Abstract
Acetylcholine (ACh) is detected in a variety of non-neuronal cells where it acts as a para/autocrine signaling molecule controlling basic cell functions such as proliferation, differentation, and maintenance of cell-cell contacts. ACh-synthesizing enzymes include choline acetyltransferase and carnitine acetyltransferase (CarAT). ACh is released through vesicular exocytosis or directly from the cytoplasm via organic cation transporters (OCT). Extracellular ACh binds to nicotinic (nAChR) and muscarinic receptors (MR). Degradation of ACh is performed by acetylcholinesterase and butyrylcholinesterase (BChE). Here, we have determined whether these molecules are expressed in osteoblast-like cells, by means of reverse transcription polymerase chain reaction and immunohistochemistry, focusing on nAChR subunits α3 and α5. RNA for CarAT, OCT-1, M2R, M5R, nAChR subunits α3, α5, α9, α10, β2, β3, and BChE were detected in human (SAOS-2) and murine (MC3T3-E1) osteoblast-like cells. Other cholinergic components were only expressed species-specifically, e.g., M3R and nAChR subunit α7. Immunhistochemistry localized the nAChR subunits α3 and α5 in osteoblasts in vitro and in vivo where they were up-regulated after application of bone morphogenetic protein-2 (BMP-2) during fracture healing in a rat model. Thus, the cholinergic system of osteoblast-like cells might be regulated by BMP-2 during bone remodeling. Osteoblast-like cells express all necessary enzymes, transporters, and receptors for ACh synthesis and recycling.
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Anderson AA, Helmering J, Juan T, Li CM, McCormick J, Graham M, Baker DM, Damore MA, Veniant MM, Lloyd DJ (2009) Pancreatic islet expression profiling in diabetes-prone C57BLKS/J mice reveals transcriptional differences contributed by DBA loci, including Plagl1 and Nnt. Pathogenetics 2:1
Arredondo J, Hall LL, Ndoye A, Nguyen VT, Chernyavsky AI, Bercovich D, Orr-Urtreger A, Beaudet AL, Grando SA (2003) Central role of fibroblast alpha3 nicotinic acetylcholine receptor in mediating cutaneous effects of nicotine. Lab Invest 83:207–225
Berse B, Szczecinska W, Lopez-Coviella I, Madziar B, Zemelk V, Kaminski R, Kozar K, Lips KS, Pfeil U, Blusztajn JK (2005) Expression of high affinity choline transporter during mouse development in vivo and its upregulation by NGF and BMP-4 in vitro. Brain Res Dev Brain Res 157:132–140
Brady KP, Dushkin H, Förnzler D, Koike T, Magner F, Her H, Gullans S, Segre GV, Green RM, Beier DR (1999) A novel putative transporter maps to the osteosclerosis (oc) mutation and is not expressed in the oc mutant mouse. Genomics 56:254–261
Buisson P, Picard F, Bertrand D (2000) Neuronal nicotinic acetylcholine receptors: from biophysical properties to human diseases. In: Clementi F, Gotti C, Fornasari D (eds) Neuronal nicotinic acetylcholine receptors. Springer, Berlin Heidelberg New York, pp 272–299
Caulfield MP, Birdsall NJM (1998) International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol Rev 50:279–290
Cho G, Youngshin L, Zand D, Golden JA (2008) Sizn1 is a novel protein that functions as a transcriptional coactivator of bone morphogenic protein signaling. Mol Cell Biol 28:1565–1572
Eiden LE (1998) The cholinergic gene locus. J Neurochem 70:2227–2240
Feitelson JB, Rowell PP, Roberts CS, Fleming JT (2003) Two week nicotine treatment selectively increases bone vascular constriction in response to norepinephrine. J Orthop Res 21:497–502
Genever PG, Birch MA, Brown E, Skerry TM (1999) Osteoblast-derived acetylcholinesterase: a novel mediator of cell-matrix interactions in bone? Bone 24:297–303
Grisaru D, Lev-Lehman E, Shapira M, Chaikin E, Lessing JB, Eldor A, Eckstain F, Soreq H (1999) Human osteogenesis involves differentiation-dependent increases in the morphogenetically active 3′ alternative splicing variant of acetylcholinesterase. Mol Cell Biol 19:788–795
Herber DL, Severance EG, Cuevas J, Morgan D, Gordon MN (2004) Biochemical and histochemical evidence of nonspecific binding of alpha7nAChR antibodies to mouse brain tissue. J Histochem Cytochem 52:1367–1376
Hoogduijn MJ, Cheng A, Genever PG (2008) Functional nicotinic and muscarinic receptors on mesenchymal stem cells. Stem Cells Dev (in press)
Inkson CA, Brabbs AC, Grewal TS, Skerry TM, Genever PG (2004) Characterization of acetylcholinesterase expression and secretion during osteoblast differentiation. Bone 35:819–827
Jositsch G, Papadakis T, Habergerger RV, Wolff M, Wess J, Kummer W (2009) Suitability of muscarinic acetylcholine receptor antibodies for immunohistochemistry evaluated on tissue sections of receptor gene-deficient mice. Naunyn Schmiedebergs Arch Pharmacol 379:389–395
Kummer W, Wiegand S, Akinci S, Wessler I, Schinkel AH, Wess J, Koepsell H, Haberberger RV, Lips KS (2006) Role of acetylcholine and polyspecific cation transporters in serotonin-induced bronchoconstriction in the mouse. Respir Res 7:65
Koepsell H, Lips K, Volk C (2007) Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications. Pharm Res 24:1227–1251
Lian JB, Shalhoub V, Aslam F, Frenkel B, Green J, Hamrah M, Stein GS, Stein JL (1997) Species-specific glucocorticoid and 1, 25-dihyroxyvitamin D responsiveness in mouse MC3T3–E1 osteoblasts: dexamethasone inhibits osteoblast differentiation and vitamin D down-regulates osteocalcin gene expression. Endocrinology 138:2117–2127
Lips KS, Volk C, Schmitt BM, Pfeil U, Arndt P, Miska D, Ermert L, Kummer W, Koepsell H (2005) Polyspecific cation transporters mediate luminal release of acetylcholine from bronchial epithelium. Am J Respir Cell Mol Biol 33:79–88
Lips KS, Wunsch J, Zarghooni S, Bschleipfer T, Schukowski K, Weidner W, Wessler I, Schwantes U, Koepsell H, Kummer W (2007) Acetylcholine and molecular components of its synthesis and release machinery in the urothelium. Eur Urol 51:1042–1053
Lopez-Coviella I, Berse B, Thies RS, Blusztajn JK (2002) Upregulation of acetylcholine synthesis by bone morphogenetic protein 9 in a murine septal cell line. J Physiol (Paris) 96:53–59
Massoulie J (2002) The origin of the molecular diversity and functional anchoring of cholinesterases. Neurosignals 11:130–143
McCarthy ID, Andhoga M, Batten JJ, Mathie RT (1997) Endothelium-dependent vasodilatation produced by the L-arginine/nitric oxide pathway in normal and ischemic bone. Acta Orthop Scand 68:361–368
Millar NS, Harkness PC (2008) Assembly and trafficking of nicotinic acetylcholine receptors. Mol Membr Biol 25:279–292
Moser N, Mechawar N, Jones I, Gochberg-Sarver A, Orr-Urtreger A, Plomann M, Salas R, Molles B, Marubio L, Roth U, Maskos U, Winzer-Serhan U, Bourgeois JP, Le Sourd AM, De Biasi M, Schröder H, Lindstrom J, Maelicke A, Changeux JP, Wevers A (2007) Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures. J Neurochem 102:479–492
Okuda T, Haga T (2003) High-affinity choline transporter. Neurochem Res 28:483–488
Pereira A, McLaren A, Bell WR, Copolov D, Dean B (2003) Potential clozapine target sites on peripheral hematopoietic cells and stromal cells of the bone marrow. Pharmacogenomics J 3:227–234
Romano SJ, Corriveau RA, Schwarz RI, Berg DK (1997) Expression of the nicotinic receptor alpha 7 gene in tendon and periosteum during early development. J Neurochem 68:640–648
Schoepfer R, Conroy WG, Whiting P, Gore M, Lindstrom J (1990) Brain alpha-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily. Neuron 5:35–48
Sekhon HS, Keller JA, Proskocil BJ, Martin EL, Spindel ER (2002) Maternal nicotine exposure upregulates collagen gene expression in fetal monkey lung. Association with alpha7 nicotinic acetylcholine receptors. Am J Respir Cell Mol Biol 26:31–41
Sudo H, Kodama HA, Amagai Y, Yamamoto S, Kasai S (1983) In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 96:191–198
Termaat MF, Den Boer FC, Bakker FC, Patka P, Haarman HJ (2005) Bone morphogenetic proteins. Development and clinical efficacy in the treatment of fractures and bone defects. J Bone Joint Surg Am 87:1367–1378
Tournier JM, Maouche K, Coraux C, Zahm JM, Cloëz-Tayarani I, Nawrocki-Raby B, Bonnomet A, Burlet H, Lebargy F, Polette M, Birembaut P (2006) Alpha3alpha5beta2-nicotinic acetylcholine receptor contributes to the wound repair of the respiratory epithelium by modulating intracellular calcium in migrating cells. Am J Pathol 168:55–68
Tucek S (1982) The synthesis and release of acetylcholine in normal and denervated rat diaphragms during incubations in vitro. J Physiol (Lond) 322:54–69
Walker LM, Preston MR, Magnay JL, Thomas PBM, El Haj AJ (2001) Nicotinic regulation of c-fos and osteopontin expression in human-derived osteoblast-like cells and human trabecular bone organ culture. Bone 28:603–608
Wenisch S, Trinkaus K, Hild A, Hose D, Herde K, Heiss C, Kilian O, Alt V, Schnettler R (2005) Human reaming debris: a source of multipotent stem cells. Bone 36:74–83
Wessler I, Kirkpatrick CJ (2008) Acetylcholine beyond neurons: the non-neuronal cholinergic system in humans. Br J Pharmacol 154:1558–1571
Wessler I, Roth E, Deutsch C, Brockerhoff P, Bittinger F, Kirkpatrick CJ, Kolbinger H (2001) Release of non-neuronal acetylcholine from the isolated human placenta is mediated by organic cation transporters. Br J Pharmacol 134:951–956
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The authors thank M. Bodenbenner, R. Braun, and I. Oberst for expert technical assistance.
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En-Nosse, M., Hartmann, S., Trinkaus, K. et al. Expression of non-neuronal cholinergic system in osteoblast-like cells and its involvement in osteogenesis. Cell Tissue Res 338, 203–215 (2009). https://doi.org/10.1007/s00441-009-0871-1
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DOI: https://doi.org/10.1007/s00441-009-0871-1