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Netrin-1-Induced Stem Cell Bioactivity Contributes to the Regeneration of Injured Tissues via the Lipid Raft-Dependent Integrin α 6 β 4 Signaling Pathway

View Article: PubMed Central - PubMed

ABSTRACT

Netrin-1 (Ntn-1) is a multifunctional neuronal signaling molecule; however, its physiological significance, which improves the tissue-regeneration capacity of stem cells, has not been characterized. In the present study, we investigate the mechanism by which Ntn-1 promotes the proliferation of hUCB-MSCs with regard to the regeneration of injured tissues. We found that Ntn-1 induces the proliferation of hUCB-MSCs mainly via Inα6β4 coupled with c-Src. Ntn-1 induced the recruitment of NADPH oxidases and Rac1 into membrane lipid rafts to facilitate ROS production. The Inα6β4 signaling of Ntn-1 through ROS production is uniquely mediated by the activation of SP1 for cell cycle progression and the transcriptional occupancy of SP1 on the VEGF promoter. Moreover, Ntn-1 has the ability to induce the F-actin reorganization of hUCB-MSCs via the Inα6β4 signaling pathway. In an in vivo model, transplantation of hUCB-MSCs pre-treated with Ntn-1 enhanced the skin wound healing process, where relatively more angiogenesis was detected. The potential effect of Ntn-1 on angiogenesis is further verified by the mouse hindlimb ischemia model, where the pre-activation of hUCB-MSCs with Ntn-1 significantly improved vascular regeneration. These results demonstrate that Ntn-1 plays an important role in the tissue regeneration process of hUCB-MSC via the lipid raft-mediated Inα6β4 signaling pathway.

No MeSH data available.


Related in: MedlinePlus

Regulatory effect of Ntn-1on stem cell proliferation.(A) hUCB-MSCs were incubated with 50 ng/mL of Ntn-1 for 48 h, and the number of cells was counted. Data represent the mean ± S.E. n = 5. *P < 0.05 vs. 0 h (B) Cells were pre-treated with DCC-function-blocking antibody (2.5 μg/mL) or a combination of Inα6- and Inβ4-function-blocking antibodies (2.5 μg/mL) for 30 min prior to Ntn-1 exposure for 24 h. Cell counting was performed. Data represent the mean ± S.E. n = 4. *P < 0.05 vs. vehicle. #P < 0.05 vs. Ntn-1 alone. (C) Cells were treated with Ntn-1 for 24 h. Gates were manually configured to determine the percentage of cells in S phase based on DNA content by using PI staining and flow cytometry. Data represent the mean ± S.E. n = 4. (D) G1/S ratios measured by flow cytometry. Data represent the mean ± S.E. n = 4. *P < 0.01 vs. vehicle. #P < 0.05 vs. Ntn-1 alone. (E) The cells were incubated in the presence of Ntn-1 for 24 h and then harvested. Total protein was extracted and blotted with Cyclin D1, CDK4, Cyclin E, and CDK2 antibodies. Data represent the mean ± S.E. n = 4. *P < 0.05 vs. 0 h. (F) The level of cell cycle proteins in cells pre-treated with DCC-function-blocking antibody (2.5 μg/mL) or a combination of Inα6- and Inβ4-function-blocking antibodies (2.5 μg/mL) for 30 min prior to Ntn-1 exposure for 24 h is shown. Data represent the mean ± S.E. n = 4. *P < 0.01 vs. vehicle. #P < 0.05 vs. Ntn-1 alone. (G) The effect of Ntn-1 on the level of cell cycle proteins in cells transfected with MMP12siRNA is shown. Cells were transfected for 24 h with specific siRNA for MMP12 prior to Ntn-1 exposure for 24 h. Non-targeting (nt) control siRNA was used as a negative control. Data represent the mean ± S.E. n = 4. *P < 0.01 vs. nt siRNA. (E–G) ROD is the abbreviation for relative optical density.
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f1: Regulatory effect of Ntn-1on stem cell proliferation.(A) hUCB-MSCs were incubated with 50 ng/mL of Ntn-1 for 48 h, and the number of cells was counted. Data represent the mean ± S.E. n = 5. *P < 0.05 vs. 0 h (B) Cells were pre-treated with DCC-function-blocking antibody (2.5 μg/mL) or a combination of Inα6- and Inβ4-function-blocking antibodies (2.5 μg/mL) for 30 min prior to Ntn-1 exposure for 24 h. Cell counting was performed. Data represent the mean ± S.E. n = 4. *P < 0.05 vs. vehicle. #P < 0.05 vs. Ntn-1 alone. (C) Cells were treated with Ntn-1 for 24 h. Gates were manually configured to determine the percentage of cells in S phase based on DNA content by using PI staining and flow cytometry. Data represent the mean ± S.E. n = 4. (D) G1/S ratios measured by flow cytometry. Data represent the mean ± S.E. n = 4. *P < 0.01 vs. vehicle. #P < 0.05 vs. Ntn-1 alone. (E) The cells were incubated in the presence of Ntn-1 for 24 h and then harvested. Total protein was extracted and blotted with Cyclin D1, CDK4, Cyclin E, and CDK2 antibodies. Data represent the mean ± S.E. n = 4. *P < 0.05 vs. 0 h. (F) The level of cell cycle proteins in cells pre-treated with DCC-function-blocking antibody (2.5 μg/mL) or a combination of Inα6- and Inβ4-function-blocking antibodies (2.5 μg/mL) for 30 min prior to Ntn-1 exposure for 24 h is shown. Data represent the mean ± S.E. n = 4. *P < 0.01 vs. vehicle. #P < 0.05 vs. Ntn-1 alone. (G) The effect of Ntn-1 on the level of cell cycle proteins in cells transfected with MMP12siRNA is shown. Cells were transfected for 24 h with specific siRNA for MMP12 prior to Ntn-1 exposure for 24 h. Non-targeting (nt) control siRNA was used as a negative control. Data represent the mean ± S.E. n = 4. *P < 0.01 vs. nt siRNA. (E–G) ROD is the abbreviation for relative optical density.

Mentions: To determine the functional role of netrin-1 (Ntn-1), hUCB-MSCs were exposed to Ntn-1 for 48 h. Ntn-1 significantly increased the number of hUCB-MSC from 24 h to 48 h at 50 ng/mL in a time-dependent manner (Fig. 1A). The activity of Ntn-1 to promote cell proliferation was significantly attenuated by a pre-treatment with a combination of Inα6- and Inβ4-function-blocking antibodies (2.5 μg/mL), but not by a DCC-function-blocking antibody (2.5 μg/mL), suggesting that Ntn-1 has receptor specificity in promoting cell proliferation (Fig. 1B). We previously confirmed that Inα6β4- and DCC-function-blocking antibodies have significant capacities to block the Inα6β4 and DCC functions when regulating stem cell motility11 and apoptosis12. A flow cytometric analysis revealed that Ntn-1 induced a G1-to-S-phase transition (Fig. 1C), resulting in a significant decrease in the G1/S ratio for 24 h in an Inα6β4-dependent manner (Fig. 1D). Cell cycle progression is regulated by protein complexes composed of cyclins and cyclin-dependent kinases (CDKs)22. Ntn-1 increased the levels of Cyclin D1, Cdk4, Cyclin E, and Cdk2 (Fig. 1E). The blocking of Inα6β4 activity, but not DCC, decreased the amounts of cyclins and CDKs (Fig. 1F). Knockdown of MMP-12, which is known to enhance cell migration in response to Ntn-111, was not found to have an effect on the levels of Cyclin D1, Cdk4, Cyclin E, or Cdk2 (Fig. 1G), indicating that Ntn-1 uniquely mediates cell-cycle transition from the G1 to the S phase via Inα6β4-dependent pathways in promoting hUCB-MSC proliferation.


Netrin-1-Induced Stem Cell Bioactivity Contributes to the Regeneration of Injured Tissues via the Lipid Raft-Dependent Integrin α 6 β 4 Signaling Pathway
Regulatory effect of Ntn-1on stem cell proliferation.(A) hUCB-MSCs were incubated with 50 ng/mL of Ntn-1 for 48 h, and the number of cells was counted. Data represent the mean ± S.E. n = 5. *P < 0.05 vs. 0 h (B) Cells were pre-treated with DCC-function-blocking antibody (2.5 μg/mL) or a combination of Inα6- and Inβ4-function-blocking antibodies (2.5 μg/mL) for 30 min prior to Ntn-1 exposure for 24 h. Cell counting was performed. Data represent the mean ± S.E. n = 4. *P < 0.05 vs. vehicle. #P < 0.05 vs. Ntn-1 alone. (C) Cells were treated with Ntn-1 for 24 h. Gates were manually configured to determine the percentage of cells in S phase based on DNA content by using PI staining and flow cytometry. Data represent the mean ± S.E. n = 4. (D) G1/S ratios measured by flow cytometry. Data represent the mean ± S.E. n = 4. *P < 0.01 vs. vehicle. #P < 0.05 vs. Ntn-1 alone. (E) The cells were incubated in the presence of Ntn-1 for 24 h and then harvested. Total protein was extracted and blotted with Cyclin D1, CDK4, Cyclin E, and CDK2 antibodies. Data represent the mean ± S.E. n = 4. *P < 0.05 vs. 0 h. (F) The level of cell cycle proteins in cells pre-treated with DCC-function-blocking antibody (2.5 μg/mL) or a combination of Inα6- and Inβ4-function-blocking antibodies (2.5 μg/mL) for 30 min prior to Ntn-1 exposure for 24 h is shown. Data represent the mean ± S.E. n = 4. *P < 0.01 vs. vehicle. #P < 0.05 vs. Ntn-1 alone. (G) The effect of Ntn-1 on the level of cell cycle proteins in cells transfected with MMP12siRNA is shown. Cells were transfected for 24 h with specific siRNA for MMP12 prior to Ntn-1 exposure for 24 h. Non-targeting (nt) control siRNA was used as a negative control. Data represent the mean ± S.E. n = 4. *P < 0.01 vs. nt siRNA. (E–G) ROD is the abbreviation for relative optical density.
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Related In: Results  -  Collection

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f1: Regulatory effect of Ntn-1on stem cell proliferation.(A) hUCB-MSCs were incubated with 50 ng/mL of Ntn-1 for 48 h, and the number of cells was counted. Data represent the mean ± S.E. n = 5. *P < 0.05 vs. 0 h (B) Cells were pre-treated with DCC-function-blocking antibody (2.5 μg/mL) or a combination of Inα6- and Inβ4-function-blocking antibodies (2.5 μg/mL) for 30 min prior to Ntn-1 exposure for 24 h. Cell counting was performed. Data represent the mean ± S.E. n = 4. *P < 0.05 vs. vehicle. #P < 0.05 vs. Ntn-1 alone. (C) Cells were treated with Ntn-1 for 24 h. Gates were manually configured to determine the percentage of cells in S phase based on DNA content by using PI staining and flow cytometry. Data represent the mean ± S.E. n = 4. (D) G1/S ratios measured by flow cytometry. Data represent the mean ± S.E. n = 4. *P < 0.01 vs. vehicle. #P < 0.05 vs. Ntn-1 alone. (E) The cells were incubated in the presence of Ntn-1 for 24 h and then harvested. Total protein was extracted and blotted with Cyclin D1, CDK4, Cyclin E, and CDK2 antibodies. Data represent the mean ± S.E. n = 4. *P < 0.05 vs. 0 h. (F) The level of cell cycle proteins in cells pre-treated with DCC-function-blocking antibody (2.5 μg/mL) or a combination of Inα6- and Inβ4-function-blocking antibodies (2.5 μg/mL) for 30 min prior to Ntn-1 exposure for 24 h is shown. Data represent the mean ± S.E. n = 4. *P < 0.01 vs. vehicle. #P < 0.05 vs. Ntn-1 alone. (G) The effect of Ntn-1 on the level of cell cycle proteins in cells transfected with MMP12siRNA is shown. Cells were transfected for 24 h with specific siRNA for MMP12 prior to Ntn-1 exposure for 24 h. Non-targeting (nt) control siRNA was used as a negative control. Data represent the mean ± S.E. n = 4. *P < 0.01 vs. nt siRNA. (E–G) ROD is the abbreviation for relative optical density.
Mentions: To determine the functional role of netrin-1 (Ntn-1), hUCB-MSCs were exposed to Ntn-1 for 48 h. Ntn-1 significantly increased the number of hUCB-MSC from 24 h to 48 h at 50 ng/mL in a time-dependent manner (Fig. 1A). The activity of Ntn-1 to promote cell proliferation was significantly attenuated by a pre-treatment with a combination of Inα6- and Inβ4-function-blocking antibodies (2.5 μg/mL), but not by a DCC-function-blocking antibody (2.5 μg/mL), suggesting that Ntn-1 has receptor specificity in promoting cell proliferation (Fig. 1B). We previously confirmed that Inα6β4- and DCC-function-blocking antibodies have significant capacities to block the Inα6β4 and DCC functions when regulating stem cell motility11 and apoptosis12. A flow cytometric analysis revealed that Ntn-1 induced a G1-to-S-phase transition (Fig. 1C), resulting in a significant decrease in the G1/S ratio for 24 h in an Inα6β4-dependent manner (Fig. 1D). Cell cycle progression is regulated by protein complexes composed of cyclins and cyclin-dependent kinases (CDKs)22. Ntn-1 increased the levels of Cyclin D1, Cdk4, Cyclin E, and Cdk2 (Fig. 1E). The blocking of Inα6β4 activity, but not DCC, decreased the amounts of cyclins and CDKs (Fig. 1F). Knockdown of MMP-12, which is known to enhance cell migration in response to Ntn-111, was not found to have an effect on the levels of Cyclin D1, Cdk4, Cyclin E, or Cdk2 (Fig. 1G), indicating that Ntn-1 uniquely mediates cell-cycle transition from the G1 to the S phase via Inα6β4-dependent pathways in promoting hUCB-MSC proliferation.

View Article: PubMed Central - PubMed

ABSTRACT

Netrin-1 (Ntn-1) is a multifunctional neuronal signaling molecule; however, its physiological significance, which improves the tissue-regeneration capacity of stem cells, has not been characterized. In the present study, we investigate the mechanism by which Ntn-1 promotes the proliferation of hUCB-MSCs with regard to the regeneration of injured tissues. We found that Ntn-1 induces the proliferation of hUCB-MSCs mainly via In&alpha;6&beta;4 coupled with c-Src. Ntn-1 induced the recruitment of NADPH oxidases and Rac1 into membrane lipid rafts to facilitate ROS production. The In&alpha;6&beta;4 signaling of Ntn-1 through ROS production is uniquely mediated by the activation of SP1 for cell cycle progression and the transcriptional occupancy of SP1 on the VEGF promoter. Moreover, Ntn-1 has the ability to induce the F-actin reorganization of hUCB-MSCs via the In&alpha;6&beta;4 signaling pathway. In an in vivo model, transplantation of hUCB-MSCs pre-treated with Ntn-1 enhanced the skin wound healing process, where relatively more angiogenesis was detected. The potential effect of Ntn-1 on angiogenesis is further verified by the mouse hindlimb ischemia model, where the pre-activation of hUCB-MSCs with Ntn-1 significantly improved vascular regeneration. These results demonstrate that Ntn-1 plays an important role in the tissue regeneration process of hUCB-MSC via the lipid raft-mediated In&alpha;6&beta;4 signaling pathway.

No MeSH data available.


Related in: MedlinePlus