Ezrin Mediates Neuritogenesis via Down-Regulation of RhoA Activity in Cultured Cortical Neurons

<div><p>Neuronal morphogenesis is implicated in neuronal function and development with rearrangement of cytoskeletal organization. Ezrin, a member of Ezrin/Radixin/Moesin (ERM) proteins links between membrane proteins and actin cytoskeleton, and contributes to maintenance of cellular function and morphology. In cultured hippocampal neurons, suppression of both radixin and moesin showed deficits in growth cone morphology and neurite extensions. Down-regulation of ezrin using siRNA caused impairment of netrin-1-induced axon outgrowth in cultured cortical neurons. However, roles of ezrin in the neuronal morphogenesis of the cultured neurons have been poorly understood. In this report, we performed detailed studies on the roles of ezrin in the cultured cortical neurons prepared from the ezrin knockdown (<i>Vil2^kd/kd</i>) mice embryo that showed a very small amount of ezrin expression compared with the wild-type (<i>Vil2^+/+</i>) neurons. Ezrin was mainly expressed in cell body in the cultured cortical neurons. We demonstrated that the cultured cortical neurons prepared from the <i>Vil2^kd/kd</i> mice embryo exhibited impairment of neuritogenesis. Moreover, we observed increased RhoA activity and phosphorylation of myosin light chain 2 (MLC2), as a downstream effector of RhoA in the <i>Vil2^kd/kd</i> neurons. In addition, inhibition of Rho kinase and myosin II rescued the impairment of neuritogenesis in the <i>Vil2^kd/kd</i> neurons. These data altogether suggest a novel role of ezrin in the neuritogenesis of the cultured cortical neurons through down-regulation of RhoA activity.</p></div

Y-27632 rescues neuritogenesis.

<p><i>A-D</i>, The <i>Vil2^+/+</i> and <i>Vil2^kd/kd</i> neurons treated with DMSO (<i>A</i>,<i>C</i>) or 40 µM Y-27632 (24 h, <i>B</i>,<i>D</i>) were fixed at 2 DIV and stained with an anti-neuronal class III β-tubulin antibody (green) and rhodamine phalloidin (red). Scale bars, 50 µm. <i>E-G</i>, The number (<i>E</i>) and length (<i>F</i>) of neurites, and length of axon (<i>G</i>) were quantified in the <i>Vil2^+/+</i> and <i>Vil2^kd/kd</i> neurons treated with DMSO (white columns, n = 30) or 40 µM Y-27632 (black columns, n = 30). Three independent experiments were performed. <i>*p</i><0.05, <i>**p</i><0.01, <i>***p</i><0.001 (DMSO-treated vs. Y-27632-treated), <i>###p</i><0.001 (DMSO-treated <i>Vil2^+/+</i> vs. DMSO-treated <i>Vil2^kd/kd</i>), Student's t test. Data represent mean ± SE.</p

Distribution of ezrin in wild-type cultured cortical neurons at the stages 1, 2 and 3 was observed by immunofluorescence.

<p>Neurons at the stages 1, 2 and 3 were stained with an anti-ezrin antibody, rhodamine phalloidin, and an anti-α-tubulin antibody, respectively. In the bottom lane, neurons were triple stained with an anti-ezrin antibody (green), rhodamine phalloidin (red) and an anti-α-tubulin antibody (blue). Scale bars, 50 µm.</p

Neuritogenesis is impaired by ezrin knockdown.

<p><i>A</i>, <i>B</i>, The <i>Vil2^+/+</i> (<i>A</i>) and <i>Vil2^kd/kd</i> (<i>B</i>) neurons were fixed at 2 DIV and stained with an anti-neuronal class III β-tubulin antibody (green) and rhodamine phalloidin (red). Scale bars, 50 µm. <i>C</i>, Stacked bar graph showing stage progression in the <i>Vil2^+/+</i> (n = 153) and <i>Vil2^kd/kd</i> (n = 162) neurons. Stage of cells were defined by the length of the longest neurite as reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105435#pone.0105435-Hirai1" target="_blank">[19]</a>. <i>D-F</i>, Quantitation of number (<i>D</i>) and length (<i>E</i>) of neurites, and length of axon (<i>F</i>) in the <i>Vil2^+/+</i> (gray columns, n = 50) and <i>Vil2^kd/kd</i> (green columns, n = 50) neurons. Three independent experiments were performed. <i>*p</i><0.05, <i>**p</i><0.01, <i>***p</i><0.001, Student's t test. Data represent mean ± SE.</p

Increased RhoA activity in the Vil2^kd/kd neurons.

<p><i>A-C</i>, The amounts of active and total RhoA (<i>A</i>), Rac1 (<i>B</i>) and Cdc42 (<i>C</i>) from cell lysates of the <i>Vil2^+/+</i> and <i>Vil2^kd/kd</i> neurons (2 DIV). Representative patterns were presented. <i>D-F</i>, The ratios of active RhoA (<i>D</i>), Rac1 (<i>E</i>) and Cdc42 (<i>F</i>) to total amount of proteins were compared between the <i>Vil2^+/+</i> (white columns) and <i>Vil2^kd/kd</i> (black columns) neurons. Each experiment was performed in triplicate. <i>*p</i><0.05 , Student's t test. Data represent mean ± SE.</p

CD9 Negatively Regulates CD26 Expression and Inhibits CD26-Mediated Enhancement of Invasive Potential of Malignant Mesothelioma Cells

<div><p>CD26/dipeptidyl peptidase IV is a cell surface glycoprotein which consists of multiple functional domains beside its ectopeptidase site. A growing body of evidence indicates that elevated expression of CD26 correlates with disease aggressiveness and invasive potential of selected malignancies. To further explore the molecular mechanisms involved in this clinical behavior, our current work focused on the interaction between CD26 and CD9, which were recently identified as novel markers for cancer stem cells in malignant mesothelioma. We found that CD26 and CD9 co-modulated and co-precipitated with each other in the malignant mesothelioma cell lines ACC-MESO1 and MSTO-211H. SiRNA study revealed that depletion of CD26 led to increased CD9 expression, while depletion of CD9 resulted in increased CD26 expression. Consistent with these findings was the fact that gene transfer of CD26 into CD26-negative MSTO-211H cells reduced CD9 expression. Cell invasion assay showed that overexpression of CD26 or gene depletion of CD9 led to enhanced invasiveness, while CD26 gene depletion resulted in reduced invasive potential. Furthermore, our work suggested that this enhanced invasiveness may be partly mediated by α5β1 integrin, since co-precipitation studies demonstrated an association between CD26 and α5β1 integrin. Finally, gene depletion of CD9 resulted in elevated protein levels and tyrosine phosphorylation of FAK and Cas-L, which are downstream of β1 integrin, while depletion of CD26 led to a reduction in the levels of these molecules. Collectively, our findings suggest that CD26 potentiates tumor cell invasion through its interaction with α5β1 integrin, and CD9 negatively regulates tumor cell invasion by reducing the level of CD26-α5β1 integrin complex through an inverse correlation between CD9 and CD26 expression. Our results also suggest that CD26 and CD9 serve as potential biomarkers as well as promising molecular targets for novel therapeutic approaches in malignant mesothelioma and other malignancies.</p></div

CD26 associates with CD9 in an inverse manner.

<p>(A). Heat map representing color-coded expression levels of differentially expressed genes. CD26/Depletion: control siRNA- and CD26 siRNA-transfectedMESO1. CD26/Over expression: MSTO-Wild and MSTO-CD26 (+) cells. Upregulated (red) or downregulated (green). (B and C). MESO1 transfectants of control siRNA, CD26 siRNA, and CD9 siRNA, or MSTO-Wild and MSTO-CD26 (+) cells were stained with anti-CD26-FITC or anti-CD9-FITC and subjected to flow cytometry. (D). MESO1 transfectants with control siRNA, CD26 siRNA, and CD9 siRNA, or MSTO-Wild and MSTO-CD26 (+) cells were lysed and probed with anti-CD26 polyclonal antibody, anti-CD9 mAb (5H9) and anti-β-actin polyclonal antibody. (E). RT-PCR was carried out for analysis of CD26 and CD9 gene expressions on MESO1 transfectants with controlsiRNA, CD26siRNA, and CD9siRNA, or on MSTO-Wild and MSTO-CD26 (+) cells. GAPDH amplification was used as internal control. These results were also confirmed by 5 separate experiments.</p

CD26 associates with CD9.

<p>(A). Flow cytometric analysis of CD26 and CD9 expression on MESO1, MSTO-Wild or MSTO-CD26 (+) cells. (B). MESO1 or MSTO-CD26 (+) cells were incubated up to 72 h at 37 °C with either control IgG (10 µg/ml) or humanized anti-CD26 mAb (10 µg/ml). These cells were stained with anti-CD26-FITC (5K76) or with anti-CD9-FITC, and subjected to flow cytometry. Intensity of modulation was indicated by mean fluorescence intensity (MFI). (C). MESO1 or MSTO-CD26 (+) cells were subjected to immunoprecipitation with control IgG, humanized anti-CD26 mAb, and anti-CD9 mAb (5H9). Immunoblot was conducted with anti-CD26 polyclonal antibody, and anti-CD9 mAb (5H9). These results were also confirmed by 5 separate experiments.</p

CD26 potentiates invasiveness through α5β1 integrin.

<p>(A).MSTO-Wild, MSTO-CD26 (+) cells were subjected to flow cytometry for CD26, α5, and β1. (B). MESO1 and MSTO-CD26 (+) cells were subjected to immunoprecipitation with control IgG, humanized anti-CD26 mAb, anti-CD9 mAb (5H9), anti-α5 mAb (2H6), or anti-β1 mAb (4B4). The immunoblot was probed with anti-CD26 polyclonal antibody, anti-CD9 mAb (5H9), anti-α5 mAb (2H6), or anti-β1 mAb (4B4). (C). Boyden chamber-based cell invasion assay of MESO1 and MSTO-CD26 (+) cells treated with anti-α5, and β1 antibodies for 24 h. (n = 5). *p<0.01, **p<0.005. (D).The cell migration assay of MESO1 and MSTO-CD26 (+) cells treated with anti-α5, and β1 antibodies.(n = 5). *p<0.05, **p<0.01.</p

CD26 potentiates tumor cell invasion.

<p>(A). Flow cytometric analysis of CD26 and CD9 expressions in MESO1. (B). Sorting of CD26⁻CD9⁺ cells and CD26⁺CD9⁺ cells, and immunoprecipitation was performed with humanized anti-CD26 mAb and anti-CD9 mAb (5H9), then probed with anti-CD26 polyclonal antibody and anti-CD9 mAb (5H9). (C and E).Tumor cell invasion was measured with the Boyden chamber-based cell invasion assay for 24 h. Number of invaded cells was represented as means ± SE (n = 5).*p<0.01, **p<0.001. (D). Flow cytometric analysis of CD26 and CD9 expressions in MESO1 and sorting of CD26⁺CD9⁺ cells and CD26⁺CD9⁻ cells. These results were also confirmed by 3 separate experiments.</p