Impaired gap junction formation and intercellular calcium signaling in urinary bladder cancer cells can be improved by Gö6976.
OBJECTIVE
Calcium wave propagation and connexin 26, 32 and 43 expression were studied in normal and malignant urothelial cells.


METHODS
Human urothelial cell cultures were established from tissue biopsies obtained from three healthy control persons and compared to human transitional cell carcinoma (TCC) cell line 5637. Fluo-3 was used to study intercellular calcium signaling in urothelial cells. The cells were stimulated mechanically in the presence of inhibitors of gap-junctional or ATP-mediated communication to determine which pathways are operative in intercellular calcium signaling. In addition, Gö6976 was used to determine the effects of PKC alpha and betaI inhibition on intercellular calcium signaling.


RESULTS
In normal urothelial cells, the primary pathway for intercellular calcium mediated cell signaling was gap junctional intercellular communication (GJIC), but the paracrine ATP-mediated signaling was also operative. In 5637 TCC cells, GJIC and ATP-mediated signaling routes were altered when compared to normal urothelial cells. More specifically, inhibition of GJIC resulted in a complete block of intercellular calcium signaling, while inhibition of ATP-mediated signaling decreased signal transduction in 5637 TCC cells. The results of the present study also demonstrated that connexin 26 was the most abundant gap junction plaque protein in cultured normal human urothelial cells and that it did not form gap junction plaques in 5637 TCC cell culture. Treatment with Gö6976 induced gap junction plaque formation by connexin 26 in 5637 TCC cells. In addition, the exposure to Gö6976 enhanced intercellular calcium mediated signaling in 5637 TCC cells, but not in normal cells.


CONCLUSIONS
The results of the present study suggest that gap junctions play a major role in intercellular calcium signaling in urothelial cells. In addition, intercellular calcium signaling is altered in urinary bladder carcinoma cells, and it can be improved by PKC alpha and betaI inhibition. (Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resources; Movie files of Fig. 2normal Gö6976-, normal Gö6976+, TCC Gö6976-, TCC Gö6976+ and image of Supplementary Figure 1).
Leinonen
Pekka
P
Department of Anatomy and Cell Biology, University of Oulu, Oulu, Finland.
Aaltonen
Vesa
V
Koskela
Sanna
S
Lehenkari
Petri
P
Korkiamäki
Timo
T
Peltonen
Juha
J
D015153:Blotting, Western; D020013:Calcium Signaling / Q000187:drug effects*; D002227:Carbazoles / Q000494:pharmacology*; D002295:Carcinoma, Transitional Cell / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D002450:Cell Communication / Q000502:physiology; D002478:Cells, Cultured; D000072259:Connexin 26; D017630:Connexins / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D017629:Gap Junctions / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology*; D007527:Isoenzymes / Q000502:physiology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D001743:Urinary Bladder / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology
D002227:Carbazoles; D017630:Connexins; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D000072259:Connexin 26; C081021:Go 6976; C496628:PRKCA protein, human; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha