decrease that can be associated to phases of cellular transition to a committed state. The existence of a serum response element on the AChE promoter may suggest that the relative high levels of AChE and the increase during the EB differentiation might be an effect of the high serum concentration used for EB culture. However, the same effect was observed on both CGR8 and D3-cell lines, although only CGR8 was switched from 10 to 20% serum, while D3 EBs were obtained by further cultivation in 15% serum medium, without LIF. CGR8 cells were induced
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L.E. Sperling et al. / Chemico-Biological Interactions 175 (2008) 156–160
Fig. 1. The expression of cholinesterases in mouse embryonic stem cells and EBs. RNA samples from embryonic stem cells (CGR8 and D3 lines) and 1–14day-old EBs were analyzed by RT-PCR for expression of AChE, BChE. RNA of mouse brain was analyzed as a positive control. Results are representative for four independent experiments. (A and B) AChE and BChE transcripts were detected in CGR8 ESC and EBs. AChE transcripts were increased after the treatment of EBs with retinoic acid, a factor that stimulates neuronal differentiation. (C) The expression of cholinesterases was confirmed in the D3-cell line. ES, embryonic stem cell; S, EB stage, the number shows days in culture; C, positive control mouse brain RNA; *the day of RA addition.
to differentiate along the neuroectodermal pathway by culturing free-floating EBs for 2 days in 10−6 M retinoic acid (RA). As cells differentiated towards a more neuronal phenotype, increased AChE expression and a more
pronounced decrease of BChE transcripts was observed (Fig. 1B). The Ellman assay was used to determine how much cholinesterase activity is expressed by EBs; inhibitors
Fig. 2. Cholinesterase activity in ESCs and cell lysates and sections of embryoid bodies as revealed by Ellman activity measurements tests (A) and Karnovsky and Roots staining (B). (A) AChE and BChE specific activity was determined in cell lysates from ESC and cultures grown as EBs for 1–10 days. Measurements are the means of three independent experiments expressed as ±S.D. *P values <0.5. (B) EBs cultured for 10 days were washed with PBS, fixed in 4% paraformaldehyde and sectioned at 10 m on a cryostat. AChE (a) and BChE (b) were visualized by Karnovsky and Roots activity staining. The two images represent serial sections of the same EB. Note that ChEs staining is distributed in patches. The pictures were taken under a 10× objective on a Zeiss Axiophot.
L.E. Sperling et al. / Chemico-Biological Interactions 175 (2008) 156–160
specific for AChE (BW284c51) or BChE (iso-OMPA) were included to determine the amount of catalytic activity for the two esterases (Fig. 2A). In line with the RT-PCR results, we found that AChE activity of EBs increases and BChE activity decreases, with AChE activity being lower than BChE until day 10 in culture (Fig. 2A). AChE and BChE secretion during differentiation, as assayed by using culture media of EBs grown under serum-free conditions, turned out to be very low (not shown). Karnovsky and Roots histochemistry on cryosections of EBs revealed that the AChE and BChE activity were localized in separate clusters (Fig. 2B) and expressed by mutually exclusive cell populations. Our results demonstrate that mouse embryonic stem cells, e.g. non-neuronal cells, express both cholinesterases; thereby, AChE and BChE expression are regulated during EB differentiation. This came not by surprise since cholinesterases are known to be expressed by various nonneuronal cells. Their physiological role in mESCs, however, remains to be clarified. The expression of non-neuronal acetylcholine (ACh) in ESCs [14] explains convincingly the expression of its degrading enzymes AChE and BChE. Nonneuronal ACh is synthesized by the majority of all cells [16,17] and serves to regulate basic cellular functions, e.g. proliferation or differentiation. On the other hand, a sequence homology of AChE with cell adhesion proteins and its binding to different proteins [18–20] combine to suggest that AChE may also play a structural role in ESCs independent of its catalytic activity [1]. BChE is present in many animal species [21] in liver, lung, smooth muscle, blood or at early developmental stages where it is likely to participate in the hydrolysis of ACh or regulate cell proliferation [10,11]. This would explain why BChE transcripts were more abundant than AChE transcripts in EBs, supporting the hypothesis that BChE is predominantly expressed by proliferating cells. In addition, an increase in the rate of transcription of the AChE gene and AChE activity was detected during the course of differentiation. This may reflect the normal differentiation process, since AChE is known to be up-regulated in differentiating cells in the CNS. On the other hand, the regulation of cholinesterase transcripts and activity may not be the result of changes in the rate of gene transcription, but the result of post-transcriptional control. Previous studies on the expression of AChE in embryonic carcinoma cells [22] support this hypothesis; similar to our results, AChE was actively transcribed in the embryonic carcinoma cell at the stem cell stage and the level of AChE increased during differentiation. Moreover, the transcription rate of the AChE remained constant, indicating that the levels of AChE mRNA and protein are regulated through stabilization of AChE mRNA [22]. Embryonic stem cells have an innate potential to differentiate into all cell types. Therefore, some of the differentiating cells in the embryoid bodies will possibly become neurons and express AChE; the AChE increase will be a result of neuronal differentiation and not to general differentiation of ESCs. However, the study was done for a short time period, up to day 14 in culture and within the first few days in vitro EBs generate populations of cells that express genes indicative of primitive
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endoderm and mesoderm [13]. Only after extended periods in culture (more than 10 days) EBs can generate cells of the hematopoietic, endothelial, muscle and neuronal lineages; thus, is reasonable that the regulation of cholinesterases expression is due to general differentiation processes and not restricted to the neuronal type. Future studies are necessary and should bring more insights into the role of cholinesterases in differentiating embryonic tissues. References [1] P.G. Layer, E. Willbold, Novel functions of cholinesterases in development, physiology and disease, Prog. Histochem. Cytochem. 29 (1995) 1–94. [2] J. Massoulié, L. Pezzementi, S. Bon, E. Krejci, F.M. Vallette, Molecular and cellular biology of cholinesterases, Prog. Neurobiol. 41 (1993) 31–91. [3] H. Soreq, S. Seidman, Acetylcholinesterase—new roles for an old actor, Nat. Rev. Neurosci. 2 (2001) 294–302. [4] P.G. Layer, T. Weikert, R. Alber, Cholinesterases regulate neurite growth of chick nerve cells in vitro by means of a non-enzymatic mechanism, Cell Tissue Res. 273 (1993) 219–226. [5] R.Y.Y. Chan, F.A. Adatia, A.M. Krupa, B.J. Jasmin, Increased expression of acetylcholinesterase T and R transcripts during hematopoietic differentiation is accompanied by parallel elevations in the levels of their respective molecular forms, J. Biol. Chem. 273 (1998) 9727–9733. [6] M. Pick, C. Flores-Flores, D. Grisaru, S. Shochat, V. Deutsch, H. Soreq, Blood-cell-specific acetylcholinesterase splice variations under changing stimuli, Int. J. Dev. Neurosci. 22 (2004) 523–531. [7] C.A. Inkson, A.C. Brabbs, T.S. Grewal, T.M. Skerry, P.G. Genever, Characterization of acetylcholinesterase expression and secretion during osteoblast differentiation, Bone 35 (2004) 819–827. [8] X.J. Zhang, L. Yang, Q. Zhao, J.P. Caen, H.Y. He, Q.H. Jin, L.H. Guo, M. Alemany, L.Y. Zhang, Y.F. Shi, Induction of acetylcholinesterase expression during apoptosis in various cell types, Cell Death Differ. 9 (2002) 790–800. [9] D. Patinkin, S. Seidman, F. Eckstein, F. Benseler, H. Zakut, H. Soreq, Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro, Mol. Cell. Biol. 10 (1990) 6046–6050. [10] A. Robitzki, A. Mack, A. Chatonnet, P.G. Layer, Transfection of reaggregating embryonic chicken retinal cells with an antisense 5 -DNA butyrylcholinesterase expression vector inhibits proliferation and alters morphogenesis, J. Neurochem. 69 (1997) 823–833. [11] A. Robitzki, F. Döll, C. Richter-Landsberg, P.G. Layer, Regulation of the rat oligodendroglia cell line OLN-93 by antisense transfection of butyrylcholinesterase, Glia 31 (2000) 195–205. [12] B. Li, J.A. Stribley, A. Ticu, W. Xie, L.M. Schopfer, P. Hammond, S. Brimijoin, S.H. Hinrichs, O. Lockridge, Abundant tissue butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse, J. Neurochem. 75 (2000) 1320–1331. [13] G. Weitzer, Embryonic stem cell-derived embryoid bodies: an in vitro model of eutherian pregastrulation development and early gastrulation, Handb. Exp. Pharmacol. 174 (2006) 21–51. [14] L.E. Paraoanu, G. Steinert, A. Koehler, I. Wessler, P.G. Layer, Expression and possible functions of the cholinergic system in a murine embryonic stem cell line, Life Sci. 80 (2007) 2375–2379. [15] M.J. Karnovsky, L. Roots, A “direct-coloring” thiocholine method for cholinesterases, J. Histochem. Cytochem. 12 (1964) 219– 221. [16] I. Wessler, C.J. Kirkpatrick, K. Racké, The cholinergic ‘pitfall’: acetylcholine, a universal cell molecule in biological systems, including humans, Clin. Exp. Pharmacol. Physiol. 26 (1999) 198– 205. [17] I. Wessler, H. Kilbinger, F. Bittinger, C.J. Kirkpatrick, The biological role of non-neuronal acetylcholine in plants and humans, Jpn. J. Pharmacol. 85 (2001) 2–10. [18] K.R. Birikh, E.H. Sklan, S. Shoham, H. Soreq, Interaction of “readthrough” acetylcholinesterase with RACK1 and PKCbeta II correlates with intensified fear-induced conflict behaviour, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 283–288.
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[19] L.E. Paraoanu, P.G. Layer, Mouse acetylcholinesterase interacts in yeast with the extracellular matrix component laminin-1beta, FEBS Lett. 576 (2004) 161–164. [20] C. Perry, M. Pick, E. Podoly, A. Gilboa-Geffen, G. Zimmerman, E.H. Sklan, Y. Ben-Shaul, S. Diamant, H. Soreq, Acetylcholinesterase/C terminal binding protein interactions modify ikaros functions, causing T lymphopenia, Leukemia 21 (2007) 1472–1480.
[21] X. Cousin, U. Strähle, A. Chatonnet, Are there non-catalytic functions of acetylcholinesterases? Lessons from mutant animal models, Bioessays 27 (2005) 189–200. [22] A.B. Coleman, P. Taylor, Regulation of acetylcholinesterase expression during neuronal differentiation, J. Biol. Chem. 271 (1996) 4410–4416.
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Characterisation of cholinesterase expression during murine embryonic stem cell differentiation L.E. Sperling a,∗ , G. Steinert a , J. Boutter a , D. Landgraf a , J. Hescheler b , D. Pollet c , P.G. Layer a a b c
Institute of Zoology, Darmstadt University of Technology, Schnittspahnstrasse 3, D-64287 Darmstadt, Germany Institute of Neurophysiology, University of Cologne, D-50931 Cologne, Germany Institute of Chemistry and Biotechnology, University of Applied Science, D-64287 Darmstadt, Germany
a r t i c l e
i n f o
Article history: Available online 6 June 2008 Keywords: Acetylcholinesterase Butyrylcholinesterase Embryoid bodies Embryonic stem cells In vitro differentiation
a b s t r a c t It is already established that cholinesterases (ChEs) appear in every embryonic blastema at a very early stage of development, independently from innervation. Embryonic butyrylcholinesterase (BChE) is typically found in cells engaged in proliferation processes, while acetylcholinesterase (AChE) is expressed by cells undergoing morphogenetic processes. In order to better define the regulation of cholinesterases during development, we examined their expressions during in vitro differentiation of two murine embryonic stem cell lines by reverse transcription polymerase chain reaction, histochemistry and enzyme activity measurements. AChE and BChE activity and mRNA were present in the undifferentiated stem cells. To test whether the ChEs expression is regulated during differentiation, we employed the embryoid bodies (EBs) culture method, allowing the cells to differentiate, to then collect them at various stages in culture. Interestingly, phases of differentiation were accompanied by increased AChE transcripts; BChE expression was constant, decreasing at later differentiation stages. Cholinesterase activities showed corresponding patterns, with AChE activity increasing at later stages in culture and BChE slightly decreasing. Histochemistry revealed that AChE and BChE activities were mutually exclusive, being expressed by different cell subpopulations. Thus, we have demonstrated that mouse embryonic stem cells express cholinesterases, the enzymes are functional and their expression is regulated during differentiation. Therefore, it appears that their functions under these conditions are not related to synaptic transmission, but for the developmental processes. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Mammals contain two cholinesterases (ChEs): acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The two enzymes differ genetically, structurally, in their expression patterns and kinetics. AChE regulates cholinergic neurotransmission by hydrolyzing acetylcholine, whereas BChEs function remains unclear; AChE is predomi-
∗ Corresponding author. Tel.: +49 6151 166105; fax: +49 6151 166548. E-mail address: [email protected] (L.E. Sperling). 0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2008.05.034
nantly expressed in neurons and muscle, while BChE occurs in non-synaptic sites, such as liver, lung, plasma or glia. However, AChE occurs also in non-neuronal and embryonic tissues like blood cells, epithelial cells and vascular endothelial cells or during neural tube development [1]. This distribution of ChEs led to the idea that ChEs have other functions unrelated to synaptic transmission [1–3]. Various experimental data strongly support a role for ChEs in the control of cell proliferation and the onset of differentiation. AChE is involved in promoting neurite growth [4], in hematopoiesis [5,6], osteogenesis [7] and apoptosis [8] and BChE is associated with thrombopoiesis and
L.E. Sperling et al. / Chemico-Biological Interactions 175 (2008) 156–160
megakaryocytopoiesis [9], or is shown to be involved in retinal and glial proliferation [10,11]. The observation that mice live without AChE and the high levels of BChE activity in tissues of AChE-knockout mice, suggests that BChE activity facilitated their survival and that BChE has an essential function as a back-up for AChE [12]. Embryonic stem cells (ESCs) represent a unique experimental model for studying the role of a specific molecule or pathway during tissue development. Embryoid bodies are aggregates of stem cells, representing an in vitro tool to investigate developmental processes. It has been suggested that during EB formation, the tissues undergo developmental patterns similar to those during in vivo growth of the embryo [13]. Our laboratory has previously demonstrated that embryonic stem cells express most of the cholinergic markers [14]. Here, we set out to characterize the expression of cholinesterases during the in vitro differentiation of ESCs by formation of EBs and we report a regulation of the ChEs expression during ESCs differentiation. 2. Materials and methods 2.1. Cell culture Two murine embryonic stem cell lines CGR8 and D3 were used for this study. Undifferentiated CGR8 cells were grown on gelatinized-flasks in Glasgow’s buffered minimal essential medium (Gibco, Germany) supplemented with 100 units/ml leukemia inhibitory factor (LIF) (Sigma, Germany), 0.05 mM -mercaptoethanol (-ME), 2 mM l-glutamine, 100 units/ml penicillin, 0.01 mg/ml streptomycin and 10% fetal calf serum (Perbio, Germany). The D3 cells were grown on gelatinized flasks (0.2% gelatine) in DMEM supplemented with 15% fetal calf serum (BiochromAG, Germany), 2 mM l-glutamine, 50 U/ml penicillin, 50 g/ml streptomycin, 1% non-essential amino acids, 0.1 M -mercaptoethanol and 1000 U/ml leukemia inhibitory factor. To induce ESC differentiation, the cells were cultured in GMEM supplemented with 20% FCS, 1% non-essential amino acids and -ME as spheres of cells referred to as EBs using the hanging-drop method. 2.2. RNA isolation and RT-PCR Total RNA from undifferentiated CGR8 and D3 cells was prepared with RNeasy kit (Qiagen, Germany) or TriReagent (Sigma, Germany) according to the manufacturer’s instructions. For reverse transcription polymerase chain reaction (RT-PCR) analysis, 1 g total RNA previously treated with DN-ase to avoid contamination with genomic DNA was reverse-transcribed for 1 h at 42 ◦ C using an oligo dT primer and following manufacturer’s protocol (Reverse Transcription Systems, Promega, Germany). For PCR reactions, the primers used to amplify and the PCR conditions were previously described [14]. Shortly, PCR for GAPDH was run for 27 cycles and for AChE and BChE for 30 cycles to avoid entering in the saturation phase of the PCR. For detecting AChE in the D3-cell line, PCR was run for 32 cycles. Additionally, PCR products were sequenced to confirm their identity.
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2.3. Protein determination, Ellman assay and statistics Protein concentrations were routinely determined according to the method of Bradford. Bovine serum albumin was used as a standard. Cholinesterase activities were determined from whole cell extracts by the Ellman assay; the detailed procedure is described elsewhere [14]. All assays were carried out at least in triplicates. The results were statistically analyzed by one-way ANOVA followed by Tuckey’s multiple comparison test using the software GraphPad Prism 3.0. P-Values <0.5 were considered significant. 2.4. Karnovsky and Roots staining, microscopy and photography Cholinesterase activity staining was described elsewhere [15]. Shortly, sections of EBs were incubated with the staining solution for 2–3 h at 37 ◦ C for AChE and up to 6 h for BChE. Acetylthiocholine was used as substrate for the AChE staining with 10−4 M iso-OMPA to inhibit BChE and butyrylthiocholine was used as substrate for BChE staining with 10−4 M BW284c51 to inhibit AChE. Stained cells were documented using a Zeiss Axiophot microscope with DIC (Nomarski) and fluorescence optics (10× and 20× objectives). Photomicrographs were taken using an Intas camera and a computer program (Diskus 1280, CH Hilgers, Königswinter). The figures were produced using Adobe Photoshop 7. 3. Results and discussion To determine if cholinesterase expression is regulated during differentiation of murine embryonic stem cells, we used the EB model. Two murine embryonic stem cell lines, CGR8 and D3, were cultured as spheres and induced to differentiate by removal of LIF from the culture media. Mouse embryonic stem (ES) cells are maintained and self-renew (proliferation without differentiation) in media containing LIF. Pluripotency of the cells (their stemness) was confirmed by examining the expression of pluripotency markers such as Oct4, Nanog and alkaline phosphatase. EBs were collected at different days in culture and used for detection of cholinesterase transcripts and enzyme activities. The RT-PCR analysis revealed that mRNA for AChE and BChE is present in stem cells and EBs (Fig. 1). Interestingly, we found that AChE transcripts are up-regulated, starting at day 6 in culture (Fig. 1A) and BChE expression was high at the beginning and was slightly down-regulated at late stages in culture. A slight decrease of AChE transcripts was observed around day 8 in culture, decrease that can be associated to phases of cellular transition to a committed state. The existence of a serum response element on the AChE promoter may suggest that the relative high levels of AChE and the increase during the EB differentiation might be an effect of the high serum concentration used for EB culture. However, the same effect was observed on both CGR8 and D3-cell lines, although only CGR8 was switched from 10 to 20% serum, while D3 EBs were obtained by further cultivation in 15% serum medium, without LIF. CGR8 cells were induced
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Fig. 1. The expression of cholinesterases in mouse embryonic stem cells and EBs. RNA samples from embryonic stem cells (CGR8 and D3 lines) and 1–14day-old EBs were analyzed by RT-PCR for expression of AChE, BChE. RNA of mouse brain was analyzed as a positive control. Results are representative for four independent experiments. (A and B) AChE and BChE transcripts were detected in CGR8 ESC and EBs. AChE transcripts were increased after the treatment of EBs with retinoic acid, a factor that stimulates neuronal differentiation. (C) The expression of cholinesterases was confirmed in the D3-cell line. ES, embryonic stem cell; S, EB stage, the number shows days in culture; C, positive control mouse brain RNA; *the day of RA addition.
to differentiate along the neuroectodermal pathway by culturing free-floating EBs for 2 days in 10−6 M retinoic acid (RA). As cells differentiated towards a more neuronal phenotype, increased AChE expression and a more
pronounced decrease of BChE transcripts was observed (Fig. 1B). The Ellman assay was used to determine how much cholinesterase activity is expressed by EBs; inhibitors
Fig. 2. Cholinesterase activity in ESCs and cell lysates and sections of embryoid bodies as revealed by Ellman activity measurements tests (A) and Karnovsky and Roots staining (B). (A) AChE and BChE specific activity was determined in cell lysates from ESC and cultures grown as EBs for 1–10 days. Measurements are the means of three independent experiments expressed as ±S.D. *P values <0.5. (B) EBs cultured for 10 days were washed with PBS, fixed in 4% paraformaldehyde and sectioned at 10 m on a cryostat. AChE (a) and BChE (b) were visualized by Karnovsky and Roots activity staining. The two images represent serial sections of the same EB. Note that ChEs staining is distributed in patches. The pictures were taken under a 10× objective on a Zeiss Axiophot.
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specific for AChE (BW284c51) or BChE (iso-OMPA) were included to determine the amount of catalytic activity for the two esterases (Fig. 2A). In line with the RT-PCR results, we found that AChE activity of EBs increases and BChE activity decreases, with AChE activity being lower than BChE until day 10 in culture (Fig. 2A). AChE and BChE secretion during differentiation, as assayed by using culture media of EBs grown under serum-free conditions, turned out to be very low (not shown). Karnovsky and Roots histochemistry on cryosections of EBs revealed that the AChE and BChE activity were localized in separate clusters (Fig. 2B) and expressed by mutually exclusive cell populations. Our results demonstrate that mouse embryonic stem cells, e.g. non-neuronal cells, express both cholinesterases; thereby, AChE and BChE expression are regulated during EB differentiation. This came not by surprise since cholinesterases are known to be expressed by various nonneuronal cells. Their physiological role in mESCs, however, remains to be clarified. The expression of non-neuronal acetylcholine (ACh) in ESCs [14] explains convincingly the expression of its degrading enzymes AChE and BChE. Nonneuronal ACh is synthesized by the majority of all cells [16,17] and serves to regulate basic cellular functions, e.g. proliferation or differentiation. On the other hand, a sequence homology of AChE with cell adhesion proteins and its binding to different proteins [18–20] combine to suggest that AChE may also play a structural role in ESCs independent of its catalytic activity [1]. BChE is present in many animal species [21] in liver, lung, smooth muscle, blood or at early developmental stages where it is likely to participate in the hydrolysis of ACh or regulate cell proliferation [10,11]. This would explain why BChE transcripts were more abundant than AChE transcripts in EBs, supporting the hypothesis that BChE is predominantly expressed by proliferating cells. In addition, an increase in the rate of transcription of the AChE gene and AChE activity was detected during the course of differentiation. This may reflect the normal differentiation process, since AChE is known to be up-regulated in differentiating cells in the CNS. On the other hand, the regulation of cholinesterase transcripts and activity may not be the result of changes in the rate of gene transcription, but the result of post-transcriptional control. Previous studies on the expression of AChE in embryonic carcinoma cells [22] support this hypothesis; similar to our results, AChE was actively transcribed in the embryonic carcinoma cell at the stem cell stage and the level of AChE increased during differentiation. Moreover, the transcription rate of the AChE remained constant, indicating that the levels of AChE mRNA and protein are regulated through stabilization of AChE mRNA [22]. Embryonic stem cells have an innate potential to differentiate into all cell types. Therefore, some of the differentiating cells in the embryoid bodies will possibly become neurons and express AChE; the AChE increase will be a result of neuronal differentiation and not to general differentiation of ESCs. However, the study was done for a short time period, up to day 14 in culture and within the first few days in vitro EBs generate populations of cells that express genes indicative of primitive
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endoderm and mesoderm [13]. Only after extended periods in culture (more than 10 days) EBs can generate cells of the hematopoietic, endothelial, muscle and neuronal lineages; thus, is reasonable that the regulation of cholinesterases expression is due to general differentiation processes and not restricted to the neuronal type. Future studies are necessary and should bring more insights into the role of cholinesterases in differentiating embryonic tissues. References [1] P.G. Layer, E. Willbold, Novel functions of cholinesterases in development, physiology and disease, Prog. Histochem. Cytochem. 29 (1995) 1–94. [2] J. Massoulié, L. Pezzementi, S. Bon, E. Krejci, F.M. Vallette, Molecular and cellular biology of cholinesterases, Prog. Neurobiol. 41 (1993) 31–91. [3] H. Soreq, S. Seidman, Acetylcholinesterase—new roles for an old actor, Nat. Rev. Neurosci. 2 (2001) 294–302. [4] P.G. Layer, T. Weikert, R. Alber, Cholinesterases regulate neurite growth of chick nerve cells in vitro by means of a non-enzymatic mechanism, Cell Tissue Res. 273 (1993) 219–226. [5] R.Y.Y. Chan, F.A. Adatia, A.M. Krupa, B.J. Jasmin, Increased expression of acetylcholinesterase T and R transcripts during hematopoietic differentiation is accompanied by parallel elevations in the levels of their respective molecular forms, J. Biol. Chem. 273 (1998) 9727–9733. [6] M. Pick, C. Flores-Flores, D. Grisaru, S. Shochat, V. Deutsch, H. Soreq, Blood-cell-specific acetylcholinesterase splice variations under changing stimuli, Int. J. Dev. Neurosci. 22 (2004) 523–531. [7] C.A. Inkson, A.C. Brabbs, T.S. Grewal, T.M. Skerry, P.G. Genever, Characterization of acetylcholinesterase expression and secretion during osteoblast differentiation, Bone 35 (2004) 819–827. [8] X.J. Zhang, L. Yang, Q. Zhao, J.P. Caen, H.Y. He, Q.H. Jin, L.H. Guo, M. Alemany, L.Y. Zhang, Y.F. Shi, Induction of acetylcholinesterase expression during apoptosis in various cell types, Cell Death Differ. 9 (2002) 790–800. [9] D. Patinkin, S. Seidman, F. Eckstein, F. Benseler, H. Zakut, H. Soreq, Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro, Mol. Cell. Biol. 10 (1990) 6046–6050. [10] A. Robitzki, A. Mack, A. Chatonnet, P.G. Layer, Transfection of reaggregating embryonic chicken retinal cells with an antisense 5 -DNA butyrylcholinesterase expression vector inhibits proliferation and alters morphogenesis, J. Neurochem. 69 (1997) 823–833. [11] A. Robitzki, F. Döll, C. Richter-Landsberg, P.G. Layer, Regulation of the rat oligodendroglia cell line OLN-93 by antisense transfection of butyrylcholinesterase, Glia 31 (2000) 195–205. [12] B. Li, J.A. Stribley, A. Ticu, W. Xie, L.M. Schopfer, P. Hammond, S. Brimijoin, S.H. Hinrichs, O. Lockridge, Abundant tissue butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse, J. Neurochem. 75 (2000) 1320–1331. [13] G. Weitzer, Embryonic stem cell-derived embryoid bodies: an in vitro model of eutherian pregastrulation development and early gastrulation, Handb. Exp. Pharmacol. 174 (2006) 21–51. [14] L.E. Paraoanu, G. Steinert, A. Koehler, I. Wessler, P.G. Layer, Expression and possible functions of the cholinergic system in a murine embryonic stem cell line, Life Sci. 80 (2007) 2375–2379. [15] M.J. Karnovsky, L. Roots, A “direct-coloring” thiocholine method for cholinesterases, J. Histochem. Cytochem. 12 (1964) 219– 221. [16] I. Wessler, C.J. Kirkpatrick, K. Racké, The cholinergic ‘pitfall’: acetylcholine, a universal cell molecule in biological systems, including humans, Clin. Exp. Pharmacol. Physiol. 26 (1999) 198– 205. [17] I. Wessler, H. Kilbinger, F. Bittinger, C.J. Kirkpatrick, The biological role of non-neuronal acetylcholine in plants and humans, Jpn. J. Pharmacol. 85 (2001) 2–10. [18] K.R. Birikh, E.H. Sklan, S. Shoham, H. Soreq, Interaction of “readthrough” acetylcholinesterase with RACK1 and PKCbeta II correlates with intensified fear-induced conflict behaviour, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 283–288.
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