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, 6 (6), e21058

SHP-2 Promotes the Maturation of Oligodendrocyte Precursor Cells Through Akt and ERK1/2 Signaling in Vitro

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SHP-2 Promotes the Maturation of Oligodendrocyte Precursor Cells Through Akt and ERK1/2 Signaling in Vitro

Xiujie Liu et al. PLoS One.

Abstract

Background: Oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes (OLs), which are responsible for myelination. Myelin is essential for saltatory nerve conduction in the vertebrate nervous system. However, the molecular mechanisms of maturation and myelination by oligodendrocytes remain elusive.

Methods and findings: In the present study, we showed that maturation of oligodendrocytes was attenuated by sodium orthovanadate (a comprehensive inhibitor of tyrosine phosphatases) and PTPi IV (a specific inhibitor of SHP-2). It is also found that SHP-2 was persistently expressed during maturation process of OPCs. Down-regulation of endogenous SHP-2 led to impairment of oligodendrocytes maturation and this effect was triiodo-L-thyronine (T3) dependent. Furthermore, over-expression of SHP-2 was shown to promote maturation of oligodendrocytes. Finally, it has been identified that SHP-2 was involved in activation of Akt and extracellular-regulated kinases 1 and 2 (ERK1/2) induced by T3 in oligodendrocytes.

Conclusions: SHP-2 promotes oligodendrocytes maturation via Akt and ERK1/2 signaling in vitro.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. SOV inhibits expression of MBP in oligodendrocytes in vitro.
(A) Identification of purified OPCs. (B) OPCs were pre-treated with different doses of SOV for 1 h every 24 h before the medium were changed to fresh differentiation medium without SOV. OPCs were immunostained with anti-MBP antibody (red) 3 days after maturation induction. The total number cells were identified by Hoechst staining (blue). The culture without SOV treatment was used as control. (C) Percentages of MBP-positive cells against the total number of cells. For each group, over 1000 cells were analyzed. *P<0.05, **P<0.01 vs controls. (D) After induction of maturation, OPCs treated with or without SOV were lysed and immunoblotted with antibody against MBP. GAPDH was used as a loading control. (E) Quantification of D was presented as fold of control. Results were from three independent experiments. *P<0.05, **P<0.01.
Figure 2
Figure 2. SOV inhibits morphological maturation of OPCs in vitro.
(A) OPCs were pre-treated with different doses of SOV. After induction of maturation for 1 day, cells were fixed and stained with O4 monoclonal antibody. O4-positive cells were then categorized into three types. The representative images of cells in three categories are shown. (B) The percentages of the cells treated with or without SOV in each category were quantified after induction of maturation. For each group, over 1000 cells were analyzed. *P<0.05, **P<0.01 vs controls. Data represent five independent experiments.
Figure 3
Figure 3. The effect of SOV on proliferation and apoptosis of the OPCs in vitro.
(A) The primary cultures of OPCs were placed on PLL-coated dishes and cultured in different concentrations of SOV for 8 h with 10 µM BrdU. Proliferated cells were labeled with anti-BrdU (green), and cells were labeled with NG2 antibody (red). The culture without SOV was used as control and quantification was presented as fold of control. Ratio of BrdU-labeled cells was calculated. Data shown are the mean ± S.D. of three independent experiments. *P<0.05 vs controls. (B) Apoptotic cells were detected by TUNEL staining (red), and total number of cells was detected with Hoechst staining (blue). The culture without SOV was used as control and quantification was presented as fold of control. Ratio of TUNEL-labeled cells was calculated. Data shown are the mean ± S.D. of three independent experiments. **P<0.01 vs controls.
Figure 4
Figure 4. PTPi IV attenuates MBP expression of oligodendrocytes in vitro.
(A) OPCs were pre-treated with different doses of PTPi IV for 6 h every 24 h before the medium were changed to fresh differentiation medium without PTPi IV. OPCs were immunostained with anti-MBP antibody (red) 3 days after induction of maturation. The total number of cells was identified by Hoechst staining (blue). The culture added DMSO was used as control. (B) Percentages of MBP-positive cells against the total number of cells. For each group, over 1000 cells were analyzed. *P<0.05, **P<0.01 vs controls. Data represent three independent experiments. (C) After induction of maturation, OPCs treated with different concentration of PTPi IV were lysed and immunoblotted with antibody against MBP with GAPDH as a loading control. (D) Quantification of C was presented as fold of Control. Results were from three independent experiments. *P<0.05, **P<0.01.
Figure 5
Figure 5. The effect of PTPi IV on proliferation and apoptosis of the OPCs in vitro.
(A) The OPCs were cultured in different concentration of PTPi IV for 8 h with 10 µM BrdU. OPCs were labeled with NG2 antibody (red), and proliferated cells were labeled with anti-BrdU (green). The culture without PTPi IV was used as control and quantification was presented as fold of control. Ratio of BrdU-labeled cells was calculated. Data shown are the mean ± S.D. of three independent experiments. (B) Apoptotic cells were detected by TUNEL staining (red), and the total number of cells were identified by Hoechst staining (blue). The culture without PTPi IV was used as control and quantification was presented as fold of control. Ratio of TUNEL-labeled cells was calculated. Data shown are the mean ± S.D. of three independent experiments.
Figure 6
Figure 6. Expression of SHP-2 in oligodendrocytes in vitro and in vivo.
(A) OPCs differentiated for 1, 3 and 5 days and analyzed protein levels of SHP-2 by western blot. GAPDH and MBP were used as control. (B) Densitometry to determine levels of SHP-2 protein, quantification presented as fold of control. Results were from three independent experiments. (C) Expression of SHP-2 in primary cultured rat OPCs. The distribution of SHP-2 (green) and NG2 (red) or MBP (red) is shown by immunofluorescence. (D) SHP-2 expression of oligodendrocytes in brain slices. The tissue slices from rat brain were immunostained with SHP-2 antibody (green) and markers of oligodendrocytes (red). Arrows indicate SHP-2 expression in oligodendrocytes.
Figure 7
Figure 7. Knockdown of SHP-2 inhibits oligodendrocytes maturation.
(A) RT-PCR analysis of SHP-2 mRNA transcription in OPCs infected with lentivirus expressing ShRNAs of SHP-2 (SHP2-si), Scramble (Control-si) or blank vector (Control). GAPDH was used as an internal standard. (B) Quantification of A, presented as fold of control. Results were from three independent experiments. **p<0.01. (C) OPCs infected with lentivirus expressing ShRNA were immunostained with anti-MBP antibody (red) after induction of maturation for 3 days. Arrows indicate MBP+ in GFP-expressing cells. The cells infected with Lentivirus expressing the control ShRNA and induced by T3 were used as control. (D) Percentages of MBP-positive cells (red) against the GFP-labeled cells, over 500 cells were analyzed for each group. Data represent three independent experiments. **p<0.01.
Figure 8
Figure 8. Overexpression of SHP-2 positively regulates oligodendrocyte maturation.
(A) OPCs were infected with lentivirus expressing SHP-2. After induction of maturation for 3 days, cells were immunostained with anti-MBP antibody (red). Arrows indicate MBP+ in GFP-expressing cells. The cells infected with Lentivirus expressing the blank vector tagged by GFP were used as control. (B) Percentages of MBP-positive cells (red) against the total number of infected cells, over 500 cells were analyzed for each group. Data represent three independent experiments. *p<0.05. (C) The overexpression of GFP-SHP2 in transfected OPCs were detected by western blot using GFP antibody.
Figure 9
Figure 9. The effect of SHP-2 knockdown on proliferation of the OPCs in vitro.
(A, B and C) OPCs were infected with the lentivirus expressing SHP-2 ShRNA and corresponding control. After 72 hours lentiviral-transfection, the medium was changed and the following supplements were added: B104 CDM (B) or 30 nM T3 (C). The basal medium (Neurobasal) was used as control (A). OPCs were incubated for 8 hours with 10 µM BrdU. Proliferated cells were labeled with anti-BrdU (red), and cells were labeled with GFP antibody (green). (D) Quantification of A was presented as -fold of control. The culture transfected with the blank vector was used as control. Ratio of BrdU-labeled cells was calculated. Data shown are the mean ± S.D. of three independent experiments. *P<0.05, **P<0.01.
Figure 10
Figure 10. SHP-2 is involved in T3-induced activation of Akt and ERK1/2 in OPCs.
(A) OPCs were infected with lentivirus expressing SHP-2 using blank vector as control. Activation of Akt and ERK1/2 in oligodendrocytes was assessed with western blot after T3 stimulation in transfected OPCs. (B, C) Quantification of A with densitometric analysis was presented as fold of control. Results were from three independent experiments. *p<0.05 and **p<0.01. (D) Activation of Akt and ERK1/2 in oligodendrocytes were assessed with western blot after T3 stimulation in OPCs transfected with SHP-2 ShRNA or blank vector. (E, F) Quantification of D with densitometric analysis was presented as fold of control. Results were from three independent experiments. *p<0.05 and **p<0.01.
Figure 11
Figure 11. Inhibition activation of Akt and ERK1/2 attenuates the promotional effect of SHP-2 on oligodendrocyte maturation.
(A) OPCs were infected with lentivirus expressing SHP-2 using blank vector as control. Cultures transfected with SHP-2 were pre-treated with 2.0 µM PD98059 (MEK1/2 inhibitor) or 20 µM LY294002 (PI3K inhibitor) for 30 min, respectively. Cells were immunostained with anti-MBP antibody (red). Arrows indicate MBP+ in GFP-expressing cells. The cells infected with Lentivirus expressing the blank vector tagged by GFP were used as control. (B) Percentages of MBP-positive cells (red) against the number of infected cells. Over 500 cells were analyzed for each group. Data represent three independent experiments. *p<0.05 and **p<0.01. (C) Activation of Akt and ERK1/2 in oligodendrocytes induced by T3 (30 nM) were assessed with western blot after inhibitors pre-treatment. (D) Pre-treatment with PD98059 (2.0 µM) or LY294002 (20 µM) did not induce significant OPCs death in cell culture system.

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