Differential localization and roles of splice variants of rat suppressor of cancer cell invasion (SCAI) in neuronal cells

https://doi.org/10.1016/j.bbrc.2020.06.064Get rights and content

Highlights

  • Expression of Scai mRNA decreases during brain development.

  • Expression of Scai mRNA is higher in neurons than in astrocytes.

  • Newly identified SCAI splice variants are shorter than full-length SCAI.

  • Localization of short SCAI splice variants is more cytoplasmic than full-length SCAI.

  • Short SCAI variants inhibit mDia-induced neurite extension less than full-length SCAI.

Abstract

Suppressor of cancer cell invasion (SCAI) is a suppressor of myocardin-related transcription factor (MRTF)-mediated transcription and cancer cell invasion. However, roles of SCAI in the brain and neuronal cells are not fully resolved. In this study, we initially investigated the distribution of Scai mRNA in the developing rat brain and in neurons. We found that, although Scai mRNA levels decreased during brain development, it was highly expressed in several brain regions and in neurons but not astrocytes. Subsequently, in addition to Scai variant 1, we identified novel rat Scai variants 2 and 3 and characterized their functions in Neuro-2a cells. The novel Scai variants 2 and 3 contain unique exons that possess stop codons and therefore encode shorter proteins compared with the full-length Scai variant 1. SCAI variants 2 and 3 possess a nuclear localization signal, but do not have an MRTF-binding site. Immunostaining of green fluorescent protein (GFP)-tagged SCAI variants revealed a nuclear localization of variant 1, whereas localization of variants 2 and 3 was throughout the cytoplasm and nucleus, suggesting that other nuclear localization signals, which act in Neuro-2a cells, exist in SCAI. All three SCAI variants suppressed the neuron-like morphological change of Neuro-2a cells induced by a Rho effector, constitutively active mDia; however, the suppressive effects of variants 2 and 3 were weaker than that of full-length SCAI variant 1, indicating that the SCAI-mediated change toward a neuronal morphology appeared to be consistent with their nuclear localization. These findings indicate that generation of multiple SCAI splice variants fines-tune neuronal morphology.

Introduction

The Rho signaling pathway is involved in cell migration, including cancer cell invasion and controlling neuronal cell shape [1,2]. Rho propagates signals via its downstream effectors, such as Rho-kinase (ROCK) and mammalian Diaphanous (mDia) and eventually regulates actin rearrangement [3,4]. Activation of Rho-mDia signaling not only causes actin-based morphological alteration but also regulates serum response factor (SRF)-mediated gene expression through nuclear translocation of myocardin-related transcription factor-A (MRTFA), a coactivator for serum response factor (SRF) [5,6]. mDia and MRTF regulate neuronal morphology [[7], [8], [9], [10]] as well as cancer metastasis [11]. Collectively, accumulating evidence suggest that the Rho-mDia-SRF-MRTF axis may play an important role in neuronal morphology.

Suppressor of cancer cell invasion (SCAI) was firstly identified as an interacting partner of Dia1 and MRTFA [12]. SCAI possesses a nuclear localization signal (NLS) in the N-terminus and a putative MRTF-binding site and is therefore thought to suppress cancer cell invasion by inhibiting MRTFA’s function in the nucleus and downregulating β1-integrin gene expression [12]. SCAI also inhibits other MRTF-mediated transcriptional activation [13]. In addition to β1-integrin, SCAI is a repressor of SM22, telokin, α-smooth muscle actin (a-SMA) and calponin expression [13,14]. These studies of SCAI function were performed in non-neuronal cells. Although Brandt et al. showed that SCAI is expressed in the brain, little is known about the expression and function of SCAI in neuronal cells. We previously found that overexpression of SCAI reduced the dendritic complexity of cortical neurons induced by activin [10]. This finding promoted us to determine the detailed expression pattern of SCAI in different cell types during brain development. We also isolated rat Scai splice variants and found them to be differentially localized and to have neuritic roles in Neuro-2a cells.

Section snippets

Animals

Male and pregnant female Sprague-Dawley (SD) rats were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan). All experiments were carried out in accordance with ARRIVE guidelines and the guidelines of the Animal Care and Experimentation Committee of University of Toyama, Sugitani Campus. The protocols were approved with permit numbers: S-2008 PHA-3, S2009 PHA-23, S2010 PHA-1, A2011 PHA-5, A2012 PHA-1, A2013PHA-4, A2016PHA-8, and A2019PHA-7. Every effort was made to minimize animal suffering.

Cell culture

Expression pattern of rat Scai mRNA

We initially examined the tissue-distribution of rat Scai mRNA by qPCR. It was highly expressed in the testis and total brain. Higher levels were seen in several brain regions, including the olfactory bulb, cerebellum, hippocampus, and cerebral cortex compared with other tissues (Fig. 1A). In the developing rat brain, Scai expression was down-regulated (Fig. 1B). Neuronal expression of Scai was higher than that in astrocytes (Fig. 1C).

Identification of rat Scai splice variants

To characterize the function of rat SCAI, we cloned it using

Discussion

In this study, we investigated the expression levels of Scai in a variety of tissues, during brain development, and in neurons and astrocytes. The highest level of expression was observed in total brain and testis (Fig. 1A). During brain development, Scai expression was down-regulated (Fig. 1B). Furthermore, we found that Scai was highly expressed in neurons rather than in astrocytes (Fig. 1C). Decreased expression of Scai is consistent with increased invasive cell migration [12]. In the

Declaration of competing interest

The authors have no conflicts of interest to declare.

Acknowledgements

We thank Dr. S. Narumiya (Kyoto University, Japan) for providing us with the constitutively active and constitutively inactive mDia expression vector. We thank Dr. D. T. Brandt (Institute of Pharmacology, Philipps-University, Germany) for his constructive suggestions. This study was funded in part by Presidential Discretionary Funds, University of Toyama 2015 (A.T.). We thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

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