pBMP-9 and SpBMP-9 activate the Smad1/5 pathway

Effect of pBMP-9 and SpBMP-9 on kinetic of Smad 1/5 phosphorylation

We have recently shown that BMP-9 (1 nM) with or without RA can activate the canonical Smad1/5 pathway within 30 min in SH-SY5Y cells8. We therefore verified the ability of both pBMP-9 and SpBMP-9 (1 nM) to activate the Smad 1/5 pathway (Fig. 1A). The total Smad1/5 (TSmad) was used as a control. Without RA, the phosphorylated Smad1/5 (pSmad) bands were detected after incubation for 30 min in cells stimulated with pBMP-9 or SpBMP-9. Both peptides in the presence of RA induced the phosphorylation of Smad1/5 at 15 min. The Smad1/5 remained phosphorylated within 240 min in the presence of pBMP-9 or SpBMP-9 with or without RA. Densitometric analysis of bands corresponding to pSmad1/5 and standardized to that of TSmad (Fig. 1A) confirmed that pSmad1/5 levels reached a plateau after 1 h.

Figure 1 Effect of pBMP-9 and SpBMP-9 on the activation of the canonical Smad1/5 pathway. (A) Western blots of phosphorylated Smad1/5 (pSmad) and densitometric analysis of pSmad1/5 as referred to total Smad (TSmad) showing the effect of 1 nM BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA in SH-SY5Y cells after incubation for 0, 15, 30, 60, 120 and 240 min. (B) Pictures showing the nucleus (blue) and the pSmad1/5 (red) in SH-SY5Y cells stimulated with an equimolar concentration of BMP-9, pBMP-9 or SpBMP-9 (1 nM) +/− 10 μM RA after incubation for 240 min. Results are representative of 2 independent experiments performed in duplicate (Bar = 100 μm). Full size image

Effect of pBMP-9 and SpBMP-9 on the nuclear translocation of phosphorylated Smad1/5

Since pBMP-9 and SpBMP-9 induced the phosphorylation of Smad 1/5 (pSmad1/5) in SH-SY5Y cells, we then analyzed the pSmad1/5 translocation into the nucleus after incubation for 240 min by labelling both DNA (nucleus) and pSmad1/5 (Fig. 1B). Without RA, cells incubated with BMP-9 (1 nM) had a strong pSmad1/5 staining compared to untreated cells (CTL) and the pSmad1/5 were mainly located at the nucleus. Both pBMP-9 and SpBMP-9 at 1 nM induced a slight pSmad1/5 labelling compared to BMP-9. Only some cells possessed a strong nuclear translocation of the pSmad1/5. The same tendencies were observed in cells treated by BMP-9 or its derived peptides in the presence of RA.

pBMP-9 and SpBMP-9 do not affect the number of SH-SY5Y cells

Effect of pBMP-9 and SpBMP-9 on cell metabolic activity and viability

We investigated the influence of pBMP-9 and SpBMP-9 with or without RA on the viability of SH-SY5Y cells. BMP-9 was used as a control. We measured the activity of the enzyme succinate dehydrogenase using the MTS assay (Fig. 2A). The control (CTL) showed no alteration in the metabolic activity either with or without RA. Incubation for 3d with 0.1 nM BMP-9 or 0.1 nM SpBMP-9 but no RA resulted in a significant increase in metabolic activity (p < 0.05) in comparison to the CTL. Similar results were obtained after incubation for 5d with 0.1 or 1 nM BMP-9 and with 0.1 nM SpBMP-9. Cells incubated with 0.1 or 1 nM BMP-9 and its derived peptides plus RA for 3d and 5d had significantly increased the metabolic activity.

Figure 2 Effect of pBMP-9 and SpBMP-9 on the metabolic enzymatic activity and indirect cell counting. (A) MTS assays of SH-SY5Y cells stimulated for 1d, 3d and 5d with equimolar concentrations (0.1 and 1 nM) of BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA in serum-free culture medium (⬨p < 0.05 and ⬨⬨⬨p < 0.001 compared with the control without RA, *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the control with RA). Results are the means ± SEM of at least two independent experiments performed in duplicate. (B) Indirect cell counting showing the relative fluorescence intensity of the stained nuclei in cells used to perform the MTS assays (*p < 0.05, ** p < 0.01, ***p < 0.001). (C) Indirect cell counting results plot in function of the MTS enzymatic assay results showing a correlation of 0.7. (D) Cell nucleus staining in SH-SY5Y cells stimulated by BMP-9, pBMP-9 or SpBMP-9 (0.1 and 1 nM) in the presence of 10 μM RA for 5d (Bar = 100 μm). Full size image

Effect of pBMP-9 and SpBMP-9 on cell number

Cell nucleus was stained with Hoechst 33342 to verify whether the observed increase in metabolic activity were caused by increases in cell numbers (Fig. 2B). In the absence of RA, there was no significant difference between experimental conditions, neither in function of time. In the presence of RA, after 5d of incubation, cells stimulated with BMP-9 (1 nM), pBMP-9 (0.1 nM) or SpBMP-9 (0.1 nM) had a significant increased amount of nucleus relative fluorescence intensity in comparison with the control, those results being in accordance with the metabolic enzymatic assay. There was a determination coefficient of 0.7 between indirect cell counting and MTS assay results, which indicates that the metabolic activity might be related to the number of cells (Fig. 2C).

However, since the cell number was evaluated indirectly by measuring the relative fluorescence intensity of the nucleus staining, we then looked at the cell nucleus in a higher magnification to evaluate if the increase in the cell number in experiments performed with RA could rather be explained by the presence of pyknotic cell nuclei (Fig. 2D). There were some pyknotic nuclei for cells stimulated for 5d in the presence of BMP-9, pBMP-9 and SpBMP-9 plus RA. From those results, it appears that cells stimulated with BMP-9, pBMP-9 and SpBMP-9, especially in the presence of RA increase their metabolic activity, which is not caused by an increase in cell number.

pBMP-9 and SpBMP-9 affect the morphology of SH-SY5Y cells and increase their neurites outgrowth

Effect of pBMP-9 and SpBMP-9 on cell morphology

The impact of BMP-9, pBMP-9 and SpBMP-9 on neuron well-being and the differentiation of cells like SH-SY5Y require an examination of the cell morphology (Fig. 3A). Cells incubated in serum free-medium with BMP-9, especially at 1 nM, reacted more like neuroblastoma cancerous cells, forming clusters with less neuron-like morphology. Cells stimulated with pBMP-9 and SpBMP-9 show an increase amount of neurite outgrowth. RA had a dramatic effect on cell morphology under all experimental conditions: it increased neurite outgrowth and displayed more neuronal morphology. Cells stimulated with pBMP-9 or pBMP-9 plus RA had more interconnected neurites than did control plus RA.

Figure 3 Effect of pBMP-9 and SpBMP-9 on the morphology of SH-SY5Y cells and neurite length. (A) Modified phase-contrast pictures of SH-SY5Y cells stimulated for 5d with equimolar concentrations (0, 0.1 and 1 nM) of BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA. Pictures are representative of at least 3 independent experiments performed in duplicate. Neurites and cell bodies were highlighted with an edge-detection filter and superimposed (blue edges) on the original phase contrast image (Bar = 100 μm). (B) Average neurite length of SH-SY5Y cells stimulated for 5d with equimolar concentration of BMP-9, pBMP-9 or SpBMP-9 (0.1 and 1 nM) +/− 10 μM RA determined as the Euclidean distance between the end of neurites and the cell body. Results are the means ± SEM of at least 3 independent experiments performed in duplicate, where a total of over 300 measurements were taken per experimental condition (*p < 0.05, **p < 0.01, ***p < 0.001). Full size image

We then measured the average length of neurites under each experimental condition (Fig. 3B). SH-SY5Y cells stimulated with pBMP-9 (at least p < 0.01) or SpBMP-9 (p < 0.001) without RA had longer neurites than those stimulated with BMP-9 or the CTL. Adding RA increased the neurite length under all experimental conditions. Cells stimulated with 0.1 nM BMP-9 or 0.1 and 1 nM pBMP-9 and SpBMP-9 plus RA had significant (p < 0.001) longer neurites than the corresponding CTL, whereas the neurites of cells stimulated with 1 nM BMP-9 plus RA were not significant different from those of CTL. The lower concentrations (0.1 nM) of both pBMP-9 (p < 0.05) and SpBMP-9 (p < 0.01) stimulated the formation of significantly longer neurites than did the higher concentration (1 nM).

pBMP-9 and SpBMP-9 increase the expression of early and late neuronal differentiation markers

Effect of stimulation time on neuron markers expression

Since pBMP-9 and SpBMP-9 had an effect on the morphology of SH-SY5Y cells inducing neurite formation characteristic of the neuron phenotype, we used Western blots to evaluate the expression of MAP-2 protein, an early marker of neuron differentiation38 (Fig. 4A). There was significantly more of MAP-2 in cells stimulated with pBMP-9 and SpBMP-9 (0.1 nM) without RA for 5d than in the CTL as shown by the densitometric analyses (p < 0.05). There was a significant increase in MAP-2 in cells stimulated for 3d with pBMP-9 or SpBMP-9 plus RA in comparison to BMP-9 plus RA (p < 0.01). Finally, the expression of MAP-2 decreased between 3d and 5d in the CTL (p < 0.05) and in cells treated with SpBMP-9 (p < 0.05), while it increased in cells stimulated with BMP-9 (p < 0.05). The MAP-2 levels in cells stimulated with pBMP-9 for 3d and 5d were the same.

Figure 4 Effect of pBMP-9 and SpBMP-9 on the differentiation of SH-SY5Y cells. (A) Western blots of MAP-2 and βactin (2 independent experiments) and densitometric analyses of MAP-2 bands normalized to βactin (means ± SEM) for SH-SY5Y cells stimulated with 0.1 nM BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA for 3d and 5d (*p < 0.05, **p < 0.01). Only cropped pictures of western blots showing the 80 kDa MAP-2 isoform were presented in order to allow a better comparison between experimental conditions. Complete gel pictures are available in the supplementary data file. (B) Merged pictures showing immunostaining for neuronal differentiation markers βIII-tubulin (Alexa Fluor® 488, green), MAP-2 (Alexa Fluor® 488, green), NeuN (FITC, green) and NSE (FITC, green), and nuclei staining (Hoechst, blue) of SH-SY5Y cells stimulated for 5d with 0, 0.1 or 1 nM BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA. Pictures are representative of at least two independent experiments (Bar = 100 μm). Full size image

Neuron markers distribution

Knowledge of the distribution of neuron differentiation markers in cells is essential for confirming the neuron phenotype. For example, NeuN is specific to neuron cells and is restricted to the cell nucleus39, 40. We used immunolabelling to determine the effect of BMP-9, pBMP-9 and SpBMP-9 on the expression and distributions of the markers of early (βIII-tubulin, MAP-2) and late neuron differentiation (NeuN and NSE) in cells incubated for 5d (Fig. 4B).

We detected βIII-tubulin under all experimental conditions, but it was most prominent in cells stimulated with RA for all experimental conditions. BMP-9 without RA produced less intense labelling regardless of its concentration. However the cells stimulated with pBMP-9 or SpBMP-9 contained more markers than did the control or those stimulated with BMP-9 with or without RA.

MAP-2 was most abundant in the neurites of SH-SY5Y cells stimulated with 0.1 or 1 nM pBMP-9 or SpBMP-9, especially in the presence of RA. Since MAP-2 is a microtubule-associated protein, an increase in its expression and its distribution in the neurites is associated with greater neuronal differentiation38. Staining for the marker of late differentiation, NeuN, in cell nuclei39, was more intense in cells stimulated with pBMP-9, and especially SpBMP-9, with or without RA, than in cells stimulated with BMP-9 or the CTL (Fig. 4B). SH-SY5Y cells stimulated with 1 nM BMP-9 were poorly stained, especially those incubated without RA. However, there was some staining in the cell body around the nucleus. This could be due to the presence of synapsin I, which is recognized by NeuN antibodies41. Finally, incubation with pBMP-9 or SpBMP-9 produced the best staining for NSE, a late marker located in the cell cytoplasm, regardless of their concentration or the presence of RA.

These results indicate that pBMP-9 and SpBMP-9 induce more rapid neuron differentiation than the whole BMP-9 protein or the CTL, especially in the presence of RA. However, greater neuron differentiation does not necessarily mean that cells are moving toward the cholinergic phenotype.

SpBMP-9 increases the expression of choline acetyltransferase, vesicular acetylcholine transporter protein and the level of intracellular acetylcholine

BMP-9 activates the cholinergic transcriptome of murine septal cells and basal forebrain cholinergic neurons and increase the levels of Ach and the vesicular acetylcholine transporter protein (VAchT)11, 25, 26. Ach is synthesized in the cell body by fusion of acetyl Co-A and choline catalysed by choline acetyltransferase (ChAT); it is then transported to vesicles by VAchT. The ChAT and VAchT genes are conserved at the same locus, which suggests that their expressions are coordinated42. We investigated the effect of pBMP-9 and SpBMP-9 on the induction and the maintenance of the cholinergic phenotype since cholinergic dysfunction is a major hallmark of AD (Figs 5–7).

Figure 5 Effect of pBMP-9 and SpBMP-9 on the expression of choline acetyltransferase. (A) Merged pictures showing immunostaining for ChAT (FITC, green) and nuclei labelling (Hoechst, blue) of SH-SY5Y cells stimulated for 5d with 0, 0.1, or 1 nM BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA (Bar = 100 μm). Pictures are representative of at least 2 independent experiments. (B) Analysis of ChAT fluorescence intensity relative to the nucleus staining was also presented. Results are the means ± SEM (***p < 0.001). Full size image

Figure 6 Effect of pBMP-9 and SpBMP-9 on the expression and the distribution of VAchT. Merged pictures showing immunostaining for VAchT (FITC, green), actin cytoskeleton (rhodamine-phalloidin, red) and nuclei labelling (Hoechst, blue) of SH-SY5Y cells stimulated for 5d with 0, 0.1 or 1 nM BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA. White squares show magnified zones, white arrows indicate VAchT vesicles in cell neurites (Bar = 100 μm). Pictures are representative of at least 2 independent experiments. Full size image

Figure 7 Effect of pBMP-9 and SpBMP-9 on the intracellular Ach and AchE. (A) Intracellular Ach in SH-SY5Y cells stimulated with 0, 0.1 or 1 nM BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA for 3d and 5d (B) AchE activity for SH-SY5Y cells stimulated with 0, 0.1 or 1 nM BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA for 5d. Results are the means ± SEM of at least 4 independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001). Full size image

Effect of pBMP-9 and SpBMP-9 on choline acetyltransferase

The ChAT enzyme responsible for converting acetyl-co-A and choline to acetylcholine in SH-SY5Y cells incubated with BMP-9 or its derived peptides with or without RA was detected by immunolabelling (Fig. 5A and B). We found ChAT immunostaining in all cell bodies under all experimental conditions (Fig. 5A). However, the intensity of labelling differed, especially when the cells were stimulated with SpBMP-9 without RA. The CTL without RA had the lowest ChAT staining, while cells incubated with 0.1 nM SpBMP-9 had the highest ones as confirmed by the relative fluorescence intensity analysis (p < 0.05) (Fig. 5B). Cells stimulated with BMP-9 and pBMP-9 had similar fluorescence intensities as the control, which were lower than that of SpBMP-9-stimulated cells. However, no difference in relative fluorescence intensities was observed when the same assays were run in the presence of RA.

Effect of incubation time and pBMP-9 or SpBMP-9 dose on intracellular acetylcholine concentration and VAchT expression and distributions within cells

VAchT in the axon terminals plays an important role in the accumulation of Ach prior to its release43. We immunolabelled VAchT to evaluate the effects of SpBMP-9 and pBMP-9 on its expression and distribution in the cells (5d, Fig. 6). The presence of labelled VAchT in small vesicles within the neurites indicates a cholinergic differentiation. Only cells stimulated with 0.1 nM or 1 nM SpBMP-9 (without RA) had VAchT vesicles in their neurites, with 1 nM being more efficient. RA alone significantly stimulated VAchT accumulation in the neurites. Cells stimulated with 0.1 nM BMP-9, 0.1 and 1 nM pBMP-9 and SpBMP-9 plus RA all had similar amounts and distributions of VAchT vesicles in the neurites. However, cells stimulated with 1 nM BMP-9 contained no vesicles.

We then measured intracellular Ach to determine the abilities of BMP-9 and its derived peptides to stimulate the synthesis of Ach in SH-SY5Y cells (Fig. 7A). Only 1 nM SpBMP-9 (p < 0.01) and pBMP-9 (p < 0.05) significantly stimulated Ach synthesis after incubation for 3d without RA. The concentration of Ach in SH-SY5Y cells stimulated with 1 nM SpBMP-9 for 5d was also higher than that in unstimulated cells (p < 0.05) or cells stimulated with 0.1 nM BMP-9 (p < 0.01). Cells incubated for 3d in the presence of RA plus 1 nM BMP-9, 0.1 or 1 nM pBMP-9 or SpBMP-9 contained more Ach than did the CTL, while those incubated with SpBMP-9 had the highest Ach concentration (p < 0.001). For the SH-SY5Y cells stimulated with BMP-9, pBMP-9 or SpBMP-9 for 5d in the presence of RA, intracellular Ach levels, except for cells stimulated with 1 nM of BMP-9, were similar to the CTL plus RA.

Effect of pBMP-9 and SpBMP-9 on acetylcholinesterase activity

We measured AchE activity to determine the action of SpBMP-9 on Ach breakdown (Fig. 7B). AchE cleaves the neurotransmitter released into the synaptic clef to give choline and acetic acid; the choline is then recycled back into the cells. Only the SH-SY5Y cells incubated for 5d with SpBMP-9, but no RA, contained AchE activity that was significantly enhanced in comparison to the CTL (p < 0.05) (Fig. 7B). However, the presence of RA increase significantly in all experimental conditions the relative AchE activity compared to the cells without RA (p < 0.001). In addition, cells incubated with BMP-9 or its derived peptides plus RA had AchE activities similar to the control plus RA.

pBMP-9 and SpBMP-9 increase the activation PI3K/Akt pathway and inactivate GSK3β

Since SpBMP-9 has been shown to induce a higher cholinergic differentiation of SH-SY5Y cells, we then evaluated the effect of SpBMP-9 on the activation state of GSK3β, a Tau kinase44. As GSK3β can be inactivated by Akt-catalysed phosphorylation of its Ser944, 45, we first analyzed the activation state of Akt in cells incubated with BMP-9, pBMP-9 or SpBMP-9 with and without RA.

Phosphorylation of Akt at Thr308

We analyzed the effect of equimolar concentration of BMP-9, pBMP-9 or SpBMP-9 (0.1 nM) with or without RA on the phosphorylation of Akt at its catalytic site (Thr308) by immunostaining (Fig. 8A). Cells stimulated with pBMP-9 or SpBMP-9 showed a higher level of fluorescence corresponding to pAkt (Thr308) compared to unstimulated cells or those incubated with BMP-9, as confirmed by relative fluorescence intensity analyses of these immunolabellings (p < 0.001). SpBMP-9 was also most effective than pBMP-9 (p < 0.01). In the presence of RA, Akt was phosphorylated at Thr308 in all experimental conditions. BMP-9 or pBMP-9 plus RA induced slightly less pAkt on Thr308 in comparison to RA alone (p < 0.001).

Figure 8 Effect of pBMP-9 and SpBMP-9 on the PI3K/Akt/GSK3β pathway. (A) Merged pictures representative of at least 2 independent experiments showing immunostaining for pAkt (Thr308) (green) and nuclei (Hoechst, blue) of SH-SY5Y cells stimulated for 2 h with 0.1 nM BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA (Bar = 100 μm) and pAkt(Thr308) fluorescence activity relative to the nucleus. (B) Western blots of phosphorylated GSK3β at Ser9 (pGSK3β) and densitometric analysis of pGSK3β bands standardized by actin showing the effect of 0.1 nM BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA in SH-SY5Y cells after incubation for 0, 15, 30, 60, 120 and 240 min. Only cropped pictures of western blots were shown in order to allow a better comparison between experimental conditions. Complete gel pictures are available in the supplementary data file. (C) Merged pictures representative of at least 2 independent experiments showing immunostaining for pGSK3β (Ser9) (FITC, green) and nuclei (Hoechst, blue) in SH-SY5Y cells stimulated for 4 h with 0.1 nM BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA (Bar = 100 μm). Analysis of pGSK3β(Ser9) fluorescence intensity relative to the nucleus staining was also presented. Results are the means ± SEM (**p < 0.01, ***p < 0.001). Full size image

Inactivation of GSK3β by its phosphorylation at Ser9

We used Western blotting to assess phosphorylated GSK3β (pGSK3β) on Ser9 to determine whether Akt activation leads to inactivation of GSK3β (Fig. 8B). BMP-9, without RA, had a transient effect as confirmed by densitometric analysis of bands corresponding to pGSK3β and standardized to that of β actin: pGSK3β (Ser9) increased between 0 and 60 min, plateaued and then decreased from 120 to 240 min. Incubation with pBMP-9 and SpBMP-9 gave a different activation pattern. The pGSK3β (Ser9) in cells incubated with either pBMP-9 or SpBMP-9 increased from 0 to 240 min. Cells incubated with pBMP-9 or SpBMP-9 plus RA had similar time-dependent increases in pGSK3β (Ser9). pGSK3β (Ser9) was detected after 240 min in cells incubated with BMP-9, pBMP-9 or SpBMP-9 plus RA. We confirmed these observations by immunostaining for pGSK3β (Ser9) at 4 h (Fig. 8C). pBMP-9 and SpBMP-9 induced a higher level of pGSK3β (Ser9) than BMP-9 or the CTL, as confirmed by relative fluorescence intensity analysis (p < 0.001 and p < 0.01 respectively). In addition, cells incubated with RA alone or combined with BMP-9 or its derived peptides contained more phosphorylated GSK3β on Ser9 than did cells stimulated without RA (p < 0.001).