Expression of leukaemia inhibitory factor/cholinergic differentiation factor is linked to adrenoceptor stimulation.

Gene 'knock-out' studies have demonstrated that the pleiotropic cytokine leukaemia inhibitory factor (LIF)/ cholinergic differentiation factor is necessary in blastocyst implantation [ 11, and in the injury response of sympathetic neurons [2]. Additional roles for LIF both as a neuronal differentiation factor 131 and in the adult brain, where LIF exhibits a highly selective pattern of expression 141, are undefined. Although it is known that interleukin1 induces LIF in sympathetic ganglia IS], investigation of the mechanisms that determine LIF expression in the nervous system have been restricted by the low level of LIF mRNA that is found in v i v a It is now shown that LIF transcripts may be readily detected, using either Northern or i n situ h y b r i d i z a t i o n a n a l y s i s , i n e x p l a n t e d neuroendocrine tissues of the rat. Furthermore, it has been possible to demonstrate regulatory pathways that control the level of LIF mRNA, including a novel mechanism of regulat ion through adrenoceptor stimulation. Tissues of adult male Sprague-Dawley ra t s were maintained in explant culture 161, and RNA analysis performed as described [7,8]. A 4 k b L I F RNA transcript, which was not observed in control tissues, was readily detected in Northern blots of explanted superior cervical ganglia, pineals and pituitary glands. After 24 h. in culture, treatment with inhibitors of protein synthesis resulted in a marked up-regulation of the level of LIP mRNA, anisomycin and cycloheximide being most effective. Previous studies 191 have reported that a post-transcriptional mechanism a t least partially mediates the super-induction of LIF by cycloheximide in human monocytes. Subsequent experiments using pineal glands showed that L I F mRNA was also up-regulated by both forskolin and phorbol 12,13-dibutyrate indicating regulation through intracellular pathways linked to CAMP and protein kinase C. Moreover, treatment with selective inhibitors of protein kinases attenuated the the effect of anisomycin on LIF expression in a manner that suggests a role for protein kinase C in the superinduction by protein synthesis inhibitors. Since norepinephrine regulates cellular phenotype in the pineal gland, further experiments investigated the effect of adrenoceptor stimulation on L I F mRNA. Although norepinephrine alone did not affect the level of LIF mRNA, norepinephrine and anisomycin together elicited a greater effect than anisomycin alone. The s ignif icant ( p < 0.05), g r e a t e r t h a n two-fold enhancement of the anisomycin-induced response was blocked by pret reatment with the adrenoceptor an tagonis t s propanolo l a n d prazosin. I n s i tu hybridization analysis of pineals t r e a t e d with norepinephrine and anisomycin revealed a subpopulation of pineal cells exhibiting abundant LIF transcripts (Fig. I ) . The demonstration of a link between adrenoceptor stimulation and LIF mRNA levels in the r a t pineal gland is interesting with regard to the described effects of LIF on the regulation of neurotransmitter gene expression and the differentiation of catecholaminergic neurons [3,5,10]. Thus, the result is indicative of a feedback mode of regulation in which the expression of neuronal differentiation factors are to some extent