Reduction of connexin43 expression and dye-coupling during neuronal differentiation of human NTera2/clone D1 cells.
Gap junctions are plasma membrane specializations that allow direct communication among adjoining cells. We used a human pluripotential teratocarcinoma cell line, NTera-2/clone D1 (NT2/D1), as a model to study gap junctions in CNS neurons and their neuronal precursors. These cells were differentiated following retinoic acid (RA) treatment for 4 weeks and antiproliferative agents for 3 weeks, respectively, to yield post-mitotic CNS neuronal (NT2-N) cells. The cytoplasmic RNA was isolated from NT2/D1 cells both before and during RA treatment and from differentiated neurons (NT2-N cells). These RNA samples were examined using Northern blot analysis with cDNA probes specific for connexin26, -32, and -43. Connexin26 and -32 mRNAs were absent in NT2/D1 and NT2-N cells. Connexin43 mRNA was expressed at high levels in NT2/D1 cells before RA treatment, but it decreased significantly during RA induction. There was no detectable connexin43 mRNA in NT2-N cells. Western blot analysis confirmed the expression of connexin43 protein in NT2/D1 cells before and during RA treatment. The protein profile detected in Western blot analysis indicated two bands representing different phosphorylation states of connexin43. Our immunocytochemistry results did not show connexin26 and -32 immunoreactivity in NT2/D1 and NT2-N cells. However, we detected connexin43 immunoreactivity in NT2/D1 cells with a decreasing pattern upon RA induction. Both Western blotting and immunocytochemistry confirmed the absence of connexin43 protein in NT2-N cells. NT2/D1 cells passed calcein readily to an average of 18 cells, confirming the functionality of gap junctions in these cells. The extent of dye-coupling decreased about 78% when NT2/D1 cells were RA treated for 4 weeks. NT2-N differentiated neurons did not pass dye to the adjacent cells. We conclude that both connexin43 expression and dye coupling capacity decrease during neuronal differentiation of NT2/D1 cells.
Bani-Yaghoub
M
M
Department of Anatomy & Cell Biology, The University of Western Ontario, London, Canada.
Bechberger
J F
JF
Naus
C C
CC
D002450:Cell Communication; D002454:Cell Differentiation / Q000187:drug effects; D000072259:Connexin 26; D018031:Connexin 43 / Q000096:biosynthesis* / Q000235:genetics; D017630:Connexins; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms / Q000556:secondary; D008297:Male; D008856:Microscopy, Fluorescence; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D009474:Neurons / Q000473:pathology*; D012333:RNA, Messenger / Q000096:biosynthesis; D012334:RNA, Neoplasm / Q000096:biosynthesis; D018243:Teratocarcinoma / Q000473:pathology* / Q000556:secondary; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000473:pathology
D018031:Connexin 43; D017630:Connexins; D005452:Fluoresceins; D005456:Fluorescent Dyes; C000607042:GJB2 protein, human; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D000072259:Connexin 26; C085925:calcein AM; D014212:Tretinoin