In vivo expression and regulation of Elk-1, a target of the extracellular-regulated kinase signaling pathway, in the adult rat brain

Abstract

The transcription factor Elk-1, a nuclear target of extracellular-regulated kinases (ERKs), plays a pivotal role in immediate early gene induction by external stimuli. Notably, the degree of phosphorylation of Elk-1 is tightly correlated with the level of activation of transcription of c-fos by proliferative signals. No data yet indicate the role of Elk-1 in the adult brain in vivo. To address this question, we have analyzed in the present work (1) Elk-1 mRNA and protein expression in the adult rat brain, and (2) the regulation of Elk-1 (i.e., its phosphorylation state) in an in vivo model of immediate early gene (IEG) induction: an electrical stimulation of the cerebral cortex leading to c-fos and zif268 mRNA induction in the striatum. Using in situ hybridization, we show that Elk-1 mRNA is expressed in various brain structures of adult rat, and that this expression is exclusively neuronal. We demonstrate by immunocytochemistry using various specific Elk-1 antisera that the protein is not only nuclear (as shown previously in transiently transfected cell lines) but is also present in soma, dendrites, and axon terminals. On electrical stimulation of the glutamatergic corticostriatal pathway, we show a strict spatiotemporal correspondence among ERK activation, Elk-1 phosphorylation, and IEG mRNA induction. Furthermore, both activated proteins, analyzed by immunocytochemistry, are found in cytosolic and nuclear comparments of neuronal cells in the activated area. Our data suggest that the ERK signaling pathway plays an important role in regulating genes controlled by serum response element sites via phosphorylation of Elk-1 in vivo.

Fig. 1.

In situ detection of Elk-1 mRNA in rat brain. Rat cerebral sections were hybridized with³³P-cRNA-Elk-1 sense (A, inset) and antisense (A–I) probes. Coronal sections (20 μm thick) were taken in the whole rostrocaudal extension of the rat brain. The cerebellum was taken according to a sagittal plane.OT, Olfactory tubercles; Cx, cerebral cortex; Acb, accumbens nucleus; CC, corpus callosum; CPu, caudate–putamen;Spt, septum; GP, globus pallidus;Thal, thalamus; CA, Amon corn layer of the hippocampus; DG, dentate gyrus of the hippocampus;SC, superior colliculi; SNr, substantia nigra pars reticulata; PAG, periaqueducal gray layer;PN, pedonculopontine nuclei; Cb, cerebellum. J, High magnification (500×) of Elk-1 mRNA hybridization in the CA1 layer of hippocampus. The cresyl violet counterstaining allows the characterization of glial (small arrows) and neuronal (large arrows) cells. Note that hybridization signals are always observed on neuronal cells. Scale bar, 17 μm. K, Elk-1 hybridization in the corpus callosum (glial cells, small arrows).

Fig. 2.

Expression of Elk-1 protein in various brain structures. A, Western blot analysis of Elk-1 expression in the striatum, the hippocampus, and the cerebellum. The COOH antibody yields a band with an apparent molecular weight of 62 kDa (protein molecular weight standards). B, Immunocytochemical detection of Elk-1 using the COOH antibody. Shown are the striatum, the CA1 layer of hippocampus, and the Purkinje cell layer of the cerebellum. Elk-1 COOH antibody was used on rat brain sections processed in parallel. Note that in all three structures, the protein is present in the nucleus and the cytosol, as well as dendritic processes (arrowhead). Arrow, Heavy immunoreactivity for Elk-1 in a subpopulation of striatal cells. Scale bar, 17 μm.

Fig. 3.

Subcellular localization of Elk-1, CREB, and STAT3 proteins in the pyramidal cell layer of the cerebral cortex.A, Serial sections were processed for immunocytochemistry with three different Elk-1 antibodies (described in the diagram at the top): anti-COOH, anti-C-terminal (C-term), and anti-Ets. Note that all three antibodies show the protein in the nucleus (thin-headed, long arrows), cytosol (short arrows), and also in dendritic processes (arrowheads) of neuronal cells. B, CREB and C, STAT3 immunocytochemistry in adjacent sections of cerebral cortex. Note the nuclear localization of CREB (B, thin-headed, long arrows) and the cytoplasmic localization of STAT3 (C, short arrows). Magnification, 500×. Scale bar, 17 μm.

Fig. 4.

Visualization of Elk-1 immunoreactivity with confocal microscopy. Two examples of Elk-1-immunoreactive striatal neurons with COOH antibody. In these two cells, the fluorescent signal predominates in the somatic (arrows) and dendritic (arrowheads) cytoplasm, and the nuclei (n) are not stained. A1, B1, Confocal sections; A2, B2, maximum intensity projection (25–28 sections, respectively). Scale bar, 5 μm.

Fig. 5.

Elk-1 protein detection in striatopallidal and striatonigral axon terminals. After unilateral kainic acid lesion of striatal cells, Elk-1 immunodetection was processed on sections corresponding to the external segment of the globus pallidus (GPe) and the substantia nigra pars reticulata (SNr) levels. Note in both cases the clear decrease (*) of the neuropil immunoreactivity in the GPe (B) and the SNr (D) ipsilateral to the lesion when compared with the contralateral side of the lesion (A, C, respectively).

Fig. 6.

c-fos andzif268 mRNA induction after unilateral electrical stimulation of the corticostriatal pathway. Adjacent brain sections were in situ-hybridized with³³P-c-fos and³³P-zif268 antisense probes. Left panels, In sham-operated (Sham) rats, c-fos and zif268 mRNAs are induced in the cortex ipsilateral to the electrode implantation. In the entire striatum, whereas c-fos is not detectable, a constitutive expression of zif268 is observable.Right panels, In stimulated (Stim) rats, c-fos and zif268 mRNAs are slightly induced in the cortex ipsilateral to the stimulation. In the lateral part of the striatum, a restricted bilateral induction is strongly observed for both messengers, when compared with the medial area.iCx, Ipsilateral cortex; cCx, contralateral cortex to the electrode implantation; mSt,lSt, medial and lateral parts of the striatum, respectively.

Fig. 7.

Phosphorylation of ERK and Elk-1 on corticostriatal activation. Adjacent striatal sections from stimulated rats (n = 3) were processed in parallel for immunocytochemistry using antibodies specific for phosphorylated forms of ERK (P-ERK) and Elk-1 (P-Elk-1) proteins. Shown are ipsilateral striata to the stimulation site at low (40×; A, E) and high (500×; B, C, F, G) magnification. Note the increase of P-ERK (A–C) and P-Elk-1 (E–G) in the lateral part of the striatum. Arrows, Increase of nuclear immunolabeling.Arrowheads, Dendritic immunolabeled processes of striatal cells. D, H, Optical density measurements of P-ERK and P-Elk-1 immunoreactivities. Striatal sections were digitized with an image analyzer (IMSTAR), and precise delineation of medial and lateral parts was performed on each striatum (4 sections per rat;n = 3 independent rats). Optical densities were measured and statistical comparisons were performed between lateral and medial parts of the striatum by a paired Student’s ttest (*p < 0.05; n = 3).mSt, lSt, Medial and lateral parts of the striatum, respectively. Scale bar, 530 μm for 16×; 17 μm for 500× magnification.

Fig. 8.

Western blot analysis of P-ERK and P-Elk-1 immunoreactivities. The medial and lateral parts of the striatum were rapidly dissected just after the end of the stimulation period, lysed, and processed for Western blot analysis. Left panels, P-ERK and ERK proteins were detected on the same blot after a stripping procedure. Note the marked increase of phosphorylated 42 and 44 kDa proteins in the lateral striatal extracts relative to those from the medial striatum and the equal amount of ERK1 and ERK2 proteins in both extracts. Right panels, The same procedure was applied for P-Elk-1 and Elk-1 immunodetection. Note the slight increase of 62 kDa phosphorylated protein in the lateral versus medial area with no variation of Elk-1 in these areas. The data presented are representative of three independent experiments, which gave similar results.

Fig. 9.

Double staining of P-Elk-1 immunoreactivity withzif268 mRNA in situ hybridization. Striatal sections from stimulated rats were coprocessed for P-Elk-1 with zif268 mRNA as described above. In both medial and lateral parts of the striatum, a constitutive expression ofzif268 mRNA is detectable, as well as a weak P-Elk-1 immunoreactivity (thin arrows). In the lateral part of the striatum, neurons presenting a strong P-Elk-1 immunoreactivity (large arrows) also show a marked increase inzif268 mRNA expression. Note the perfect correlation between strong P-Elk-1 immunoreactivity and zif268 mRNA expression.