Transcriptional basis for the inhibition of neural stem cell proliferation and migration by the TGFβ-family member GDF11.

Signalling through EGF, FGF and endocannabinoid (eCB) receptors promotes adult neurogenesis, and this can be modelled in culture using the Cor-1 neural stem cell line. In the present study we show that Cor-1 cells express a TGFβ receptor complex composed of the ActRIIB/ALK5 subunits and that a natural ligand for this receptor complex, GDF11, activates the canonical Smad2/3 signalling cascade and significantly alters the expression of ∼4700 gene transcripts within a few hours of treatment. Many of the transcripts regulated by GDF11 are also regulated by the EGF, FGF and eCB receptors and by the MAPK pathway - however, in general in the opposite direction. This can be explained to some extent by the observation that GDF11 inhibits expression of, and signalling through, the EGF receptor. GDF11 regulates expression of numerous cell-cycle genes and suppresses Cor-1 cell proliferation; interestingly we found down-regulation of Cyclin D2 rather than p27kip1 to be a good molecular correlate of this. GDF11 also inhibited the expression of numerous genes linked to cytoskeletal regulation including Fascin and LIM and SH3 domain protein 1 (LASP1) and this was associated with an inhibition of Cor-1 cell migration in a scratch wound assay. These data demonstrate GDF11 to be a master regulator of neural stem cell transcription that can suppress cell proliferation and migration by regulating the expression of numerous genes involved in both these processes, and by suppressing transcriptional responses to factors that normally promote proliferation and/or migration

GDF11 significantly attenuates Cor-1 cell proliferation in a dose-dependent manner.

<p>Cor-1 cells were grown in 96-well plates with cell number determined relative to control cultures using the MTS assay (see Methods). A representative experiment showing GDF11-dependent inhibition of Cor-1 cell proliferation over a 72 hr period is shown in (<b>A</b>). Each data point shows the mean relative optical density (+/−SEM) from 16 replicate cultures. (<b>B</b>) Incubation with either 25 ng/ml or 50 ng/ml of GDF11 significantly inhibits Cor-1 cell proliferation as monitored by MTS assay at the 48 hr time point. Data were normalised to control values for each of 5 independent experiment (mean ± SEM; ***<i>p</i><0.001, n = 5). (<b>C</b>) The inhibition of proliferation caused by GDF11 (50 ng/ml) measured at the 48 hr time point can be prevented in a dose-dependent manner by the GDF8 propeptide (PP). Shown here are results from a representative experiment, with each data point representing the mean +/− SEM relative to the control determined from 16 replicate cultures.</p

Volcano plots of the transcriptional responses to GDF11.

<p>Volcano plots are scatter plots of the log₂ fold change against the negative log₂ probability calculated by Student’s t-test. The points in red correspond to those probe sets passing the p<0.05 significance and 10% fold change thresholds. GDF11 treatment clearly elicits a substantial response that is skewed towards a downregulation of transcript levels (top). The relative transcriptional response to GDF11 can be compared to the other canonical pathways by superimposing the volcano plots (bottom). Here, pixels (δlog₂(prob) = 1, δlog₂(fold) = 0.1) are coloured if they contain more than 10 points. The ‘density plot’ illustrates the similar magnitude of GDF11 treatment (green/yellow) and EGF receptor inhibition (red,yellow), which both dominate the FGF receptor inhibition response (black).</p

GDF11 inhibits expression of migratory transcripts and migration in Cor-1 cells.

<p>(<b>A</b>) Gene Ontology analysis of the GDF11 microarray data indicates suppression of a large number of transcripts encoding proteins associated with cell migration. Shown here are the top 11 downregulated responders. (<b>B</b>) Q-PCR analysis of Fascin and LASP1 transcript levels in Cor-1 cells incubated with either control vehicle or 25 ng/ml GDF11 for 6 hr confirmed the microarray results. Expression was normalised against GAPDH (mean ± SEM; *<i>p</i><0.05; **<i>p</i><0.01, n = 3). (<b>C</b>) Cor-1 cells were plated in a 24-well plate to achieve a confluent monolayer. Approximately 8 hr after plating, cells were incubated with GDF11 overnight as indicated. The following day, a wound was produced in the centre of each well and images were taken every 2 hr to monitor wound closure. Images show the initial scratch wound mask (black) and the area infiltrated by migrating cells (grey) 6 and 12 hr after scratching for control and GDF11-treated samples. (<b>D</b>) Quantification of the wound area infiltrated by migrating cells at 12 hr normalized to the control reveals a significant inhibition of migration in GDF11-treated samples (mean ± SEM; **<i>p</i><0.01, ***<i>p</i><0.001, n = 3).</p

GDF11 inhibits Cyclin D2 expression.

<p>(<b>A</b>) Q-PCR analysis showing a significant decrease in Cyclin D2 levels in Cor-1 cells after treatment with 25 ng/ml of GDF11 for 3 hr. Cyclin D2 transcript levels are expressed relative to the levels of the house-keeping gene GAPDH (mean ± SEM; **<i>p</i><0.01, n = 6). (<b>B</b>) Densitometric analysis of western blots of lysates from Cor-1 cells treated with 25 or 50 ng/ml of GDF11 for 24 hr shows a significant decrease in Cyclin D2 protein levels (mean ± SEM; **<i>p</i><0.01, n = 3). Cyclin D2 protein levels were normalised to actin, with a representative blot probed for Cyclin D2 and actin shown in (<b>C</b>).</p

Pathway enrichment analysis for the GDF11 transcriptional response.

<p>Sets of genes whose expression was significantly altered by GDF11 treatment were scored for enrichment against the GSEA MSigDB pathway gene sets. Responder sets were considered if including transcripts with significant (p<0.05) expression changes of more than 10%. Enrichment significance was defined as the probability of obtaining the same or better enrichment score with a random set of genes. 18 of the pathways that are regulated by GDF11 are shown in (<b>A</b>) with “N” giving the number of genes assigned to the pathway and “n” showing the number regulated by the treatment. (<b>B</b>) Overlap between the pathways regulated by GDF11 and the EGF, FGF and cannabinoid receptors (CB) is shown as a simple Venn diagram. We next determined if the transcripts that are co-regulated by GDF11 and the EGF, FGF and cannabinoid receptors, or the MAPK pathway, are regulated in the same or opposite direction by determining the Pearson co-efficient <i>r</i> and statistical significance of the regression analysis as indicated in (<b>C</b>). Here, EGF receptor responders were identified by use of selective EGF receptor inhibitors (EGFR-inhib), or by direct stimulation of starved cells with EGF (EGFR-stim). See text for details.</p

GDF11 does not induce neuronal nor astrocytic differentiation.

<p>(<b>A</b>) Cor-1 cells incubated with 50 ng/ml of GDF11 for 48 hr are negative for either the early neuronal marker Tuj1 (left) or the glial marker GFAP (centre). Incubation with 10 ng/ml of BMP4 for 48 hr induces astrocytic differentiation revealed by positive GFAP immunoreactivity. Hoechst dye (blue) was used to stain cell nuclei. Scale bars are 40 µm. Consistent with the immunocytochemistry results, Tuj1 (<b>B</b>) and GFAP (<b>C</b>) cannot be detected by Western blot in lysates of Cor-1 cells exposed to either 25 or 50 ng/ml GDF11 for 24 or 48 hr. As positive controls, Tuj1 is detected in Cor-1 cells plated on laminin and maintained for 48 hr in medium lacking EGF (<b>B</b>, first lane on the left) while GFAP is detected in Cor-1 cells undergoing astrocytic differentiation after incubation with BMP4 for 48 hr (<b>C</b>, first lane on the left).</p

GDF11 regulates the EGF signal transduction pathway.

<p>(<b>A</b>) Q-PCR analysis of EGFR transcript levels in Cor-1 cells incubated with either control vehicle or 25 ng/ml GDF11 for 3 hr. EGF receptor expression is normalised against two housekeeping genes, GAPDH or HPRT. GDF11 significantly downregulates EGF receptor transcript levels (mean ± SEM; **<i>p</i><0.01, n = 6). (<b>B</b>) Cor-1 cells incubated with 25 or 50 ng/ml GDF11 for either 24 or 48 hr were lysed and analysed for EGFR levels by Western blot. Exposure to GDF11 significantly decreases EGF receptor protein levels as shown by densitometric analysis relative to actin (mean ± SEM; *<i>p</i><0.05, **<i>p</i><0.01, n = 3) and by a representative blot. (<b>C</b>) Cor-1 cells were transiently co-transfected with a luciferase reporter construct and a <i>Renilla</i> plasmid by nucleofection. Twenty hours later cells were pre-incubated with 50 ng/ml GDF11 for 36 h before being starved of EGF and FGF for 6 h to reduce endogenous pCREB levels. Cells were then challenged with 10 ng/ml EGF and luciferase expression was subsequently measured by addition of a luciferase substrate. Transfection efficiency and cell numbers were taken into account by normalising luciferase readings at 570 nm against <i>Renilla</i> activity (detected by measuring light emission at 480 nm) (mean ± SEM; ***<i>p</i><0.001, n = 3).</p

Cor-1 cells express the ALK5/ActRIIb receptor complex and respond appropriately to GDF11.

<p>(<b>A</b>) Cor-1 cells were grown until near confluence. Lysates were analysed for the presence of the ActRIIb and ALK5 receptors by western blotting. Bands at the appropriate molecular weights (70 kDa and 56 kDa, respectively) were readily detected. (<b>B</b>) Cor-1 cells were treated as indicated with GDF11 (+: 10 ng/ml, ++: 25 ng/ml) with or without the GDF8 propeptide (PP) (1 µg/ml). Lysates were probed by western blot to detect phospo-Smad2, phospho-Smad3 and total Smad2/3. EGF/FGF2 stimulation was used as a negative control.</p

Transcriptional Basis for the Inhibition of Neural Stem Cell Proliferation and Migration by the TGFβ-Family Member GDF11

Signalling through EGF, FGF and endocannabinoid (eCB) receptors promotes adult neurogenesis, and this can be modelled in culture using the Cor-1 neural stem cell line. In the present study we show that Cor-1 cells express a TGFβ receptor complex composed of the ActRIIB/ALK5 subunits and that a natural ligand for this receptor complex, GDF11, activates the canonical Smad2/3 signalling cascade and significantly alters the expression of ∼4700 gene transcripts within a few hours of treatment. Many of the transcripts regulated by GDF11 are also regulated by the EGF, FGF and eCB receptors and by the MAPK pathway – however, in general in the opposite direction. This can be explained to some extent by the observation that GDF11 inhibits expression of, and signalling through, the EGF receptor. GDF11 regulates expression of numerous cell-cycle genes and suppresses Cor-1 cell proliferation; interestingly we found down-regulation of Cyclin D2 rather than p27kip1 to be a good molecular correlate of this. GDF11 also inhibited the expression of numerous genes linked to cytoskeletal regulation including Fascin and LIM and SH3 domain protein 1 (LASP1) and this was associated with an inhibition of Cor-1 cell migration in a scratch wound assay. These data demonstrate GDF11 to be a master regulator of neural stem cell transcription that can suppress cell proliferation and migration by regulating the expression of numerous genes involved in both these processes, and by suppressing transcriptional responses to factors that normally promote proliferation and/or migration