Transcription factor CREB and its stimulus-dependent phosphorylation in cell and explant cultures of the bovine subcommissural organ.

The subcommissural organ (SCO) is an ependymal brain gland that synthesizes and secretes glycoproteins. Very little is known about the signal transduction cascades operating in this organ and their impact on gene expression. An important transcription factor that regulates gene expression in glial cells and neurons is the cyclic-AMP-responsive element binding protein (CREB), which is activated by phosphorylation of the serine residue 133. Here, we analyzed the presence of CREB in bovine SCO cells and its phosphorylation by drugs that activate cyclic-AMP-dependent or calcium-dependent signal transduction pathways. We also investigated the effects of three natural signaling molecules, serotonin (5HT), substance P (SP) and ATP, on CREB phosphorylation and on the second messengers cyclic AMP and calcium. Investigations were performed with cell and explant cultures by using immunocytochemistry, immunoblot, enzyme-linked immunosorbent assay, and the Fura-2 technique. A strong immunosignal for total (phosphorylated and unphosphorylated) CREB was found in virtually all SCO cells. Total CREB levels did not change upon stimulation. Phosphorylated (p)CREB levels were low in unstimulated cells and significantly elevated by drugs that increase the levels of cyclic AMP or free calcium ions. pCREB was also induced by SP and ATP; both substances increased the intracellular calcium concentration but did not affect the formation of intracellular cyclic AMP. 5HT did not influence the phosphorylation of CREB, the intracellular calcium concentration, or the formation of cyclic AMP. Our data identify CREB as an SCO transcription factor that can be activated by the second messengers cAMP and calcium. SP and ATP stimulate the phosphorylation of CREB apparently via a calcium-dependent mechanism and are thus involved in the control of gene expression in the bovine SCO.