Method of transfection affects the cAMP-mediated induction of the RIIbeta subunit of protein kinase A in Sertoli cells: inhibition of response by increase in intracellullar calcium.
mRNA for the regulatory subunit RIIbeta of cAMP-dependent protein kinase is stimulated more than 50-fold by cAMP in primary cultures of rat Sertoli cells. We have previously shown that this induction involves regulation of transcriptional activation as well as mRNA stabilization. The rat RIIbeta gene contains no cAMP response element (CRE), and the induction of RIIbeta mRNA is slow and requires on-going protein synthesis. When a construct containing the 5'-flanking region of the RIIbeta gene upstream of a CAT reporter was transfected into Sertoli cells by the calcium phosphate method, low and variable responses to cAMP (three- to fivefold) were observed, whereas a 15- to 20-fold increase in reporter activity by cAMP was observed after lipofectamine transfection. Interestingly, when a vector containing CRE elements upstream of a reporter gene was transfected into Sertoli cells, the responses to cAMP were similar regardless of the transfection method used. We have also demonstrated that increased intracellular levels of calcium by A23187 and thapsigargin dramatically inhibit cAMP-mediated induction of RIIbeta mRNA, but not the mRNA for the CRE-containing RIalpha gene. Furthermore, decreased cAMP responsiveness of endogenous RIIbetamRNA (but not RIalpha) was also observed in calcium phosphate-transfected Sertoli cells but not in lipofectamine-transfected cells. Thus, calcium-mediated reduction in cAMP response appears to be a gene-specific phenomenon.
Institute of Medical Biochemistry, University of Oslo, N-0317 Oslo, Norway.
L M
LM
Gronning
H K
HK
Knutsen
W
W
Eskild
V
V
Hansson
K
K
Taskén
K A
KA
Taskén
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002130:Calcium Phosphates; D002411:Cation Exchange Resins; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008055:Lipids; D008297:Male; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012045:Regulatory Sequences, Nucleic Acid; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection / Q000379:methods*
D002130:Calcium Phosphates; D002411:Cation Exchange Resins; D004791:Enzyme Inhibitors; D007476:Ionophores; D008055:Lipids; C086724:Lipofectamine; D012333:RNA, Messenger; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D000001:Calcimycin; D019284:Thapsigargin; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C485829:calcium phosphate, dibasic, anhydrous; D002118:Calcium