The hypothesis that a sequential linkage exists between neuropeptide-elicited, calcium-dependent phosphorylation of the proto-oncogene, ets-1, and subsequent effects on synthesis and/or release of neurotrophic factors is being investigated. The ets-1 protein has been identified as a putative sequence-specific transcriptional regulator with a highly conserved domain that mediates binding to DNA. Within the nervous system, this factor has been shown to be expressed in glial, but not neuronal cells. Serine phosphorylation of ets-1 is rapidly stimulated in glial cells by a variety of transmitter substances which cause an increase in the levels of intracellular calcium. Phosphorylation of ets-1 appears to destabilize its DNA binding affinity. Using a monoclonal antibody raised specifically against p51-ets, we have detected ets-1 protein in a variety of glial cell types including primary astrocytes, human astrocytoma and C6 glioma cells, as well as RN-22 schwannoma cells derived from a Schwann cell tumor of the peripheral nervous system. Experiments have been performed that expand the list of ligands found to promote ets-1 phosphorylation. In addition to carbachol, norepinephrine and the nonapeptide, bradykinin, we have demonstrated that the neurotransmitter, serotonin and the potent vasoactive peptide, endothelin (ET), are also capable of stimulating ets-1 phosphorylation in a variety of glial cell types. More detailed analysis of ET-mediated, ets-phosphorylation using three distinct isoforms, ET-1, ET-2, and ET-3 suggest that the ETA-type endothelin receptor is responsible for promoting the ET-induced phosphorylation of ets-1. Pretreatment with the cell permeant, myosin light chain kinase-selective inhibitor, KT5926, inhibits ET-stimulated, ets-1 phosphorylation, although in vitro kinase assays involving purified forms of myosin light chain kinase and ets-1 failed to confirm that this is, in fact, the kinase responsible for mediating ET-elicited serine phosphorylation. These initial findings have been summarized in an abstract to be presented at the Society for Neuroscience annual meeting (1996). Additional studies involving pharmacologic inhibition of myosin light chain kinase to block calcium-dependent ets-1 phosphorylation, as well as administration of endothelin-selective receptor antagonists, will be used to investigate the possible connection between ets-1 phosphorylation and expression of glial cell line-derived neurotrophic factor. The significance of a linkage between endothelin and ets-1 phosphorylation is emphasized by observations indicating that endothelin is capable of inducing the transcription factor, c-fos; stimulating DNA synthesis, and upregulating the expression of the neurotrophin, nerve growth factor, in astrocytes. Understanding regulation of neurotrophic factor expression is of significant therapeutic importance, particularly since delivery of neurotrophic factors to sites in the the brain is complicated by blood-brain barrier exlusion properties.
