PKA II mediates cAMP-dependent phosphorylation of CREB and activation of gene transcription in differentiated rat thyroid cells

Thyroid stimulating hormone (TSH) is the key modulator of thyroid cell function. Binding of TSH to its receptor (TSHR) results in an increase of cAMP intracellular concentration, which activates protein kinase A (PKA) to phosphorylate CREB and other substrates. Eventually, this leads to gene transcription modification associated with both proliferation and differentiation. Two major types of PKA (PKA I and II) exist in mammal cells, but their specific role in TSH signaling is poorly understudy so far. Therefore, the aim of this study was to dissect PKA I/II effects on gene transcription in thyroid cells. In order to selectively activate PKA I or II, pairs of cAMP analogs with different affinities for sites A and B of PKA regulatory subunits were used. The effect of PKA I or II activation on the transcription of early genes, genes involved in cell replication and genes associated with thyroid differentiated function was evaluated in FRTL5 cells by quantitative real-time PCR. The results indicate that PKA II, but not PKA I, stimulation can mimic the effect of TSH treatment on these genes. In addition, pre-treatment with a PKA II inhibitor was able to abolish the effect of TSH. Since CREB is the major transcriptional mediator of cAMP signal, we also evaluated the effect of PKA I or II activation on CREB phosphorylation. Also in this case, only PKA II activation lead to efficient CREB phosphorylation, and TSH-dependent activation was abolished by pre-incubation with the PKA II inhibitor. Finally, silencing of PKA regulatory subunit 2B (Prkar2b) by RNA interference resulted in down-regulation of proliferative genes. These results indicate that PKA II is the principal mediator of cAMP transcriptional effects in thyroid cells. Further studies are needed to evaluate the role of PKA I, which could play a role in post-transcriptional modifications rather than directly affecting gene transcription.