4.1 TRIP6 is expressed by NSCs in the embryonic and adult mouse brain
To study the role of TRIP6 in the nervous system, we asked whether it was present in the brains of embryonic and adult mice. For this, we first examined the specificity of the TRIP6 antibody, which has been reported to be able to detect endogenous TRIP6 by immunobloting (Xu et al., 2004) and immunostaining (Takizawa et al., 2006). In 3T3 cells, this antibody detected endogenous TRIP6 at focal adhesions in the scrambled shRNA-expressing cell, but not in the cell expressing shTRIP6 (Fig. 1A).
Our TRIP6 antibody could not detect TRIP6 signal in
shTRIP6-transfected 3T3 cells (Fig. 1B). These results confirm the specificity of our TRIP6 antibody.
From the whole brain extract, we found that TRIP6 mRNA was expressed in embryonic day 15.5–16.5 (E15.5–16.5) mouse brains, but the level was low or undetectable in adult ones (Fig. 1C). Consistent with the mRNA result, TRIP6 protein was only detected in embryonic, but not in adult brains (Fig. 1D). These results implicate that the role of TRIP6 in the nervous system may be mainly in neural development. After the nervous system is developed, the expression of TRIP6 is down-regulated.
Since we found that TRIP6 was abundant in the embryonic brain (Fig.
1C, D) and TRIP6 has been reported to be enriched in NSCs (Ramalho-Santos et al., 2002), we carried out immunofluorescence of TRIP6 with E16.5 forebrain sections to investigate whether embryonic NSCs express TRIP6. We found that there was a ventricle-to-mantle gradient of TRIP6
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expression in the dorsal and ventral forebrain, where the primordium of cerebral cortex and striatum reside, respectively (Fig. 2A). Since
embryonic NSCs are located at the VZ, we double labeled forebrain sections with antibodies against TRIP6 and the neural stem cell marker SOX2. We found that most of the TRIP6-expressing cells were also SOX2-positive in the dorsal and ventral VZ (Fig. 2B, C). The percentage of TRIP6-positive cells that also express SOX2 was 90.2 ± 2.7% (Fig.
2E). During the embryonic stage, NSCs are actively dividing. To examine whether TRIP6-positive cells are proliferative, E16.5 forebrain sections were double-labeled with antibodies against TRIP6 and the cell cycle marker Ki67 (Fig. 2D). We found that 34.1 ± 2.1% of TRIP6-positive cells were labeled with Ki67 (Fig. 2E). These results suggest that TRIP6 is mainly expressed by NSCs in the embryonic forebrain.
The central nervous system is mostly developed during the embryonic stage, but neurogenesis persists in the SVZ-olfactory bulb pathway and the DG of adult brains. Since we found that embryonic NSCs expressed TRIP6, it is possible that TRIP6 may also be expressed by postnatal NSCs. Although we did not find TRIP6 expression in the adult brain by Western blot analysis, its mRNA was detected in some of the analyzed adult brains (Fig. 1C, D). Due to relatively fewer NSCs present in the adult nervous system, it is difficult to detect TRIP6 signal from whole brain extracts. Therefore, immunofluorescence can provide a better resolution of TRIP6-expressing cells in the adult brain. Indeed, we found that TRIP6 was expressed in the SVZ (Fig. 3A, C–H), but not in the SGZ (Fig. 3B). Consistent with the finding from embryonic brains, most
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TRIP6-expressing cells in the adult SVZ were also SOX2-positive (62.7 ± 1.6%; Fig. 3C, D, D’, I). However, only 8.0 ± 0.3% of TRIP6-positive cells were labeled with Ki67 (Fig. 3E, F, I). Since adult NSCs are mostly quiescent, it is reasonable that there are fewer TRIP6 and Ki67 double-positive cells in the adult SVZ. It is also proposed that adult NSCs are ependymal cells lining the lateral ventricle (Spassky et al., 2005). We also double labeled adult SVZ sections with antibodies against TRIP6 and the ependymal cell marker S100β, and found that 44.4 ± 1.5% of TRIP6-expressing cells were also S100β-positive (Fig. 3G, H, I). These results suggest that TRIP6 is mainly expressed by adult NSCs in the SVZ.
To examine whether the progeny of adult NSCs also expresses TRIP6, we double-labeled adult SVZ sections with antibodies against TRIP6 and the neuroblast marker, DCX, the neuronal maker, microtubule-associated protein 2 (MAP2), or the astrocyte marker, GFAP. We found that only 6.7 ± 1.8% of TRIP6-expressing cells were DCX-positive (Fig. 4A).
However, MAP2-positive neurons do not express TRIP6 (Fig. 4B).
Interestingly, TRIP6 was not expressed by GFAP-positive astrocytes in the striatum and the corpus callosum (Fig. 4C and data not shown). To further examine whether TRIP6 is expressed by microglia, we double-labeled SVZ sections with antibodies against TRIP6 and the microglia marker, ionized calcium-binding adapter molecule 1 (Iba1, Fig. 4D). We found that TRIP6 was not expressed by Iba1-positive microglia, either.
Taken together, TRIP6 is mainly expressed by NSCs, ependymal cells, and some of the neuroblasts in the adult SVZ, but not in more
differentiated cells including neurons, astrocytes, and microglia. These
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results implicate that TRIP6 may regulate NSC properties.
4.2 TRIP6 maintains self-renewal and proliferation of NSCs Postnatal NSCs from the SVZ can be cultured in vitro in suspension condition supplied with mitogenic growth factors to form primary
neurospheres (1’ NS). 1’ NS can be dissociated and cultured in the same condition to form secondary NS (2’ NS). After growth factor removal, NS can differentiate into neurons, astrocytes and oligodendrocytes.
Therefore, the rate of 2’ NS formation and neural differentiation are indices of self-renewal and multipotency of NSCs, respectively. To investigate whether TRIP6 is required for the self-renewal ability of NSCs, 1’ NS derived from postnatal day 7 (P7) SVZ were dissociated, transfected with scrambled shRNA (Ctrl) or shTRIP6 together with GFP plasmids, and cultured to form 2’ NS for 5 days. Scrambled control transfected cells formed 2’ NS (Fig. 5A). However, shTRIP6 ones did not (Ctrl: 55 ± 3.6 spheres/106 cells, TRIP6: 0.33 ± 0.3 spheres/106 cells, p<0.01; Fig. 5B, C). To further confirm this result, we also performed gain-of-function experiment by overexpression of TRIP6. 1’ NS were dissociated, transfected with control (Ctrl) or TRIP6 together with GFP vectors, and cultured to form 2’ NS. Transfected cells formed 2’ NS in both the control and TRIP6 groups (Fig. 5D, E). The number of 2’ NS was not increased by TRIP6 overexpression (TRIP6: 41 ± 4.93
spheres/106 cells). This could be due to the abundance of endogenous TRIP6 in neural stem cells. Therefore, we measured the sphere diameter as another index of self-renewal. For the control of the knockdown
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experiment, the size of the 2’ NS tended to be slightly larger than that in the control of the overexpression experiment, which could be due to different vectors transfected. Still, TRIP6 shifted the distribution toward larger spheres where TRIP6 significantly decreased the number of small-sized spheres (100–150 mm; p<0.05) and increased the number of large-sized ones (250–300 mm; p<0.05; Fig. 5F). Together, these results suggest that TRIP6 is necessary and sufficient for self-renewal of NSCs.
Since TRIP6 increases the neurosphere size, it is possible that it
positively regulates cell proliferation of NSCs. To test this hypothesis, 1’
NS were dissociated, transfected with scrambled shRNA (Ctrl) or shTRIP6, and cultured in differentiation condition for 1 day when cells were still actively dividing. BrdU, the thymidine analog, was added 2 hr before fixation. We found that BrdU-positive cells more significantly decreased in the shTRIP6 group than those in the control group (Ctrl: 100
± 0%, shTRIP6: 77.3 ± 5.8%, p<0.05; Fig. 6A–C). We also overexpressed TRIP6 in dissociated 1’ NS and cultured them in differentiation condition for 2 days when cells started to exit cell cycle and underwent
differentiation. BrdU was again added 2 hr before fixation. We found that BrdU-positive cells significantly increased in the TRIP6 group (Ctrl: 100
± 0%, TRIP6: 132.7 ± 5.4%, p<0.05; Fig. 6D–F). These results together suggest that TRIP6 positively regulates NSC/neural progenitor cell proliferation.
4.3 TRIP6 inhibits differentiation of postnatal NSCs
Since TRIP6 positively regulates self-renewal of postnatal NSCs (Fig. 5),
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TRIP6 may also be involved in NSC differentiation. To test whether TRIP6 modulates NSC differentiation, we started with the loss-of-function approach by knockdown of TRIP6. In our culture condition, NSCs mostly differentiate into astrocytes and neurons labeled by GFAP and class III b-tubulin (Tuj1) antibodies, respectively (Fig. 7), and a few differentiate into oligodendrocytes (data not shown). Therefore, we examined neuronal and astroglial differentiation after manipulation of TRIP6 expression. 1’ NS derived from P7 SVZ were dissociated, co-transfected with GFP and shTRIP6 constructs, and cultured in
differentiation condition for 3 days. We found that knockdown of TRIP6 significantly enhanced neuronal differentiation of NSCs (Ctrl: 100 ± 0%, shTRIP6: 208 ± 20.8%, p<0.05; Fig. 7A, B, and E) at the expense of glial differentiation (Ctrl: 100 ± 0%, shTRIP6: 66.5 ± 3.3%, p<0.01; Fig. 7C, D, and E), suggesting that TRIP6 negatively regulates neuronal
differentiation. To further confirm the role of TRIP6 in NSC differentiation, we transfected 1’ NS with control (Ctrl) or TRIP6 constructs and cultured them in differentiation condition for 3 days.
Overexpression of TRIP6 decreased neuronal differentiation (Ctrl: 100 ± 0%, TRIP6: 50.8 ± 3.7%; p<0.01; Fig. 7F, G, and J) and reduced glial differentiation (Ctrl: 100 ± 0%, TRIP6: 71.2 ± 6.9%, p<0.05; Fig. 7H–J), suggesting that TRIP6 inhibits differentiation. Taken together, our results demonstrate that TRIP6 mainly maintains NSC identity and negatively regulates NSC differentiation.
4.4 TRIP6 activates the Notch signaling pathway
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Notch, Shh, and Wnt pathways have been reported to be involved in NSC maintenance (Ming and Song, 2011). Since we found that TRIP6 is
necessary and sufficient to regulate self-renewal and negatively regulates differentiation of postnatal NSCs (Fig. 5, 7), it is plausible that these signaling pathways mediate the function of TRIP6. To examine these possibilities, we tested whether TRIP6 regulates Notch, Shh, or Wnt activity by Luciferase reporter assays in P19 mouse embryonic carcinoma cell line. P19 cells are pluripotent cells, which can be induced to
differentiate into neurons by retinoic acid (RA) treatment or expression of neural basic helix-loop-helix (bHLH) transcription factors (Farah et al., 2000; Jones-Villeneuve et al., 1982; Jones-Villeneuve et al., 1983). First, we tested whether TRIP6 interacted with the Notch pathway. Upon ligand binding, Notch intracellular domain (NICD) is cleaved from the cell membrane and translocated into the nucleus to increase the transcription of genes containing CBF binding sites (Hsieh et al., 1996). We
transfected P19 cells with Luciferase reporter constructs containing wild-type (WT) or mutant (MT) CBF binding sites and found that TRIP6
significantly promoted the Luciferase activity with WT CBF binding sites up to 2-fold (Ctrl: 100 ± 0%, TRIP6: 192.5 ± 26.2%, p<0.05; Fig. 8A).
However, TRIP6 did not promote Luciferase activities with Gli or TCF binding sites, which are consensus binding sites for Shh and Wnt
pathways, respectively (data not shown). To rule out the possibility that this is a P19 cell-specific effect, we repeated the same experiment with the SH-SY5Y neuroblastoma cell line, which can be induced to
differentiate into neurons with RA (Biedler et al., 1978). Consistent with
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the result from P19 cells, TRIP6 also significantly increased Notch activity in SH-SY5Y cells (Ctrl: 100 ± 0%, TRIP6: 260.4 ± 32.6%, P<0.05; Fig. 8B). Taken together, our results suggest that TRIP6 may activate the Notch pathway to maintain NSC identity.
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5. Discussion
As a focal adhesion molecule, studies of TRIP6 have been focused on its role in regulating cell migration, including cancer metastasis and pathogen invasiveness in the body. Its functions in progression and survival of cancer cells have also been reported (Lai et al., 2010; Lin et al., 2013). Since a microarray analysis of different lineages of stem cells reveals that TRIP6 may serve as a stemness gene (Ramalho-Santos et al., 2002), here we investigate its role in NSCs. We found that TRIP6 was expressed by NSCs in the embryonic and adult forebrain. It was required for their self-renewal and proliferation and inhibited differentiation. In addition, TRIP6 activated the Notch signaling pathway.
Sox1, 2, and 3 belonging to SoxB1 transcription factors are expressed by NSCs (Wang et al., 2006; Zappone et al., 2000). SOXB1 transcription factors and the Notch pathway are well known for their roles in
maintaining NSCs in an undifferentiated state (Bylund et al., 2003; Ferri et al., 2004; Ross et al., 2003). Moreover, the Notch pathway has been shown to activate Sox2 expression and inhibit neural differentiation (Ehm et al., 2010). It has also been reported that the Notch signaling is
downregulated in RA-induced differentiation of glioblastoma stem cells, further suggesting that the Notch pathway is required to maintain stem cells in an undifferentiated state (Ying et al., 2011). Here, we found that TRIP6 was expressed by SOX2-positive NSCs in the embryonic and adult forebrain (Figs. 2, 3). TRIP6 maintained self-renewal ability and inhibited differentiation of postnatal NSCs (Figs. 5, 7). More importantly, TRIP6 increased the Notch activity (Fig. 8). In addition, the Notch signaling is
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reported to inhibit the neuronal fate while inducing astrocyte
differentiation in embryonic and postnatal neural stem cells (Grandbarbe et al., 2003; Tanigaki et al., 2001). Moreover, the Notch pathway has been shown to maintain adherens junctions of neural stem cells (Hatakeyama et al., 2014). Loss of adherens junctions promotes detachments of neural progenitor cells and induces precocious
neurogenesis (Hatakeyama et al., 2014; Rousso et al., 2012). Here, we found that knockdown of TRIP6 increased neuronal differentiation at the expense of glial differentiation (Fig. 7). This result further supports that TRIP6 may act upstream of the Notch-SOX2 signaling pathway to maintain NSC identity and regulate their differentiation.
During neurogenesis, adhesion status of NSCs regulates the balance of self-renewal and differentiation. NSCs form a self-supporting niche by adhering to the luminal surface of ventricle and neighboring progenitor cells (Meng and Takeichi, 2009; Zhang et al., 2010a). Loss of adhesion status correlates with differentiation, while secured adhesion promotes the self-renewal of NSCs (Malaguti et al., 2013; Rousso et al., 2012).
These reports mainly concentrate on adherens junctions and cadherin proteins. However, studies of focal adhesion molecules in neurogenesis are limited and most of them focus on cell migration. Interestingly, the dynamics of focal adhesions has been shown to correlate with differentiation of NSCs (Lyu et al., 2013) and deficiency of a focal adhesion molecule, vinculin, disrupts neural tube formation (Xu et al., 1998). Here, we provide evidence that TRIP6 is involved in
regulating of NSC self-renewal and differentiation. Taken together, this
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finding suggests that focal adhesion molecules also play a role in neurogenesis.
Previously, we found that TRIP6 regulates not only cell motility, but also cell proliferation and survival in cancer cells (Lai et al., 2005; Lai et al., 2010; Lin et al., 2013). It activates ERK, Akt, and NFkB pathways to promote cell motility, proliferation, and survival in ovarian cancer and glioblastoma cells (Lai et al., 2005; Lai et al., 2010; Lin et al., 2013).
During neurogenesis, ERK, Akt, and NF-kB signaling pathways are also important regulators for proliferation and survival of NSCs (Shioda et al., 2009; Widera et al., 2006; Zhou and Miller, 2006). Therefore, TRIP6 may also regulate properties of NSCs through these pathways.
In the adult SVZ, TRIP6 is also expressed by ependymal cells (Fig. 3).
Ependymal cells are one kind of glial cells that remain in the VZ during neural development (Spassky et al., 2005). Johansson et al. have reported that ependymal cells serve as NSCs that give rise to new neurons in the adult olfactory bulb (Johansson et al., 1999). Ependymal cells can also respond to spinal cord injury and generate migratory astrocytes to the injured sites (Johansson et al., 1999). Our results show that TRIP6 is expressed in these cells and SOX2-positive cells, suggesting that TRIP6 is majorly expressed in cells with stem cell properties and may maintain the stemness features of these cells.
Ependymoma is a glioma transformed from ependymal cells.
According to the microarray data from the Neale Multi-cancer data set, TRIP6 mRNA expression is also higher in ependymoma and anaplastic ependymoma. In addition, we have studied the role of TRIP6 in glioma
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tumorigenesis and found that TRIP6 is highly expressed in glioblastoma compared to control tissues (Lai et al., 2010). Moreover, TRIP6 enhances the anti-apoptotic ability of glioblastoma cells and promotes tumor
growth in vivo (Lai et al., 2010; Lin et al., 2013). Consistent with this, the expression level of TRIP6 is reversely correlated with the overall survival of patients; poor prognosis of patients usually has a higher level of TRIP6 (Lin et al., 2013). These studies suggest that TRIP6 may be a key factor in various gliomas. Since our results showed that mature astrocytes do not express TRIP6 (Fig. 4C) but NSCs do (Figs. 2, 3), it is possible that the ectopic expression of TRIP6 in differentiated glial cells may transform them into cancer cells and make them tumorigenic. Therefore, TRIP6 may not only serve as a biomarker, but also be a therapeutic target for malignant brain diseases.
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