Balla Peter, Maros Mate Elod, Barna Gabor, Antal Imre, Papp Gergo, Sapi Zoltan, Athanasou Nicholas Anthony, Benassi Maria Serena, Picci Pierro, Krenacs Tibor
1st Department of Pathology & Experimental Cancer Research, Semmelweis University Budapest, Hungary.
1st Department of Pathology & Experimental Cancer Research, Semmelweis University Budapest, Hungary; Department of Neuroradiology, University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany.
PLoS One. 2015 May 1;10(5):e0125316. doi: 10.1371/journal.pone.0125316. eCollection 2015.
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.
编码缝隙连接通道蛋白连接蛋白43（Cx43）的GJA1基因错义突变会导致骨骼畸形，进而引发眼牙指发育不良（ODDD），而GJA1基因缺失和ODDD突变小鼠会出现骨质减少。在本研究中，我们调查了骨巨细胞瘤（GCTB）中Cx43的表达和通道功能，GCTB是一种局部侵袭性溶骨性病变，其进展情况尚不确定。通过免疫组织化学评估的Cx43蛋白水平与89例原发性和34例复发性GCTB病例的组织微阵列中的GCTB细胞类型、临床放射学分期及无进展生存期相关。还对培养的肿瘤性GCTB基质细胞与骨髓基质细胞或HDFa成纤维细胞（作为对照）之间的Cx43表达、磷酸化、亚细胞分布及缝隙连接偶联进行了研究和比较。在GCTB组织中，大多数Cx43由CD163阴性的肿瘤性基质细胞产生，而由CD163阳性的反应性单核细胞/巨噬细胞或巨细胞产生的较少。在α-平滑肌肌动蛋白阳性的基质细胞中检测到的Cx43明显少于α-平滑肌肌动蛋白阴性的基质细胞，在富含破骨细胞的肿瘤巢中检测到的Cx43也明显少于相邻的反应性基质。GCTB中Cx43产生的逐渐减少与临床放射学分期的进展及更差的无进展生存期显著相关。在肿瘤性GCTB基质细胞培养物中，大多数Cx43蛋白定位于核旁高尔基体区域，而在骨髓基质细胞和HDFa成纤维细胞中，Cx43蛋白集中在细胞膜上。在蛋白质免疫印迹中，对照细胞中检测到的与丝氨酸残基（Ser369、Ser372或Ser373）相关的碱性磷酸酶敏感条带在GCTB基质细胞中缺失。Cx43通道细胞膜定位缺陷与GCTB基质细胞之间622 Da荧光素钙黄绿素染料转移的显著减少一致。我们的结果表明，肿瘤性基质细胞中Cx43表达和缝隙连接偶联的显著下调与GCTB的临床进展及更差的预后相关。
