Kinase inhibit region of SOCS3 attenuates IL6‐induced proliferation and astrocytic differentiation of neural stem cells via cross talk between signaling pathways

Abstract Aims Efficiency of neural stem cells (NSCs) therapy for brain injury is restricted by astrogliosis around the damaged region, in which JAK2/STAT3 signaling plays a key role. The SOCS3 that can directly inhibit JAK/STAT3 pathway. Here, we investigated the effects of a fusion peptide that combined kinase inhibitory region (KIR) of SOCS3 and virus trans‐activator of transcription (TAT) on biological behavior of cultured NSCs under inflammatory conditions. Methods NSCs were isolated from embryonic brain of SD rats, TAT‐KIR was synthesized, and penetration rate was evaluated by flow cytometry (FACS). CCK8, immunostaining, and FACS were used to detected of TAT‐KIR on the proliferation of NSCs. The expressions of GFAP and β tubulin III positive cells induced by IL6 with/without TAT‐KIR were examined by immunostaining and Western blotting to observe the NSCs differentiation, and the effect of TAT‐KIR on signaling cross talk was observed by Western blotting. Results Penetration rate of TAT‐KIR into primary cultured NSCs was up to 94%. TAT‐KIR did not affect the growth and viability of NSCs. It significantly reduced the NSCs proliferation that enhanced by IL‐6 stimulation via blocking the cell cycle progression from the G0/G1 to S phase. In addition, TAT‐KIR attenuated astrocytic differentiation and kept high level of neuronal differentiation derived from IL‐6‐induced NSCs. The fate of NSCs differentiation under inflammatory conditions was affected by TAT‐KIR, which was associated with synchronous inhibition of STAT3 and AKT, while promoting JNK expression. Conclusion TAT‐KIR mimetic of SOCS3 could be a promising approach for brain repair via regulating the biological behaviors of exogenous NSCs.


| INTRODUC TI ON
Brain injury is a common disease with poor prognosis. The outcomes of brain injury result from the degree of destruction or degeneration of neuronal plasticity. 1,2 NSCs are capable of self-renewal and differentiating into diverse types of neural cells. It not only provides a cellular reservoir for the replacement of lost/damaged cells, but also possesses several intrinsic capacities to release some neurotrophic factors. 3,4 Therefore, NSCs transplantation has been considered as an ideal therapeutic strategy for brain injury. According to the characteristics of NSCs, during neural development, NSCs differentiate into specific types of neural cells in response to the local developmental cues. 5 Along with the maturation, the endogenous NSCs get fewer and quiescent. Up to adulthood, the amount of NSCs is limited and insufficient to compensate for the cell loss after injury. 6 Therefore, exogenous NSCs are required and considered as a key role for cell replacement after brain injury. 2,7 Although some studies have presented that neuronal functions could be improved after NSCs implantation, NSC-based cell therapy for brain injury still faces multiple challenges. 2,[7][8][9] One of the challenges is that the local inflammatory environment induces astrogliosis and hinders the neuronal regeneration and limits the therapeutic effect of NSCs transplantation. 10 Therefore, effective alleviation of local inflammatory astrogliosis and promotion of neuronal regeneration could be more stirring for neuronal function restoration after brain injury. 11,12 Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway is implicated in a variety of inflammatory response under multiple physiological and pathological conditions. JAK2/STAT3 is highly expressed during development and plays important roles in embryonic cell growth. 13,14 In adulthood, JAK2/STAT3 pathway is also responsible for proliferation and differentiation of exogenous neural cells in vivo and in vitro treating for most neural disorders, including epilepsy, brain cancer, lesion, ischemia, and neurodegenerative disease. [10][11][12] It is involved in both neurogenesis and neuroregeneration to regulate NSCs' biological behaviors. [15][16][17][18] Activation of JAK2/STAT3 pathway by inflammatory cytokines, such as interleukin 6 (IL-6), leukemia inhibit factor (LIF) and interferons (IFNs), 10,19 triggers the differentiation of NSCs into glial cells, particularly astrocytes. Conversely, repressing JAK2/STAT3 could reduce astrogliosis and enhance neuronal differentiation. 18,[20][21][22] The one of most useful elements for regulation JAK2/STAT3 pathway is inevitably involved in suppressor of cell signaling 3 (SOCS3). [23][24][25] SOCS3 is a 225 amino acid protein, and it is known to suppress JAK2 activity through direct binding to the JAK2 catalytic center and promotion of the proteasome degradation of JAK2. 25 It directly inhibits IL-6-induced activation of JAK2/STAT3 inflammatory pathway via SOCS3/JAK2/gp130 complexes. 23,26 Therefore, regulation of SOCS3 is considered as a candidate approach to reduce brain astrogliosis and promote neuron survival both in vitro and in vivo. 21,22,[27][28][29] As well known, the kinase inhibitory region (KIR) is the central domain of SOCS3 and consists of 8-12 amino acid sequences that directly inhibit JAK2. 23 Currently, there are no studies on direct use of this small peptide fragment KIR to inhibit the differentiation of NSCs into glial cells in exogenous NSCs niche.
In current study, we directly applied KIR of SOCS3 to inhibit the activation of JAK2/STAT3 signaling and then subsequently to observe the biological behaviors of NSCs in inflammatory condition.
A fusion peptide was constructed by using TAT to lead the penetration of cell membrane, 30 and the IL-6 was used to induce the inflammatory responses. We found that peptide TAT-KIR as the mimetic of SOCS3 has the capacity to enter NSCs and help to inhibit the excessive proliferation of NSCs in inflammatory condition. TAT-KIR attenuates astrocytic differentiation of NSCs by inhibiting STAT3 and AKT in sync, while promoting neuronal differentiation via upregulation of JNK2 in inflammatory condition. This study tested the feasibility of TAT-KIR applied to NSCs' replacement therapy in vitro to provide a potential strategy for repair of brain injury.

| Embryonic NSCs isolation and culture
Tissues were dissected from the cerebral cortex of SD rat embryos on embryonic day 14 (E14). Single-cell suspension was acquired by gently trituration with pipette. Cells were cultured in DMEM/F12 (the Dulbecco's modified Eagle's medium and Ham's F12, 1:1) serumfree growth medium that contained 10 ng/ml of the basic fibroblast growth factor, 20 ng/ml of the epidermal growth factor, 1% penicillin, 1% streptomycin, 1% N2, 2% B27 supplement (all from Invitrogen), and incubated in 5% CO 2 at 37°C, following the standard protocol 31 and optimized in our laboratory. 32,33 Cells were sub-cultured after 5-7 days in vitro (DIV) when the spheres were clearly visible.

| NSCs identification
For observation of spontaneous differentiation, the cell aggregates were trypsinized into single cells and seeded on poly-L-lysinecoated coverslips at a density of 0.5 × 10 4

| Penetrating rate of TAT-KIR into NSCs analysis
The penetration rate of the fusion peptides was measured via FACS analysis. NSCs 2 × 10 5 cells/well were cultured in 6-well plate with growth medium for 3 days. NSCs were randomly allocated in the experimental groups: TAT-KIR-FITC and KIR-FITC. Fusion peptides were added into the NSCs (3 μM) and incubated for 30 min in dark.
Then NSCs were washed with PBS to remove the non-penetrated peptides before the measuring of FITC fluorescence by FACS.
All NSCs seeded into 96-well plates at 5 × 10 4 cells/ml and cultured in growth medium for 24 h. IL-6 in the concentration of 100 ng/ ml was applied to mimic inflammatory condition. Thirty minutes later, TAT-KIR or TAT-scramble was added into the culture system.

Cell viability of each group was observed by Universal Microplate
Spectrophotometer (QuantTM, BioTek) at first, third, and fifth day after peptide administration ( Figure 3A). Optical density (OD) values at 450 nm were measured. Immunocytochemistry staining with ki67 (#ab16667, Abcam, 1:300) was used to assess NSC proliferation. Cell cycles were analyzed by FACS at third day after peptide administration. The rates of cells in G0/G1, S, and G2/M were measured, and the proliferation index (PI) {PI = (S + G2/M)/(G1/G1 + S + G2/M) × 100%} was calculated, respectively. In each independent experiment, the procedures were carried out in triplicate.

| NSCs differentiation assessment
NSCs at 1 × 10 5 cells/ml were seeded into 24-well plates allocated randomly in Control, TAT-KIR, IL-6, and TAT-KIR+IL6 groups. After 24 h culture with differentiation media, IL-6 and TAT-KIR were added into the medium as above order and reapplied every other day ( Figure 4A). Cells were harvested at 7 DIV. Immunocytochemical staining was performed with different antibodies, including GFAP and β Tublin III. Stained cells were observed with fluorescence microscope (BX-51, Olympus) at an operating temperature below 25°C.
Then the numbers of GFAP positive and β Tubulin III positive cells were counted, and the percentages of differentiated cells were calculated by using Image J (version 1.61).

| Western blotting analysis
NSCs at 1 × 10 6 cells/ml in 6-well plate were treated with IL-6 and TAT-KIR as above. Cells were harvested at 3 h, 6 h, 12 h, 24 h, or

| Statistical analysis
All experiments were performed in triplicate. All data were shown as mean ± standard deviation and analyzed with SPSS 18.0 software.
All statistical analyses were performed using Student's unpaired t test and one-way analysis of variance (ANOVA). If the data were homogeneous variance, Least Significant Difference test was used for multiple comparisons of ANOVA; if data were non-homogeneous variance, Dunnett T3 was performed. Nonparametric Kruskal-Wallis tests were performed with data that were not normally distributed.
p < 0.05 was considered as statistically significant. Origin (version 11.0) was used for bar graph plotting.

| Fusion peptide TAT-KIR penetrates into NSCs efficiently
As a key role for cell replacement after brain injury, a sufficient number of NSCs are required. 2,7 According to Fred Gage's research studies and our previous study, NSCs were isolated from developing cerebral cortex of rat embryos on embryonic day 14.5 ( Figure 1A) and cultured in DMEM/F12 (1:1) growth medium. 31,33 Cell aggregates (also called neurospheres) in different sizes developed over a period of 5 days ( Figure 1B). The majority of cells in neurospheres were nestin positive NSCs ( Figure 1C). Those cells could differentiate into β-tubulin III-positive neurons, GFAP-positive astrocytes, and O4positive oligodendrocytes, respectively, after 7 days culture in the differentiation medium ( Figure 1D,E). These results confirmed that the cells we cultured are NSCs.
The inflammatory environment induces astrogliosis and impedes the neuronal differentiation of transplanted NSCs in local regions. 10 Therefore, effective alleviation of local inflammation plays a vital role in NSC-based therapy. 11,12 Since SOCS3 plays a critical role in the inhibition of IL-6-induced activation of JAK2/STAT3 inflammatory pathway, and KIR is the central role domain of SOCS3. 23,26,[34][35][36] In the current study, we constructed a fusion peptide KIR (12 amino acid) with TAT (9 amino acid) and labeled with FITC in N-terminal, 94.73 ± 1.51% as shown by flow cytometry (FACS) analysis. It was significantly higher than penetration rate of KIR-FITC (3.53 ± 0.25%, p = 0.000) ( Figure 2C), while the growth and viability of NSCs had not been affected as shown by the CCK-8 assay ( Figure 2D).

| TAT-KIR inhibits the IL-6-induced excessive proliferation of NSCs
It has been reported that the level of inflammatory cytokines, such as IL-6, LIF, and IFNs, significantly elevated after brain injury. 10 In the current study, IL-6 was used to mimic the inflammatory response after brain injury and to active JAK2/STAT3 pathway 10,19 ( Figure 3A). Consistent with the existed reports that IL-6 could enhance the proliferation of neural progenitors. 37 In our study, the primary cultured embryonic NSCs' proliferation was dramatically promoted after IL-6 induction for 3 days, as shown by the percentage of the Ki67 positive proliferating cell (54.33 ± 10.29% vs. control 24.27 ± 6.06%, p = 0.000). No significant difference between normal control (24.27 ± 6.06%) and TAT-KIR-treated group (20.67 ± 3.74%).
However, the elevation induced by IL-6 was significantly reduced after TAT-KIR application (41.33 ± 7.72% vs. IL6 group, p = 0.047; Figure 3B,C). Similar results were observed in regarding to NSCs growth as shown by CCK-8 assay ( Figure 3D). IL-6-induced OD values of NSCs were strikingly increased from normal level at third and fifth day after drug application (OD value of third day 0.81 ± 0.06, The result of flow cytometry showed the penetration rate of TAT-KIR-FITC was approximately 94%. It was significantly higher than the rate of KIR-FITC by nonparametric test (p = 0.000). Data were expressed as Mean ± SD (n = 3 independent experiments) and were analyzed by Student's unpaired t test. ***p < 0.001 compared to the KIR-FITC group. (D) NSCs viability was measured by CCK-8 assay, and no significant effect was observed after TAT-KIR application. Data were expressed as Mean ± SD and were analyzed by one-way ANOVA followed by Least Significant Difference's test (n = 3).   Figure 3D).
NSCs were cultured in differentiation medium for 7 days. In normal culture condition, TAT-KIR did not affect NSCs differentiation ( Figure 4C-E 36.78 ± 6.81% vs. IL-6 (p = 0.003), and vs. con (p = 0.298; Figure 4D). Together, the excessive astrocytic differentiation of NSCs induced by IL-6 was significantly attenuated by TAT-KIR as we expected, while the effect on neuronal differentiation requires further investigation.

| Cross talk of different signaling pathways during TAT-KIR affecting NSCs biological behaviors
As the key inhibitory region of SOCS3, KIR can directly inhibit activity of JAK2 and lead to a secondary reduce of STAT3 phosphorylation. 23 Our results showed that in the normal culture environment, TAT-KIR did not affect the phosphorylation of STAT3 as well as the astrocytic differentiation of NSCs. Nevertheless, it significantly reduced the IL-6-induced excessive elevated p-STAT3 and the inflammatory reactive proliferation/astrocytic differentiation of NSCs ( Figure 4B-E). Since the mitogen-activated protein kinase (MAPK) signaling pathway is essential in regulating many cellular processes including inflammation, cell stress response, cell division, proliferation, and differentiation. 38 In order to see the potential cross talk between JAK2/STAT3 and MAPK signaling pathways during TAT- is much easier and feasible. However, no study has yet directly used KIR of SOCS3 to regulate neural cellular functions.
In this study, we hypothesized that KIR could be the mimetic of SOCS3 and to inhibit the activation of STAT3 signaling, alter the inflammatory responses, and regulate the biological behaviors of NSCs. A fusion peptide TAT-KIR (24 amino acids) was constructed and applied to alter the IL-6-induced inflammatory effects on NSCs.
TAT is an efficient transmembrane peptide widely used for drug delivery. 30 It could successfully carry various proteins into NSCs and used for neural disorders treatment due to its competitive ability of crossing blood-brain barrier (BBB) and lower cell toxicity. 41 In the current study, the penetration rate of TAT-KIR to NSCs was 94%, and no cell toxicity was found. It absolutely met our requirements.
The effects of TAT-KIR on survival and proliferation of NSCs were explored. Taken the results of cell viability, cell cycle assays, and Ki67 immunostaining together, TAT-KIR significantly reduced the excessive alteration of NSCs division and proliferation that induced by IL-6. Notably, more NSCs arrested in G0/G1 phase, and fewer cells moved to S phase after treated with TAT-KIR. It is consistent with previous reports that stimulation of cytokine receptor enhanced embryonic stem (ES) cell differentiation 14 and G1 to S cellcycle transition. 42 Activation of STAT3 also exhibited a tendency toward ES cell differentiation. 14 This may relate to that p-STAT3 dimer is involved in synthesis of cyclin D1 and mediated the entry of S phase. 14,42,43 Conversely, inhibition of STAT3 activation resulted in the growth of ES cells as undifferentiated clones. 14 In current study, our results suggested that KIR helps to keep NSCs in quiescence in normal environment and could reduce the excessive alteration of NSCs in IL-6-induced inflammatory environment. We then sent out to observe the effect of KIR on NSCs differentiation. After induced by IL-6, astrocytic differentiation of NSCs dramatically increased accompanied with the activation of JAK2/STAT3 signaling pathway. It is consistent with other reports that IL-6 can activate JAK2/STAT3 signaling pathway and lead to reactive astrogliosis and scar formation after neural injury. 37 Regulation of reactive astrocytes in inflammatory microenvironment is pivotal for functional performance of NSCs after transplantation. Several methods that regulating IL-6 and Stat3 had been used to inhibit astrogliosis. IL-6deficient mice have remarkable reduction of activated astrocytes and increase of late neuronal response after axotomy. 16 After Stat3 deletion, NSCs favor to differentiate into neurons rather than astrocytes. 20 Conditional deletion of Stat3 mice had a notable lower level astrocytes reaction, which help axons extending in the early stage after contusive spinal cord injury. 21 Since many reports have used SOCS3 to hinder reactive astrocytes generation, 21,22,[27][28][29]44 in this study, we directly used KIR, the catalytic region of SOCS3, to observe the alteration of NSC biological behaviors in both normal and inflammatory environments.
Our data presented that KIR was able to abolish the IL-6-induced generation of astrocytes successfully. It demonstrated that KIR played an anti-inflammatory role via impeding the activation and regeneration of astrocytes. As regards the effect on neuronal differentiation of NSCs, further investigation is required, although the ascent tendency of expression of β tubulin III was observed after TAT-KIR or IL-6 treatment.  In addition to JAK2/SATA3, MAPK and PI3K/AKT signaling pathways have also been considered as regulators of proliferation and differentiation of several types of cells. 37,40 Our results showed that there were cross talks between JAK2/SATA3 and MAPK, PI3K/AKT signaling pathways. ERK is involved in the regulation of both cell proliferation and differentiation. 43,45 KIR, as the inhibitor of JAK2/ SATA3 signals, could reduce the activation of ERK both in normal and inflammatory conditions. While it kept the level of p-ERK at a balance between normal and inflammatory states. This change was coincided with NSCs proliferation.
JNK and p38 MAPK are the other two members of the MAPKs.
They have been implicated in toxicant-induced apoptosis of neurons. 46 JNK can keep redox homeostasis in stress signal transduction. 47 It also is a key regulator for early neuronal differentiation of NSCs via the cross talk with STAT3 pathway in vitro and in vivo. 48,49 In our study, JNK was activated in a specific way. When the JAK2/ PI3K/AKT pathway has been implicated in proliferation and differentiation of NSCs. Activation of PI3K/AKT could upregulate cell cycle progression mediating G1 to S phase of mitotic cycle in ES cells. 42,43 In the current study, we demonstrated KIR inhibits the phosphorylation of AKT under inflammatory conditions. This change was coincided with phosphorylation of STAT3 and astrocytic differentiation of NSCs.
One of the main limitations in our study is that we only explored the effects of TAT-KIR on NSCs behavior in vitro, there is a lack of in vivo evaluation to confirm the in vitro conclusions. The approaches of exogenous stem cell for CNS tissue repair have many challenges. 50 (1) Drug safety. Although cellular assay of TAT was demonstrated low toxicity, 41 safety assessment of TAT-KIR in vivo is necessary. (2) Drug delivery. In vivo experiments, local injection of TAT-KIR into the injury region of brain would be considered first. However, TAT is competitive in crossing the BBB, 30 intravenous administration should also be attempted as another attractive method that is easy to use clinically. (3) Additionally, there may be differences between in vitro and in vivo results. Therefore, the therapeutic effect of TAT-KIR on transplanted NSCs should be evaluated in vivo to provide more support for future clinical trials.

| CON CLUS ION
In current work, we evaluated the small molecular fusion peptide TAT-KIR has the capacity to enter NSCs and helps to keep NSCs in quiescent in non-inflammatory condition. It significantly impedes the proliferation and astrocytic differentiation of NSCs in IL-6induced inflammatory environment. Meanwhile, TAT-KIR tends to promote the neuronal differentiation. TAT-KIR regulates the activation of ERK, JNK, and AKT by inhibiting JAK2/SATA3 signaling pathway, which changes the proliferation and differentiation of NSCs in vitro (Graphical abstract). Our results provided TAT-KIR could be considered as a promising therapeutic approach for brain repair via regulating the biological behaviors of exogenous NSCs.

ACK N OWLED G M ENTS
This work was supported by the National Nature Science Foundation of China (Grant No. 81571205 and 81500724) and Newton International fellowship (NIF\R1\181649). We thank Prof. X-L C for kindly reading this manuscript. The authors would like to acknowledge X-H Z for technical assistance.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.