Connexin43high prostate cancer cells induce endothelial connexin43 up-regulation through the activation of intercellular ERK1/2-dependent signaling axis

Abstract

Connexin(Cx)43 regulates the invasive potential of prostate cancer cells and participates in their extravasation. To address the role of endothelial Cx43 in this process, we analyzed Cx43 regulation in human umbilical vein endothelial cells in the proximity of Cx43 (DU-145 and MAT-LyLu) and Cx43 prostate cancer cells (PC-3 and AT-2). Endothelial Cx43 up-regulation was observed during the diapedesis of DU-145 and MAT-LyLu cells. This process was attenuated by transient Cx43 silencing in cancer cells and by chemical inhibition of ERK1/2-dependent signaling in endothelial cells. Cx43 expression in endothelial cells was insensitive to the inhibition of gap junctional intercellular coupling between Cx43 prostate cancer and endothelial cells by 18α-glycyrrhetinic acid. Instead, endothelial Cx43 up-regulation was correlated with the local contraction of endothelial cells and with their activation in the proximity of Cx43 DU-145 and MAT-LyLu cells. It was also sensitive to pro-inflammatory factors secreted by peripheral blood monocytes, such as TNFα. In contrast to Cx43 AT-2 cells, Cx43 PC-3 cells produced angioactive factors that locally activated the endothelial cells in the absence of endothelial Cx43 up-regulation. Collectively, these data show that Cx43 and Cx43 prostate cancer cells can adapt discrete, Cx43-independent and Cx43-dependent strategies of diapedesis. Our observations identify a novel strategy of prostate cancer cell diapedesis, which depends on the activation of intercellular Cx43/ERK1/2/Cx43 signaling axis at the interfaces between Cx43 prostate cancer and endothelial cells.

Fenofibrate Reduces the Asthma-Related Fibroblast-To-Myofibroblast Transition by TGF-Β/Smad2/3 Signaling Attenuation and Connexin 43-Dependent Phenotype Destabilization

“…The averaged signal for at least 50 stress fibers was compared between the conditions. Cytofluorimetric analyses of Cx43 levels were performed using the same excitation/exposure settings as described previously [ 23 , 57 ].…”

“…HBF cultures were lysed as described previously [ 23 , 30 ], and the protein content in the supernatant was determined using the Bradford method. Protein samples (30 µg/lane) were electrophoresed on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Bio Rad, Hercules, CA, USA) as described previously [ 23 , 57 ]. Next, after blocking, the membranes were incubated overnight at 4 °C with primary antibodies: rabbit polyclonal IgG against p(Ser467)-Smad2 (1:500), rabbit polyclonal IgG against Cx43 (1:2000), mouse monoclonal IgG against α-SMA (1:2000), mouse monoclonal IgG against vinculin (1:1000), mouse monoclonal IgG against β-tubulin (1:1000), and mouse monoclonal IgG against glyceraldehyde-3-phosphate dehydrogenase GAPDH (1:3000), all from Sigma-Aldrich, St. Louis, MO, USA; rabbit monoclonal IgG against Smad2 (D43B4), rabbit monoclonal IgG against p(Ser465/467)-Smad2 (138D4), rabbit monoclonal IgG against Smad3 (C67H9), rabbit monoclonal IgG against p(Ser423/425)-Smad3 (C25A9), rabbit polyclonal IgG against Focal adhesion kinase (FAK), rabbit polyclonal IgG against p(Thr202/Tyr204)-Erk1/2, and rabbit polyclonal IgG against Erk1/2 (all: 1:500; Cell Signaling Technology ® Danvers, MA, USA).…”

Invasive bronchial fibroblasts derived from asthmatic patients activate lung cancer A549 cells in�vitro

“…Movement of A549 cells in co-cultures with HBFs and in the conditioned media was registered using Leica DMI6000B time-lapse videomicroscopy system (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a temperature/CO 2 chamber and Integrated Modulation Contrast (IMC) optics. Sequences of cell centroid positions were recorded at 300 s time intervals for 6 h using a dry 20×, NA-0.75 objective ( 16 ). The averaged total length of cell trajectory (i.e., the ‘Distance’ covered by the cells during the registration time; µm) and the total length of cell displacement (‘Displacement’, i.e., the distance from the starting point directly to the cell's final position; µm) were quantified from cell trajectories with the purpose-designed Hiro program.…”

Connexin-dependent intercellular stress signaling in tissue homeostasis and tumor development

“…Cx43 function during this process may be attributed to the disturbance of endothelial calcium homeostasis by GJIC-mediated calcium fluxes from tumor to endothelial cells (Lewalle et al, 1998). We have recently demonstrated that prostate cancer cells can also activate the "inside-out" stress signaling axis with endothelial cells in Cx43-dependent, GJIC-independent manner (Piwowarczyk et al, 2015;Piwowarczyk et al, 2017). Other studies showed increased apoptosis of endothelial cells in the proximity of tumor cells, however the involvement of connexin in this process still requires experimental verification.…”