Background/Aims: There is an urgent need to identify the molecular factors involved in osteoblast proliferation and differentiation in order to improve bone formation and treat bone disease. Recent studies demonstrate that many ‘axon guidance’ molecules play important roles in the development and remodeling of bone through their actions regulating osteoblast or osteoclast differentiation. The expressions and roles in bone tissue of the neuron guidance molecules slit and robo have not been reported previously. The objective of the current study is to investigate the expression and the roles of slit and robo in osteoblastic cells. Methods: The mRNAs slit2 and robo were detected in primary cell lines during their osteoblastic differentiation. The role of slit2 was studied using the recombinant proteins slit2 and hemagglutinin (HA)-roboN by transfecting the 293 cells. Results: The data indicated that the mRNAs of slit2, robo1 and robo2 were expressed during the osteoblastic differentiation of rat osteoblasts (calvarial osteoblasts derived from newborn rats), the nontransformed preosteogenic cell line MC3T3-E1, rat marrow stromal cells and the murine mesenchymal progenitor cell line C3H10T1/2. The exogenous slit2 protein inhibited the differentiation of rat osteoblasts and MC3T3-E1. Furthermore, the osteoblastic differentiation was improved when the endogenous slits were antagonized by recombinant HA-roboN, which is the extracellular domain of the rat robo1 protein. The inhibition of slit2 in osteogenic differentiation was independent of the RhoA/ROCK pathway. Conclusion: Slit2 plays a role in regulating in vitro osteoblast differentiation. This has opened a new avenue to understand the molecular events involved in the osteoblastic differentiation.

Brose, K., K.S. Bland, K.H. Wang, D. Arnott, W. Henzel, C.S. Goodman, M. Tessier-Lavigne, T. Kidd (1999) Slit proteins bind robe receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96: 795–806.
Delorme, G., F. Saltel, E. Bonnelye, P. Jurdic, I. Machuca-Gayet (2005) Expression and function of semaphorin 7A in bone cells. Biol Cell 97: 589–597.
Ducy, P., G. Karsenty (1998) Genetic control of cell differentiation in the skeleton. Curr Opin Cell Biol 10: 614–619.
Hall, A. (1998) Rho GTPases and the actin cytoskeleton. Science 279: 509–514.
Harmey, D., G. Stenbeck, C.D. Nobes, A.J. Lax, A.E. Grigoriadis (2004) Regulation of osteoblast differentiation by Pasteurella multocida toxin (PMT): a role for Rho GTPase in bone formation. J Bone Miner Res 19: 661–670.
Harper, J., L.C. Gerstenfeld, M. Klagsbrun (2001) Neuropilin-1 expression in osteogenic cells: down-regulation during differentiation of osteoblasts into osteocytes. J Cell Biochem 81: 82–92.
Hinck, L. (2004) The versatile roles of ‘axon guidance’ cues in tissue morphogenesis. Developmental Cell 7: 783–793.
Holmes, G., L. Niswander (2001) Expression of slit-2 and slit-3 during chick development. Dev Dyn 222: 301–307.
Holmes, G.P., K. Negus, L. Burridge, S. Raman, E. Algar, T. Yamada, M.H. Little (1998) Distinct but overlapping expression patterns of two vertebrate slit homologs implies functional roles in CNS development and organogenesis. Mech Dev 79: 57–72.
Itoh, A., T. Miyabayashi, M. Ohno, S. Sakano (1998) Cloning and expressions of three mammalian homologues of Drosophila slit suggest possible roles for Slit in the formation and maintenance of the nervous system. Brain Res Mol Brain Res 62: 175–186.
Koh, J.M., B. Oh, J.Y. Lee, J.K. Lee, K. Kimm, G.S. Kim, B.L. Park, H.S. Cheong, H.D. Shin, J.M. Hong, T.H. Kim, E.K. Park, S.Y. Kim (2006) Association study of semaphorin 7a (sema7a) polymorphisms with bone mineral density and fracture risk in postmenopausal Korean women. J Hum Genet 51: 112–117.
Lai, J.M., C.L. Hsieh, Z.F. Chang (2003) Caspase activation during phorbol ester-induced apoptosis requires ROCK-dependent myosin-mediated contraction. J Cell Sci 116: 3491–3501.
Li, H.S., J.H. Chen, W. Wu, T. Fagaly, L.J. Zhou, W.L. Yuan, S. Dupuis, Z.H. Jiang, W. Nash, C. Gick, D.M. Ornitz, J.Y. Wu, Y. Rao (1999) Vertebrate slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons. Cell 96: 807–818.
Li, Y.S.J., J.H. Haga, S. Chien (2005) Molecular basis of the effects of shear stress on vascular endothelial cells. J Biomech 38: 1949–1971.
Loes, S., K. Luukko, I.H. Kvinnsland, P. Kettunen (2001) Slit1 is specifically expressed in the primary and secondary enamel knots during molar tooth cusp formation. Mech Dev 107: 155–157.
Mackie, E.J. (2003) Osteoblasts: novel roles in orchestration of skeletal architecture. Int J Biochem Cell Biol 35: 1301–1305.
Malek, A.M., S. Izumo (1996) Mechanism of endothelial cell shape change and cytoskeletal remodeling in response to fluid shear stress. J Cell Sci 109: 713–726.
Marie, P.J. (2003) Fibroblast growth factor signaling controlling osteoblast differentiation. Gene 316: 23–32.
McBeath, R., D.M. Pirone, C.M. Nelson, K. Bhadriraju, C.S. Chen (2004) Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell 6: 483–495.
Morimoto, Y., T. Ohba, S. Kobayashi, T. Haneji (1997) The protein phosphatase inhibitors okadaic acid and calyculin A induce apoptosis in human osteoblastic cells. Exp Cell Res 230: 181–186.
Nakashima, K., B. de Crombrugghe (2003) Transcriptional mechanisms in osteoblast differentiation and bone formation. Trends Genet 19: 458–466.
Nakayama, M., D. Nakajima, T. Nagase, N. Nomura, N. Seki, O. Ohara (1998) Identification of high-molecular-weight proteins with multiple EGF-like motifs by motif-trap screening. Genomics 51: 27–34.
Orriss, I.R., G.E. Knight, S. Ranasinghe, G. Burnstock, T.R. Arnett (2006) Osteoblast responses to nucleotides increase during differentiation. Bone 39: 300–309.
Riha, G.M., X.W. Wang, H. Wang, H. Chai, H. Mu, P.H. Lin, A.B. Lumsden, Q.Z. Yao, C.Y. Chen (2007) Cyclic strain induces vascular smooth muscle cell differentiation from murine embryonic mesenchymal progenitor cells. Surgery 141: 394–402.
Rodan, G.A. (1992) Introduction to bone biology. Bone 13: S3–S6.
Schiller, P.C., G. D’Ippolito, W. Balkan, B.A. Roos, G.A. Howard (2001) Gap-junctional communication mediates parathyroid hormone stimulation of mineralization in osteoblastic cultures. Bone 28: 38–44.
Shamir, D., S. Keila., M. Weinreb (2004) A selective EP4 receptor antagonist abrogates the stimulation of osteoblast recruitment from bone marrow stromal cells by prostaglandin E-2 in vivo and in vitro. Bone 34: 157–162.
Sun, H.L., C.T. Wu, K.R. Dai, J. Chang, T.T. Tang (2006) Proliferation and osteoblastic differentiation of human bone marrow-derived stromal cells on akermanite-bioactive ceramics. Biomaterials 27: 5651–5657.
Tamagnone, L., S. Giordano (2006) Semaphorin pathways orchestrate osteogenesis. Nat Cell Biol 8: 545–547.
Thomas, C.H., J.H. Collier, C.S. Sfeir, K.E. Healy (2002) Engineering gene expression and protein synthesis by modulation of nuclear shape. Proc Natl Acad Sci USA 99: 1972–1977.
Wu, J.Y., L.L. Feng, H.T. Park, N. Havlioglu, L. Wen, H. Tang, K.B. Bacon, Z.H. Jiang, X.C. Zhang, Y. Rao (2001) The neuronal repellent Slit inhibits leukocyte chemotaxis induced by chemotactic factors. Nature 410: 948–952.
Xiao, Y., H.H. Fu, I. Prasadam, Y.C. Yang, J.O. Hollinger (2007) Gene expression profiling of bone marrow stromal cells from juvenile, adult, aged and osteoporotic rats: with an emphasis on osteoporosis. Bone 40: 700–715.
Yuan, W.L., L.J. Zhou, T.H. Chen, J.Y. Wu, Y. Rao, D.M. Ornitz (1999) The mouse SLIT family: secreted ligands for ROBO expressed in patterns that suggest a role in morphogenesis and axon guidance. Dev Biol 212: 290–306.
Zamurovic, N., D. Cappellen, D. Rohner, M. Susa (2004) Coordinated activation of Notch, Wnt, and transforming growth factor-beta signaling pathways in bone morphogenic protein 2-induced osteogenesis Notch: target gene Hey1 inhibits mineralization and Runx2 transcriptional activity. J Biol Chem 279: 37704–37715.
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.