Abstract
Recent studies indicate multiple mechanisms are involved in Ca2+ stimulation of gene expression. We have used cell-permeable, specific inhibitors of calmodulin-dependent protein kinases (CaM kinases) and phosphatase (calcineurin) to investigate the involvement of these enzymes in transcriptional regulation of three immediate early genes in PC12 cells stimulated with A23187 or KCl. Preincubation of PC12 cells with the CaM kinase inhibitor KN-62 blocked autophosphorylation of CaM kinase II in response to stimulation by the Ca2+ ionophore A23187. KN-62 treatment also resulted in a 60-70% inhibition of Ca(2+)-dependent transcription of c-fos, NGFI-A (zif 268), and NGFI-B (nur 77) as assessed by either Northern or nuclear run-on analyses. Preincubation with the calcineurin inhibitors FK-506 or cyclosporin A strongly enhanced expression of NGFI-A and blocked transcription of NGFI-B, but it had no significant effect on Ca(2+)-stimulated transcription of c-fos. Both FK-506 and KN-62 were specific for Ca(2+)-stimulated transcription as neither effected transcription in response to forskolin or phorbol ester (12-O-tetradecanoylphorbol-13-acetate) treatment. This is the first report of CaM kinase and calcineurin involvement in transcriptional regulation of NGFI-A and NGFI-B. Activation of CaM kinases and calcineurin, in response to elevated intracellular Ca2+, would exert antagonistic effects on transcription of NGFI-A. Since inhibition of either the kinase or phosphatase decreased transcription of NGFI-B by 60-90%, this suggests that each enzyme is necessary but not sufficient for Ca2+ stimulation. These results indicate that CaM kinases and calcineurin can mediate broad and complex regulation of Ca(2+)-stimulated gene expression.
Highlights
268),and NGFI-B(nur77)as assessed by either Northern terized systemis the CAMPresponse, which requires phosphoor nuclear run-on analyses
Lated transcription of c-fos.Both FK-506 and KN-62 were CREB can alsobe phosphorylatedon Ser133by several members specific for Ca2+-stimulatedtranscription as neither ef- of the CaM kinase family (9, lo),and this habseen proposed as fected transcription in response to forskolin or phorbol a mechanism to explain Ca2+-dependent regulation of CRE
Blocks Ca2+induction of a transfected coninhibition of either thekinase or phosphatase decreased struct containing thgelucagon CRE in frontof a reporter gene transcription ofNGFI-Bby60-90%, this suggests that [11].KN-62 inhibits many membersof the CaM kinase family each enzymeis necessary but not sufficient for Ca2+ [10, 12] competitively with Ca2+/CaM,but it does not inhibit stimulation. These results indicate that CaM kinases and calcineurin can mediate broad and complex regulation of Ca2+-stimulategdene expression
Summary
The newly synthesized RNA was separated from free. Cell Culture-PC12 cells were grown in Dulbecco's modified Eagle's nucleotides by ethanol precipitation, andspecific message was detected medium (DMEM) supplemented wit5h% fetal calf serum and5% horse by hybridization of the RNA with nitrocellulose membranes on which serum on Primaria dishes.Cell were maintained in 5a% CO, incubator cDNAs for eachgenehad been immobilized. The cells were incubated in serum-free washed and subjected to autoradiography. The32Pincorporation was bated with ["P]orthophosphate (0.45 mCi) in 1.5 ml of phosphate-free quantified by densitometric scanning Thecells were thawed and scraped wit1hml of radioimmune tyl-1-methylxanthine,and TPA were from Sigma; KN-62 was from precipitation buffer Phenylmethylsulfonyl fluoride, 6 &ml leupeptin, 10 pg/ml aprotinin, 10 pg/ml pepstatin A, 0.1% SDS, 1p~ microcystin-LR), the lysates were
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