XLMR protein related to neurite extension (Xpn/KIAA2022) regulates cell–cell and cell–matrix adhesion and migration
Introduction
Several genes have been identified as members of the X-linked mental retardation (XLMR) family (Ropers, 2006). A previous study reported an inactivating disruption of the X-linked KIAA2022 gene by a chromosomal rearrangement in two male patients with severe mental retardation (Cantagrel et al., 2004). Furthermore, a recent study reported that patients with KIAA2022 mutations had intellectual disability with autistic features and strabismus (Van Maldergem et al., 2013). Thus, it was suggested that the expression level of KIAA2022 was related to the pathogenesis of XLMR. KIAA2022 encodes a large protein of 1516 amino acids. Previous reports have shown that KIAA2022 has no functional motifs or significant homology with other known proteins (Cantagrel et al., 2004, Cantagrel et al., 2009). KIAA2022 mRNA is highly expressed in the brain, particularly during the late embryonic and perinatal stages of development. Recent studies show that KIAA2022 participates in neurite outgrowth in PC12 cells and neurons, suggesting a role for Xpn in brain development. Therefore, we termed KIAA2022 as Xpn (XLMR protein related to neurite extension) (Ishikawa et al., 2012, Van Maldergem et al., 2013). However, few reports are available on the functional role of Xpn in neuronal development.
The cadherins are a superfamily of proteins that regulate many cellular functions, including cell adhesion and motility (Takeichi, 1991, Tepass et al., 2000). Cadherins are important for neuronal development, with many members of this family being expressed in the brain (Tepass et al., 2000). N-cadherin is involved in the formation of both adherens and synaptic junctions in the nervous system. Moreover, recent reports have shown that members of the cadherin family are associated with neuropsychiatric disorders, including mental retardation (Pedrosa et al., 2008, Redies et al., 2012, Wang et al., 2009). Integrins are transmembrane receptors that function in the recognition of extracellular matrix and cell-surface proteins (Hynes, 2002). Integrins have been shown to be important in cell migration, axonal guidance and proper synapse formation during the development of the central and peripheral nervous systems (Reichardt and Tomaselli, 1991, Schmid and Anton, 2003).
In the present study, to investigate the functional role of Xpn, we studied the effect of Xpn knockdown on cell–cell and cell–matrix adhesion and migration. We report that Xpn regulates cellular adhesion and migration of PC12 cells by modulating N-cadherin and β1-integrin expression.
Section snippets
Cell culture
PC12 and 293T cells were maintained in tissue culture dishes (Thermo Fisher Scientific, Rochester, NY, USA). The PC12 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies Inc., Grand Island, NY, USA) containing 5% heat inactivated fetal bovine serum and 10% horse serum. The 293T cells were cultured in DMEM containing 10% heat inactivated fetal bovine serum. The cultures were maintained at 37 °C in an atmosphere of 95% air and 5% CO2. The cells were observed under a
Xpn knockdown promotes cellular adhesion
To investigate the physiological functions of Xpn, RNA interference analysis was used to knock down endogenous Xpn expression in PC12 cells. The siRNA sequences targeting the rat Xpn gene have already been shown to effectively suppress rat Xpn mRNA expression in PC12 cells (Ishikawa et al., 2012). Using qRT-PCR analysis, we confirmed that the siRNA targeting Xpn suppressed the expression of Xpn mRNA. The expression level of Xpn mRNA in the siRNA transfected cells was reduced to 50% compared
Discussion
In this study, we demonstrated that knockdown of Xpn expression enhanced cell–cell and cell–matrix adhesion, which are mediated by N-cadherin and β1-integrin, respectively, in PC12 cells (Fig. 1). Knockdown of Xpn increased N-cadherin and β1-integrin expression at both the protein and mRNA levels (Fig. 2). Overexpressed Xpn protein was strongly expressed in the nuclei of PC12 and 293T cells (Fig. 3). Knockdown of Xpn impaired the migration of PC12 cells in a wound healing assay (Fig. 4).
Acknowledgements
The authors declare no conflict of interest. This work was supported in part by the MEXT Strategic Research Program for Brain Sciences, a Grant-in-Aid for Scientific Research on Innovative Areas “Neural Diversity and Neocortical Organization” from MEXT and by a grant from Dainippon Sumitomo Pharma Co., Ltd. We thank Mrs. Moriya and Mrs. Ohashi for preparing this manuscript.
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These authors contributed equally to this work.