Activity‐dependent somatostatin gene expression is regulated by cAMP‐dependent protein kinase and Ca2+‐calmodulin kinase pathways

Abstract

Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels (L-VSCC) is required for K⁺-induced somatostatin (SS) mRNA. Increase in intracellular Ca²⁺ concentration leads to the activation of cyclic AMP-responsive element binding protein (CREB), a key regulator of SS gene transcription. Several different protein kinases possess the capability of driving CREB upon membrane depolarization. We investigated which of the signalling pathways involved in CREB activation mediates SS gene induction in response to membrane depolarization in cerebrocortical cells exposed to 56 mM K⁺. Activity dependent phosphorylation of CREB in Ser¹³³ was immunodetected. Activation of CREB was biphasic showing two peaks at 5 and 60 min. The selective inhibitors of extracellular signal related protein kinase/mitogen-activated protein kinase (ERK/MAPK) PD098059, cyclic-AMPdependent protein kinase (cAMP/PKA) H89 and RpcAMPS, and Ca²⁺/calmodulin-dependent protein kinases (CaMKs) pathways KN62 and KN93 were used to determine the signalling pathways involved in CREB activation. Here we show that the early activation of CREB was dependent on cAMP/PKA along with CaMKs pathways whereas the ERK/MAPK and CaMKs were implicated in the second peak. We observed that H89, RpcAMPS, KN62 and KN93 blocked K⁺-induced SS mRNA whereas PD098059 did not. These findings indicate that K⁺-induced SSmRNA is mediated by the activation of cAMP/PKA and CaMKs pathways, thus suggesting that the early activation of CREB is involved in the induction of SS by neuronal activity. We also demonstrated, using transient transfections of cerebrocortical cells, that K⁺ induces the transcriptional regulation of the SS gene through the cAMP-responsive element (CRE) sequence located in the SS promoter.