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. 1993 May;91(5):1888–1896. doi: 10.1172/JCI116406

Connexin43 mediates direct intercellular communication in human osteoblastic cell networks.

R Civitelli 1, E C Beyer 1, P M Warlow 1, A J Robertson 1, S T Geist 1, T H Steinberg 1
PMCID: PMC288182  PMID: 8387535

Abstract

We have examined cell coupling and expression of gap junction proteins in monolayer cultures of cells derived from human bone marrow stromal cells (BMC) and trabecular bone osteoblasts (HOB), and in the human osteogenic sarcoma cell line, SaOS-2. Both HOB and BMC cells were functionally coupled, since microinjection of Lucifer yellow resulted in dye transfer to neighboring cells, with averages of 3.4 +/- 2.8 (n = 131) and 8.1 +/- 9.3 (n = 51) coupled cells per injection, respectively. In contrast, little diffusion of Lucifer yellow was observed in SaOS-2 monolayers (1.4 +/- 1.8 coupled cells per injection, n = 100). Dye diffusion was inhibited by octanol (3.8 mM), an inhibitor of gap junctional communication. All of the osteoblastic cells expressed mRNA for connexin43 and connexin45, but not for connexins 26, 32, 37, 40, or 46. Whereas all of the osteoblastic cells expressed similar quantities of mRNA for connexin43, the poorly coupled SaOS-2 cells produced significantly less Cx43 protein than either HOB or BMC, as assessed by immunofluorescence and immunoprecipitation. Conversely, more Cx45 mRNA was expressed by SaOS-2 cells than by HOB or BMC. Thus, intercellular coupling in normal and transformed human osteoblastic cells correlates with the level of expression of Cx43, which appears to mediate intercellular communication in these cells. Gap junctional communication may serve as a means by which osteoblasts can work in synchrony and propagate locally generated signals throughout the skeletal tissue.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ashton B. A., Abdullah F., Cave J., Williamson M., Sykes B. C., Couch M., Poser J. W. Characterization of cells with high alkaline phosphatase activity derived from human bone and marrow: preliminary assessment of their osteogenicity. Bone. 1985;6(5):313–319. doi: 10.1016/8756-3282(85)90321-7. [DOI] [PubMed] [Google Scholar]
  2. Atkinson M. M., Sheridan J. D. Altered junctional permeability between cells transformed by v-ras, v-mos, or v-src. Am J Physiol. 1988 Nov;255(5 Pt 1):C674–C683. doi: 10.1152/ajpcell.1988.255.5.C674. [DOI] [PubMed] [Google Scholar]
  3. Beyer E. C., Kistler J., Paul D. L., Goodenough D. A. Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J Cell Biol. 1989 Feb;108(2):595–605. doi: 10.1083/jcb.108.2.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beyer E. C., Paul D. L., Goodenough D. A. Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol. 1987 Dec;105(6 Pt 1):2621–2629. doi: 10.1083/jcb.105.6.2621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beyer E. C., Reed K. E., Westphale E. M., Kanter H. L., Larson D. M. Molecular cloning and expression of rat connexin40, a gap junction protein expressed in vascular smooth muscle. J Membr Biol. 1992 Apr;127(1):69–76. doi: 10.1007/BF00232759. [DOI] [PubMed] [Google Scholar]
  6. Beyer E. C., Steinberg T. H. Evidence that the gap junction protein connexin-43 is the ATP-induced pore of mouse macrophages. J Biol Chem. 1991 May 5;266(13):7971–7974. [PubMed] [Google Scholar]
  7. Bignami M., Rosa S., Falcone G., Tató F., Katoh F., Yamasaki H. Specific viral oncogenes cause differential effects on cell-to-cell communication, relevant to the suppression of the transformed phenotype by normal cells. Mol Carcinog. 1988;1(1):67–75. doi: 10.1002/mc.2940010113. [DOI] [PubMed] [Google Scholar]
  8. Boland C. J., Fried R. M., Tashjian A. H., Jr Measurement of cytosolic free Ca2+ concentrations in human and rat osteosarcoma cells: actions of bone resorption-stimulating hormones. Endocrinology. 1986 Mar;118(3):980–989. doi: 10.1210/endo-118-3-980. [DOI] [PubMed] [Google Scholar]
  9. Brissette J. L., Kumar N. M., Gilula N. B., Dotto G. P. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the ras oncogene modulate expression and phosphorylation of gap junction proteins. Mol Cell Biol. 1991 Oct;11(10):5364–5371. doi: 10.1128/mcb.11.10.5364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Burt J. M., Spray D. C. Single-channel events and gating behavior of the cardiac gap junction channel. Proc Natl Acad Sci U S A. 1988 May;85(10):3431–3434. doi: 10.1073/pnas.85.10.3431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chanson M., Bruzzone R., Bosco D., Meda P. Effects of n-alcohols on junctional coupling and amylase secretion of pancreatic acinar cells. J Cell Physiol. 1989 Apr;139(1):147–156. doi: 10.1002/jcp.1041390121. [DOI] [PubMed] [Google Scholar]
  12. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  13. Civitelli R., Kim Y. S., Gunsten S. L., Fujimori A., Huskey M., Avioli L. V., Hruska K. A. Nongenomic activation of the calcium message system by vitamin D metabolites in osteoblast-like cells. Endocrinology. 1990 Nov;127(5):2253–2262. doi: 10.1210/endo-127-5-2253. [DOI] [PubMed] [Google Scholar]
  14. Doty S. B. Morphological evidence of gap junctions between bone cells. Calcif Tissue Int. 1981;33(5):509–512. doi: 10.1007/BF02409482. [DOI] [PubMed] [Google Scholar]
  15. Duncan R., Misler S. Voltage-activated and stretch-activated Ba2+ conducting channels in an osteoblast-like cell line (UMR 106). FEBS Lett. 1989 Jul 17;251(1-2):17–21. doi: 10.1016/0014-5793(89)81420-6. [DOI] [PubMed] [Google Scholar]
  16. Jeansonne B. G., Feagin F. F., McMinn R. W., Shoemaker R. L., Rehm W. S. Cell-to-cell communication of osteoblasts. J Dent Res. 1979 Apr;58(4):1415–1423. doi: 10.1177/00220345790580042101. [DOI] [PubMed] [Google Scholar]
  17. Jüppner H., Abou-Samra A. B., Uneno S., Gu W. X., Potts J. T., Jr, Segre G. V. The parathyroid hormone-like peptide associated with humoral hypercalcemia of malignancy and parathyroid hormone bind to the same receptor on the plasma membrane of ROS 17/2.8 cells. J Biol Chem. 1988 Jun 25;263(18):8557–8560. [PubMed] [Google Scholar]
  18. Kanter H. L., Saffitz J. E., Beyer E. C. Cardiac myocytes express multiple gap junction proteins. Circ Res. 1992 Feb;70(2):438–444. doi: 10.1161/01.res.70.2.438. [DOI] [PubMed] [Google Scholar]
  19. Long M. W., Williams J. L., Mann K. G. Expression of human bone-related proteins in the hematopoietic microenvironment. J Clin Invest. 1990 Nov;86(5):1387–1395. doi: 10.1172/JCI114852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Marcus R. Normal and abnormal bone remodeling in man. Annu Rev Med. 1987;38:129–141. doi: 10.1146/annurev.me.38.020187.001021. [DOI] [PubMed] [Google Scholar]
  21. McSheehy P. M., Chambers T. J. 1,25-Dihydroxyvitamin D3 stimulates rat osteoblastic cells to release a soluble factor that increases osteoclastic bone resorption. J Clin Invest. 1987 Aug;80(2):425–429. doi: 10.1172/JCI113089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McSheehy P. M., Chambers T. J. Osteoblast-like cells in the presence of parathyroid hormone release soluble factor that stimulates osteoclastic bone resorption. Endocrinology. 1986 Oct;119(4):1654–1659. doi: 10.1210/endo-119-4-1654. [DOI] [PubMed] [Google Scholar]
  23. Morris C. A., Mitnick M. E., Weir E. C., Horowitz M., Kreider B. L., Insogna K. L. The parathyroid hormone-related protein stimulates human osteoblast-like cells to secrete a 9,000 dalton bone-resorbing protein. Endocrinology. 1990 Mar;126(3):1783–1785. doi: 10.1210/endo-126-3-1783. [DOI] [PubMed] [Google Scholar]
  24. Musil L. S., Beyer E. C., Goodenough D. A. Expression of the gap junction protein connexin43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation. J Membr Biol. 1990 Jun;116(2):163–175. doi: 10.1007/BF01868674. [DOI] [PubMed] [Google Scholar]
  25. Musil L. S., Cunningham B. A., Edelman G. M., Goodenough D. A. Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines. J Cell Biol. 1990 Nov;111(5 Pt 1):2077–2088. doi: 10.1083/jcb.111.5.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Narbaitz R., Stumpf W. E., Sar M., Huang S., DeLuca H. F. Autoradiographic localization of target cells for 1 alpha, 25-dihydroxyvitamin D3 in bones from fetal rats. Calcif Tissue Int. 1983;35(2):177–182. doi: 10.1007/BF02405028. [DOI] [PubMed] [Google Scholar]
  27. Parfitt A. M. The coupling of bone formation to bone resorption: a critical analysis of the concept and of its relevance to the pathogenesis of osteoporosis. Metab Bone Dis Relat Res. 1982;4(1):1–6. doi: 10.1016/0221-8747(82)90002-9. [DOI] [PubMed] [Google Scholar]
  28. Paul D. L., Ebihara L., Takemoto L. J., Swenson K. I., Goodenough D. A. Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes. J Cell Biol. 1991 Nov;115(4):1077–1089. doi: 10.1083/jcb.115.4.1077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Paul D. L. Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol. 1986 Jul;103(1):123–134. doi: 10.1083/jcb.103.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rifas L., Halstead L. R., Peck W. A., Avioli L. V., Welgus H. G. Human osteoblasts in vitro secrete tissue inhibitor of metalloproteinases and gelatinase but not interstitial collagenase as major cellular products. J Clin Invest. 1989 Aug;84(2):686–694. doi: 10.1172/JCI114216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Robey P. G., Termine J. D. Human bone cells in vitro. Calcif Tissue Int. 1985 Sep;37(5):453–460. [PubMed] [Google Scholar]
  32. Rodan G. A., Martin T. J. Role of osteoblasts in hormonal control of bone resorption--a hypothesis. Calcif Tissue Int. 1981;33(4):349–351. doi: 10.1007/BF02409454. [DOI] [PubMed] [Google Scholar]
  33. Schiller P. C., Mehta P. P., Roos B. A., Howard G. A. Hormonal regulation of intercellular communication: parathyroid hormone increases connexin 43 gene expression and gap-junctional communication in osteoblastic cells. Mol Endocrinol. 1992 Sep;6(9):1433–1440. doi: 10.1210/mend.6.9.1331776. [DOI] [PubMed] [Google Scholar]
  34. Schirrmacher K., Schmitz I., Winterhager E., Traub O., Brümmer F., Jones D., Bingmann D. Characterization of gap junctions between osteoblast-like cells in culture. Calcif Tissue Int. 1992 Oct;51(4):285–290. doi: 10.1007/BF00334489. [DOI] [PubMed] [Google Scholar]
  35. Shen V., Rifas L., Kohler G., Peck W. A. Prostaglandins change cell shape and increase intercellular gap junctions in osteoblasts cultured from rat fetal calvaria. J Bone Miner Res. 1986 Jun;1(3):243–249. doi: 10.1002/jbmr.5650010302. [DOI] [PubMed] [Google Scholar]
  36. Shupnik M. A., Tashjian A. H., Jr Epidermal growth factor and phorbol ester actions on human osteosarcoma cells. Characterization of response and nonresponsive cell lines. J Biol Chem. 1982 Oct 25;257(20):12161–12164. [PubMed] [Google Scholar]
  37. Silve C. M., Hradek G. T., Jones A. L., Arnaud C. D. Parathyroid hormone receptor in intact embryonic chicken bone: characterization and cellular localization. J Cell Biol. 1982 Aug;94(2):379–386. doi: 10.1083/jcb.94.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stewart W. W. Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer. Cell. 1978 Jul;14(3):741–759. doi: 10.1016/0092-8674(78)90256-8. [DOI] [PubMed] [Google Scholar]
  39. Thomson B. M., Mundy G. R., Chambers T. J. Tumor necrosis factors alpha and beta induce osteoblastic cells to stimulate osteoclastic bone resorption. J Immunol. 1987 Feb 1;138(3):775–779. [PubMed] [Google Scholar]
  40. Thomson B. M., Saklatvala J., Chambers T. J. Osteoblasts mediate interleukin 1 stimulation of bone resorption by rat osteoclasts. J Exp Med. 1986 Jul 1;164(1):104–112. doi: 10.1084/jem.164.1.104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Willecke K., Heynkes R., Dahl E., Stutenkemper R., Hennemann H., Jungbluth S., Suchyna T., Nicholson B. J. Mouse connexin37: cloning and functional expression of a gap junction gene highly expressed in lung. J Cell Biol. 1991 Sep;114(5):1049–1057. doi: 10.1083/jcb.114.5.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Xia S. L., Ferrier J. Propagation of a calcium pulse between osteoblastic cells. Biochem Biophys Res Commun. 1992 Aug 14;186(3):1212–1219. doi: 10.1016/s0006-291x(05)81535-9. [DOI] [PubMed] [Google Scholar]
  43. Zhang J. T., Nicholson B. J. Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA. J Cell Biol. 1989 Dec;109(6 Pt 2):3391–3401. doi: 10.1083/jcb.109.6.3391. [DOI] [PMC free article] [PubMed] [Google Scholar]

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