GDF11 induces differentiation and apoptosis and inhibits migration of C17.2 neural stem cells via modulating MAPK signaling pathway

GDF11, a member of TGF-β superfamily, has recently received widespread attention as a novel anti-ageing/rejuvenation factor to reverse age-related dysfunctions in heart and skeletal muscle, and to induce angiogenesis and neurogenesis. However, these positive effects of GDF11 were challenged by several other studies. Furthermore, the mechanism is still not well understood. In the present study, we evaluated the effects of GDF11 on C17.2 neural stem cells. GDF11 induced differentiation and apoptosis, and suppressed migration of C17.2 neural stem cells. In addition, GDF11 slightly increased cell viability after 24 h treatment, showed no effects on proliferation for about 10 days of cultivation, and slightly decreased cumulative population doubling for long-term treatment (p < 0.05). Phospho-proteome profiling array displayed that GDF11 significantly increased the phosphorylation of 13 serine/threonine kinases (p < 0.01), including p-p38, p-ERK and p-Akt, in C17.2 cells, which implied the activation of MAPK pathway. Western blot validated that the results of phospho-proteome profiling array were reliable. Based on functional analysis, we demonstrated that the differentially expressed proteins were mainly involved in signal transduction which was implicated in cellular behavior. Collectively, our findings suggest that, for neurogenesis, GDF11 might not be the desired rejuvenation factor, but a potential target for pharmacological blockade.

154 cells were served as control. 155 The cell morphology and viability were examined using LIVE/DEAD ® viability / 156 cytotoxicity kit (catalog No.L3224) for mammalian cells (Invitrogen, USA) according to 157 the manufacturer's instructions under inverted fluorescence microscope (AXIO, Zeiss, Jena, 158 Germany). The live cells were stained with calcein AM in green, and the dead cells were stained 159 with ethidium homodimer-1(EthD-1) in red. 161 Cell viability was assessed by CCK-8 assay. Briefly, 10 μL of CCK-8 agent was added to each 162 well 2 h before the termination of the experiment. The optical density (OD) values at 450 nm 163 were determined using SpectraMax M2 e (Molecular Devices, Sunnyvale, USA). Then, by 164 comparing the absorbance of GDF11-treated and untreated cells, percentage viability was 165 calculated.

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For proliferation assay, 1*10 4 /mL cells were seeded in 12-well plates in triplicates. When the 167 cell cultured to ~80% confluence (generally 3 days), cells were trypsinized and manually 168 counted using a haemocytometer. Cell population doubling (PD) was calculated using the 169 following formulae:

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(1)PD = log2 (N/N 0 ), 171 where N 0 represents the number of cells seeded at the initial passage, N is the final number of 172 cells.  C17.2 cells were cultured with complete medium in a 48-well plate at a density of 5 × 182 10 4 cells/well. After reaching ~80% confluence, a single uniform scratch was made by using a 183 200μL pipette tip along the center of each monolayer. The scratch was lightly washed with PBS 184 twice to remove the detached cells, and then starved medium supplemented with various 185 concentrations of GDF11 was added (0ng/mL, 12.5ng/mL, 25ng/mL, 50ng/mL and 100ng/mL, 186 respectively). The scratches were monitored at 0h, 12h and 36h after scratching by taking photos 221 2.9 Western Blot analysis and validation 222 C17.2 cells were cultured in 6-well dishes in starved medium with or without GDF11 for 24h. 223 Then, the cells were lysed in RIPA buffer containing 1× phosphatase inhibitor cocktail and 1× 224 protease inhibitor cocktail on ice for 30 min, and centrifuged at 14000 g for 5 min at 4°C. The 225 supernatants were collected, and the protein concentration was determined by BCA protein assay 226 kit. The samples were mixed with 4× NuPAGE LDS loading buffer, separated on NuPAGE 4-12% 227 Bis-Tris gels, and subsequently transferred to PVDF membranes by a wet transfer apparatus 228 (Bio-Rad, Hercules, USA). Following blocking with superblock at room temperature for 2h, the 229 membranes were incubated with rabbit anti-β-actin (1:1000), anti-Smad2/3 (1:1000), anti-p-   WoLF PSORT (a subcellular localization predication tool, version of PSORT/PSORT II) and 242 SubLoc ( http://www.bioinfo.tsinghua.edu.cn/SubLoc/ ) were used to predict subcellular 243 localization of all identified differentially expressed proteins.

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The domain functional description of the differentially expressed proteins were annotated by 245 InterProScan (a sequence analysis application) based on protein sequence alignment method, and 246 the InterPro domain database was used (http://www.ebi.ac.uk/interpro/).

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GO annotation was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/), 248 and the differentially expressed proteins were classified by GO annotation based on the three 249 categories (GO term level 1): biological process, cellular component and molecular function. 250 According to GO annotation information of the identified proteins, we summed up the amount of 251 the differentially expressed proteins in each GO term of level 2.

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The protein-protein interaction networks and pathways were annotated by Kyoto Encyclopedia 253 of Genes and Genomes (KEGG) database.
The results were presented as the mean ± standard error (SE). Multi-group comparisons were 256 performed by one-way ANOVA followed by Tukey's post hoc test. Paired analysis of control 257 and treatment was accomplished using two-tailed unpaired or unpaired Student's t -tests when 258 appropriate. In addition, Statistical analyses were conducted using SPSS statistics 259 software,version 17.0 (SPSS Inc., Chicago, USA), and p < 0.05 was considered statistically 260 significant.. When compared with the counts of C17.2 cells initially seeded, both GDF11-and vehicle-265 treated cells significantly proliferated after 72h of cultivation ( Supplementary Fig. S1). Imaging 266 revealed that GDF11 significantly altered the morphology of C17.2 cells (Fig.1a). Cells without 267 GDF11 treatment remained their native neural stem cell state (Fig.1a, control), whereas cells 268 treated with various concentrations of GDF11 showed visual outgrowth of neuritis, displaying 269 phenotypes similar to neuron-and astrocyte-like cells (Fig.1a, GDF11). Remarkably, compared 270 to the control, supplement with high concentrations of GDF11 (50 and 100 ng/mL) significantly 271 resulted in morphological changes (differentiation and apoptosis) (Supplementary Fig.S1a).

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To investigate the effect of GDF11 on cell viability, C17.2 cells were treated with indicated 273 concentrations of GDF11 (0, 12.5, 25, 50 and 100 ng/mL) for a 72h period, followed by CCK-8 274 assays. GDF11 slightly increased (less than 10%, p<0.05) cell viability after 24h treatment, 275 whereas it did not affect the cell viability after 72h treatment (Fig.1c). 276 As displayed in Fig.1d, all groups of C17.2 cells showed robust proliferation for the 6-passage 277 duration. GDF11 showed no effect on C17.2 cell proliferation till the 4 th passages. From the 5 th 278 passage, the low concentrations of GDF11 (12.5 and 25 ng/mL) still didn't affect the 279 proliferation of C17.2 cells, whereas higher concentrations of GDF11 (50 and 100 ng/mL) 280 significantly inhibited cell proliferation (p<0.05) and the exposure of C17.2 cells to 100 ng/mL 281 GDF11 resulted in the lowest cumulative population doubling level during the 6 passages of 282 cultivation amongst the 5 groups, which was approximately 17% lower than control (p<0.05). 283 Next, we detected the mRNA expression of cyclin D1 and cyclin D2, the cell cycle-related 284 proteins. GDF11 slightly but not significantly attenuated the expression of cyclin D1 and cyclin 285 D2 in the mRNA levels ( Fig. 2d; p>0.05). These provide a potential molecular basis for the 286 effects of GDF11 on C17.2 cell viability and proliferation.

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Together, these results revealed that GDF11 slightly increased cell viability after a short-term 288 (24h) cultivation and showed no effect on cell viability from 1 st to 4 th passage of cultivation 289 (approximately 10 days), whereas high concentrations of GDF11 significantly suppressed 290 cumulative population doubling for a long-term treatment.

3.2 GDF11 induced differentiation and apoptosis of C17.2 cells 292
The mRNA levels of the neural progenitor cell marker, nestin, were noticeably decreased after 293 being treated with GDF11, as compared to control levels ( Fig. 2a; p<0.01). By contrast, the 294 GDF11-treated groups showed significant increase in βIII-tubulin (neuronal biomarker) and 295 GFAP (astrocytic biomarker) mRNA expression as compared to the control (Fig. 2a; p<0.05). 296 These all indicated the maturation and differentiation of C17.2 neural stem cells.
The 297 differences in nestin mRNA expression among the groups of GDF11 treatment were, however, 298 not significant, similar to βIII-tubulin and GFAP. Concomitantly with the mRNA expression, the 299 protein levels of nestin, βIII-tubulin and GFAP confirmed the similar results by western blot (Fig.  300 2b and c). When compared with the control, GDF11-treated cells showed the protein level of 301 nestin was significantly attenuated whereas βIII-tubulin and GFAP were up-regulated (Fig. 2b  302 and c), further indicating that GDF11 induced neuronal and astrocytic differentiation. However, 303 no dose-dependent effect of GFD11 was observed.

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The results of Annexin V-FITC/PI dual staining revealed that GDF11 substantially induced 305 apoptosis of C17.2 cells. As shown in Fig. 1b and e, the number of total (both early and late) 306 apoptotic cells significantly increased in a GDF11 dose-dependent manner. After 72h of 307 cultivation, the apoptotic cells were negligible in C17.2 cells without GDF11-treated, whereas 308 there were 2.1%, 9.8%, 13.1% and 17.7% of cells exhibiting apoptosis as a result of exposure to 309 12.5, 25, 50 and 100ng/mL GDF11, respectively (p<0.05). Meanwhile, the amount of necrotic 310 cells showed a slight but significant increase when treated with GDF11. The migration of C17.2 cells was performed by a "scratch wound healing'' assay. The wound 313 closure data are shown in Fig.3. It was observed that the wound closure increased as cell 314 migration progressed over time. After 12 h, the wound area had little difference compared to the 315 initial scratch area. As compared with that of 0h, wound area of 36h significantly decreased, 316 displaying 25.1% (0 ng/mL GDF11), 64.9% (12.5 ng/mL GDF11), 60.4% (25 ng/mL GDF11), 317 70.9% (50 ng/mL GDF11) and 75.7% (100 ng/mL GDF11) wound area, respectively (Fig.3b). 318 These implied wound closure was significantly inhibited when cells were treated with GDF11. 319 Of note, it was revealed that GDF11 showed slight but significant dose-dependent effects in the 320 inhibition of the migration. Together, these results demonstrated that GDF11 significantly 321 suppressed (but not completely abolished) the migratory potential of C17.2 neural stem cells. 322 3.4 GDF11 activated phosphorylation levels of selected signaling kinases 323 We deduced that, in C17.2 cells, GDF11 transmitted signals through phosphorylation of 324 Smads, as GDF11 belongs to TGF-β superfamily. First of all, we analyzed the effects of GDF11 As shown in Table 1, the differentially expressed proteins were mainly classified as 356 cytoplasmic (n=8), nuclear (n=7) and mitochondrial (n=1) proteins.

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For an overview of the differentially expressed proteins, GO annotation was carried out to 358 identify the significantly enriched GO functions. According to the analysis, the 15 differentially 359 expressed proteins between GDF11-treated cells and control were mainly clustered into 38 360 functional groups, including 18 biological processes, 12 cellular components, and 8 molecular 361 functions (Fig.6a).  N=3, *p<0.05versus with vehicle control and **p<0.01versus with vehicle control.