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Primary Cultures of Chick Osteocytes Retain Functional Gap Junctions between Osteocytes and between Osteocytes and Osteoblasts

Published online by Cambridge University Press:  15 February 2007

Hiroshi Kamioka ,
Yoshihito Ishihara ,
Hans Ris ,
Sakhr A. Murshid ,
Yasuyo Sugawara ,
Teruko Takano-Yamamoto  and
Soo-Siang Lim
Hiroshi Kamioka
Affiliation:
Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Yoshihito Ishihara
Affiliation:
Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Hans Ris
Affiliation:
Department of Zoology and Integrated Microscopy Resource, University of Wisconsin, Madison, WI 53706-1794, USA
Sakhr A. Murshid
Affiliation:
Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Yasuyo Sugawara
Affiliation:
Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Teruko Takano-Yamamoto
Affiliation:
Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Soo-Siang Lim
Affiliation:
National Science Foundation, Arlington, VA 22230, USA
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Abstract

The inaccessibility of osteocytes due to their embedment in the calcified bone matrix in vivo has precluded direct demonstration that osteocytes use gap junctions as a means of intercellular communication. In this article, we report successfully isolating primary cultures of osteocytes from chick calvaria, and, using anti-connexin 43 immunocytochemistry, demonstrate gap junction distribution to be comparable to that found in vivo. Next, we demonstrate the functionality of the gap junctions by (1) dye coupling studies that showed the spread of microinjected Lucifer Yellow from osteoblast to osteocyte and between adjacent osteocytes and (2) analysis of fluorescence replacement after photobleaching (FRAP), in which photobleaching of cells loaded with a membrane-permeable dye resulted in rapid recovery of fluorescence into the photobleached osteocyte, within 5 min postbleaching. This FRAP effect did not occur when cells were treated with a gap junction blocker (18α-glycyrrhetinic acid), but replacement of fluorescence into the photobleached cell resumed when it was removed. These studies demonstrate that gap junctions are responsible for intercellular communication between adjacent osteocytes and between osteoblasts and osteocytes. This role is consistent with the ability of osteocytes to respond to and transmit signals over long distances while embedded in a calcified matrix.

Keywords

osteocytesosteoblastsgap junctionsintercellular communicationdye couplingFRAP
Type
BIOLOGICAL APPLICATIONS
Information
Microscopy and Microanalysis , Volume 13 , Issue 2 , April 2007 , pp. 108 - 117
Copyright
© 2007 Microscopy Society of America

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References

REFERENCES

Davidson, J.S. & Baumgarten, I.M. (1988). Glycyrrhetinic acid derivatives: A novel class of inhibitors of gap-junctional intercellular communication. Structure–activity relationships. J Pharmacol Exp Ther 246, 1104–1107.Google Scholar
Donahue, H.J., McLeod, K.J., Rubin, C.T., Andersen, J., Grine, E.A., Hertzberg, E.L. & Brink, P.R. (1995). Cell-to-cell communication in osteoblastic networks: Cell line-dependent hormonal regulation of gap junction function. J Bone Miner Res 10, 881–889.Google Scholar
Doty, S.B. (1981). Morphological evidence of gap junctions between bone cells. Calcif Tissue Int 33, 509–512.Google Scholar
Flagg-Newton, J., Simpson, I. & Loewenstein, W.R. (1979). Permeability of the cell-to-cell membrane channels in mammalian cell junction. Science 205, 404–407.Google Scholar
Gaietta, G., Deerinck, T.J., Adams, S.R., Bouwer, J., Tour, O., Laird, D.W., Sosinsky, G.E., Tsien, R.Y. & Ellisman, M.H. (2002). Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 503–507.Google Scholar
Holtrop, M.E. & Weinger, J.M. (1975). The ultrastructure of bone. Ann Clin Lab Sci 5, 264–271.Google Scholar
Ilvesaro, J., Vaananen, K. & Tuukkanen, J. (2000). Bone-resorbing osteoclasts contain gap-junctional connexin-43. J Bone Miner Res 15, 919–926.Google Scholar
Jones, S.J., Gray, C., Sakamaki, H., Arora, M., Boyde, A., Gourdie, R. & Green, C. (1993). The incidence and size of gap junctions between the bone cells in rat calvaria. Anat Embryol (Berl) 187, 343–352.Google Scholar
Jorgensen, N.R., Geist, S.T., Civitelli, R. & Steinberg, T.H. (1997). ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells. J Cell Biol 139, 497–506.Google Scholar
Kamioka, H., Honjo, T. & Takano-Yamamoto, T. (2001). The three-dimensional distribution of osteocyte processes revealed by the combination of confocal laser scanning microscopy and differential interference contrast microscopy. Bone 28, 145–149.Google Scholar
Kamioka, H., Sugawara, Y., Honjo, T., Yamashiro, T. & Takano-Yamamoto, T. (2004). Terminal differentiation of osteoblasts to osteocytes is accompanied by dramatic changes in the distribution of actin-binding proteins. J Bone Miner Res 19, 471–478.Google Scholar
Kato, Y., Windle, J.J., Koop, B.A., Mundy, G.R. & Bonewald, L.F. (1997). Establishment of an osteocyte-like cell line, MLO-Y4. J Bone Miner Res 12, 2014–2023.Google Scholar
Laing, J.G., Manley-Markowski, R.N., Koval, M., Civitelli, R. & Stenberg, T.H. (2001). Connexin 45 interacts with zonula occulens-1 and connexin43 in osteoblastic cells. J Biol Chem 276, 23051–23055.Google Scholar
Nijweide, P.J. & Mulder, R.J. (1986). Identification of osteocytes in osteoblast-like cell cultures using a monoclonal antibody specifically directed against osteocytes. Histochemistry 84, 342–347.Google Scholar
Ris, H. (1985). The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections. J Cell Biol 100, 1474–1487.Google Scholar
Schiller, P.C., Mehta, P.P., Roos, B.A. & Howard, G.A. (1992). Hormonal regulation of intercellular communication: Parathyroid hormone increases connexin 43 gene expression and gap-junctional communication in osteoblastic cells. Mol Endocrinol 6, 1433–1440.Google Scholar
Schwarzmann, G., Wiegandt, H., Rose, B., Zimmerman, A., Ben, H.D. & Loewenstein, W.R. (1981). Diameter of the cell-to-cell junctional membrane channels as probed with neutral molecules. Science 213, 551–553.Google Scholar
Shapiro, F. (1997). Variable conformation of GAP junctions linking bone cells: A transmission electron microscopic study of linear, stacked linear, curvilinear, oval, and annular junctions. Calcif Tissue Int 61, 285–293.Google Scholar
Shiokawa-Sawada, M., Mano, H., Hanada, K., Kakudo, S., Kameda, T., Miyazawa, K., Nakamaru, Y., Yuasa, T., Mori, Y., Kumegawa, M. & Hakeda, Y. (1997). Down-regulation of gap junctional intercellular communication between osteoblastic MC3T3-E1 cells by basic fibroblast growth factor and a phorbol ester (12-O-tetradecanoylphorbol-13-acetate). J Bone Miner Res 12, 1165–1173.Google Scholar
Sugawara, Y., Kamioka, H., Honjo, T., Tezuka, K. & Takano-Yamamoto, T. (2005). Three-dimensional reconstruction of chick calvarial osteocytes and their cell processes using confocal microscopy. Bone 36, 877–883.Google Scholar
Tanaka-Kamioka, K., Kamioka, H., Ris, H. & Lim, S. (1998). Osteocyte shape is dependent on actin filaments and osteocyte processes are unique actin-rich projections. J Bone Miner Res 13, 1555–1568.Google Scholar
Vander Molen, M.A., Rubin, C.T., McLeod, K.J., McCauley, L.K. & Donahue, H.J. (1996). Gap junctional intercellular communication contributes to hormonal responsiveness in osteoblastic networks. J Biol Chem 271, 12165–12171.Google Scholar
Wade, M.H., Trosko, J.E. & Schindler, M. (1986). A fluorescence photobleaching assay of gap junction-mediated communication between human cells. Science 232, 525–528.Google Scholar
Xia, S.L. & Ferrier, J. (1992). Propagation of a calcium pulse between osteoblastic cells. Biochem Biophys Res Commun 186, 1212–1219.Google Scholar
Yellowley, C.E., Li, Z., Zhou, Z., Jacobs, C.R. & Donahue, H.J. (2000). Functional gap junctions between osteocytic and osteoblastic cells. J Bone Miner Res 15, 209–217.Google Scholar