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Kdm6b and Pmepa1 as Targets of Bioelectrically and Behaviorally Induced Activin A Signaling

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Abstract

The transforming growth factor-β (TGF-β) family member activin A exerts multiple neurotrophic and protective effects in the brain. Activin also modulates cognitive functions and affective behavior and is a presumed target of antidepressant therapy. Despite its important role in the injured and intact brain, the mechanisms underlying activin effects in the CNS are still largely unknown. Our goal was to identify the first target genes of activin signaling in the hippocampus in vivo. Electroconvulsive seizures, a rodent model of electroconvulsive therapy in humans, were applied to C57BL/6J mice to elicit a strong increase in activin A signaling. Chromatin immunoprecipitation experiments with hippocampal lysates subsequently revealed that binding of SMAD2/3, the intracellular effectors of activin signaling, was significantly enriched at the Pmepa1 gene, which encodes a negative feedback regulator of TGF-β signaling in cancer cells, and at the Kdm6b gene, which encodes an epigenetic regulator promoting transcriptional plasticity. Underlining the significance of these findings, activin treatment also induced PMEPA1 and KDM6B expression in human forebrain neurons generated from embryonic stem cells suggesting interspecies conservation of activin effects in mammalian neurons. Importantly, physiological stimuli such as provided by environmental enrichment proved already sufficient to engender a rapid and significant induction of activin signaling concomitant with an upregulation of Pmepa1 and Kdm6b expression. Taken together, our study identified the first target genes of activin signaling in the brain. With the induction of Kdm6b expression, activin is likely to gain impact on a presumed epigenetic regulator of activity-dependent neuronal plasticity.

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Acknowledgments

We thank Iwona Izydorczyk and Birgit Vogler for technical assistance.

Compliance with ethical standards

ᅟ

Funding

This study was funded by the Deutsche Forschungsgemeinschaft (DFG AL 294/10-1 and INST 90/675-1 FUGG to C.A.), the Johannes und Frieda Marohn-Stiftung (to F.Z. and C.A.), the Emerging Field Initiative of the University Erlangen-Nürnberg (to C.A.), the Dr. Ernst und Anita Bauer Stiftung (to A. L.), the Jürgen Manchot Stiftung (to A. L.), the Studienstiftung des deutschen Volkes (to S. L.), the Swiss National Science Foundation (310030_132884 to S.W.) and the Swiss Cancer League (KFS-2822-08-2011 to S.W.). Financial support to B.W. came from the German Federal Ministry of Education and Research (BMBF, 01GQ113), the Interdisciplinary Centre for Clinical Research (IZKF, University Hospital of Erlangen), and the Bavarian Ministry of Education and Culture, Science and the Arts in the framework of the BioSysNet and ForIPS.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Institute of Physiology and Pathophysiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Universitätsstr. 17, 91054, Erlangen, Germany

    Andrea S. Link, Sandra Lehnert, Tobias Huth, Fang Zheng & Christian Alzheimer

  2. Department of Biology, Institute of Molecular Health Sciences, ETH Zurich, 8093, Zurich, Switzerland

    Svitlana Kurinna & Sabine Werner

  3. IZKF Junior Research Group and BMBF Research Group Neuroscience, IZKF, Friedrich-Alexander-Universität Erlangen-Nürnberg, Glückstr. 6, 91054, Erlangen, Germany

    Steven Havlicek & Beate Winner

  4. Department of Psychiatry and Psychotherapy, University Hospital, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schwabachanlage 6, 91054, Erlangen, Germany

    Martin Reichel & Johannes Kornhuber

  5. Present address: Stem Cell and Regenerative Biology, Genome Institute of Singapore, A*STAR, 60 Biopolis Street, 138672, Singapore, Singapore

    Steven Havlicek

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  1. Andrea S. Link
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Correspondence to Christian Alzheimer.

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Figure S1

Analysis of TGF-β/activin signaling after ECS. Hippocampal mRNA levels of Bdnf (a), TGF-β subfamily ligands that activate SMAD2/3 signaling (b-e), their inhibitors (f,k-l), and activin receptors (g-j) were analyzed by RT-qPCR after electroconvulsive seizures (ECS) and were normalized to the mean mRNA levels of Tbp, Hprt and Rpl13a. Error bars represent mean +/- SEM, n ≥ 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way ANOVA and Tukey multiple comparison test. (DOC 351 kb)

Figure S2

siRNA-mediated knock-down of Pmepa1 does not significantly enhance activin-induced SMAD2/3 phosphorylation in HT22 cells. (a) Dose-dependent effect of activin A on SMAD2/3 phosphorylation in HT22 cells. Cells were treated for 24 h with 10 or 50 ng/ml activin A and analyzed by western blotting for the levels of total and phosphorylated SMAD2/3. The ratio between P-SMAD2/3 and total SMAD2/3 is shown. (b) HT22 cells were transfected with different concentrations of two different Pmepa1 siRNAs or scrambled siRNA as indicated and treated with 10 ng/ml activin A 24 h later. Pmepa1 mRNA levels were determined after another 24 h. Transfection with siRNA against Pmepa1 significantly suppressed the activin-induced increase in Pmepa1 mRNA levels (grey bars) in comparison to cells transfected with scrambled siRNA (black bar). Error bars represent mean +/- SEM; *p < 0.05, **p < 0.01, ****p < 0.0001 using one-way ANOVA and Tukey test for multiple comparison. (c) Densitometric quantification of Westernblots assessing levels of total and phosphorylated SMAD2/3 revealed no significant increase in SMAD2/3 phosphorylation after transfection with Pmepa1-specific siRNAs and activin treatment. The concentration of the siRNA is indicated at the bottom of the grey bars. (d) Representative Westernblot demonstrating that SMAD2/3 phosphorylation levels were almost back to baseline (first lane) after 24 h of activin treatment (10 ng/ml, second lane) and were largely unaffected by transfection with Pmepa1 siRNA (third lane). (DOC 362 kb)

Figure S3

Impact of ECS on H3K27 trimethylation. (a) The amount of the histone mark H3K27me3 was analyzed at different time points after ECS or sham treatment (sh) by Westernblot and normalized to histone 3 (H3) protein levels. β-actin was used as independent loading control. Densitometric quantification (bar graph) did not show significant changes in global H3K27me3 methylation relative to the corresponding sham controls (white bars) using one-way ANOVA and Tukey test for multiple comparison. n = 3. Error bars represent mean +/- SEM. (b) Immunofluorescence staining of H3K27me3 (visualized in red, left panel) did not reveal obvious local changes in H3K27 trimethylation in the hippocampus at 0 h (upper row) and 24 h (middle row) after ECS. DAPI was used to counterstain nuclei (blue, middle panel). Merged pictures are shown in the right panel. Primary antibody was omitted to control for background staining (control, bottom row). Scale bar: 500 μm. (DOC 1604 kb)

Figure S4

Expression analysis of several TGF-β family members during environmental enrichment (EE). Hippocampal mRNA levels of Tgfb1 (a), Tgfb2 (b), Tgfb3 (c), Inhbb (d) and Inha (e) were analyzed by RT-qPCR and did not markedly change during exposure to EE. mRNA levels were normalized to the mean of Tbp, Hprt and Rpl13a. Experiments were conducted twice except for Inhbb (n = 3). Error bars represent mean +/- SEM. One-way ANOVA and Tukey multiple comparison test was used to test for significant changes. (DOC 377 kb)

Figure S5

Analysis of Inhba and Kdm6b mRNA levels 0.5 h after ECS. While Inhba (a) mRNA expression was still unaltered shortly after ECS, Kdm6b (b) mRNA levels had already increased significantly. mRNA levels were normalized to the mean of Tbp, Hprt and Rpl13a. n = 3. Error bars represent mean +/- SEM; ***p < 0.001, ****p < 0.0001 using one-way ANOVA and Tukey test for multiple comparison. (DOC 120 kb)

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Link, A.S., Kurinna, S., Havlicek, S. et al. Kdm6b and Pmepa1 as Targets of Bioelectrically and Behaviorally Induced Activin A Signaling. Mol Neurobiol 53, 4210–4225 (2016). https://doi.org/10.1007/s12035-015-9363-3

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