Valproate, an anticonvulsant drug used to treat bipolar disorder, was studied for its ability to promote neurogenesis from embryonic rat cortical or striatal primordial stem cells. Six days of valproate exposure increased by up to fivefold the number and percentage of tubulin b III-immunopositive neurons, increased neurite outgrowth, and decreased by fivefold the number of astrocytes without changing the number of cells. Valproate also promoted neuronal differentiation in human fetal forebrain stem cell cultures. The neurogenic effects of valproate on rat stem cells exceeded those obtained with the neurotrophins brain-derived growth factor (BDNF) or NT-3, and slightly exceeded the effects obtained with another mood stabilizer, lithium. No effect was observed with carbamazepine. Most of the newly formed neurons were GABAergic, as shown by 10-fold increases in neurons that immunostained for GABA and the GABA-synthesizing enzyme GAD65/67. Double immunostaining for bromodeoxyuridine and tubulin b III showed that valproate increased by four-to fivefold the proliferation of neuronal progenitors derived from rat stem cells and increased cyclin D2 expression. Valproate also regulated the expression of survival genes, Bad and Bcl-2, at different times of treatment. The expression of prostaglandin E synthase, analyzed by quantitative RT-PCR, was increased by ninefold as early as 6 h into treatment by valproate. The enhancement of GABAergic neuron numbers, neurite outgrowth, and phenotypic expression via increases in the neuronal differentiation of neural stem cell may contribute to the therapeutic effects of valproate in the treatment of bipolar disorder.

The cytokine ciliary neurotrophic factor (CNTF) and transforming growth factor-␤ (TGF-␤) both induce transcription of the vasoactive intestinal peptide (VIP) gene through a 180-base pair cytokine response element (CyRE) in the VIP promoter. While CNTF induces STAT and AP-1 proteins to bind to cognate sites in the VIP CyRE, the mechanism through which TGF-␤ acts to induce VIP gene transcription is not known. Here we show that Smad3 and Smad4 proteins can bind to two distinct sites within the VIP CyRE. These sites are absolutely required for the induction of VIP CyRE transcription by TGF-␤. TGF-␤ induces endogenous Smad-containing complexes to bind to these sites in human neuroblastoma cells. CNTF and TGF-␤ synergize to induce VIP mRNA expression and transcription through the VIP CyRE. This synergy is dependent on the Smad, STAT, and AP-1 sites, suggesting that these two independent cytokine pathways synergize through the cooperation of pathway-specific transcription factors binding to distinct sites within the VIP CyRE.

Activin, a member of the transforming growth factor-beta superfamily, can regulate neuropeptide gene expression in the nervous system and in neuroblastoma cells. Among the neuropeptide genes whose expression can be regulated by activin is the gene encoding the neuropeptide vasoactive intestinal peptide (VIP). To investigate the molecular mechanisms by which activin regulates neuronal gene expression, we have examined activin's regulation of VIP gene expression in NBFL neuroblastoma cells. We report here that NBFL cells respond to activin by increasing expression of VIP mRNA. Activin regulates VIP gene transcription in NBFL cells through a 180-bp element in the VIP promoter that was previously characterized to be necessary and sufficient to mediate the induction of VIP by the neuropoietic cytokines and termed the cytokine response element (CyRE). We find that the VIP CyRE is necessary and sufficient to mediate the transcriptional response to activin. In addition, ciliary neurotrophic factor (CNTF), a neuropoietic cytokine, synergizes with activin to increase VIP mRNA expression and transcription through the VIP CyRE. Mutations in either the Stat (signal transducer and activator of transcription) or AP-1 sites within the CyRE that reduce the response to CNTF, also reduce the response to activin. However, mutating both the Stat and AP-1 sites within the wild-type CyRE, while reducing the separate responses to either activin or CNTF, eliminates the synergy between them. These data suggest that activin and CNTF, two factors that appear to signal though distinct pathways, activate VIP gene transcription through a common transcriptional element, the VIP CyRE.