Abstract
Missense mutations of the GJA1 gene encoding the gap junction channel protein connexin43 (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 aggressive osteolytic lesion with uncertain progression. Cx43 protein levels assessed by immunohistochemistry 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 osteoclast-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 membranes both in bone marrow stromal cells and HDFa fibroblasts. In Western blots, alkaline phosphatase sensitive bands, linked to serine residues (Ser369, Ser372 or Ser373) detected in control cells, were missing in GCTB stromal cells. Defective cell membrane localization 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 downregulation of Cx43 expression and gap junction coupling in neoplastic stromal cells are associated with the clinical progression and worse prognosis in GCTB.
Highlights
Connexins, in particular connexin43 (Cx43) and their cell membrane channels, play crucial roles in bone development including the regulation of osteoblast proliferation and differentiation, and the coordination of osteocyte adaptation to mechanical loading and soluble growth factors [1,2,3]
In this study of 89 primary and 34 recurrent giant cell tumor of bone (GCTB) cases we show that significant downregulation of Cx43 protein correlates with reduced progression free survival (PFS) and advanced clinico-radiological stages in GCTB
Image analysis of double immunofluorescence labeling revealed that significantly more Cx43 reaction belonged to the CD163 negative neoplastic stromal cells (81,7%, standard deviation (SD):±12.56%) than to CD163 positive monocyte/marcophages (p
Summary
In particular connexin (Cx43) and their cell membrane channels, play crucial roles in bone development including the regulation of osteoblast proliferation and differentiation, and the coordination of osteocyte adaptation to mechanical loading and soluble growth factors [1,2,3]. In giant cell tumor of bone (GCTB), which is a benign but locally aggressive osteolytic lesion with unpredictable progression, neoplastic stromal cells of osteoblast origin promote pathological osteolysis [5,6,7]. Cx43 expression was tested in primary and recurrent GCTB cases and in isolated neoplastic stromal cells and correlated with the clinico-radiological tumor stages and progression free patient survival. GCTB constitutes 5–20% of bone tumors in the Western and South-Asian population, respectively [5,8]. It arises mainly in the epi-metaphyseal region of long bones of young adults (20–45 years of age) and is associated with progressive bone destruction [9,10]. In 10% of cases GCTB can show malignant transformation, and in 1–4% it can form benign lung implants, which are called metastases [12,13,14]
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