Attenuation of cAMP-mediated responses in MA-10 Leydig tumor cells by genetic manipulation of a cAMP-phosphodiesterase.
In order to assess the effect of increased cAMP degradation on the responsiveness on an endocrine cell, we have obtained stable transfectants of MA-10 Leydig tumor cells that overexpress a mammalian cAMP-phosphodiesterase. Two novel cell lines, designated MA-10(P+8) and MA-10(P+29), that express high levels of the transfected enzyme were characterized. Although the basal levels of cAMP in the mutant cell lines are comparable to those of the wild-type cells, the increase in cAMP accumulation elicited by human choriogonadotropin (hCG) is severely blunted. Further studies with MA-10(P+29) show that the ability of hCG to stimulate adenylyl cyclase activity is normal. The failure of MA-10(P+29) cells to accumulate cAMP in response to hCG can be correlated with a similar reduction in hCG-stimulated steroidogenesis. On the other hand, the maximal steroidogenic response of MA-10(P+29) cells to dibutyryl cAMP, a cAMP analogue that is fairly resistant to phosphodiesterase degradation, is normal. We also show that the ability of these cells to respond to hCG with increased cAMP accumulation and steroid synthesis can be restored with a specific phosphodiesterase inhibitor. These results demonstrate that overexpression of a cAMP-phosphodiesterase in MA-10 cells limits the levels of cAMP attained under hCG stimulation and supresses the steroidogenic response of these cells to hCG. Since gonadotropins increase the cAMP-phosphodiesterase activity in their target cells, these findings also provide evidence that this regulation plays a major role in the modulation of cell responsiveness. Last, these new cell lines should be valuable in the study of the actions of cAMP because they express a conditional and reversible cAMP-resistant phenotype.
Swinnen
J V
JV
Department of Pediatrics, University of North Carolina, Chapel Hill 27599.
D'Souza
B
B
Conti
M
M
Ascoli
M
M
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics* / Q000378:metabolism; D015152:Blotting, Northern; D003994:Bucladesine / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP / Q000378:metabolism*; D006801:Humans; D007985:Leydig Cells / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D012313:RNA / Q000032:analysis; D014162:Transfection; D014407:Tumor Cells, Cultured
D006063:Chorionic Gonadotropin; D012313:RNA; D003994:Bucladesine; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases