Prognostic Impact of Reduced Connexin43 Expression and Gap Junction Coupling of Neoplastic Stromal Cells in Giant Cell Tumor of Bone

Marie, Pierre J and Balla, Peter and Maros, Mate Elod and Barna, Gabor and Antal, Imre and Papp, Gergo and Sapi, Zoltan and Athanasou, Nicholas Anthony and Benassi, Maria Serena and Picci, Pierro and Krenacs, Tibor (2015) Prognostic Impact of Reduced Connexin43 Expression and Gap Junction Coupling of Neoplastic Stromal Cells in Giant Cell Tumor of Bone. PLOS ONE, 10 (5). e0125316. ISSN 1932-6203

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Abstract

Missense mutations of the GJA1 gene encoding the gap junction channel protein con- nexin43 (Cx43) cause bone malformations resulting in oculodentodigital dysplasia (ODDD), while GJA1 null and ODDD mutant mice develop osteopenia. In this study we investigated Cx43 expression and channel functions in giant cell tumor of bone (GCTB), a locally aggres- sive osteolytic lesion with uncertain progression. Cx43 protein levels assessed by immuno- histochemistry were correlated with GCTB cell types, clinico-radiological stages and progression free survival in tissue microarrays of 89 primary and 34 recurrent GCTB cases. Cx43 expression, phosphorylation, subcellular distribution and gap junction coupling was also investigated and compared between cultured neoplastic GCTB stromal cells and bone marow stromal cells or HDFa fibroblasts as a control. In GCTB tissues, most Cx43 was produced by CD163 negative neoplastic stromal cells and less by CD163 positive reactive monocytes/macrophages or by giant cells. Significantly less Cx43 was detected in α - smooth muscle actin positive than α -smooth muscle actin negative stromal cells and in oste- oclast-rich tumor nests than in the adjacent reactive stroma. Progressively reduced Cx43 production in GCTB was significantly linked to advanced clinico-radiological stages and worse progression free survival. In neoplastic GCTB stromal cell cultures most Cx43 protein was localized in the paranuclear-Golgi region, while it was concentrated in the cell mem- branes both in bone marrow stromal cells and HDFa fibroblasts. In Western blots, alkaline phosphatase sensitive bands, linked to serine residues (Ser369, Ser372 or Ser373) de- tected in control cells, were missing in GCTB stromal cells. Defective cell membrane locali- zation of Cx43 channels was in line with the significantly reduced transfer of the 622 Da fluorescing calcein dye between GCTB stromal cells. Our results show that significant PLOS ONE | DOI:10.1371/journal.pone.0125316 May 1, 2015 1/18 OPENACCESS Citation: Balla P, Maros ME, Barna G, Antal I, Papp G, Sapi Z, et al. (2015) Prognostic Impact of Reduced Connexin43 Expression and Gap Junction Coupling of Neoplastic Stromal Cells in Giant Cell Tumor of Bone. PLoS ONE 10(5): e0125316. doi:10.1371/ journal.pone.0125316 Academic Editor: Pierre J Marie, Inserm U606 and University Paris Diderot, FRANCE Received: November 12, 2014 Accepted: March 12, 2015 Published: May 1, 2015 Copyright: © 2015 Balla et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by EuroBoNet an EU FP6 funded Network of Excellence Programme (LSHC-CT-2006-018814). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. downregulation of Cx43 expression and gap junction coupling in neoplastic stromal cells are associated with the clinical progression and worse prognosis in GCTB.

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