β-adrenergic stimulation of VEGF gene expression : intracellular signalling pathways

Abstract: In the present study, the intracellular signalling pathway mediating ß-adrenergic stimulation of the expression of the vascular endothelial growth factor (VEGF) gene was investigated.VEGF is an activator of angiogenesis, the formation of new blood vessels, and is considered to be a major regulator of both physiologically and pathologically related angiogenesis, as well as being essential for development of the vascular system. At the outset of the present investigation, it was already known that several factors - such as hypoxia, certain growth factors and phorbol esters - were potent stimulators of Vegf expression; however, it was unknown whether activation of ß-adrenergic receptors could influence the expression of the VEGF gene. Thus, to investigate this issue, cultured primary brown adipocytes, which express ß-adrenergic receptors, were isolated from mouse brown adipose tissue and used as a model system. The present cell system is here thought of as a relevant model for elucidation of angiogenesis-related processes - such as regulation of Vegf expression - since brown adipose tissue is a highly vascularised tissue, demonstrating high angiogenesis activity during tissue recruitment which is induced as a response to cold exposure or norepinephrine stimulation.With Ucp1-ablated mice - the brown adipocytes of which were demonstrated to lack the capacity for adrenergically induced oxygen consumption (thermogenesis) - results were obtained that provide support for the idea that the physiological activator of ß-adrenergic receptors, norepinephrine, is the factor that directly mediates physiological stimulation of Vegf expression in brown adipose tissue during recruitment, i.e. without involvement of hypoxic conditions being generated in the tissue.It was demonstrated that norepinephrine-induced activation of ß-adrenergic receptors mediates a potent stimulation of Vegf expression in brown adipocytes. For elucidation of the intracellular signalling factors activated via the ß-receptors, differentiated brown adipocytes, which possess ß3-adrenergic receptors, were used. It was found that for mediation of the ß-adrenergic signal, high intracellular levels of cAMP, and subsequent activation of PKA, are the exclusive mediating pathway for adrenergic stimulation; involvement of other adrenergic signalling mechanisms were excluded. In connection with investigations of PKA-mediated signalling via ß3-adrenergic receptors, the PKA inhibitor H89 was used and it was concluded that it functioned as intended - to abolish ß3-adrenergically induced Vegf and Ucp1 expression and thermogenesis by PKA inhibition - and did not act as a ß3-adrenergic antagonist.Further, adrenergic activation of the Erk1/2 MAP kinase signalling pathway in brown adipocytes was elucidated and shown to proceed through Src tyrosine kinases. ß-Adrenergic activation of Src, via a cAMP/PKA-mediated pathway, was demonstrated to partially mediate ß-adrenergic stimulation of Vegf expression, without dependence on further mediation via the downstream-acting Erk1/2, thus indicating a branch-point in the ß-adrenergically activated Src-Erk1/2 signalling cascade which further connects to the VEGF gene.In conclusion, the present investigation has provided further insight into the mechanisms by which stimulation of Vegf expression can be mediated, and has contributed information on the involvement of ß-adrenergic receptors in this process.