RA and TPA up-regulated expression of Nrf2 and its downstream genes
SH-SY5Y cells were treated with 10 µM of RA for different time periods and expression of Nrf2, NQO1, and NF-M was analyzed by immunoblot analysis. Induction of Nrf2 was observed as early as 2 hr and remained elevated up to 48 h, with the maximal induction at 24 hr (). Elevated expression of NQO1 was observed at 24 hr and 48h. As expected, NF-M was also up-regulated 12 hr following RA treatment and remained elevated at 48 hr, whereas, the levels of control α-tubulin remained the same (). These data indicate that there might be a positive correlation between Nrf2 activation and NF-M expression. Another parallel set of samples treated with 10µM of RA was also evaluated for mRNA expression of Nrf2, NQO1, and NF-M by real-time qPCR. While there was only a marginal induction of Nrf2 at the mRNA level, expression of NQO1 and NF-M mRNA showed a significant increase (). Next, the 24 hr time point was chosen for induction of Nrf2, NQO1, and NF-M by different doses of RA. As shown in , RA induced the protein levels of Nrf2 and NQO1 at all the doses used. Similarly, RA had no effect on Nrf2 transcription while NQO1 mRNA expression was up-regulated significantly (). Induction of NF-M mRNA was observed with any treatment and reached peak with 10 µM RA treatment (). In order to understand whether Nrf2 induction is due to the differentiation processes, or is specific to RA treatment, induction of Nrf2 by TPA, which induces neuronal differentiation through a different mechanism, was also determined. Similar to RA, TPA enhanced Nrf2 and NQO1 protein levels (). Furthermore, TPA-induced Nrf2 activation correlated with enhanced expression of NF-M (). Collectively, these data indicate that the Nrf2 signaling pathway is up-regulated during neuronal differentiation processes.
Fig. 1 RA up-regulated expression of Nrf2 and its downstream genes. (A) Endogenous Nrf2 increased in a time-dependent manner following 10µM RA treatment. SH-SY5Y cells were treated with 10µM RA for the indicated time periods. Cells were lysed (more ...)
RA up-regulated Nrf2 through enhancing the rate of Nrf2 protein synthesis
Next, the mechanism of RA-mediated up-regulation of Nrf2 was explored. SH-SY5Y cells were treated with two doses of tBHQ, RA, or in combination as indicated, and mRNA expression of Nrf2 and NQO1 was measured by qRT-PCR. Nrf2 mRNA expression was not affected by any treatment (, upper panel). However, the mRNA expression of NQO1 was enhanced slightly by RA treatment alone (, lower panel, bar 2). Cotreatment of RA and tBHQ resulted in a higher induction of NQO1 (, compare lane 2 to lane 4 or lane 6). To test any change in the amount of Nrf2 bound to the ARE, parallel-treated cells were subjected to the ARE affinity immunoprecipitation analysis. Nrf2 expression in the total lysate was shown () and the mutant ARE was included as a negative control (, lanes 1 and 8). RA treatment alone increased Nrf2 binding to the ARE (, compare lane 3 with lane 2) and combined treatment further enhanced binding of Nrf2 with the ARE (, lane 4–7). To demonstrate that there is more Nrf2 bound to the ARE after RA treatment in vivo, we performed chromatin immunoprecipitation analysis (ChIP). tBHQ induced Nrf2 binding to the ARE significantly while RA enhanced Nrf2 binding to the ARE about 2 fold (). These data indicate that RA is able to activate the Nrf2-mediated signaling pathway by increasing the total protein level of Nrf2 and, thus, facilitating more Nrf2 binding to the ARE. Previously, it has been well-demonstrated that most Nrf2-inducers, including tBHQ and sulforaphane, upregulate Nrf2 by suppressing Nrf2 degradation. Therefore, the half-life of Nrf2 in response to RA treatment was measured using the cycloheximide/immunoblot method. To our surprise, RA did not affect the half-life of Nrf2 and the half-life was approximately 18 min in the absence or presence of RA (), whereas tBHQ increased the half-life of Nrf2 to 39 min. Next, protein synthesis and degradation were measured by the pulse chase method (). The half-life of Nrf2 measured by the pulse chase method was consistent with that measured by the cycloheximide/immunoblot method. Interestingly, the newly synthesized proteins in the 30-min pulse period were higher in the RA-treated sample, compared to that in the non-treated control (, lane 1, compare upper panel to lower panel). These data indicate that RA is able to upregulate the Nrf2-signaling pathway by enhancing the rate of Nrf2 protein synthesis.
Fig. 2 RA up-regulated Nrf2 by enhancing the rate of Nrf2 protein synthesis. (A) RA potentiated the effect of tBHQ on mRNA levels of Nrf2 and NQO1 in SH-SY5Y cells. Cells were treated with tBHQ (10µM or 20µM) and/or RA (10µM) simultaneously (more ...)
RA-induced neuronal outgrowth was accompanied by enhanced expression and nuclear translocation of Nrf2
To test the coordinated up-regulation of Nrf2 with a neuron-like morphological change in response to RA treatment, an indirect double immunofluorescence stain with anti-Nrf2 and anti-MAP-2 was performed in SH-SY5Y cells in the absence or presence of RA. In the undifferentiated cells, MAP-2 expression was low but increased upon treatment with RA (, compare panel A with B). The cell density was low in the RA-treated sample, compared to the non-treated sample, even though the same amount of cells were seeded for the two groups (, compare panel A with B). This is due to a reduced growth rate after RA stimulation. In addition, neurite extension was clearly seen following RA treatment (, compare panel A with B). Cellular localization of Nrf2 was switched from the whole cell to the nucleus in response to RA treatment, (, compare panel C with D). Furthermore, the intensity of Nrf2 was enhanced markedly, indicating an increase in the Nrf2 protein level in response to RA treatment (, compare panel C with D; panel E with F). show a phase-contrast image of the same set of samples taken prior to the immunofluorescence staining. The percentage of differentiated cells was counted and the mean neurite length was measured using phase-contrast images. Both parameters were significantly increased in the RA-treated samples (). Clearly, RA induced both Nrf2 and neuronal cell differentiation.
Fig. 3 RA-induced neuronal outgrowth was accompanied by enhanced expression and nuclear translocation of Nrf2. (A) RA induced morphological changes and up-regulated MAP-2 and Nrf2. Cells were treated with 10µM RA for 4 days, fixed in methanol, and immunostained (more ...)
Stable-overexpression of Nrf2 promoted neuronal differentiation
Next, the possible correlation between Nrf2 up-regulation and neuronal cell differentiation was tested by stable overexpression of Nrf2. A SH-SY5Y-derived stable cell line expressing HA-Nrf2 was established using a lentivirus delivery and puromycin selection system. As shown in , HA-Nrf2 was expressed as detected by immunoblot analysis with anti-HA antibodies (). Similarly, overall expression of Nrf2 was enhanced in this stable cell line as detected with anti-Nrf2 antibodies (). Overexpression of Nrf2 resulted in enhanced expression of NQO1, indicating up-regulation of Nrf2 and its downstream events (). In this cell line, overexpression of Nrf2 promoted RA-induced up-regulation of NF-M although it had no effect on the levels of NF-M in the absence of RA (, compare lane 1 with lane 3, lane 2 with 4), indicating that upregualtion of the Nrf2 signal pathway itself is insufficient to initiate neuronal differentiation processes. Morphological differences in the vector-transfected and HA-Nrf2 transfected cells in the absence or presence of RA were also determined. Overexpression of HA-Nrf2 had no effect on neurite outgrowth in the absence of RA, but potentiated neuronal differentiation in RA-treated samples as determined by the indirect immunofluorescence stain with MAP-2 (, compare panel A with C; panel B with D). Similarly, neurite outgrowth promoted by Nrf2 overexpression was observed in phase-contrast images (, compare panel B with panel D). Moreover, the conclusion that Nrf2 positively regulates neuronal differentiation was confirmed by quantifying the percentage of differentiated cells and the mean neurite length ().
Fig. 4 Stable-overexpression of Nrf2 promoted neuronal differentiation. (A) A SH-SY5Y-derived stable cell line has enhanced expression of Nrf2 and its downstream gene, NQO1. SH-SY5Y cells were infected using a lentivirus delivery system to express HA-Nrf2 and (more ...)
Up-regulation of endogenous Nrf2 potentiated differentiation
Next, we tested whether up-regulation of endogenous Nrf2 by tBHQ is able to modulate neuronal differentiation. SH-SY5Y cells were treated with tBHQ, RA, or in combination as indicated and cell lysates were subjected to immunoblot analysis. Although tBHQ up-regulated Nrf2 and NQO1 ( lanes 3–6), tBHQ alone had no effect on NF-M expression (, lanes 1, 3, and 5). Significantly, tBHQ enhanced the RA-mediated upregualtion of NF-M protein levels (, compare lanes 4 and 6 with lane 2), demonstrating the positive role of Nrf2 in potentiating neuronal differentiation processes. Phase contrast images were taken before cells were lysed (), and these images were used for quantification of the percentage of differentiated cells and the mean neurite length (). Co-treatment of tBHQ with RA increased the percentage of differentiated cells slightly and significantly increased the mean neurite length, compared with RA-only treatment (). Furthermore, tBHQ had no effect in the absence of RA (). tBHQ treatment enhanced Nrf2 (, compare panels G and H with panels E and F) and MAP-2 staining (, panel D). Together, these data demonstrate that induction of endogenous Nrf2 facilitates RA-induced neuronal differentiation, although Nrf2 activation itself is insufficient to induce neuronal differentiation.
Fig. 5 Up-regulation of endogenous Nrf2 potentiated differentiation. (A) tBHQ enhanced RA-mediated up-regulation of NF-M. SH-SY5Y cells undifferentiated or RA-differentiated and/or the indicated concentration of tBHQ were lysed and subjected to immunoblot analysis (more ...)
Down-regulation of Nrf2 compromised neuronal differentiation
To further verify the importance of Nrf2 in neuronal differentiation, the possible requirement of Nrf2 in neuronal differentiation was tested. Two approaches were used to knockdown the expression of endogenous Nrf2. In one set of experiments, Nrf2-siRNA or control siRNA were transfected into SH-SY5Y cells and levels of Nrf2 were measured. Transfection of Nrf2-siRNA for 48 hr decreased levels of Nrf2 to 50% (, compare lane 1 and 2, Nrf2 panel). At 48 hr post-transfection, cells were further treated with RA for an additional 24 hr and NF-M expression was determined by immunoblot analysis. Knockdown of Nrf2 expression itself had no effect on NF-M levels (, compare lane 1 with 2; 3 and 5; NF-M panel). However, Nrf2-siRNA blocked induction of NF-M in response to RA (, compare lane 4 with lane 6). Morphological analysis showed retarded neurite outgrowth in cells transfected with Nrf2 siRNA, but not with the control siRNA, in response to RA treatment (6B, compare panel B with panel D; panel A with panel B). In addition, knockdown of Nrf2 expression was clearly shown by the reduced Nrf2-fluorescence staining (, compare panels G and H with panels E and F). The phase contrast images of these cells were taken before cells were fixed for immunofluorescence staining (). Changes in the percentage of differentiated cells and the mean neurite length by RA treatment were diminished in Nrf2-siRNA transfected cells (). The fact that inhibition of Nrf2 by Nrf2-siRNA was unable to completely block the RA-mediated neurite outgrowth can be due to two reasons: (i) Nrf2 is not absolutely required for the neuronal differentiation process; (ii) complete inhibition of Nrf2 is not achieved and Nrf2-siRNA only reduced Nrf2 expression by 50%. Another approach was also used to inhibit expression of Nrf2 by overexpression of Keap1, the substrate adapter for E3 ubiquitin ligase responsible for Nrf2 degradation. SH-SY5Y cells were infected with lentivirus carrying HA-Nrf2 and Keap1-CBD. Transient overexpression of Nrf2 and Keap1 was confirmed by immunoblot analysis with anti-HA and anti-CBD antibodies (data not shown). In response to RA treatment, overexpression of Nrf2 resulted in further up-regulation of NF-M, whereas knockdown of Nrf2 by Keap1 overexpression suppressed upregualtion of NF-M by RA (, compare lanes 4 and 6 with lane 2). Together, these data demonstrate that inhibition of Nrf2 expression compromised neuronal differentiation. Noticeably, up- or down-regulation of Nrf2 had no effect on the levels of NF-M in the absence of RA treatment, indicating that Nrf2 itself neither can initiate neuronal differentiation nor is absolutely required for neuronal differentiation.
Fig. 6 Down-regulation of Nrf2 compromised neuronal differentiation. (A) Nrf2 siRNA effectively decreased Nrf2 protein levels and blocked induction of NF-M in response to RA. SH-SY5Y cells were transiently transfected for 48 hr with either control siRNA or Nrf2 (more ...)
Primary neurons isolated from Nrf2-null mice were retarded in neurite outgrowth
To further verify the important function of Nrf2 in regulating neuronal cell differentiation, primary neurons were isolated from wild-type and Nrf2-null mice. The primary neurons were allowed to grow in culture and morphological changes were observed by taking phase contrast images every 24 hr for 5 consecutive days (). The neuron mean length at day 3, 4 and 5 was measured (, bar graphs). The mean neurite length was significantly reduced in primary neurons from Nrf2-null mice, especially at day 3, demonstrating a slow differentiation process in neurons that are deficient in Nrf2 expression. Primary neurons that had been growing in culture for three days were subjected to immunofluorescence staining with anti-MAP-2 (). Expression of MAP-2 was lower in neurons from Nrf2-null mice as shown by a reduced intensity of MAP-2 staining (, compare panel A with panel B). In addition, primary neurons isolated from Nrf2-null mice had less neurite outgrowth (, compare panel A with panel B). Similar amounts of cells were seeded in these two groups as shown by nuclear staining (, panel C and D), indicating that the difference in neurite outgrowth is not due to a difference in cell confluency, but rather, it is due to the status of Nrf2. These results clearly demonstrate the functional role of Nrf2 in potentiating neuronal differentiation.
Fig. 7 Primary neurons isolated from Nrf2-null mice were retarded in neurite outgrowth. (A) Cell morphology and neurite lengths were compromised in Nrf2-null mice compared to wild-type. Primary neurons were isolated from wild-type and Nrf2-null mice and grown (more ...)