Characterization of reproductive steroid receptors and response to estrogen in a rat serotonergic cell line

Study of the cellular and molecular consequences of steroid hormone action in the serotonin neural system will provide new avenues for pharmacotherapeutic intervention in mental illness related to reproductive function. However, it is difficult to probe intracellular mechanisms with whole animal models. We sought the steroid receptor compliment and estrogen response of two rat serotonin cell lines in order to determine if they could be of future assistance in this matter. Immunohistochemistry with a panel of antibodies, RT-PCR and a serotonin ELISA were utilized to characterize the RN46A-V1 cells (herein called RN46A), and the subclone RN46A-B14 (herein called B14) that is stably transfected with brain derived neurotrophic factor (BDNF). RN46A and B14 cells express estrogen receptor beta (ERbeta), androgen receptors (AR) and nuclear factor kappa B (NFkappaB) but not estrogen receptor alpha (ERalpha) or progestin receptors (PR). RT-PCR confirmed the presence of ERbeta and the absence of ERalpha and PR in both cell lines. B14 cells contain more immunodetectable BDNF and serotonin than the RN46A parent line. In addition, immunofluorescence for the serotonin reuptake transporter (SERT) was observed in the cell body region of undifferentiated B14 cells. After differentiation at a nonpermissive temperature, SERT immunostaining was observed in both the cell body region and along the extent of the axons. Serotonin content as determined by ELISA was higher in B14 than RN46A cells. Estrogen (0.1 and 1.0 nM) stimulated serotonin in the B14 cells in serum free medium. In summary, the RN46A cells and the B14 subclone contain the same compliment of nuclear steroid receptors as rat raphe serotonin neurons and thus may provide a convenient in vitro model for study of intracellular mechanisms of action of steroid hormones in the context of a serotonin neuron.