Endogenous SOLOIST/MRTFB i4, a Neuronal Isoform of MKL2/MRTFB, Positively and Negatively Regulates SRF Target Immediate Early Genes in Neuro-2a Cells

Abstract

Megakaryoblastic leukemia 2 (MKL2)/myocardin-related transcription factor-B (MRTFB) is a serum response factor (SRF) cofactor that is enriched in the brain and controls SRF target genes and neuronal morphology. There are at least four isoforms of MKL2/MRTFB. Among these, MKL2/MRTFB isoform 1 and spliced neuronal long isoform of SRF transcriptional coactivator (SOLOIST)/MRTFB isoform 4 (MRTFB i4) are highly expressed in neurons. Although, when overexpressed in neurons, isoform 1 and SOLOIST/MRTFB i4 have opposing effects on dendritic morphology and differentially regulate SRF target genes, it is unknown how endogenous SOLOIST/MRTFB i4 regulates gene expression. Using isoform-specific knockdown, we investigated the role of endogenous SOLOST/MRTFB i4 in regulating the expression of other MKL2/MRTFB isoforms and SRF-target genes in Neuro-2a cells. Knockdown of SOLOIST/MRTFB i4 downregulated SOLOIST/MRTFB i4, while it upregulated isoform 1 without affecting isoform 3. Knockdown of SOLOIST/MRTFB i4 downregulated the SRF target immediate early genes egr1 and Arc, while it upregulated c-fos. Double knockdown of isoform 1 and SOLOIST/MRTFB i4 inhibited c-fos expression. Taken together, our findings in Neuro-2a cells suggest that endogenous SOLOIST/MRTFB i4 positively regulates egr1 and Arc expression. In addition, endogenous SOLOIST/MRTFB i4 may negatively regulate c-fos expression, possibly by downregulating isoform 1 in Neuro-2a cells.

INTRODUCTION

The transcription factor serum response factor (SRF) plays fundamental roles in regulating immediate early genes (IEGs) and other genes involved in cell motility, morphology and circadian rhythms in a variety of cell types.^1,2) Megakaryoblastic leukemia (MKL)/myocardin-related transcription factor (MRTF) is a transcriptional cofactor that interacts with SRF to control target gene expression.³⁾ MKL1/MRTFA and MKL2/MRTFB, both of which are expressed in the brain,^4,5) are critical regulators of gene expression⁶⁾ as well as brain structure⁷⁾ and neuronal morphology.^4,5) Transcription of MKL2/MRTFB gene gives rise to at least four isoforms of MKL2/MRTFB, including isoforms 1, 2, and 3, and spliced neuronal long isoform of SRF transcriptional coactivator/MRTF isoform 4 (SOLOIST/MRTFB i4).⁸⁾ Among these, isoform 1 and SOLOIST/MRTFB i4 are highly expressed in the brain and neurons, but not astrocytes.⁸⁾ Although overexpression of MKL2/MRTFB isoform 1 and SOLOIST/MRTFB i4 promotes the induction of the immediate early genes c-fos, egr1 and Arc, they have converse effects on dendritic morphology—isoform 1 increases dendritic complexity, while SOLOIST/MRTFB i4 reduces complexity.⁸⁾ These findings suggest that the various MKL2/MRTFB isoforms play differential roles in the regulation of gene expression and neuronal morphology. However, the role of endogenous SOLOIST/MRTFB i4 in regulating other isoforms and SRF target genes is still unclear. In this study, we confirmed endogenous expression of MKL2/MRTFB isoforms in Neuro-2a cells. We then specifically knocked down SOLOIST/MRTFB i4 to evaluate its role in regulating the expression of the different MKL2/MRTFB isoforms and SRF target genes.

MATERIALS AND METHODS

Cell Culture

Neuro-2a cells were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). The culture protocol was as described previously.⁸⁾ The culture medium contained Dulbecco’s modified Eagle Medium (DMEM) (11885-084, Gibco/Life Technologies, Carlsbad, CA, U.S.A.), 50 U/mL penicillin, 50 µg/mL streptomycin (15070-063, Invitrogen, Carlsbad, CA, U.S.A.) and 10% fetal bovine serum (26140-079, Invitrogen). For PCR, cells were seeded at 1.5 × 10⁵/well in six-well plates.

RNA Isolation and Quantitative (q)PCR

Total RNA was extracted from Neuro-2a cells at 72 h after transfection using TRIsure (BIO-38032, Bioline, London, U.K.) and cDNA was synthesized from total RNA as previously described.⁸⁾ PCR was used to evaluate the expression of MKL2/MRTFB isoforms in Neuro-2a cells. The primers for isoform 1, isoform 3 and SOLOIST/MRTFB i4 are shown in Supplementary Table S1, and the positions of the primers are illustrated in Fig. 1A. The PCR was performed in a 25-µL reaction mix including 1 µL cDNA solution, 0.625 U GoTaq DNA polymerase (M8291, Promega, Madison, WI, U.S.A.), 0.2 mM deoxyribonucleotide triphosphate (dNTP) and 0.5 µM of each primer. The amplified bands were resolved by 2% agarose gel electrophoresis. To detect the different MKL2/MRTFB isoforms using qPCR, isoform-specific primers were used (the sequences are shown in Supplementary Table S1). The positions of the primers are illustrated in Fig. 1B. To confirm the specificity of the primers, the PCR was performed with plasmid DNA carrying MKL2/MRTFB isoforms as template instead of cDNA.

Fig. 1. Endogenous MKL2/MRTFB Isoform Expression and the Impact of SOLOIST/MRTFB i4 or Isoform 1-Specific Knockdown in Neuro-2a Cells

(A) Endogenous expression of MKL2/MRTFB isoforms in Neuro-2a cells. Primer positions are shown as arrows in the exon-intron structures of MKL2/MRTFB isoforms 1, 2 and 3 and SOLOIST/MRTFB i4.⁸⁾ S: sense primer; AS: antisense primer. RNA was isolated from Neuro-2a cells and subjected to RT-PCR with the primers shown on the left. Amplified bands on the agarose gel indicate endogenous MKL2/MRTFB isoforms. (B) Primer specificity for detection of MKL2/MRTFB isoforms. Primer positions are shown as arrows in the exon-intron structures of MKL2/MRTFB isoforms 1 and 3 and SOLOIST/MRTFB i4.⁸⁾ S: sense primer; AS: antisense primer. PCR was performed in the mixture containing the vector for MKL2/MRTFB isoform 1 or 3 or SOLOIST/MRTFB i4 as template. Specific amplification with each isoform primer was detected on agarose gels. (C) Design of the vector expressing small hairpin RNA to specifically knock down SOLOIST/MRTFB i4. The target sequence shown in the box is located within the specific exon of SOLOIST/MRTFB i4. (D) Effect of SOLOIST/MRTFB i4-specific knockdown on the expression of MKL2/MRTFB isoforms in Neuro-2a cells. The shR-luc control vector or the vector shown in Fig. 1C (shSOLOIST/MRTFB i4) (4 µg/well) was transfected into Neuro-2a cells. Then, RNA was isolated and qPCR was performed with the primers shown in Fig. 1B. ** p < 0.05; NS, not significant vs. shR-luc (n = 5). (E) Design of the vector expressing small hairpin RNA to specifically knockdown isoform 1. The target sequence shown in the box is located within the specific exon of isoform 1. (F) Effect of isoform 1-specific knockdown on the expression of MKL2/MRTFB isoforms in Neuro-2a cells. The same experimental procedure as in (D) was used. ** p < 0.01; NS, not significant vs. shR-luc (n = 3).

To investigate the expression levels of the various MKL2/MRTFB isoforms, IEGs and other SRF target genes, qPCR was performed using 20 µL 1 × SYBR Select Master Mix (4472908, Thermo Fisher Scientific, Waltham, MA, U.S.A.) with 0.2 µM of each primer (sequences are shown in Supplementary Table S1) and 2 µL cDNA solution. Thermocycling conditions are summarized in Supplementary Table S2. As an internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used.

Plasmids

The control vector shR-luc targets the Renilla luciferase gene, has been previously described.⁹⁾ To specifically knock down SOLOIST/MRTFB i4 without affecting isoforms 1 and 3, we chose a target sequence corresponding to the exon specific to SOLOIST/MRTFB i4 (Fig. 1C). To construct the vector expressing small hairpin RNA, annealed oligonucleotides (60-mers) 5′-GATCCCCGTATAGTCTTTGGGGAGTCTTCAAGAGAGACTCCCCAAAGACTATACTTTTTA-3′ and 5′-AGCTTAAAAAGTATAGTCTTTGGGGAGTCTCTCTTGAAGACTCCCCAAAGACTATACGGG-3′ were subcloned into the pSUPER vector after digestion with the HindIII and BglII (OligoEngine Platform, Seattle, WA, U.S.A.). Additionally, an isoform 1-specific knockdown vector was constructed using the KOD-Plus mutagenesis kit (SMK-101, Toyobo, Osaka, Japan) for sequence insertion in pSUPER with the following primers: 5′-GAGATCTGGGAAGCCTTGTGGCTGCTTTTTAAGCTTATCGATACCGTCGACCTC-3′ and 5′-TTGAATCTGGGAAGCCTTGTGGCTGCGGGGATCTGTGGTCTCATACAGAACT-3′.

Transfection

Neuro-2a cells were transfected with plasmid DNA together with 6 µL of Lipofectamine (18324-012, Invitrogen) and 4 µL of Plus reagent (10964-021, Invitrogen). After incubation with transfection reagents for 4 h, the conditioned medium was removed and fresh medium was added.

Statistical Analysis

All data are shown as mean ± standard error (S.E.) (n = 3–5). For statistical analysis, Microsoft Excel 2013 [Version: (15.0.5127.1000)] was used. After the values were log-converted, significant differences were evaluated using a paired t-test (p < 0.05) or paired t-test with Bonferroni’s correction (p < 0.05/2 in Fig. 2D).

Fig. 2. Effect of SOLOIST/MRTFB i4-Specific Knockdown on the Expression of SRF Target Immediate Early Genes and Isoform 1-Specific Knockdown-Mediated Suppression of c-fos Expression Induced by Knockdown of SOLOIST/MRTFB i4

The shR-luc control vector or the vector shSOLOIST/MRTFB i4 was transfected into Neuro-2a cells. Then, RNA was isolated and qPCR was performed with primers for c-fos (A), egr1 (B) or Arc (C). * p < 0.05 vs. shR-luc (n = 5). (D) The shR-luc control vector or the vector shSOLOIST/MRTFB i4 (4 µg/well) and shR-luc control vector or shisoform 1 (2 µg/well) were transfected into Neuro-2a cells. RNA was isolated and qPCR was performed with primers for c-fos. * p < 0.05/2 vs. shisoform 1(−)/shSOLOIST/MRTFB i4(−) and ^# p < 0.05/2 vs. shisoform 1(−)/shSOLOIST/MRTFB i4(+) (n=4).

RESULTS AND DISCUSSION

Previously, we reported that primary cultured cortical neurons expressed MKL2/MRTFB isoforms 1 and 3 and SOLOIST/MRTFB i4.⁸⁾ We initially investigated whether these isoforms are endogenously expressed in Neuro-2a cells. We performed PCR using cDNA from Neuro-2a cells. As shown in Fig. 1A, bands corresponding to isoforms 1 and 3 and SOLOIST/MRTFB i4 were present, while isoform 2 was not, suggesting that Neuro-2a cells express isoforms 1 and 3 and SOLOIST/MRTFB i4, but not isoform 2. This result is consistent with the undetectable level of isoform 2 in mouse tissues.⁸⁾ To optimize the primers for qPCR, we redesigned the primers, as shown in Fig. 1B. We confirmed that these primers specifically detected each different MKL2/MRTFB isoform using plasmid vector as template (Fig. 1B). Next, we constructed a vector expressing small hairpin (sh) RNA against SOLOIST/MRTFB i4 (shSOLOIST/MRTFB i4). Figure 1C shows that SOLOIST/MRTFB i4 possesses a specific exon that neither isoform 1 nor 3 contains (Figs. 1A, B). Therefore, we chose the sequence within the specific exon as a target sequence (Fig. 1C). We found that transfection of the sh-expressing vector into Neuro-2a cells reduced SOLOIST/MRTFB i4 mRNA expression, but did not alter mRNA expression of isoform 3 (Fig. 1D). In contrast, SOLOIST/MRTFB i4 knockdown upregulated isoform 1 (Fig. 1D). This finding suggests that endogenous SOLOIST/MRTFB i4 negatively regulates isoform 1 in Neuro-2a cells. We also constructed a sh isoform 1-expressing vector (shisoform 1) (Fig. 1E) and confirmed that the vector showed isoform 1-specific knockdown (Fig. 1F).

Next, we investigated whether c-fos, egr1 and Arc, representative SRF target IEGs, were altered in Neuro-2a cells with SOLOIST/MRTFB i4-specific knockdown. The expression of c-fos was increased (Fig. 2A). In contrast, the mRNA levels of egr1 and Arc were decreased, compared with the control (Figs. 2B, C). Collectively, these findings suggest that endogenous SOLOIST/MRTFB i4 negatively regulates the mRNA levels of isoform 1 and c-fos in Neuro-2a cells. Knockdown of isoform 1 suppressed c-fos expression induced by knockdown of SOLOIST/MRTFB i4 (Fig. 2D), suggesting that isoform 1 acts as a transcriptional coactivator for inducing c-fos gene expression in Neuro-2a cells. Overexpression of SOLOIST/MRTFB i4 also induced the c-fos gene in Neuro-2a cells,⁸⁾ suggesting that distinct mechanisms (e.g., different protein–protein interactions) might be working following c-fos gene induction under the conditions of overexpressed and endogenous SOLOIST/MRTFB. Since expression of SOLOIST/MRTFB i4 is highly neuronal, rather than astrocytic, and its expression is gradually induced during brain development,⁸⁾ SOLOIST/MRTFB i4-driven IEG expression might play critical roles in neuronal development and plasticity.

MKL/MRTF regulates srf and cytoskeletal genes.^10–13) Therefore, we determined whether endogenous SOLOIST/MRTFB i4 regulates the expression of srf and cytoskeletal SRF-target genes such as actinin α1, gelsolin, and β-actin. The mRNA levels of these genes were not affected by knockdown of SOLOIST/MRTFB i4 (Supplementary Fig. S1).

We previously reported that isoform 1 upregulated dendritic complexity while SOLOIST/MRTFB i4 downregulated complexity in cortical neurons.⁸⁾ Therefore, we investigated whether SOLOIST/MRTFB i4 overexpression and knockdown influenced cell morphology of Neuro-2a cells. We found that neither condition was associated with obvious morphological alterations (Supplementary Fig. S2). Knockdown of SOLOIST/MRTFB i4 should be performed in cortical neurons in a future study to confirm whether the same phenomenon is observed. Here, we found that SOLOIST/MRTFB i4 knockdown impacted IEG expression and MKL2/MRTFB isoform 1 expression in Neuro-2a cells, without affecting srf or cytoskeletal genes. Furthermore, the various MKL2/MRTFB isoforms may interact with each other, perhaps to fine-tune target gene expression.

Acknowledgments

This study was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (KAKENHI Grant numbers: JP26460064, JP18K06625 to A.T., 20K15989 to D.I.) and the Smoking Research Foundation (A.T.). We thank Barry Patel, PhD, for editing a draft of this manuscript. Parts of the exon-intron illustration in Figs. 1A and B have been published previously in Ishibashi et al. J. Neurochem. 159(4):762–777 (2021).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Olson EN, Nordheim A. Linking actin dynamics and gene transcription to drive cellular motile functions. Nat. Rev. Mol. Cell Biol., 11, 353–365 (2010).
• 2) Tabuchi A, Ihara D. SRF in neurochemistry: overview of recent advances in research on the nervous system. Neurochem. Res., 47, 2545–2557 (2022).
• 3) Pipes GC, Creemers EE, Olson EN. The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration and myogenesis. Genes Dev., 20, 1545–1556 (2006).
• 4) Shiota J, Ishikawa M, Sakagami H, Tsuda M, Baraban JM, Tabuchi A. Developmental expression of the SRF co-activator MAL in brain: role in regulating dendritic morphology. J. Neurochem., 98, 1778–1788 (2006).
• 5) Ishikawa M, Nishijima N, Shiota J, Sakagami H, Tsuchida K, Mizukoshi M, Fukuchi M, Tsuda M, Tabuchi A. Involvement of the serum response factor coactivator megakaryoblastic leukemia (MKL) in the activin-regulated dendritic complexity of rat cortical neurons. J. Biol. Chem., 285, 32734–32743 (2010).
• 6) Tabuchi A, Ihara D. Regulation of dendritic synaptic morphology and transcription by the SRF Cofactor MKL/MRTF. Front. Mol. Neurosci., 14, 767842 (2021).
• 7) Mokalled MH, Johnson A, Kim Y, Oh J, Olson EN. Myocardin-related transcription factors regulate the Cdk5/Pctaire1 kinase cascade to control neurite outgrowth, neuronal migration and brain development. Development, 137, 2365–2374 (2010).
• 8) Ishibashi Y, Shoji S, Ihara D, Kubo Y, Tanaka T, Tanabe H, Hakamata T, Miyata T, Satou N, Sakagami H, Mizuguchi M, Kikuchi K, Fukuchi M, Tsuda M, Takasaki I, Tabuchi A. Expression of SOLOIST/MRTFB i4, a novel neuronal isoform of the mouse serum response factor coactivator myocardin-related transcription factor-B, negatively regulates dendritic complexity in cortical neurons. J. Neurochem., 159, 762–777 (2021).
• 9) Kikuchi K, Ihara D, Fukuchi M, Tanabe H, Ishibashi Y, Tsujii J, Tsuda M, Kaneda M, Sakagami H, Okuno H, Bito H, Yamazaki Y, Ishikawa M, Tabuchi A. Involvement of SRF coactivator MKL2 in BDNF-mediated activation of the synaptic activity-responsive element in the Arc gene. J. Neurochem., 148, 204–218 (2019).
• 10) Ramanan N, Shen Y, Sarsfield S, Lemberger T, Schütz G, Linden DJ, Ginty DD. SRF mediates activity-induced gene expression and synaptic plasticity but not neuronal viability. Nat. Neurosci., 8, 759–767 (2005).
• 11) Weinl C, Castaneda Vega S, Riehle H, Stritt C, Calaminus C, Wolburg H, Mauel S, Breithaupt A, Gruber AD, Wasylyk B, Olson EN, Adams RH, Pichler BJ, Nordheim A. Endothelial depletion of murine SRF/MRTF provokes intracerebral hemorrhagic stroke. Proc. Natl. Acad. Sci. U.S.A., 112, 9914–9919 (2015).
• 12) Alberti S, Krause SM, Kretz O, Philippar U, Lemberger T, Casanova E, Wiebel FF, Schwarz H, Frotscher M, Schütz G, Nordheim A. Neuronal migration in the murine rostral migratory stream requires serum response factor. Proc. Natl. Acad. Sci. U.S.A., 102, 6148–6153 (2005).
• 13) Sotiropoulos A, Gineitis D, Copeland J, Treisman R. Signal-regulated activation of serum response factor is mediated by changes in actin dynamics. Cell, 98, 159–169 (1999).