SRF and SRF Cofactor mRNA Expression Is Differentially Regulated by BDNF Stimulation in Cortical Neurons

Abstract

Serum response factor (SRF) is a transcription factor that plays essential roles in multiple brain functions in concert with SRF cofactors such as ternary complex factor (TCF) and megakaryoblastic leukemia (MKL)/myocardin-related transcription factor (MRTF), which comprises MKL1/MRTFA and MKL2/MRTFB. Here, we stimulated primary cultured rat cortical neurons with brain-derived neurotrophic factor (BDNF) and investigated the levels of SRF and SRF cofactor mRNA expression. We found that SRF mRNA was transiently induced by BDNF, whereas the levels of SRF cofactors were differentially regulated: mRNA expression of Elk1, a TCF family member, and MKL1/MRTFA were unchanged, while in contrast, mRNA expression of MKL2/MRTFB was transiently decreased. Inhibitor experiments revealed that BDNF-mediated alteration in mRNA levels detected in this study was mainly due to the extracellular signal-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. Collectively, BDNF mediates the reciprocal regulation of SRF and MKL2/MRTFB at the mRNA expression level through ERK/MAPK, which may fine-tune the transcription of SRF target genes in cortical neurons. Accumulating evidence regarding the alteration of SRF and SRF cofactor levels detected in several neurological disorders suggests that the findings of this study might also provide novel insights into valuable therapeutic strategies for the treatment of brain diseases.

INTRODUCTION

Serum response factor (SRF) is a transcription factor that, in concert with SRF cofactors, contributes to the regulation of immediate early genes (IEGs) and the genes encoding regulatory proteins involved in alteration of cell shape and function, as well as the formation of neuronal circuits, memory, addiction, epilepsy, and stress response.¹⁾

Among SRF cofactors, the ternary complex factor (TCF) family and megakaryoblastic leukemia (MKL)/myocardin-related transcription factor (MRTF) family have been well known to exert biological functions in the brain.¹⁾ Elk1, a member of the TCF family, interacts with SRF homodimer to form a ternary complex for the induction of IEGs.¹⁾ MKL/MRTF, which comprises MKL1/MRTFA and MKL2/MRTFB,²⁾ interacts with SRF to regulate target genes.^1,3) MKL/MRTF is abundant in the brain and regulates the morphology of dendrites and synapses.^2,4–6) MKL/MRTF double knock out (KO) in the brain revealed a phenotype that was similar to that of SRF KO in the brain.⁷⁾

We previously isolated various MKL1/MRTFA and MKL2/MRTFB isoforms and found that their mRNA expression patterns were different during brain development.^8–10) MKL1/MRTFA isoforms include full-length MKL1 (FLMKL1), MKL1met, basic SAP coiled-coil domain (BSAC), and MKL1-elongated derivative of yield (MELODY).^8,9) MKL2/MRTFB isoforms include isoforms 1, 2, 3 and spliced neuronal long isoform of SRF transcriptional coactivator (SOLOIST)/MRTFB isoform 4 (MRTFB i4).¹⁰⁾

Upregulation of Elk1 mRNA was observed in the dentate gyrus in patients with depression.¹¹⁾ Reduced levels of MKL1/MRTFA were detected in rat Alzheimer’s disease models triggered by amyloid β_25–35.¹²⁾ Collectively, these findings suggest that expression of SRF and its cofactors is altered not only under physiological conditions but also in pathological conditions. Therefore, investigation of the regulatory mechanisms of SRF and SRF cofactor mRNA expression evoked by extracellular stimuli may be valuable. Brain-derived neurotrophic factor (BDNF) is a representative neurotrophin that leads to higher brain function and acts as a regulatory ligand for the regulation of a variety of genes.¹³⁾ Thus, we examined the effect of BDNF on SRF and its cofactor mRNA expression and analyzed the mechanism in cultured cortical neurons.

MATERIALS AND METHODS

Animals

Pregnant female Sprague-Dawley rats were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan). All animal experiments in this study were conducted under the guidelines of the Animal Care and Experimentation Committee of University of Toyama, Sugitani Campus and the ARRIVE guidelines. The animal experimental protocols were permitted with the following approval numbers: A2022PHA-6, A2019PHA-7, A2016PHA-8, A2013PHA-4, A2012PHA-1, and A2011PHA-5.

Reagents

Human recombinant BDNF protein was a gift from Sumitomo Pharma Co., Ltd. (Osaka, Japan). U0126 was purchased from Calbiochem (La Jolla, CA, U.S.A., 662005). LY294002 (L9908) and U73122 (U6756) were from Sigma-Aldrich (St. Louis, MO, U.S.A.).

Cell Culture

Primary cortical neurons from rat brains on embryonic day 17 were cultured according to a previously reported method.¹⁰⁾ Cells (2 × 10⁶ cells per well in 6-well plates) were maintained in neurobasal medium (21103-049, Invitrogen, Carlsbad, CA, U.S.A.), containing 1x B27 supplement (17504-044, Invitrogen), 0.5 mM glutamine (25030-081, Invitrogen), and 2 µg/mL gentamicin (15750-060, Invitrogen). Half of the medium was exchanged for fresh medium every 3 d.

RNA Isolation and Quantitative (q)PCR

RNA isolation and qPCR were carried out according to the methods reported previously.¹⁴⁾ In brief, total RNA was isolated using TRIsure (BIO-38032, Bioline, London, U.K.). Complementary DNA was synthesized from isolated RNA with SuperScript II (18064-014, Invitrogen). Primer positions for MKL1/MRTFA and MKL2/MRTFB isoforms are shown in Supplementary Fig. S1. To detect FLMKL1, BSAC, MELODY, MRTFB isoform 1, 3, and SOLOIST/MRTFB i4, Brilliant II SYBR Green QPCR Master Mix (600828, Agilent Technologies, St. Clara, CA, U.S.A.) was used to qPCR in accordance with the manufacturer’s protocols. To detect glyceraldehyde-3-phosphate dehydrogenase (GAPDH), SRF, and Elk1, SYBR Select Master Mix (4472908; Applied Biosystems, Waltham, MA, U.S.A.) was used, according to manufacturer’s protocol. We amplified the standard vectors harboring the DNA fragments of interest as well as cDNA samples. The level of GAPDH was used as an internal control. Primer sequences are shown in Supplementary Table S1.

Statistical Analysis

The data represent the mean ± standard error (n = 4). Significant difference was designated as p < 0.05/x (where x was the number of tests). Microsoft Excel 2013 [Version: (15.0.5127.1000)] was used for statistical analysis. Statistical analyses were performed using paired t-test with Bonferroni’s correction (See Figure legends).

RESULTS AND DISCUSSION

Previous studies have demonstrated that BDNF increases SRF protein expression in cortical neurons.³⁾ In this study, we initially investigated the time course of SRF mRNA induction by BDNF. We found that SRF mRNA expression increased at 1 h after BDNF treatment, but the induction was transient (Fig. 1A). Previous studies have shown that the CArG boxes located upstream of the transcriptional start site of the SRF gene are critical for serum-mediated activation of the SRF gene, which suggests that SRF may regulate the SRF gene transcription itself via CArG boxes.¹⁵⁾ Taken together, these findings suggest that, like an IEG, the SRF gene transiently responds to BDNF.

Fig. 1. Time–Course of SRF and Its Cofactor mRNA Expression Levels in Neurons Stimulated with BDNF

The mRNA expression levels of SRF (A), Elk1 (B), FLMKL1 (C), BSAC (D), MELODY (E), MKL2/MRTFB isoform 1 (F), MKL2/MRTFB isoform 3 (G), and SOLOIST/MRTFB i4 (H). Cortical neurons (DIV 9) were treated with vehicle or 100 ng/mL BDNF. Total RNA was prepared at the indicated times and subjected to qPCR. * p < 0.05/5 (vs. control at the same time point).

Next, we investigated the effect of BDNF on the mRNA levels of Elk1 and MKL/MRTF. Decreased Elk1 mRNA level is associated with depression and is altered by administration of antidepressant in human patients,¹¹⁾ although Elk1 mRNA expression was unchanged by BDNF in this study (Fig. 1B). Regarding the MKL/MRTF isoforms, BDNF did not influence the mRNA levels of FLMKL1 (the most abundant, Fig. 1C), BSAC (the second most abundant, Fig. 1D), and MELODY (the lowest, Fig. 1E), but MKL2/MRTFB isoforms 1 (the most abundant, Fig. 1F) and 3 (the second most abundant, Fig. 1G), and SOLOIST/MRTFB i4 (the lowest, Fig. 1H) were transiently downregulated at 3 h by BDNF. Since our previous study demonstrated that neuronal expression of MKL2/MRTFB isoform 2 is not detectable by qPCR,¹⁰⁾ we did not examine MKL2/MRTFB isoform 2. We also previously reported that MKL2/MRTFB contributes to BDNF-mediated activation of the distal enhancer in the activity-regulated cytoskeleton-associated protein (Arc) gene and the Arc mRNA expression was increased 1 h after BDNF stimulation in neurons.¹⁴⁾ Thus, the reduction of MKL2/MRTFB mRNA may occur after MKL2/MRTFB proteins are activated by BDNF.

BDNF binds to tropomyosin-related kinase B (TrkB) receptors and activates extracellular signal-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K), and phospholipase C (PLC) in neurons.¹³⁾ The transient increase in SRF mRNA and the decrease in MKL2/MRTFB mRNA expression caused by BDNF were inhibited by U0126, a MAPK/ERK kinase (MEK) inhibitor (Figs. 2A–D). PI3K inhibitor, LY294002, and PLC inhibitor, U73122, did not drastically influence the alterations of SRF and MKL2/MRTFB mRNA levels induced by BDNF (Supplementary Fig. S2). These findings suggest that ERK/MAPK mainly mediates BDNF-induced alteration of SRF and MKL2/MRTFB mRNA expression. Because ERK/MAPK also downregulates a subset of genes through direct phosphorylation and activation of CtBP transcriptional corepressor in some cases,¹⁶⁾ a similar mechanism may also act for BDNF-mediated MKL2/MRTFB gene regulation in this study. It is suggested that SRF gene induction might be mediated by MKL1/MRTFA and BDNF promotes phosphorylation of MKL1/MRTFA through ERK/MAPK.²⁾ Therefore, a BDNF-ERK/MAPK-MKL1/MRTFA-SRF axis might be important for maintaining the expression of SRF itself in a positive feedback manner. Since overexpression of MKL2/MRTFB isoforms upregulated several IEGs in Neuro-2a cells,¹⁰⁾ the transient decrease of MKL2/MRTFB mRNA mediated by the BDNF-ERK/MAPK cascade might be a phenomenon to avoid the excessive and sustained induction of these IEGs.

Fig. 2. Effect of U0126 on the mRNA Levels of SRF and MKL2/MRTFB Isoforms

The mRNA expression levels of SRF (A), MKL2/MRTFB isoform 1 (B), MKL2/MRTFB isoform 3 (C), and SOLOIST/MRTFB i4 (D). U0126 (20 µM) was added 30 min before 100 ng/mL BDNF stimulation in cortical neurons (DIV 9). Total RNA was prepared 1 h (SRF) or 3 h (MKL2/MRTFB) after BDNF stimulation and subjected to qPCR. * p < 0.05/2 and ** p < 0.01/2 (vs. control), ^# p < 0.05/2 and ^## p < 0.01/2 (vs. BDNF alone).

As MKL/MRTF can form heterodimer or homodimer,¹⁷⁾ it is speculated that possible dimer formation with MKL/MRTF isoforms could further fine-tune SRF-mediated transcription.

Recently, a study in mice and humans revealed that FGF17 ameliorates cognitive dysfunction in aged mice likely through SRF signaling.¹⁸⁾ Thus, further elucidation of the mechanism underlying upregulation of SRF expression and function might aid in the development of therapies for neurological disorders.

Acknowledgments

We thank Sumitomo Pharma Co., Ltd. for providing us with BDNF. This study was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (KAKENHI Grant Nos. JP26460064, JP18K06625 to A.T., 20K15989 to D.I.) and the Smoking Research Foundation (A.T.). We thank Emma Longworth-Mills, Ph.D., for editing a draft of this manuscript. Supplementary Figs. S1A and S1B have been published previously in Ishikawa et al. FEBS Open Bio 3: 387–393 (2013) under a CC BY license and Ishibashi et al. J. Neurochem. 159(4): 762–777 (2021), respectively.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

Notes

Masaaki Tsuda (Deceased on 15th February, 2022.)

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