Stressor and Glucocorticoid-Dependent Induction of the Immediate Early Gene Krü ppel-Like Factor 9: Implications for Neural Development and Plasticity

Krü ppel-like factor 9 (KLF9) is a thyroid hormone-induced, immediate early gene implicated in neural development in vertebrates. We analyzed stressor and glucocorticoid (GC)-dependent regulation of KLF9 expression in the brain of the frog Xenopus laevis, and investigated a possible role for KLF9 in neuronal differentiation. Exposure to shaking/confinement stressor increased plasma corticosterone (CORT) concentration, and KLF9 immunoreactivity in several brain regions, which included the medial amygdala and bed nucleus of the stria terminalis, anterior preoptic area (homologous to the mammalian paraventricular nucleus), and optic tectum (homologous to the mammalian superior colliculus). The stressor-induced KLF9 mRNA expression in the brain was blocked by pretreatment with the GC receptor antagonist RU486, or mimicked by injection of CORT. Treatment with CORT also caused a rapid and dose-dependent increase in KLF9 mRNA in X. laevis XTC-2 cells that was resistant to inhibition of protein synthesis. The action of CORT on KLF9 expression in XTC-2 cells was blocked by RU486, but not by the mineralocorticoid receptor antagonist spironolactone. To test for functional consequences of up-regulation of KLF9, we introduced a KLF9 expression plasmid into living tadpole brain by electroporation-mediated gene transfer. Forced expression of KLF9 in tadpole brain caused an increase in Golgi-stained cells, reflective of neuronal differentiation/maturation. Our results support that KLF9 is a direct, GC receptor target gene that is induced by stress, and functions as an intermediary in the actions of GCs on brain gene expression and neuronal structure. S tress can have profound and complex effects on brain function and morphology, and consequently on learning, memory, and behavior. The effects of stress can be facilitatory or inhibitory, the outcome dependent on the duration and type of stressor, and the behavioral context within which the stress is experienced. Chronic stress leads to neurodegeneration and impairment of learning and memory (1-3). By contrast, short-term stress may enhance neurotransmission, promote long-term potentiation (LTP), increase dendritic spine density in the hippocampus, and facilitate memory consolidation and reconsolidation (1-5). Stress hormone actions on the brain lead to changes in neuronal structure, but little is known about the transcriptional mechanisms that underlie cellular changes after exposure to a stressor (3). The primary vertebrate stress hormones, the glucocorticoids (GCs), are responsible for many effects of stress on the brain, and their actions are mediated by two nuclear receptors, the mineralocorticoid receptor (MR) and the GC receptor (GR) (6, 7). Receptors located in the plasma membrane transduce rapid, nongenomic actions of GCs on neural function (8 -10). The initial, facilitatory effects of GCs on neurotransmission and induction of LTP may be mediated by membrane GRs, whereas subsequent genomic actions, dependent on the nuclear GR, may reverse and normalize the enhanced excitability (2) and generate structural changes in neurons (3). This is considered to be an adaptive mechanism for limiting the stress response (i.e. through negative feedback) and facilitating information storage. Earlier, we showed that a member of the Sp/Krü ppel-like family of zinc-finger domain transcription factors (11, 12), the Krü ppellike factor 9 (KLF9) [also basic transcription element binding protein 1 (13)], plays a key role in thyroid hormone-dependent actions on neurite extension and branching (14 -18). Forced expression of KLF9 in Neuro-2a cells caused a dose-dependen