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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

Inhibition of CaSR reversed the promotive effects of [Ca2+]o on the proliferation of pBMSCs. (a) Effects of NPS2143 (0.1 μM), an antagonist of CaSR, on the proliferation of pBMSCs after 5-day incubation (n = 8). (b) Effects of 4 mM [Ca2+]o and/or 0.1 μM NPS2143 on the cell cycle progression of pBMSCs. After pBMSCs were exposed to 4 mM [Ca2+]o with or without NPS2143 (0.1 μM) for 5 days, the cells were collected and treated according to the protocol in Materials and Methods. The DNA contents were measured with FACScan flow cytometry. (c) Analysis of proliferation index (PI) and the percentage of cells in G0/G1, S, and G2/M phases. (d) The mRNA expression levels of cyclins (cyclin A2, cyclin D3, and cyclin E2), PCNA, and p21 in response to 4 mM [Ca2+]o and/or 0.1 μM NPS2143. (e) Western blot analysis of cyclin D1 and p21 in pBMSCs after 5-day culture. β-actin was used as loading control. (f) Mean ± SEM of immunoblotting bands of cyclin D1 and p21; the intensities of the bands were expressed as the arbitrary units (n = 4). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus 1 mM [Ca2+]o group (control); #P < 0.05, ##P < 0.01, and ###P < 0.001 versus 4 mM [Ca2+]o group.
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fig2: Inhibition of CaSR reversed the promotive effects of [Ca2+]o on the proliferation of pBMSCs. (a) Effects of NPS2143 (0.1 μM), an antagonist of CaSR, on the proliferation of pBMSCs after 5-day incubation (n = 8). (b) Effects of 4 mM [Ca2+]o and/or 0.1 μM NPS2143 on the cell cycle progression of pBMSCs. After pBMSCs were exposed to 4 mM [Ca2+]o with or without NPS2143 (0.1 μM) for 5 days, the cells were collected and treated according to the protocol in Materials and Methods. The DNA contents were measured with FACScan flow cytometry. (c) Analysis of proliferation index (PI) and the percentage of cells in G0/G1, S, and G2/M phases. (d) The mRNA expression levels of cyclins (cyclin A2, cyclin D3, and cyclin E2), PCNA, and p21 in response to 4 mM [Ca2+]o and/or 0.1 μM NPS2143. (e) Western blot analysis of cyclin D1 and p21 in pBMSCs after 5-day culture. β-actin was used as loading control. (f) Mean ± SEM of immunoblotting bands of cyclin D1 and p21; the intensities of the bands were expressed as the arbitrary units (n = 4). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus 1 mM [Ca2+]o group (control); #P < 0.05, ##P < 0.01, and ###P < 0.001 versus 4 mM [Ca2+]o group.

Mentions: To further elucidate the role of CaSR in [Ca2+]o-stimulated pBMSCs proliferation, NPS2143, a CaSR antagonist, was used to inhibit CaSR in the present study. As shown in Figure 2(a), NPS2143 (0.1 μM) alone had no effect on pBMSCs proliferation. However, NPS2143 significantly (P < 0.001) abolished the promotion of pBMSCs proliferation induced by 4 mM [Ca2+]o. In addition, the results of cell cycle progression analysis via flow cytometry revealed that 4 mM [Ca2+]o markedly (P < 0.05) increased the proportion of pBMSCs in the S phase and the proliferation index (PI) of pBMSCs and decreased the proportion of pBMSCs in the G0/G1 phase. However, NPS2143 eliminated the effects of [Ca2+]o on cell cycle distribution and PI (Figures 2(b) and 2(c)). Furthermore, 4 mM [Ca2+]o significantly increased the mRNA expression levels of cyclins (cyclin A2, cyclin D3, and cyclin E2) and PCNA but decreased the mRNA expression of p21, the inhibitor of cyclin-dependent kinase (Figure 2(d)). Moreover, the elevated mRNA expression levels of cyclins and PCNA and the decreased mRNA levels of p21 induced by 4 mM [Ca2+]o were reversed by NPS2143. Similarly, the increased protein expression of cyclin D1 and the decreased protein level of p21 induced by 4 mM [Ca2+]o were also abolished by NPS2143 (Figures 2(e) and 2(f)). These results showed that the inhibition of CaSR reversed the promotive effects of [Ca2+]o on pBMSCs proliferation, thereby indicating the essential role of CaSR in this process.


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)

Inhibition of CaSR reversed the promotive effects of [Ca2+]o on the proliferation of pBMSCs. (a) Effects of NPS2143 (0.1 μM), an antagonist of CaSR, on the proliferation of pBMSCs after 5-day incubation (n = 8). (b) Effects of 4 mM [Ca2+]o and/or 0.1 μM NPS2143 on the cell cycle progression of pBMSCs. After pBMSCs were exposed to 4 mM [Ca2+]o with or without NPS2143 (0.1 μM) for 5 days, the cells were collected and treated according to the protocol in Materials and Methods. The DNA contents were measured with FACScan flow cytometry. (c) Analysis of proliferation index (PI) and the percentage of cells in G0/G1, S, and G2/M phases. (d) The mRNA expression levels of cyclins (cyclin A2, cyclin D3, and cyclin E2), PCNA, and p21 in response to 4 mM [Ca2+]o and/or 0.1 μM NPS2143. (e) Western blot analysis of cyclin D1 and p21 in pBMSCs after 5-day culture. β-actin was used as loading control. (f) Mean ± SEM of immunoblotting bands of cyclin D1 and p21; the intensities of the bands were expressed as the arbitrary units (n = 4). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus 1 mM [Ca2+]o group (control); #P < 0.05, ##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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fig2: Inhibition of CaSR reversed the promotive effects of [Ca2+]o on the proliferation of pBMSCs. (a) Effects of NPS2143 (0.1 μM), an antagonist of CaSR, on the proliferation of pBMSCs after 5-day incubation (n = 8). (b) Effects of 4 mM [Ca2+]o and/or 0.1 μM NPS2143 on the cell cycle progression of pBMSCs. After pBMSCs were exposed to 4 mM [Ca2+]o with or without NPS2143 (0.1 μM) for 5 days, the cells were collected and treated according to the protocol in Materials and Methods. The DNA contents were measured with FACScan flow cytometry. (c) Analysis of proliferation index (PI) and the percentage of cells in G0/G1, S, and G2/M phases. (d) The mRNA expression levels of cyclins (cyclin A2, cyclin D3, and cyclin E2), PCNA, and p21 in response to 4 mM [Ca2+]o and/or 0.1 μM NPS2143. (e) Western blot analysis of cyclin D1 and p21 in pBMSCs after 5-day culture. β-actin was used as loading control. (f) Mean ± SEM of immunoblotting bands of cyclin D1 and p21; the intensities of the bands were expressed as the arbitrary units (n = 4). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus 1 mM [Ca2+]o group (control); #P < 0.05, ##P < 0.01, and ###P < 0.001 versus 4 mM [Ca2+]o group.
Mentions: To further elucidate the role of CaSR in [Ca2+]o-stimulated pBMSCs proliferation, NPS2143, a CaSR antagonist, was used to inhibit CaSR in the present study. As shown in Figure 2(a), NPS2143 (0.1 μM) alone had no effect on pBMSCs proliferation. However, NPS2143 significantly (P < 0.001) abolished the promotion of pBMSCs proliferation induced by 4 mM [Ca2+]o. In addition, the results of cell cycle progression analysis via flow cytometry revealed that 4 mM [Ca2+]o markedly (P < 0.05) increased the proportion of pBMSCs in the S phase and the proliferation index (PI) of pBMSCs and decreased the proportion of pBMSCs in the G0/G1 phase. However, NPS2143 eliminated the effects of [Ca2+]o on cell cycle distribution and PI (Figures 2(b) and 2(c)). Furthermore, 4 mM [Ca2+]o significantly increased the mRNA expression levels of cyclins (cyclin A2, cyclin D3, and cyclin E2) and PCNA but decreased the mRNA expression of p21, the inhibitor of cyclin-dependent kinase (Figure 2(d)). Moreover, the elevated mRNA expression levels of cyclins and PCNA and the decreased mRNA levels of p21 induced by 4 mM [Ca2+]o were reversed by NPS2143. Similarly, the increased protein expression of cyclin D1 and the decreased protein level of p21 induced by 4 mM [Ca2+]o were also abolished by NPS2143 (Figures 2(e) and 2(f)). These results showed that the inhibition of CaSR reversed the promotive effects of [Ca2+]o on pBMSCs proliferation, thereby indicating the essential role of CaSR in this process.

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