Increased Expression of Acetylcholinesterase T and R Transcripts during Hematopoietic Differentiation Is Accompanied by Parallel Elevations in the Levels of Their Respective Molecular Forms

Differentiation of hematopoietic cells is known to be accompanied by profound changes in acetylcholinesterase (AChE) enzyme activity, yet the basic mechanisms underlying this developmental regulation remain unknown. We initiated a series of experiments to examine the molecular mechanisms involved in regulating AChE expression during hematopoiesis. Differentiation of murine erythroleukemia (MEL) cells using dimethyl sulfoxide resulted in a 5- and 10-fold increase in intracellular and secreted AChE enzyme activity, respectively. Interestingly, these increases resulted from a preferential induction of the globular molecular form G1 and a slight increase in G4 instead of an increase in the levels of the G2 membrane-bound form, a molecular form expressed in mature erythrocytes. Concomitantly, expression of the two predominant AChE transcripts (R and T, for read-through and tail, respectively) in MEL cells was induced to a similar extent with differentiation. Nuclear run-on assays performed with nuclei isolated from inducedversus uninduced MEL cells revealed that in contrast to the large increases seen in the transcription of the β-globin gene, the transcriptional activity of the AChE gene remained largely unaffected after differentiation. Determination of the half-lives of the R and T transcripts demonstrated that they both exhibited an increase in stability in induced MEL cells. Taken together, results from these studies indicate that post-transcriptional regulatory mechanisms account for the increased expression of AChE in differentiated hematopoietic cells. Differentiation of hematopoietic cells is known to be accompanied by profound changes in acetylcholinesterase (AChE) enzyme activity, yet the basic mechanisms underlying this developmental regulation remain unknown. We initiated a series of experiments to examine the molecular mechanisms involved in regulating AChE expression during hematopoiesis. Differentiation of murine erythroleukemia (MEL) cells using dimethyl sulfoxide resulted in a 5- and 10-fold increase in intracellular and secreted AChE enzyme activity, respectively. Interestingly, these increases resulted from a preferential induction of the globular molecular form G1 and a slight increase in G4 instead of an increase in the levels of the G2 membrane-bound form, a molecular form expressed in mature erythrocytes. Concomitantly, expression of the two predominant AChE transcripts (R and T, for read-through and tail, respectively) in MEL cells was induced to a similar extent with differentiation. Nuclear run-on assays performed with nuclei isolated from inducedversus uninduced MEL cells revealed that in contrast to the large increases seen in the transcription of the β-globin gene, the transcriptional activity of the AChE gene remained largely unaffected after differentiation. Determination of the half-lives of the R and T transcripts demonstrated that they both exhibited an increase in stability in induced MEL cells. Taken together, results from these studies indicate that post-transcriptional regulatory mechanisms account for the increased expression of AChE in differentiated hematopoietic cells. Acetylcholinesterase (AChE) 1The abbreviations used are: AChE, acetylcholinesterase; A, asymmetric; G, globular; MEL, murine erythroleukemia; Me2SO, dimethyl sulfoxide; RT-PCR, reverse transcription-polymerase chain reaction; T, tail; H, hydrophobic; R, read-through. is responsible for inactivating acetylcholine at cholinergic synapses in both central and peripheral nervous systems. This enzyme displays a rich polymorphism because it exists as a variety of molecular forms that may be classified as either homomeric or heteromeric on the basis of their association with specialized structural subunits (1Massoulié J. Pezzementi L. Bon S. Krejci E. Vallette F.-M. Prog. Neurobiol. 1993; 41: 31-91Crossref PubMed Scopus (1051) Google Scholar). Homomeric forms include the globular (G)1 monomer and G2 dimer as well as a glycophospholipid-linked dimer. Conversely, heteromers consist of the asymmetric forms A4, A8, or A12 in which one, two, or three soluble G4tetramers attach to a collagenic structural subunit, respectively; and amphiphilic tetramers G4-linked to a 20-kDa hydrophobic anchor. Although the functional significance of this polymorphism is still elusive, it has been suggested that it allows the placement of catalytically active subunits in distinct cell types and subcellular locations to perform site-specific functions. In mammals, for example, the asymmetric forms of AChE are expressed exclusively in differentiated muscle and neuronal cells, whereas glycophospholipid-linked dimers are found preferentially in tissues of hematopoietic origin. Such varied patterns of expression suggest that expression of AChE involves several levels of regulatory mechanisms ranging from tissue-specific transcriptional control to highly regulated post-translational events. An interesting feature of AChE is that its expression is indeed not restricted to cholinergic tissues. In fact, its presence in non-cholinergic cells such as specific neuronal populations (2Landwehrmeyer B. Probst A. Palacois J.M. Mengod G. Neuroscience. 1993; 57: 615-634Crossref PubMed Scopus (33) Google Scholar, 3Hammond P. Rao R. Koenbigsberger C. Brimijoin S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10933-10937Crossref PubMed Scopus (32) Google Scholar, 4Bernard V. Legay C. Massoulié J. Bloch B. Neuroscience. 1995; 64: 995-1005Crossref PubMed Scopus (24) Google Scholar) and hematopoietic cells (5Paulus J.M. Maign J. Keyhani E. Blood. 1981; 58: 1100-1106Crossref PubMed Google Scholar, 6Ott P. Lustig A. Brodbeck U. Rosenbusch J.P. FEBS Lett. 1982; 138: 187-189Crossref PubMed Scopus (44) Google Scholar, 7Szelenyi J.G. Bartha E. Hollan S.R. Br. J. Haematol. 1982; 50: 241-245Crossref PubMed Scopus (80) Google Scholar) has led to the proposal that AChE may participate in additional cellular functions distinct from its well known catalytic activity (for review, see Ref. 8Layer P.G. Willbold E. Prog. Histochem. Cytochem. 1995; 29: 1-94PubMed Google Scholar). For example, converging lines of evidence indicate that the process of terminal differentiation in specific lineages of hematopoietic cells is accompanied by large increases in the levels of AChE activity (see Ref.9Paoletti F. Mocali A. Vannucchi A.M. Blood. 1992; 79: 2873-2879Crossref PubMed Google Scholar). Moreover, it has been suggested that AChE may participate directly in the regulation of bone marrow cell development by reducing the proliferative and expansive capacity of pluripotent stem cells committed to erythropoiesis, megakaryopoiesis, and macrophage production and by promoting apoptosis in their progenies (10Soreq H. Lev-Lehman E. Patinkin D. Grifman M. Ehrlich D. Ginzberg D. Eckstein F. Zakut H. Quinn D.M. Balasubramanian A.S. Doctor B.P. Taylor P. Enzymes of the Cholinesterase Family. Plenum Press, New York1995: 1-6Crossref Google Scholar, 11Lev-Lehman E. Hornreich G. Ginzberg D. Meytes D. Zakut H. Gene Therapy. 1994; 1: 127-135PubMed Google Scholar, 12Soreq H. Patinkin D. Lev-Lehman E. Grifman M. Ginzberg D. Eckstein F. Zakut H. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 7907-7911Crossref PubMed Scopus (100) Google Scholar). Finally, deletion of the AChE locus at 7q22 has been shown to be associated with both myelodysplastic syndromes and acute myeloid leukemia, suggesting that AChE may in fact act as a tumor suppressor gene (14Ehrlich G. Viegas-Pequignot E. Ginzberg D. Sindel L. Soreq H. Zakut H. Genomics. 1992; 13: 1192-1197Crossref PubMed Scopus (47) Google Scholar, 15Stephenson J. Czepulkowski B. Hirst W. Mufti G.J. Leukemia Res. 1996; 20: 235-241Crossref PubMed Scopus (48) Google Scholar). In light of these recent findings, it becomes important to elucidate the cellular and molecular mechanisms presiding over AChE expression in hematopoietic cells since it may provide additional insights into its physiological function in this cell type. Additionally, a thorough understanding of these mechanisms in skeletal muscle as well as in neuronal and hematopoietic cells will not only contribute to our knowledge of the regulatory mechanisms controlling tissue-specific expression of AChE, but it should also lead to the elucidation of the factors implicated in its highly controlled developmental regulation. The goal of the present study was to examine the molecular mechanisms involved in regulating AChE expression during the differentiation of hematopoietic cells. We initiated our studies using murine erythroleukemia (MEL) cells because they can be induced easily to undergo terminal hematopoietic differentiation accompanied by hemoglobinization after exposure to inducing agents such as dimethyl sulfoxide (Me2SO) (16Marks P.A. Rifkind R.A. Annu. Rev. Biochem. 1978; 47: 419-448Crossref PubMed Scopus (576) Google Scholar). This cell line represents an excellent model with which to study AChE expression during hematopoiesis because induced cells are known to express high levels of AChE compared with their uninduced counterparts (9Paoletti F. Mocali A. Vannucchi A.M. Blood. 1992; 79: 2873-2879Crossref PubMed Google Scholar, 17Conscience J.F. Miller R.A. Henry J. Ruddle F.H. Exp. Cell Res. 1977; 105: 401-412Crossref PubMed Scopus (49) Google Scholar). Friend MEL cells were grown in suspension in minimum essential medium supplemented with 5% fetal bovine serum and antibiotics (100 units/ml penicillin G and 100 μg/ml streptomycin sulfate) at 37 °C in a water-saturated atmosphere containing 5% CO2. Differentiation of these cells was induced by adding 1.5% Me2SO to the medium. The number of cells was determined using a hemocytometer. Cell viability was assessed using the trypan blue exclusion test. Extraction of intracellular AChE was performed on cells washed twice with phosphate-buffered saline and resuspended in 350–700 μl of a high salt detergent buffer containing antiproteolytic agents (10 mm Tris-HCl, pH 7.0; 10 mm EDTA; 1 m NaCl; 1% Triton X-100; 1.0 mg/ml bacitracin; 2.5 mg/ml aprotinin). The cells were homogenized for 30 s with a Polytron and then centrifuged at 14,000 rpm for 15 min at 4 °C. The resulting supernatants were transferred to fresh tubes and stored at −80 °C for further analysis. To assay for secreted AChE, MEL cells were cultured in medium containing serum in which endogenous esterase activity was inhibited by treatment with diisopropyl fluorophosphate as described except for some Taylor P. J. 1996; PubMed Scopus Google Scholar). fetal bovine serum was with fluorophosphate at on a The serum was then and at 4 °C for 10 at which 1% of the AChE activity The serum activity AChE or cell and differentiation. AChE activity was determined using a of the of V. Biochem. PubMed Scopus Google Scholar) in the presence of which as described V. J. PubMed Google Scholar). AChE activity was by in the or presence of the was determined using a assay The of AChE molecular forms in induced cells and in the medium was determined by as described in Taylor P. J. 1996; PubMed Scopus Google Scholar, V. J. PubMed Google Scholar). were containing either Triton or were centrifuged in a at rpm for at 4 °C. were from the of the tubes and for AChE of activity to distinct molecular forms were to the of Bon S. M. Massoulié J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) on the basis of their Cell were centrifuged at rpm for The cell were then in 1.0 of and were in an of of was reverse to The reverse transcription mm 1 buffer mm 10 mm Tris-HCl, pH 1 mm of of murine reverse and 2.5 of reverse transcription in which was by To AChE for of the T, H, and R transcripts were used C. M. Massoulié J. J. 1995; PubMed Scopus Google Scholar). assays were performed as described in 1993; PubMed Scopus Google Scholar, W. J. Cell 1994; PubMed Scopus Google Scholar). μl of the reverse was to μl of a containing of a mm of of the and and and buffer at of mm and 1 respectively. The involved at °C for 1 min and and at °C for min for were using either 1 or 1.5% containing and using 1.5% containing The of were using a For of the of the AChE of reverse from MEL cell were performed in with of an control as which an AChE with a deletion by for see Ref. C. M. Massoulié J. J. 1995; PubMed Scopus Google Scholar). The of AChE transcripts was determined by the of the from MEL cell a from the of using of the control We used a similar to the of AChE at the (see Ref. 1993; PubMed Scopus Google Scholar). To the stability of the AChE or was used to transcription in uninduced and induced MEL cell was from these at and by MEL cells were washed in phosphate-buffered and in 10 of buffer (10 mm Tris-HCl, pH 10 mm NaCl; mm by for 10 s and on for 10 were then by at for min at 4 °C and washed twice in were resuspended in μl of buffer mm Tris-HCl, pH mm mm and used or stored in transcription assays were performed as described in C. P. J. Biochem. 1994; PubMed Scopus Google Scholar). was used to transcripts for 30 min at °C. and was using and to containing 10 of AChE, and The of the was using a were to used as an because transcriptional of this gene been shown to remain during differentiation of MEL cells C. P. J. Biochem. 1994; PubMed Scopus Google Scholar). a exposure to Me2SO, differentiation of MEL cells be easily by their by the of not with uninduced control cells, expression of AChE was increased in induced MEL cells In fact, intracellular and secreted AChE activity increased by 5- and in induced cells shown in it also that in both uninduced and induced MEL cells, of the enzyme activity was in the that these cells large of neuronal cells Taylor P. J. 1996; PubMed Scopus Google the of enzyme secreted remained during hematopoietic differentiation of MEL cells. To differentiation was accompanied by increases in the of specific molecular by using of the cell and from both uninduced and induced MEL cells and the AChE enzyme activity in shown in and uninduced MEL cell and only a of the G1 of in induced cell high levels of G1 with of G4 In these also a high of G1 in the medium of induced MEL cells these results indicate that the large increases in and secreted AChE enzyme activity are for by increases in the levels of the globular forms of In also determined the amphiphilic of the G1 expressed by these cells using containing Although present in the not that of the form in either uninduced or induced cells a their amphiphilic and the basis of these it that both uninduced and induced MEL cells express two of AChE We the increase in AChE enzyme activity which during the differentiation of MEL cells was accompanied by increases in the levels of AChE Although AChE is by a gene, three transcripts T, H, and R are by (1Massoulié J. Pezzementi L. Bon S. Krejci E. Vallette F.-M. Prog. Neurobiol. 1993; 41: 31-91Crossref PubMed Scopus (1051) Google Scholar, P. Annu. Rev. 1994; PubMed Scopus Google Scholar). We using specific that transcripts to their C. M. Massoulié J. J. 1995; PubMed Scopus Google Scholar). For these AChE levels in uninduced and induced MEL cells were at over 4 MEL cell the levels of both T and R transcripts increased from 1 to the of these levels of the in uninduced MEL cells as by S. D. Taylor P. J. PubMed Google Scholar, S. Taylor P. J. 1993; PubMed Google Scholar). differentiation not increase expression of this shown in and expression of both T and R transcripts increased in induced MEL of AChE T and R levels by of the control that an AChE were in with from uninduced and induced MEL cells for and for the of the T and R respectively. that 1 of from MEL cell was used for of the T whereas was used for the R. are of from the the number of and from uninduced and induced MEL cell For of the were in containing and using a from the were as of indicate the levels of the transcripts in uninduced and induced MEL cells. that the of both transcripts increased by results are from To the of the T and R transcripts in uninduced and induced MEL cells, determined the of in by using the control (see Ref. C. M. Massoulié J. J. 1995; PubMed Scopus Google Scholar). 1 of of 1 of was to the to the T in that in uninduced MEL cells was of AChE T experiments revealed that of from uninduced cells of AChE R that as by assays S. D. Taylor P. J. PubMed Google S. Taylor P. J. 1993; PubMed Google the R is the T in uninduced MEL cells are for shown in this differentiation increased the expression of both transcripts by the of R transcripts In with with muscle (see Ref. 1993; PubMed Scopus Google these experiments also that both AChE are to transcripts in MEL cells. is the transcriptional regulation of the AChE gene in hematopoietic cells. the presence of a for a transcription important for and has been in the regulatory of both 1995; Scholar) and R. S. R. M. Zakut H. Soreq H. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar) AChE In experiments the increase in AChE which in MEL cells was by a increase in the transcriptional activity of the AChE Nuclear run-on assays performed with nuclei isolated from MEL cells at as (see Ref. C. P. J. Biochem. 1994; PubMed Scopus Google a large 10-fold increase in the transcriptional activity of the β-globin gene in inducedversus uninduced cells these experiments demonstrated that transcription of the AChE gene remained largely unaffected in differentiated MEL cells. with (see Ref. Taylor P. 1993; PubMed Scopus Google of the AChE was to transcripts to experiments using inhibited transcription in both that AChE resulted from the activity of the not these that the increased AChE levels seen in induced cells not from an in the transcriptional activity of the AChE in the levels of AChE in induced MEL cells may also post-transcriptional regulatory We differentiation of MEL cells was accompanied by an increase in the stability of AChE For these experiments inhibited the activity of the by adding to the of uninduced and induced cells and the levels of T and R the predominant of AChE in MEL cells. was at and the levels of these two transcripts were determined by shown in the half-lives of the R and T transcripts were only and respectively. on the basis of our run-on the stability of both AChE transcripts increased in induced MEL cells. In a similar on the of the R and T transcripts using not is well that AChE is expressed in hematopoietic cells and that changes in enzyme activity the process of terminal hematopoietic differentiation. is known the cellular and molecular mechanisms underlying the expression of AChE in hematopoietic cells as well as its induction during differentiation. We MEL cells as a model to these In the present study on the of AChE molecular forms and the transcripts expressed during hematopoiesis as well as on the of transcriptional post-transcriptional regulatory mechanisms in the induction of AChE of MEL cells to Me2SO, an of resulted in large increases in and secreted AChE activity as described (9Paoletti F. Mocali A. Vannucchi A.M. Blood. 1992; 79: 2873-2879Crossref PubMed Google Scholar, 17Conscience J.F. Miller R.A. Henry J. Ruddle F.H. Exp. Cell Res. 1977; 105: 401-412Crossref PubMed Scopus (49) Google Scholar). Interestingly, these changes in AChE expression were for by increases in the levels of G1 by changes in the of the glycophospholipid-linked which is highly expressed in mature (1Massoulié J. Pezzementi L. Bon S. Krejci E. Vallette F.-M. Prog. Neurobiol. 1993; 41: 31-91Crossref PubMed Scopus (1051) Google Scholar). uninduced and induced MEL cells are to and which are in the bone marrow C. W. J.G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google our suggest that expression of AChE is a feature in mature hematopoietic cells. In of this of has revealed the presence of AChE activity the cells on the cell M. J. PubMed Scopus Google Scholar). recent studies of that AChE is E. V. A. Soreq H. Blood. PubMed Google Scholar). MEL cell the increases in AChE activity were accompanied by increases in the levels of AChE transcripts as revealed by our analysis. Although AChE is by a gene, by to three distinct transcripts T, H, and R. studies shown that the T is the only expressed in muscle C. M. Massoulié J. J. 1995; PubMed Scopus Google Scholar) and from of 4 to the results from of 4 to and exclusively in cells of hematopoietic whereas the R results from the of 4 into the (see J. Pezzementi L. Bon S. Krejci E. Vallette F.-M. Prog. Neurobiol. 1993; 41: 31-91Crossref PubMed Scopus (1051) Google and P. Annu. Rev. 1994; PubMed Scopus Google Scholar). In with S. D. Taylor P. J. PubMed Google Scholar, S. Taylor P. J. 1993; PubMed Google in uninduced MEL cells that of the AChE to the R and the of the T levels of these transcripts are controlled by their of and our a similar for both of these transcripts in uninduced MEL cells suggest that the of the R is at a is that transcription of the AChE gene for the of AChE from 4 to for T is a in uninduced MEL cells. the is in these cells (see S. D. Taylor P. J. PubMed Google and S. Taylor P. J. 1993; PubMed Google and this the from 4 to for its as the used for of AChE This of during MEL cell differentiation as the of R to T transcripts remained in induced MEL cells the increases in the levels of both of AChE Interestingly, this is not to such as uninduced and induced MEL cells. may in fact to hematopoietic cells because assays that the of AChE in MEL cells was similar to that in the bone marrow S. D. Taylor P. J. PubMed Google which a of hematopoietic cells. the the glycophospholipid-linked dimer of AChE which is expressed in mature (1Massoulié J. Pezzementi L. Bon S. Krejci E. Vallette F.-M. Prog. Neurobiol. 1993; 41: 31-91Crossref PubMed Scopus (1051) Google the of this in both uninduced and induced MEL cells that the for of this is at a of hematopoietic differentiation. this the that of AChE is regulated in tissues as in skeletal muscle C. M. Massoulié J. J. 1995; PubMed Scopus Google Scholar) and specific molecular forms of AChE which are to distinct subcellular locations are at of hematopoietic differentiation. will be interesting to examine the is the AChE transcripts in mature such as In both uninduced and induced MEL cells, of the AChE enzyme activity to secreted in the medium. Interestingly, the R that this was only the T these results suggest that the R may be the T it is also that the secreted AChE, intracellular AChE, is not to by the the cells. To the molecular mechanisms regulating AChE gene expression been in cultured muscle Taylor P. 1993; PubMed Scopus Google Scholar, Taylor P. J. 1994; PubMed Google Scholar) and neuronal cells Taylor P. J. 1996; PubMed Scopus Google Scholar). In the cell it has been demonstrated that the increase in the levels of AChE the of into results from an increase in the stability of transcripts as to a transcriptional of the AChE gene Taylor P. 1993; PubMed Scopus Google Scholar). post-transcriptional regulatory mechanisms also been suggested to account for the large induction in the levels of AChE in cells induced to into neuronal and cells Taylor P. J. 1996; PubMed Scopus Google Scholar). MEL cells as a model to study the molecular mechanisms underlying the increase in AChE expression during in the present study that the increase in AChE levels also results from an increase in the stability of post-transcriptional at the of as a regulatory levels of AChE in cell types known to express of this the of these post-transcriptional mechanisms in the control of AChE levels in cell it is still of to examine the of specific AChE to the transcriptional regulation of the AChE gene, as it to tissue-specific In recent has been evidence that the stability of a in the basic mechanisms underlying gene expression (for review, see J.G. G. of Press, Google and 1993; PubMed Scopus Google Scholar). Such regulatory mechanisms not only distinct factors but also the of both and the of mature the basis of our it that the expressed AChE transcripts during the of a in the activity of an in induced MEL cells. This can be because our studies shown that the stability of the is not differentiation of MEL cells C. P. J. Biochem. 1994; PubMed Scopus Google increased AChE stability to be a specific hematopoietic differentiation. In muscle cells the of has been to the increased stability of AChE transcripts Taylor P. J. 1994; PubMed Google Scholar). Although not been found in MEL cells, a of intracellular in these cells after exposure to Me2SO P. J.F. J. 1993; PubMed Scopus Google Scholar). treatment with the has been shown to an and in the levels of regulation has been implicated in the control of hematopoietic and differentiation A. M. U. F. H. J. 1994; PubMed Google Scholar). it to be a in controlling AChE stability in hematopoietic cells. In cells, an increase in AChE levels after has led to the that some are involved in the of AChE Taylor P. 1993; PubMed Scopus Google Scholar). uninduced MEL cells may provide an interesting model in the of specific for AChE We are to for and We also for the MEL cell line and Legay for the for