From adrenergic signalling to gene expression : a study in brown adipocytes

Sammanfattning: Many external stimuli influence gene expression in cells by first interacting with cell surface receptors. For alteration of the expression of a gene, the signal has to be passed into the nucleus. This is facilitated by particular proteins/molecules in a signal transduction cascade. Brown adipose tissue function is mainly regulated by norepinephrine released from sympathetic nerves within the tissue. Norepinephrine controls both proliferation and differentiation of brown adipocytes, i.e. by controlling gene expression. To understand the transduction of signals from the concerted action of nor-epinephrine on various adrenergic receptors and the different signal trans-duction pathways activated, I have studied transcription factor activation and gene expression. Particular focus has been placed on the proto-oncogene c-fos and on cAMP-responsive element-mediated transcription. Stimulation of b-adrenergic receptors is mostly associated with the production of the second messenger cAMP. Elevation of cAMP activates protein kinase A, which can phosphorylate the nuclear transcription factor CREB. Stimulation of a1-adrenergic receptors is associated with activation of IP3/DAG-signalling pathways. Also, additional pathways, such as MAPK-pathways, are activated by norepinephrine stimulation in brown adipocytes, through both a1- andb-pathways.Activation of the transcription factor CREB, via phosphorylation of serine 133, was also found to be via stimulation of either a1- or b-adrenergic receptors. The kinase involved in the a1-mediated stimulation of CREB phosphorylation has not been identified, but the involvement of either a Ca2+/calmodulin-dependent kinase or a "classical" MAPK-pathway activated kinase is unlikely since there is no evidence for the existence of CaMK-proteins in brown adipocytes. Even though the MAPK-pathway is activated in our system and has been shown to mediate CREB-phosphorylation in other systems, specific inhibitors were not capable of inhibiting norepinephrine induced CREB-phosphorylation.cAMP-mediated transcription can be negatively controlled by the occurrence of repressors. ICER is one such repressor that we found to be expressed in brown adipose tissue. The expression of ICER is strictly cAMP-mediated and it may be argued that it therefore negatively regulates the CRE-mediated expression of certain genes in brown adipocytes. Since ICER is expressed in cells during proliferation, the previously proposed role of ICER as a tumour suppressor must be questioned.The expression of c-fos was found to depend on both a1- and b-adrenergic stimulation. The a1-component was sensitive to TPA-pretreatment and may thus involve protein kinase C, whereas theb-component involves the elevation of cAMP. These pathways work in synergy to induce c-fos, although the mechanism of the synergy is not understood.In conclusion, this study provides further information concerning norepinephrine signalling leading to gene transcription in brown adipocytes.