Alterations in the synthesis of insulin-like growth factor binding proteins and insulin-like growth factors in rat C6 glioma cells transfected with a gap junction connexin43 cDNA.
When C6 glioma cells are stably transfected with a connexin43 cDNA and gap junctions are increased, the rate of cellular proliferation is decreased. To determine if this phenomenon is related to alterations in IGFBP and IGF synthesis, we have compared IGFBPs and IGFs in the conditioned media from primary rat astroglia, C6, and transfected C6 clones Cx43-13 (high expresser), and Cx43-12 and Cx43-14 (intermediate expressers). Primary astroglia produced IGFBP-2 (34 kDa) and IGFBP-3 (40-45 kDa). C6 cells synthesized high levels of IGFBP-3 and low levels of IGFBP-2, and a 24 kDa IGFBP (IGFBP-4). Cx43-13 cells did not synthesize IGFBP-3, but produced low levels of IGFBP-2 and high levels of IGFBP-4. Cx43-12 and Cx43-14 secreted IGFBP profiles similar to the parent C6 line, but with reduced levels of IGFBP-2. The lack of IGFBP-3 in Cx43-13 cells was not due to the presence of proteases. Northern analysis showed IGFBP-2 mRNA to be readily detectable only in the primary astroglia. IGFBP-3 mRNA was detected in the primary astroglia, C6, Cx43-12 and Cx43-14, but not in Cx43-13. In contrast, IGFBP-4 mRNA was readily detected only in the Cx43-13. IGF-II concentrations in the media were low to undetectable for both C6 and transfected cells. IGF-I concentrations were significantly lower in the media from transfected cells compared to the C6 cells. Stable mRNA levels for IGF-I were lower in transfected cells, with the lowest levels observed in the Cx43-13 cells. Although C6 cells did not respond mitogenically to exogenous IGF-I or IGF-II, Cx43-13 cells responded to IGF-I or IGF-II in a dose dependent manner. Conditioned media from Cx43-13 cells decreased the DNA synthesis of C6 cells, and this effect could be reversed by the addition of IGF-II. The decreased synthesis of the autocrine/paracrine growth factor IGF-I together with decreased levels of a positive modulator IGFBP-3, and the increased levels of a negative modulator IGFBP-4 in the extracellular milieu, may be responsible for the reduced proliferative capacity in cells expressing abundant connexin43.
Bradshaw
S L
SL
MRC Group in Fetal and Neonatal Health and Development, University of Western Ontario, Lawson Research Institute, London, Canada.
Naus
C C
CC
Zhu
D
D
Kidder
G M
GM
D'Ercole
A J
AJ
Han
V K
VK
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D018031:Connexin 43 / Q000096:biosynthesis / Q000378:metabolism*; D017077:Culture Media, Conditioned; D018076:DNA, Complementary / Q000378:metabolism; D004273:DNA, Neoplasm / Q000096:biosynthesis; D015870:Gene Expression; D005910:Glioma / Q000378:metabolism*; D018971:Insulin-Like Growth Factor Binding Protein 2; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D018031:Connexin 43; D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D004273:DNA, Neoplasm; D018971:Insulin-Like Growth Factor Binding Protein 2; D018969:Insulin-Like Growth Factor Binding Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D013936:Thymidine