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Netrin-1 Signaling Mediates NO-Induced Glial Precursor Migration and Accumulation Cell Research (2010) 20:238-241

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Netrin-1 Signaling Mediates NO-Induced Glial Precursor Migration and Accumulation Cell Research (2010) 20:238-241 npg Netrin-1 mediates NO-induced migration and accumulation Cell Research (2010) 20:238-241. 238 © 2010 IBCB, SIBS, CAS All rights reserved 1001-0602/10 $ 32.00 npg LETTER TO THE EDITOR www.nature.com/cr Netrin-1 signaling mediates NO-induced glial precursor migration and accumulation Cell Research (2010) 20:238-241. doi: 10.1038/cr.2010.7; published online 19 January 2010 − Dear Editor, accumulation. Interestingly, more NO2 was detected in the group comprising GPs treated with SNP than in the In the central nervous system, cell migration and ac- group with SNP in the absence of GPs (Figure 1Avi), in- cumulation occur widely to construct a complex neural dicating that additional endogenous NO was synthesized network during mammalian development [1]. Previous within the cells. studies reported that both the gaseous messenger mol- To test whether the NO-induced cell migration and ecule nitric oxide (NO) [2-4] and widely studied axon accumulation could be mediated by axon guidance/mi- guidance/migration cues such as Netrins [5, 6] regulated gration cues, we first analyzed the expression profile of the migratory behavior of cells in the nervous system. Netrins in the GPs before and after SNP treatment (Figure However, little is known about whether there is any 1Bi-v) because Netrins were reported to be expressed in crosstalk between NO and guidance/migration cues dur- glial lineage cells [8, 9]. The semi-quantitative RT-PCR ing neural cell migration and accumulation. Here, we and immunocytochemistry results indicated that both report novel evidence indicating that NO induces glial Netrin-1 and Netrin-4 were up-regulated in the GPs after precursor (GP) cell migration and accumulation in vitro, SNP treatment (Figure 1Bi-v). In contrast, DCC (De- and that this process is mediated by Netrin-1 signaling. leted in Colorectal Cancer), one of the specific receptors In preliminary tests, A2B5+ GPs (Figure 1Ai) derived of Netrins [10], was expressed in the GPs regardless of from rhesus embryonic stem (ES) cells were found to whether the cells were treated with SNP or not (Figure give different responses to sodium nitroprusside (SNP, an 1Bi, ii). NO donor, 0-6.25 × 103 µM), including differentiation, To investigate the possibility that Netrin signaling motility, and apoptosis/necrosis (not shown). Interesting- might mediate the cell migration and accumulation trig- ly, the cells were able to migrate and accumulate when gered by NO, RNAi was used to determine the role of stimulated by 10-250 μM SNP (Figure 1Aii-iv). In such Netrins throughout the cell migration and accumula- conditions, GPs gradually extended neurites (Supple- tion processes. When Netrin-1 was knocked down by mentary information, Movies S1, S2) with active growth RNAi in GPs (Figure 1Bvi), there were fewer cells with cone-like structures at the tip where abundant β-actin polarized morphology and in migrating chains (Figure was organized (Figure 1Aii, 24 h), polarized, migrated in 1Bvii) than in the negative and blank controls (Figure chains (Figure 1Aiii, 36 h; Supplementary information, 1Bviii, ix). In addition, the number of accumulated cell Movie S3), and eventually accumulated into gliospheres aggregates decreased by > 50% in the Netrin-1 RNAi (Figure 1Aiv, 72 h). The migratory behavior of GPs was group in comparison with the control groups (P < 0.05), not observed in regular expansion culture conditions suggesting the GPs migrated more slowly after Netrin-1 without SNP treatment (Supplementary information, knockdown. No significant difference was observed be- Movie S4) or with treatment by photodegraded SNP tween the test group and control groups after Netrin-4 (Supplementary information, Figure S1). Further analy- was knocked down (not shown). Hence, Netrin-1 but not sis indicated that the number of gliospheres accumulated Netrin-4 was required to mediate the cell movement and was related to the SNP concentration in a dose-dependent accumulation regulated by NO. manner (Figure 1Av). In order to verify the existence To further determine whether Netrin-1 mediated the − of NO released from SNP, NO2 in the medium was ex- NO-induced cell migration and accumulation through its amined by the Griess method [7]. The results confirmed specific receptor DCC, DCC blockade was carried out − that there was prolonged NO2 generation after SNP ad- using its antibody (Figure 1C). The results demonstrated dition (Figure 1Avi), implying continuous generation that blockade of DCC impaired the GP cell migration and of NO from exogenous SNP during cell migration and accumulation in a dose-dependent manner (Figure 1C). Cell Research | Vol 20 No 2 | February 2010 Hongwei Chen et al. npg 239 i A2B5/Hoechst ii b-actin/PI iii iv v 500 ** 30 +SNP –GPs 450 vi A 25 400 –SNP +GPs 350 20 +SNP +GPs 300 M) 15 250 m ] ( 10 200 – ## 2 150 5 100 [NO Gliosphere No. 50 0 0 –5 0 50 100 0 1 12 24 48 SNP (mM) –10 Time (h) B –SNP +SNP –SNP +SNP i Netrin-1/DCC ii Netrin-1/DCC iii Netrin-4 iv Netrin-4 v –RT –SNP +SNP Netrin-1 453 bp Netrin-4 475 bp GAPDH 449 bp vi vii viii ix –RT pSilencer-shNetrin-1pSilencer-NegativepSilencer- Netrin-1 453 bp GAPDH 449 bp C SNP-DCC Ab SNP+1 mg/ml DCC Ab SNP+4 mg/ml DCC Ab SNP+10 mg/ml DCC Ab 0 h 24 h 48 h 72 h 0 h 1 h 6 h 12 h 18 h 24 h i 30 h 42 h 48 h 54 h 60 h 72 h D F-actin F-actin BAPTA 0.1 0.5 1 5 10 15 20 -AM (mM) ii iii PI Merge iv PI Merge Result MA++ MA++ MA+ MA– AN+ AN++ AN++ (24 h) SNP+DMSO (0.02%) SNP+BAPTA-AM (5 mM) www.cell-research.com | Cell Research npg Netrin-1 mediates NO-induced migration and accumulation 240 The GPs completely failed to migrate and accumulate Ca2+ participated in regulating NO-induced cell motility. when treated with a high concentration of DCC antibody However, it is still not clear whether intracellular Ca2+ (10 µg/ml, Figure 1C). However, the cell migration and directly mediates the NO signaling before or after Netrin/ accumulation could be rescued when DCC antibody was DCC signaling as it has been reported to be the down- washed out (data not shown), suggesting that the DCC stream element in Netrin/DCC signaling [5]. Excessive receptor was vital to the NO-induced cell migration and Ca2+ chelating caused cell apoptosis/necrosis (Supple- aggregation resulting from NO treatment. Therefore, the mentary information, Figure S2), indicating that it was Netrin-1/DCC pathway is necessary for mediating the also necessary for regulating other important physiologi- NO-induced GP cell migration and accumulation. cal functions. Although cyclic nucleotides have been regarded as In conclusion, we show that NO induces cell migra- principal mediators of NO-orchestrated neurite out- tion and accumulation of GPs derived from the rhesus growth, and migration of enteric neurons and glial cells monkey ES cells in vitro. Furthermore, the NO-induced in invertebrates [4], pharmacological changes to intra- cell migration and accumulation are mediated by Ne- cellular cGMP did not influence the NO-induced GP trin-1/DCC signaling, and likely also involve Ca2+. This cell migration and accumulation (data not shown). We novel mechanism implicates NO as a potential signifi- therefore examined the changes in intracellular Ca2+, cant regulator of neuron-glia interaction, neural network another second messenger, to determine whether it was establishment, and even nerve cell repair. a downstream regulator in NO signaling. The Flura-3 (Experimental materials and methods are depicted in fluorescence results indicated that after SNP addition, the Supplementary information, Data S1) 2+ [Ca ]i gradually increased until gliosphere formation 2+ (Figure 1Di). A higher level of [Ca ]i was observed in Acknowledgments cells in migrating chains than in non-migratory cells in 2+ We thank Drs Bin Li and Jian Li (Kunming Institute of Zool- aggregates (Figure 1Di), suggesting that Ca might be ogy) for their help with time-lapse video imaging. This work was involved in regulating NO-induced cell motility. 2+ supported by research grants from the National Basic Research To test this hypothesis, intracellular Ca was chelated Program (2006CB701505) and the Chinese Academy of Sciences by its chelator BAPTA-AM. Moderate chelation of intra- (KSCX2-YW-R-47). cellular Ca2+ influenced the NO-induced cell migration and accumulation in a dose-dependent manner (Figure Hongwei Chen1, 2, 3, Qiang Wei1, 2, 3, Jing Zhang1, 2, 3, 1Dii). The GPs lost their ability to assemble F-actin (Fig- Chuankun Xu4, Tao Tan1, 2, 3, Weizhi Ji1, 3 ure 1Div), and failed to migrate and accumulate when 1 treated with SNP and 5 µM BAPTA-AM, confirming that Kunming Primate Research Center and Laboratory of Repro- Figure 1 Netrin-1 signaling mediates NO-induced glial precursor migration and accumulation. (A) NO induced the migration and accumulation of the glial precursors. A2B5+ glial precursors (i, 0 h) were induced to migrate and accumulate by sodium nitroprusside (SNP, ii–iv). Neuritic outgrowth with abundant β-actin distribution at the growth cone-like structures (ii, arrow, 24 h), cell polarization, migration in chains (iii, 36 h), and eventual accumulation into gliospheres (iv, 72 h) gradually occurred. (v) The relationship between SNP concentration and the number of gliospheres accumulated in 48 h after SNP treatment (n = 5 – in three independent experiments; **, ##, P < 0.01). (vi) [NO2 ] measurement of conditioned media using the Griess method (n ≥ 6 in three independent experiments). (B) Netrin-1 mediated the NO-induced cell migration and accumulation. Gene ex- pression analysis by immunostaining (i-iv) and semi-quantitative RT-PCR (v) showed that Netrins (Netrin-1, ii, v; Netrin-4, iv, v) were up-regulated in the glial precursors after SNP treatment compared to the cells without SNP treatment (i, iii, v).
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