Poor neuronal features in neuroblastoma cells with low PAX3 expression
To explore the differentiation defect in neuroblastoma cells we have used SK-N-SH and its derived SH-SY5Y cell lines (Kovalevich and Langford 2013). SH-SY5Y cells were widely used for neuronal differentiation analysis and shows more neuronal phenotypes than the parental SK-N-SH cells (Kovalevich and Langford 2013). To further confirm this we have analyzed the neuronal features of these cells by immunostaining with neuronal marker gene class III beta 3 tubulin (TUBB3) and its protein expression by western blot analysis. Immunofluorescence results showed more neuron-like features in SH-SY5Y cells than in SK-N-SH (Figure 1A). The western blot analysis revealed increased expression of TUBB3 in SH-SY5Y cells than in SK-N-SH (Figure 1B). RT-qPCR analysis of neuronal marker genes including TUBB3, MAP2, NEFL, NEUROG2 and SYP showed increased expression in SH-SY5Y cells than SK-N-SH cells (Figure 1C). Thus, these data confirm that SH-SY5Y cells were more neurogenic than the parental SK-N-SH cells that showed poor neuron-like features (Figure 1A-C).
We have analyzed PAX3 expression at RNA and protein levels and its functional status in both cells. Here we have found an increased level of PAX3 mRNA and protein in SH-SY5Y compared to SK-N-SH cells (Figure 1D and E). PAX3 functional status is assessed by the expression of its reported target genes (TGFA, MET and NCAM1). The results revealed the expression of PAX3 target genes in SH-SY5Y and their level was relatively lower in SK-N-SH (Figure 1F). Thus we observed poor neuron-like phenotypes in SK-N-SH with low PAX3 levels compared to SH-SY5Y with functional PAX3 (Figure 1).
Retinoic acid induces neuronal differentiation in high PAX3 expressing neuroblastoma cells
Retinoic acid (RA) is a well-known agent to induce neuronal differentiation in various cells (Xun et al. 2012; Tan et al. 2015; Park and Rhee 2018). We have analyzed the effect of retinoic acid in inducing neuronal differentiation in these neuroblastoma cell lines. Firstly, we have used immunofluorescence to detect TUBB3 expression and assess the morphological changes by RA treatment. In SH-SY5Y cells, TUBB3 expression was found in both GM and DM conditions whereas in SK-N-SH cells we observed its reduced level in DM conditions and it is also reflected in western blot analysis (Figure 2A, D). The results also revealed that RA can induce neuronal outgrowth in SH-SY5Y not in SK-N-SH cells under DM conditions (Figure 2A). Quantification of neurite length and the number of cells with two or more neurites confirmed RA-mediated induction of neuron-like morphological features only in SH-SY5Y cells (Figure 2B and C). Then mRNA expression analysis showed induction of TUBB3, MAP2 and NEFL and reduction of SYP in RA-treated SH-SY5Y cells and no difference in NEUROG2 expression (Figure 2E). On the contrary, none of these neuronal marker genes were induced in SK-N-SH cells by RA (Figure 2F). Collectively these evidence suggest that RA can enhance neurogenic differentiation in PAX3 functional SH-SY5Y cells not in SK-N-SH that had low PAX3 level/activity (Figure 1, 2).
Ectopic PAX3 expression fails to induce neurogenesis in SK-N-SH cells
Here we tested whether ectopic expression of PAX3 can enhance neuronal differentiation in SK-N-SH cells or not. For this, we have generated stable SK-N-SH cells that express PAX3 cDNA (SK-PAX3) or vector (SK-vec) by retroviral mediated transduction. After confirmation of ectopic PAX3 in these cells (Figure 3A), we analyzed the neurogenic features by TUBB3 immunostaining and the results revealed slightly improved neuronal outgrowth (Figure 3B-D). However, western blot analysis showed no changes in the expression of TUBB3 in both SK-PAX3 and SK-vec cells (Figure 3E). Further, the expression of other neuronal marker genes also showed no notable change in these cells (Figure 3F). Moreover, the ectopically expressed PAX3 in these cells was functionally inactive as shown by its target genes expression (Figure 3G). Thus, these results revealed that ectopic PAX3 in SK-N-SH is unable to rescue neuronal differentiation due to its functional inactivation (Figure 3).
Depletion of PAX3b isoform alone is not sufficient to induce neuronal differentiation in SK-N-SH cells
There were eight PAX3 protein isoforms were identified and they varied in their structural features (Boudjadi et al. 2018). Among these isoforms, PAX3a and PAX3b have only paired and no homeo and transactivation domains (Tsukamoto et al. 1994). PAX3c (a well-studied isoform of PAX3), PAX3d and PAX3e have all three functional domains and PAX3g, PAX3h and PAX3i have truncated transactivation domains (Tsukamoto et al. 1994; Boudjadi et al. 2018). The inactivation status of PAX3 observed in SK-PAX3 cells (Figure 3G) could be due to interference of transcriptionally inactive PAX3 isoforms. To verify this we have analyzed the expression of various PAX3 isoforms in SH-SY5Y and SK-N-SH cells using semiquantitative RT-PCR. Here, in SH-SH5Y cells we could observe both PAX3b and PAX3c isoform expression whereas in SK-N-SH cells we could see only PAX3b expression (Figure 4A). We could not detect any other PAX3 isoforms in these cells (Figure 4A).
Defective neuronal features observed in SK-N-SH cells (Figure 1) could be due to the presence of PAX3b (Figure 4A). To verify this, we generated stable PAX3b-depleted SK-N-SH (SK-shPAX3b) and scramble control (SK-shScr) cells by lentiviral mediated transduction and the PAX3b depletion was confirmed by semiquantitative RT-PCR analysis (Figure 4B). Immunofluorescence analysis showed slightly enhanced neuronal morphology in SK-shPAX3b compared to SK-shScr cells (Figure 4C-E) with no change in TUBB3 expression and the same is confirmed in western blot analysis (Figure 4F). RT-qPCR analysis showed a reduction of all of the neuronal markers in SK-shPAX3b compared to control cells except NEUROG2 (Figure 4G). PAX3 target genes were also reduced in PAX3b-depleted cells compared to scramble control (Figure 4H). These data support that PAX3b-depletion alone is not sufficient to induce neuronal differentiation in SK-N-SH cells (Figure 4).
Functional PAX3 can induce neuronal differentiation in PAX3b-depleted SK-N-SH cells
The defect in inducing neuronal differentiation in SK-shPAX3b cells could be due to the absence of functional PAX3 (Figure 4) and restoration of functional PAX3 can induce neuronal differentiation in these cells. To test this, we have transiently introduced plasmids containing PAX3 cDNA or vector into SK-shPAX3b cells. After 48 hours of post-transfection, these cells were subjected to PAX3 target gene expression analysis. The ectopic PAX3 expression in post-transfected cells was confirmed by western blot analysis (Figure 5A). Ectopic PAX3 induced expression of PAX3 target genes in SK-shPAX3b cells (Figure 5B). Immunofluorescence analysis confirmed the increased expression of TUBB3 and morphological differentiation in PAX3 transfected SK-shPAX3b cells (Figure 5C). We have also noticed increased neuronal outgrowth in PAX3 ectopically expressed PAX3b-depleted cells (Figure 5D and E). Western blot analysis also confirms the induced expression of neuronal marker TUBB3 in PAX3 transfected cells (Figure 5F). Besides, we have observed the induction of neuronal marker genes in these cells (Figure 5G). Overall, these data suggest that restoration of functional PAX3 in PAX3b-depleted SK-N-SH cells can restore neuronal differentiation (Figure 5).