In the present study, we found that NPCs undergo neuronal differentiation and inhibition of proliferation following treatment with 1 mM of the commonly prescribed antiepileptic drug VPA . We chose 1 mM VPA because this concentration is non-toxic to the NPCs in our current study and to hippocampal neuronal progenitors . The VPA amount applied to the animals, 200 mg/kg, is identical to that used to achieve whole-brain levels of 1.0–1.5 mM by chronic application .
We investigated the mechanism by which VPA regulates differentiation and inhibition of proliferation. The role of VPA in the exclusive in vivo regulation of differentiation and proliferation of NPCs suggest an application for VPA in the production of functional neurons for therapeutic use in patients. Our study also provides a mechanism that may aid in validating the proposed use of VPA as an anti-tumor and neuroprotective agent [3, 4, 33]. VPA induced both differentiation and inhibition of proliferation in NPCs by overcoming the effect of bFGF, a factor which promotes growth/proliferation and suppresses differentiation, through the common ERK-p21Cip/WAF1 pathway. The VPA-induced inhibition of proliferation was suppressed by the MEK inhibitor PD98059, indicating a role for activation of the ERK pathway in the inhibition of proliferation by VPA. Participation of the ERK pathway in inhibition of proliferation is frequently accompanied by induction of the cell cycle inhibitory factor p21Cip/WAF1 [6, 31, 34, 35]; p21Cip/WAF1 was also induced in the VPA-treated NPCs. The role of p21Cip/WAF1 in inhibition of proliferation by VPA is also shown by release of the VPA-induced inhibition of proliferation by p21Cip/WAF1 siRNA. The role of p21Cip/WAF1 as a potent anti-proliferation factor was further shown by loss of BrdU incorporation in most cells in which p21Cip/WAF1 had been induced by VPA.
A significant decrease in the level of Tuj1 following siRNA-mediated p21Cip/WAF1 knockdown, demonstrated by both biochemical and immunocytochemical analyses, suggests that p21Cip/WAF1 may also be involved in the regulation of differentiation and inhibition of proliferation. It is clear that both inhibition of proliferation and differentiation of NPCs stimulated by VPA occur through the ERK pathway-dependent induction of p21Cip/WAF1.
We have shown that activation of the ERK-p21Cip/WAF1 pathway by VPA did not occur via the segment of the pathway involving EGFR but via the segment involving β-catenin. Although EGFR has been identified as a target of Wnt/β-catenin in liver , EGFR was reduced by VPA in NPCs. The mechanism by which EGFR transcription is inhibited by VPA is unknown; however, it has been established that EGFR transcription is repressed by bone morphogenetic protein 4 (BMP4), an alternative transcription target of β-catenin , in NPCs [38, 39]. We observed significant induction of BMP4 in the VPA-treated NPCs grown in the presence of bFGF (data not shown). These data suggest that the VPA-induced decrease in EGFR in NPCs may be acquired through induction of BMP4.
VPA directly inhibits GSK3β resulting in activation of the β-catenin signaling pathway [33, 40, 41] and β-catenin is, in turn, involved in regulation of the ERK pathway [21, 22]. Evidence for the role of β-catenin in VPA-induced activation of the ERK-p21Cip/WAF1 pathway, and subsequent effects on differentiation and inhibition of proliferation in NPCs, was seen in the reduction of the effects of VPA, including ERK activation and induction of p21Cip/WAF1 and Tuj1, following siRNA-mediated β-catenin knockdown. The β-catenin-mediated activation of the ERK-p21Cip/WAF1 pathway following VPA treatment may be attributed to upregulation of Ras, suggested by the increase in the level of Ras seen after β-catenin overexpression. The VPA-induced increase in the level of Ras may be due to the stabilization of β-catenin as a result of inhibition of GSK3?. Increases in Ras following modulation of the Wnt/β-catenin signaling pathway have been demonstrated in various cell types, including primary hepatocytes, and β-catenin has been identified as an important mediator of that process [21, 22]. Regulation of Ras protein levels by the Wnt/β-catenin system is mediated by polyubiquitination and proteasomal degradation [Kim et al., 2008, Journal of Cell Science, In print]. Differentiation and proliferation occur independently in the cerebellar cortex of the developing embryo [42–44]; however, the mechanism(s) underlying the differential regulation of the two processes has not been described. In this study, we found that differentiation and proliferation occurred independently in regions of the developing brain of embryos treated with VPA. We saw no BrdU-positive cells among Tuj1-positive NPCs following VPA treatment. We also did not observe any proliferating cells among differentiated NPCs stimulated to differentiate by β-catenin overexpression or by VPA treatment to express increased levels of p21Cip/WAF1. These results indicate that mutually exclusive patterns of differentiation and proliferation during neuronal differentiation and development may be regulated via the common Ras-ERK-p21Cip/WAF1 pathway involving β-catenin.
However, the effects of VPA, particularly its effect on proliferation, were modest or only partially inhibited by increases in p21Cip/WAF1 or siRNA-mediated β-catenin knockdown in several different cases. These results indicate the possibility that VPA-induced differentiation and inhibition of proliferation occur in part via different routes, including, e.g., the pathway affected by inhibition of HDAC . Although we improved the efficiency of transfection by making modifications to the standard method, the limited effectiveness of siRNAs in general may also be a contributing factor in the weak effects seen on differentiation and inhibition of proliferation (see Additional file 7). The concomitant stimulation of differentiation and inhibition of proliferation in NPCs and the developing rat embryo by VPA treatment indicate potential utility for VPA in the treatment of neuroblastomas  and/or in neuronal regeneration.