Pharmacological modulation of nerve growth factor synthesis: a mechanistic comparison of vitamin D receptor and beta(2)-adrenoceptor agonists

Abstract

Increasing nerve growth factor (NGF) in the PNS is a rational strategy for treating certain neurodegenerative disorders. The present studies were undertaken to compare two compounds, a vitamin D(3) analogue (CB1093) with minimal calcaemic effects, and clenbuterol, a long-acting beta(2)-adrenoceptor agonist, both of which induce NGF synthesis in vivo. Clenbuterol caused significant increases in both NGF mRNA and protein in 3T3 cells; with maxima at 10 nM and at 8-12 h exposure. Effects of clenbuterol on NGF mRNA were antagonized by propranolol. Mobility shift assays on whole cell extracts showed that clenbuterol increased AP1 binding in 3T3 cells prior to increasing NGF synthesis. Clenbuterol was without effect on NGF mRNA levels in L929 cells, whereas CB1093 caused significant increases in both NGF mRNA and protein levels in both 3T3 and L929 cells. Stimulation was almost maximal at 24 h exposure and was sustained for at least 72 h. The magnitude of the increase was much greater in L929 (700% increase) than in 3T3 cells (80%). Binding to the vitamin D nuclear receptor (VDR), which acts as a transcription factor itself, was increased as early as 30 min after exposure to of CB1093 and maintained up to 24 h. Increased VDR binding preceded increased NGF mRNA. A 150% increase in AP-1 binding was also evident. This study demonstrates that CB1093 and clenbuterol stimulate NGF levels in vitro and that AP-1 binding could be a commonality between the mechanism of NGF induction of these two compounds.