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Enhanced Proliferation of Porcine Bone Marrow Mesenchymal Stem Cells Induced by Extracellular Calcium is Associated with the Activation of the Calcium-Sensing Receptor and ERK Signaling Pathway.

Ye J, Ai W, Zhang F, Zhu X, Shu G, Wang L, Gao P, Xi Q, Zhang Y, Jiang Q, Wang S - Stem Cells Int (2016)

Bottom Line: In addition, [Ca(2+)]o resulted in a significant elevation of intracellular calcium and an increased ratio of p-ERK/ERK.However, inhibition of calcium-sensing receptor (CaSR) by its antagonist NPS2143 abolished the aforementioned effects of [Ca(2+)]o.In conclusion, our findings provided evidence that the enhanced pBMSCs proliferation in response to [Ca(2+)]o was associated with the activation of CaSR and ERK1/2 signaling pathway, which may be useful for the application of pBMSCs in future clinical studies aimed at tissue regeneration and repair.

View Article: PubMed Central - PubMed

Affiliation: College of Animal Science and National Engineering Research Center for Breeding Swine Industry, South China Agricultural University, Guangzhou 510642, China; Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding and ALLTECH-SCAU Animal Nutrition Control Research Alliance, South China Agricultural University, Guangzhou 510642, China.

ABSTRACT
Porcine bone marrow mesenchymal stem cells (pBMSCs) have the potential for application in regenerative medicine. This study aims to investigate the effects of extracellular calcium ([Ca(2+)]o) on pBMSCs proliferation and to explore the possible underlying mechanisms. The results demonstrated that 4 mM [Ca(2+)]o significantly promoted pBMSCs proliferation by reducing the G0/G1 phase cell percentage and by increasing the S phase cell proportion and the proliferation index of pBMSCs. Accordingly, [Ca(2+)]o stimulated the expression levels of proliferative genes such as cyclin A2, cyclin D1/3, cyclin E2, and PCNA and inhibited the expression of p21. In addition, [Ca(2+)]o resulted in a significant elevation of intracellular calcium and an increased ratio of p-ERK/ERK. However, inhibition of calcium-sensing receptor (CaSR) by its antagonist NPS2143 abolished the aforementioned effects of [Ca(2+)]o. Moreover, [Ca(2+)]o-induced promotion of pBMSCs proliferation, the changes of proliferative genes expression levels, and the activation of ERK1/2 signaling pathway were effectively blocked by U0126, a selective ERK kinase inhibitor. In conclusion, our findings provided evidence that the enhanced pBMSCs proliferation in response to [Ca(2+)]o was associated with the activation of CaSR and ERK1/2 signaling pathway, which may be useful for the application of pBMSCs in future clinical studies aimed at tissue regeneration and repair.

No MeSH data available.


Related in: MedlinePlus

CaSR inhibition blocked the increase of [Ca2+]i in response to [Ca2+]o during the proliferation of pBMSCs. (a) [Ca2+]i of live cells were analyzed by flow cytometry. The percentage of gated positive cells represented the cells stained with Fluo 3-AM. Representation of three independent experiments with similar results. (b) The relative FITC fluorescence intensity of gated positive cells. (c) Western blot analysis of CaSR in pBMSCs after 5-day culture in the presence of 4 mM [Ca2+]o and/or 0.1 μM NPS2143. β-actin was used as loading control. (d) Mean ± SEM of immunoblotting bands of CaSR; the intensities of the bands were expressed as the arbitrary units (n = 4). ∗∗∗P < 0.001 versus 1 mM [Ca2+]o group (control); ##P < 0.01 and ###P < 0.001 versus 4 mM [Ca2+]o group.
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fig3: CaSR inhibition blocked the increase of [Ca2+]i in response to [Ca2+]o during the proliferation of pBMSCs. (a) [Ca2+]i of live cells were analyzed by flow cytometry. The percentage of gated positive cells represented the cells stained with Fluo 3-AM. Representation of three independent experiments with similar results. (b) The relative FITC fluorescence intensity of gated positive cells. (c) Western blot analysis of CaSR in pBMSCs after 5-day culture in the presence of 4 mM [Ca2+]o and/or 0.1 μM NPS2143. β-actin was used as loading control. (d) Mean ± SEM of immunoblotting bands of CaSR; the intensities of the bands were expressed as the arbitrary units (n = 4). ∗∗∗P < 0.001 versus 1 mM [Ca2+]o group (control); ##P < 0.01 and ###P < 0.001 versus 4 mM [Ca2+]o group.

Mentions: The high level of [Ca2+]o always resulted in the increase of [Ca2+]i levels. In order to confirm the occurrence of this phenomenon and to explore the role of CaSR in this process, we examined [Ca2+]i in pBMSCs in the presence of 4 mM [Ca2+]o and/or NPS2143 (0.1 μM). As expected, [Ca2+]i, which was indicated as the percentage of gated cells (Figure 3(a)) or relative fluorescence (Figure 3(b)), was significantly (P < 0.001) increased in response to 4 mM [Ca2+]o, with an increase pattern similar to that of CaSR protein expression induced by 4 mM [Ca2+]o (Figures 3(c) and 3(d)). However, as shown in Figures 3(a) and 3(b), the significant elevation of [Ca2+]i in response to 4 mM [Ca2+]o was blocked by CaSR inhibition with NPS2143, which also abolished the promotive effect of 4 mM [Ca2+]o on CaSR protein expression (Figures 3(c) and 3(d)). These data demonstrated that CaSR inhibition blocked the elevation of [Ca2+]i induced by 4 mM [Ca2+]o in pBMSCs, thereby suggesting that the CaSR-mediated increase in [Ca2+]i in response to [Ca2+]o might be involved in the [Ca2+]o-promoted pBMSCs proliferation.


Enhanced Proliferation of Porcine Bone Marrow Mesenchymal Stem Cells Induced by Extracellular Calcium is Associated with the Activation of the Calcium-Sensing Receptor and ERK Signaling Pathway.

Ye J, Ai W, Zhang F, Zhu X, Shu G, Wang L, Gao P, Xi Q, Zhang Y, Jiang Q, Wang S - Stem Cells Int (2016)

CaSR inhibition blocked the increase of [Ca2+]i in response to [Ca2+]o during the proliferation of pBMSCs. (a) [Ca2+]i of live cells were analyzed by flow cytometry. The percentage of gated positive cells represented the cells stained with Fluo 3-AM. Representation of three independent experiments with similar results. (b) The relative FITC fluorescence intensity of gated positive cells. (c) Western blot analysis of CaSR in pBMSCs after 5-day culture in the presence of 4 mM [Ca2+]o and/or 0.1 μM NPS2143. β-actin was used as loading control. (d) Mean ± SEM of immunoblotting bands of CaSR; the intensities of the bands were expressed as the arbitrary units (n = 4). ∗∗∗P < 0.001 versus 1 mM [Ca2+]o group (control); ##P < 0.01 and ###P < 0.001 versus 4 mM [Ca2+]o group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: CaSR inhibition blocked the increase of [Ca2+]i in response to [Ca2+]o during the proliferation of pBMSCs. (a) [Ca2+]i of live cells were analyzed by flow cytometry. The percentage of gated positive cells represented the cells stained with Fluo 3-AM. Representation of three independent experiments with similar results. (b) The relative FITC fluorescence intensity of gated positive cells. (c) Western blot analysis of CaSR in pBMSCs after 5-day culture in the presence of 4 mM [Ca2+]o and/or 0.1 μM NPS2143. β-actin was used as loading control. (d) Mean ± SEM of immunoblotting bands of CaSR; the intensities of the bands were expressed as the arbitrary units (n = 4). ∗∗∗P < 0.001 versus 1 mM [Ca2+]o group (control); ##P < 0.01 and ###P < 0.001 versus 4 mM [Ca2+]o group.
Mentions: The high level of [Ca2+]o always resulted in the increase of [Ca2+]i levels. In order to confirm the occurrence of this phenomenon and to explore the role of CaSR in this process, we examined [Ca2+]i in pBMSCs in the presence of 4 mM [Ca2+]o and/or NPS2143 (0.1 μM). As expected, [Ca2+]i, which was indicated as the percentage of gated cells (Figure 3(a)) or relative fluorescence (Figure 3(b)), was significantly (P < 0.001) increased in response to 4 mM [Ca2+]o, with an increase pattern similar to that of CaSR protein expression induced by 4 mM [Ca2+]o (Figures 3(c) and 3(d)). However, as shown in Figures 3(a) and 3(b), the significant elevation of [Ca2+]i in response to 4 mM [Ca2+]o was blocked by CaSR inhibition with NPS2143, which also abolished the promotive effect of 4 mM [Ca2+]o on CaSR protein expression (Figures 3(c) and 3(d)). These data demonstrated that CaSR inhibition blocked the elevation of [Ca2+]i induced by 4 mM [Ca2+]o in pBMSCs, thereby suggesting that the CaSR-mediated increase in [Ca2+]i in response to [Ca2+]o might be involved in the [Ca2+]o-promoted pBMSCs proliferation.

Bottom Line: In addition, [Ca(2+)]o resulted in a significant elevation of intracellular calcium and an increased ratio of p-ERK/ERK.However, inhibition of calcium-sensing receptor (CaSR) by its antagonist NPS2143 abolished the aforementioned effects of [Ca(2+)]o.In conclusion, our findings provided evidence that the enhanced pBMSCs proliferation in response to [Ca(2+)]o was associated with the activation of CaSR and ERK1/2 signaling pathway, which may be useful for the application of pBMSCs in future clinical studies aimed at tissue regeneration and repair.

View Article: PubMed Central - PubMed

Affiliation: College of Animal Science and National Engineering Research Center for Breeding Swine Industry, South China Agricultural University, Guangzhou 510642, China; Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding and ALLTECH-SCAU Animal Nutrition Control Research Alliance, South China Agricultural University, Guangzhou 510642, China.

ABSTRACT
Porcine bone marrow mesenchymal stem cells (pBMSCs) have the potential for application in regenerative medicine. This study aims to investigate the effects of extracellular calcium ([Ca(2+)]o) on pBMSCs proliferation and to explore the possible underlying mechanisms. The results demonstrated that 4 mM [Ca(2+)]o significantly promoted pBMSCs proliferation by reducing the G0/G1 phase cell percentage and by increasing the S phase cell proportion and the proliferation index of pBMSCs. Accordingly, [Ca(2+)]o stimulated the expression levels of proliferative genes such as cyclin A2, cyclin D1/3, cyclin E2, and PCNA and inhibited the expression of p21. In addition, [Ca(2+)]o resulted in a significant elevation of intracellular calcium and an increased ratio of p-ERK/ERK. However, inhibition of calcium-sensing receptor (CaSR) by its antagonist NPS2143 abolished the aforementioned effects of [Ca(2+)]o. Moreover, [Ca(2+)]o-induced promotion of pBMSCs proliferation, the changes of proliferative genes expression levels, and the activation of ERK1/2 signaling pathway were effectively blocked by U0126, a selective ERK kinase inhibitor. In conclusion, our findings provided evidence that the enhanced pBMSCs proliferation in response to [Ca(2+)]o was associated with the activation of CaSR and ERK1/2 signaling pathway, which may be useful for the application of pBMSCs in future clinical studies aimed at tissue regeneration and repair.

No MeSH data available.


Related in: MedlinePlus