Phosphorylation of CREB in ovine pars tuberalis is regulated both by cyclic AMP-dependent and cyclic AMP-independent mechanisms

This study used a combination of Western blotting and immunocytochemistry to test whether signalling pathways independent of cyclic AMP have the potential to induce phospho-CREB (pCREB)-like immunoreactivity (-ir) in the oPT. Western blot analysis of extracts of primary cultures of oPT using an antiserum against CREB, revealed a major band of CREB-ir at 44 KDa. The intensity of this band did not vary systematically with treatment. In extracts from untreated cells, Western blot analysis revealed a major band of pCREB-ir at 42 KDa which was not sensitive to agonist treatment. Treatment of cells with forskolin (10(-6) M) increased the intensity of a number of other pCREB-ir bands at between ca. 38 and 44 KDa. The band at 44 KDa probably represented native pCREB whilst the other bands induced by forskolin probably represented pCREB-like proteins, Melatonin (10(-6) M) alone had no effect on pCREB-ir, but it did inhibit the effect of forskolin on the ca. 38 and 44 KDa pCREB-ir bands. Treatment with lamb serum (1%) consistently increased the intensity of the ca. 38 and 44 KDa pCREB-ir bands relative to control cells, as assessed by Western blot. However, Western blot analysis did not reveal a consistent effect of melatonin on the pCREB-ir response to serum. The effect of serum on pCREB-ir in oPT cells was characterized further by immunocytochemical analysis. In contrast to experiments utilizing Western blotting, untreated cells did not possess detectable pCREB-ir. In serum-starved oPT and oPD cultures, treatment with serum induced exclusively nuclear pCREB-ir. A large majority of oPT cells (greater than or equal to 90%) were sensitive to serum (1%), and serum caused a time- and dose-dependent increase of nuclear pCREB-ir. Melatonin attenuated the response to serum in the oPT. This inhibition of the response to serum was not apparent in the oPD, demonstrating that the effect of melatonin was specific for a tissue known to express melatonin receptors. In oPT cultures, physiological concentrations of melatonin (10(-9) M) partially reversed (ca. 70%) the inductive effect of 0.1% serum on nuclear pCREB-ir. However, in contrast to studies applying forskolin, the induction of pCREB-ir by serum occurred in the absence of measurable changes in the concentration of cyclic AMP, indicating that components of serum are able to stimulate the phosphorylation of CREB in the oPT through mechanisms independent of cyclic AMP. Both adenosine and prostaglandin E2 (PGE2) also induced nuclear pCREB-ir in the absence of increased levels of cyclic AMP. These results demonstrate that transcriptional activities in the oPT which are under the control of CREB may be modulated by convergent cyclic AMP-dependent and cyclic AMP-independent pathways. Regulation of these pathways by melatonin and other factors present in serum may be an important control-point in the generation of seasonal neuroendocrine cycles.