Tumor-induced loss of mural Connexin 43 gap junction activity promotes endothelial proliferation

BMC Cancer

Research Article

L. Douglas Case¹  Keith D. Barlow²  Wenhong Chen³  Christine Naczki³  Mayur Choudhary⁴  Linda J. Metheny-Barlow⁵ 

[1] Department of Biostatistical Sciences, Wake Forest School of Medicine, Medical Center Boulevard, 27157, Winston-Salem, NC, USA;Wake Forest Comprehensive Cancer Center, Wake Forest School of Medicine, Medical Center Boulevard, 27157, Winston-Salem, NC, USA;Department of Cancer Biology, Wake Forest School of Medicine, Medical Center Boulevard, 27157, Winston-Salem, NC, USA;Department of Radiation Oncology, Wake Forest School of Medicine, Medical Center Boulevard, 27157, Winston-Salem, NC, USA;Department of Radiation Oncology, Wake Forest School of Medicine, Medical Center Boulevard, 27157, Winston-Salem, NC, USA;Department of Cancer Biology, Wake Forest School of Medicine, Medical Center Boulevard, 27157, Winston-Salem, NC, USA;Current address: Duke Eye Center, 2351 Erwin Road, AERI Room 4000, 27705, Durham, NC, USA;Department of Radiation Oncology, Wake Forest School of Medicine, Medical Center Boulevard, 27157, Winston-Salem, NC, USA;Department of Cancer Biology, Wake Forest School of Medicine, Medical Center Boulevard, 27157, Winston-Salem, NC, USA;Wake Forest Comprehensive Cancer Center, Wake Forest School of Medicine, Medical Center Boulevard, 27157, Winston-Salem, NC, USA;

关键词: Breast cancer;    Connexin 43;    Gap junction communication;    Pericyte;    Mural cell;    Endothelial cell;    Angiogenesis;   

DOI  :  10.1186/s12885-015-1420-9

 received in 2014-11-19, accepted in 2015-05-06,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundProper functional association between mural cells and endothelial cells (EC) causes EC of blood vessels to become quiescent. Mural cells on tumor vessels exhibit decreased attachment to EC, which allows vessels to be unstable and proliferative. The mechanisms by which tumors prevent proper association between mural cells and EC are not well understood. Since gap junctions (GJ) play an important role in cell-cell contact and communication, we investigated whether loss of GJ plays a role in tumor-induced mural cell dissociation.MethodsMural cell regulation of endothelial proliferation was assessed by direct co-culture assays of fluorescently labeled cells quantified by flow cytometry or plate reader. Gap junction function was assessed by parachute assay. Connexin 43 (Cx43) protein in mural cells exposed to conditioned media from cancer cells was assessed by Western and confocal microscopy; mRNA levels were assessed by quantitative real-time PCR. Expression vectors or siRNA were utilized to overexpress or knock down Cx43. Tumor growth and angiogenesis was assessed in mouse hosts deficient for Cx43.ResultsUsing parachute dye transfer assay, we demonstrate that media conditioned by MDA-MB-231 breast cancer cells diminishes GJ communication between mural cells (vascular smooth muscle cells, vSMC) and EC. Both protein and mRNA of the GJ component Connexin 43 (Cx43) are downregulated in mural cells by tumor-conditioned media; media from non-tumorigenic MCF10A cells had no effect. Loss of GJ communication by Cx43 siRNA knockdown, treatment with blocking peptide, or exposure to tumor-conditioned media diminishes the ability of mural cells to inhibit EC proliferation in co-culture assays, while overexpression of Cx43 in vSMC restores GJ and endothelial inhibition. Breast tumor cells implanted into mice heterozygous for Cx43 show no changes in tumor growth, but exhibit significantly increased tumor vascularization determined by CD31 staining, along with decreased mural cell support detected by NG2 staining.ConclusionsOur data indicate that i) functional Cx43 is required for mural cell-induced endothelial quiescence, and ii) downregulation of Cx43 GJ by tumors frees endothelium to respond to angiogenic cues. These data define a novel and important role for maintained Cx43 function in regulation of vessel quiescence, and suggest its loss may contribute to pathological tumor angiogenesis.

【 授权许可】

Unknown   
© Choudhary et al. 2015. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.