Role of Egr-1 in Cholinergic Stimulation of Phenylethanolamine N-Methyltransferase Promoter

Abstract: The effects of the cholinergic agonist carbachol on phenylethanolamine N -methyltransferase promoter activity and Egr-1 mRNA expression in PC12-derived RS1 cells were examined to investigate the potential involvement of Egr-1 in the neural regulation of phenylethanolamine N -methyltransferase gene expression. Carbachol stimulated luciferase expression in cells transfected with a rat phenylethanolamine N -methyltransferase promoter-luciferase reporter gene construct and also elevated Egr-1 mRNA levels in untransfected cells. Maximum induction of Egr-1 mRNA by carbachol was rapid (0.5 h), whereas by comparison, peak luciferase activity was delayed (6 h). In addition, carbachol stimulation of both luciferase and Egr-1 mRNA expression could be completely inhibited by atropine but not hexamethonium. Furthermore, bethanechol but not nicotine could mimic the effects of carbachol, indicating that carbachol activation was medicated through muscarinic cholinergic receptors. Finally, carbachol failed to stimulate luciferase expression in cells transfected with a mutant construct, in which the Egr-1 binding element in the phenylethanolamine N -methyltransferase promoter was mutated. These results suggest that carbachol activates the phenylethanolamine N -methyltransferase promoter through stimulation of Egr-1 expression, and are consistent with the potential involvement of Egr-1 in the cholinergic activation of the phenylethanolamine N -methyltransferase gene.     

High level expression of the bioactive human interleukin-10 in milk of transgenic mice

Human interleukin-10 (hIL-10) has wide spectrum of anti-inflammatory activities and has shown a potential to be used for treatment of inflammatory or immune illness. In this study, transgenic mice that over-express human interleukin-10 (IL-10) in their milk were generated using a bovine beta-casein/human IL-10 hybrid gene. After cloning of the IL-10 gene, a 22 kb hybrid gene was constructed by linking a 10 kb promoter sequence of the bovine beta-casein gene to the cloned 12 kb IL-10 gene. In six of the eight transgenic mice, the transgene RNA was expressed only in the mammary gland and in the other two mice, it was also slightly expressed in the lung. The highest human IL-10 level in milk was 1620 microg x ml(-1). Notably, transgenes in all the eight transgenic mice were expressed regardless of the integration site even though no correlation was shown between the copy numbers of the transgene and expression level. These results suggest that the genomic sequence of the human IL-10 gene can induce the IL-10 expression at high levels under the control of the bovine beta-casein promoter.