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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.

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Regulatory effect of Ntn-1 on SP1 activation and VEGF expression.(A) The number of cells transfected with PKCαsiRNA prior to Ntn-1 exposure for 24 h is shown. Data represent the mean ± S.E. n = 3. *P < 0.05 vs. nt siRNA alone. #P < 0.05 vs. nt siRNA + Ntn-1 alone. (B) Activation of PKCα in cells treated with NAC (10 μM) for 30 min prior to Ntn-1 exposure for 60 min is shown. Data represent the mean ± S.E. n = 3. *P < 0.01 vs. vehicle. #P < 0.01 vs. Ntn-1 alone. (C) Phosphorylation of SP1 is shown. Data represent the mean ± S.E. n = 3. *P < 0.05 vs. 0 h. (D) The number of cells transfected with SP1siRNA prior to Ntn-1 exposure for 60 min is shown. (E) p-SP1 expression (green) was determined by confocal microscopy. Propidium iodide (PI) was used for nuclear counterstaining (red). Scale bars, 100 μm (magnification, x400). n = 3. (F) Phosphorylation of SP1 in cells transfected with PKCαsiRNA prior to Ntn-1 exposure for 60 min is shown. (G) The level of cell cycle proteins in cells transfected with SP1siRNA or NF-κBsiRNA is shown. (H) The level of VEGF is shown. Data represent the mean ± S.E. n = 3. *P < 0.05 vs. 0 h. (I) The amount of VEGF in cells transfected with SP1siRNA or NF-κBsiRNA prior to Ntn-1 exposure for 24 h is shown. (J) Cells were treated with DCC-function-blocking antibody, a combination of Inα6- and Inβ4-function-blocking antibodies, and NAC, or transfected with PKCαsiRNA prior to Ntn-1 exposure for 12 h. The binding of p-SP1 to VEGF promoter was determined by ChIP assay. n = 3. Normal mouse IgG was used as negative control for the ChIP. n = 3. (D,F,G,I) Data represent the mean ± S.E. n = 5. *P < 0.05 vs. nt siRNA. #P < 0.05 vs. nt siRNA + Ntn-1. (B,C,F–H,I) ROD is the abbreviation for relative optical density.
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f4: Regulatory effect of Ntn-1 on SP1 activation and VEGF expression.(A) The number of cells transfected with PKCαsiRNA prior to Ntn-1 exposure for 24 h is shown. Data represent the mean ± S.E. n = 3. *P < 0.05 vs. nt siRNA alone. #P < 0.05 vs. nt siRNA + Ntn-1 alone. (B) Activation of PKCα in cells treated with NAC (10 μM) for 30 min prior to Ntn-1 exposure for 60 min is shown. Data represent the mean ± S.E. n = 3. *P < 0.01 vs. vehicle. #P < 0.01 vs. Ntn-1 alone. (C) Phosphorylation of SP1 is shown. Data represent the mean ± S.E. n = 3. *P < 0.05 vs. 0 h. (D) The number of cells transfected with SP1siRNA prior to Ntn-1 exposure for 60 min is shown. (E) p-SP1 expression (green) was determined by confocal microscopy. Propidium iodide (PI) was used for nuclear counterstaining (red). Scale bars, 100 μm (magnification, x400). n = 3. (F) Phosphorylation of SP1 in cells transfected with PKCαsiRNA prior to Ntn-1 exposure for 60 min is shown. (G) The level of cell cycle proteins in cells transfected with SP1siRNA or NF-κBsiRNA is shown. (H) The level of VEGF is shown. Data represent the mean ± S.E. n = 3. *P < 0.05 vs. 0 h. (I) The amount of VEGF in cells transfected with SP1siRNA or NF-κBsiRNA prior to Ntn-1 exposure for 24 h is shown. (J) Cells were treated with DCC-function-blocking antibody, a combination of Inα6- and Inβ4-function-blocking antibodies, and NAC, or transfected with PKCαsiRNA prior to Ntn-1 exposure for 12 h. The binding of p-SP1 to VEGF promoter was determined by ChIP assay. n = 3. Normal mouse IgG was used as negative control for the ChIP. n = 3. (D,F,G,I) Data represent the mean ± S.E. n = 5. *P < 0.05 vs. nt siRNA. #P < 0.05 vs. nt siRNA + Ntn-1. (B,C,F–H,I) ROD is the abbreviation for relative optical density.

Mentions: ROS dominantly regulates PKC activation. Given that Ntn-1 specifically activates the PKCα isoform11, we further assessed whether PKCα activation is involved in cell proliferation. The knockdown of PKCα by PKCαsiRNA inhibited cell proliferation as induced by Ntn-1 (Fig. 4A). Moreover, a pre-treatment with the antioxidant NAC significantly blocked PKCα activation (Fig. 4B). These results demonstrate that ROS production is linked to PKCα activation in promoting hUCB-MSC proliferation. We further examined the role of Ntn-1 in the activation of a transcription factor, SP1, as an important PKC signaling intermediate involved in stem cell proliferation28. Ntn-1 induced SP1 phosphorylation for 60 min (Fig. 4C). The knockdown of SP1 with SP1siRNA significantly blocked Ntn-1-induced cell proliferation (Fig. 4D). The increased accumulation of SP1 phosphorylation in the nucleus was further confirmed by immunofluorescence staining and counter-labeling with propidium iodide (PI) (Fig. 4E). Moreover, SP1 phosphorylation evoked by Ntn-1 was markedly attenuated by a treatment with PKCαsiRNA (Fig. 4F), suggesting that PKCα-mediated SP1 activation is a key step in the promotion of hUCB-MSC proliferation as induced by Ntn-1. The knockdown of SP1 with SP1siRNA significantly decreased the levels of Cyclin D1, Cdk4, Cyclin E, and Cdk2 induced by Ntn-1 (Fig. 4G). Importantly, however, the knockdown of NF-κB, which is known to be required for Ntn-1-mediated cell migration11, did not have any effect on the level of cyclins and CDKs, indicating that the Ntn-1-mediated transition from the G1 to the S phase in stem cells is uniquely mediated by the phosphorylation of SP1 (Fig. 4G). In addition to cell proliferation, it is plausible that SP1 has the ability to regulate the gene expression activities involved in angiogenesis with regard to the regeneration of ischemic or injured tissues. We found that Ntn-1 significantly increased the amount of the vascular endothelial growth factor (VEGF) (Fig. 4H). The level of VEGF was decreased by a treatment with SP1siRNA, whereas the knockdown of NF-κB failed to regulate the protein level (Fig. 4I). These results indicate that SP1 is required for cell proliferation and for the angiogenic capacity of hUCB-MSC primed by Ntn-1. To determine whether SP1 directly regulates VEGF mRNA expression, we undertook a VEGF promoter region analysis using Alggen Promo2930. The VEGF promoter contains five putative SP1 binding sites which are located between the −250 bp upstream site and the transcription start site. We performed a chromatin immunoprecipitation (ChIP) assay in hUCB-MSC treated with Ntn-1 to determine the regulatory effect of Ntn-1 on the binding of SP1 to the VEGF promoter. We tested a primer that includes five putative VEGF binding sites at the region proximal −2 bp to −248 bp of the start site of the VEGF promoter. We found that our primer sets result in an amplicon from the anti-pSP1 immunoprecipitates and importantly that the interaction of SP1 with the VEGF promoter was enhanced by the Ntn-1 treatment (Fig. 4J). Interestingly, however, the level of SP1 binding to the VEGF promoter was significantly inhibited by a pre-treatment with the combination of the Inα6- and Inβ4-function-blocking antibodies, NAC, and the knockdown of PKCα (Fig. 4J). These results suggest that Ntn-1 acting on Inα6β4 transcriptionally regulates SP1 binding to the VEGF promoter via lipid raft-mediated ROS production for cell 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-1 on SP1 activation and VEGF expression.(A) The number of cells transfected with PKCαsiRNA prior to Ntn-1 exposure for 24 h is shown. Data represent the mean ± S.E. n = 3. *P < 0.05 vs. nt siRNA alone. #P < 0.05 vs. nt siRNA + Ntn-1 alone. (B) Activation of PKCα in cells treated with NAC (10 μM) for 30 min prior to Ntn-1 exposure for 60 min is shown. Data represent the mean ± S.E. n = 3. *P < 0.01 vs. vehicle. #P < 0.01 vs. Ntn-1 alone. (C) Phosphorylation of SP1 is shown. Data represent the mean ± S.E. n = 3. *P < 0.05 vs. 0 h. (D) The number of cells transfected with SP1siRNA prior to Ntn-1 exposure for 60 min is shown. (E) p-SP1 expression (green) was determined by confocal microscopy. Propidium iodide (PI) was used for nuclear counterstaining (red). Scale bars, 100 μm (magnification, x400). n = 3. (F) Phosphorylation of SP1 in cells transfected with PKCαsiRNA prior to Ntn-1 exposure for 60 min is shown. (G) The level of cell cycle proteins in cells transfected with SP1siRNA or NF-κBsiRNA is shown. (H) The level of VEGF is shown. Data represent the mean ± S.E. n = 3. *P < 0.05 vs. 0 h. (I) The amount of VEGF in cells transfected with SP1siRNA or NF-κBsiRNA prior to Ntn-1 exposure for 24 h is shown. (J) Cells were treated with DCC-function-blocking antibody, a combination of Inα6- and Inβ4-function-blocking antibodies, and NAC, or transfected with PKCαsiRNA prior to Ntn-1 exposure for 12 h. The binding of p-SP1 to VEGF promoter was determined by ChIP assay. n = 3. Normal mouse IgG was used as negative control for the ChIP. n = 3. (D,F,G,I) Data represent the mean ± S.E. n = 5. *P < 0.05 vs. nt siRNA. #P < 0.05 vs. nt siRNA + Ntn-1. (B,C,F–H,I) ROD is the abbreviation for relative optical density.
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f4: Regulatory effect of Ntn-1 on SP1 activation and VEGF expression.(A) The number of cells transfected with PKCαsiRNA prior to Ntn-1 exposure for 24 h is shown. Data represent the mean ± S.E. n = 3. *P < 0.05 vs. nt siRNA alone. #P < 0.05 vs. nt siRNA + Ntn-1 alone. (B) Activation of PKCα in cells treated with NAC (10 μM) for 30 min prior to Ntn-1 exposure for 60 min is shown. Data represent the mean ± S.E. n = 3. *P < 0.01 vs. vehicle. #P < 0.01 vs. Ntn-1 alone. (C) Phosphorylation of SP1 is shown. Data represent the mean ± S.E. n = 3. *P < 0.05 vs. 0 h. (D) The number of cells transfected with SP1siRNA prior to Ntn-1 exposure for 60 min is shown. (E) p-SP1 expression (green) was determined by confocal microscopy. Propidium iodide (PI) was used for nuclear counterstaining (red). Scale bars, 100 μm (magnification, x400). n = 3. (F) Phosphorylation of SP1 in cells transfected with PKCαsiRNA prior to Ntn-1 exposure for 60 min is shown. (G) The level of cell cycle proteins in cells transfected with SP1siRNA or NF-κBsiRNA is shown. (H) The level of VEGF is shown. Data represent the mean ± S.E. n = 3. *P < 0.05 vs. 0 h. (I) The amount of VEGF in cells transfected with SP1siRNA or NF-κBsiRNA prior to Ntn-1 exposure for 24 h is shown. (J) Cells were treated with DCC-function-blocking antibody, a combination of Inα6- and Inβ4-function-blocking antibodies, and NAC, or transfected with PKCαsiRNA prior to Ntn-1 exposure for 12 h. The binding of p-SP1 to VEGF promoter was determined by ChIP assay. n = 3. Normal mouse IgG was used as negative control for the ChIP. n = 3. (D,F,G,I) Data represent the mean ± S.E. n = 5. *P < 0.05 vs. nt siRNA. #P < 0.05 vs. nt siRNA + Ntn-1. (B,C,F–H,I) ROD is the abbreviation for relative optical density.
Mentions: ROS dominantly regulates PKC activation. Given that Ntn-1 specifically activates the PKCα isoform11, we further assessed whether PKCα activation is involved in cell proliferation. The knockdown of PKCα by PKCαsiRNA inhibited cell proliferation as induced by Ntn-1 (Fig. 4A). Moreover, a pre-treatment with the antioxidant NAC significantly blocked PKCα activation (Fig. 4B). These results demonstrate that ROS production is linked to PKCα activation in promoting hUCB-MSC proliferation. We further examined the role of Ntn-1 in the activation of a transcription factor, SP1, as an important PKC signaling intermediate involved in stem cell proliferation28. Ntn-1 induced SP1 phosphorylation for 60 min (Fig. 4C). The knockdown of SP1 with SP1siRNA significantly blocked Ntn-1-induced cell proliferation (Fig. 4D). The increased accumulation of SP1 phosphorylation in the nucleus was further confirmed by immunofluorescence staining and counter-labeling with propidium iodide (PI) (Fig. 4E). Moreover, SP1 phosphorylation evoked by Ntn-1 was markedly attenuated by a treatment with PKCαsiRNA (Fig. 4F), suggesting that PKCα-mediated SP1 activation is a key step in the promotion of hUCB-MSC proliferation as induced by Ntn-1. The knockdown of SP1 with SP1siRNA significantly decreased the levels of Cyclin D1, Cdk4, Cyclin E, and Cdk2 induced by Ntn-1 (Fig. 4G). Importantly, however, the knockdown of NF-κB, which is known to be required for Ntn-1-mediated cell migration11, did not have any effect on the level of cyclins and CDKs, indicating that the Ntn-1-mediated transition from the G1 to the S phase in stem cells is uniquely mediated by the phosphorylation of SP1 (Fig. 4G). In addition to cell proliferation, it is plausible that SP1 has the ability to regulate the gene expression activities involved in angiogenesis with regard to the regeneration of ischemic or injured tissues. We found that Ntn-1 significantly increased the amount of the vascular endothelial growth factor (VEGF) (Fig. 4H). The level of VEGF was decreased by a treatment with SP1siRNA, whereas the knockdown of NF-κB failed to regulate the protein level (Fig. 4I). These results indicate that SP1 is required for cell proliferation and for the angiogenic capacity of hUCB-MSC primed by Ntn-1. To determine whether SP1 directly regulates VEGF mRNA expression, we undertook a VEGF promoter region analysis using Alggen Promo2930. The VEGF promoter contains five putative SP1 binding sites which are located between the −250 bp upstream site and the transcription start site. We performed a chromatin immunoprecipitation (ChIP) assay in hUCB-MSC treated with Ntn-1 to determine the regulatory effect of Ntn-1 on the binding of SP1 to the VEGF promoter. We tested a primer that includes five putative VEGF binding sites at the region proximal −2 bp to −248 bp of the start site of the VEGF promoter. We found that our primer sets result in an amplicon from the anti-pSP1 immunoprecipitates and importantly that the interaction of SP1 with the VEGF promoter was enhanced by the Ntn-1 treatment (Fig. 4J). Interestingly, however, the level of SP1 binding to the VEGF promoter was significantly inhibited by a pre-treatment with the combination of the Inα6- and Inβ4-function-blocking antibodies, NAC, and the knockdown of PKCα (Fig. 4J). These results suggest that Ntn-1 acting on Inα6β4 transcriptionally regulates SP1 binding to the VEGF promoter via lipid raft-mediated ROS production for cell 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