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BMP6 Regulates Proliferation and Apoptosis of Human Sertoli Cells Via Smad2/3 and Cyclin D1 Pathway and DACH1 and TFAP2A Activation

View Article: PubMed Central - PubMed

ABSTRACT

Sertoli cells are essential for regulating normal spermatogenesis. However, the mechanisms underlying human Sertoli cell development remain largely elusive. Here we examined the function and signaling pathways of BMP6 in regulating human Sertoli cells. RT-PCR, immunocytochemistry and Western blots revealed that BMP6 and its multiple receptors were expressed in human Sertoli cells. CCK-8 and EDU assays showed that BMP6 promoted the proliferation of Sertoli cells. Conversely, BMP6 siRNAs inhibited the division of these cells. Annexin V/PI assay indicated that BMP6 reduced the apoptosis in human Sertoli cells, whereas BMP6 knockdown assumed reverse effects. BMP6 enhanced the expression levels of ZO1, SCF, GDNF and AR in human Sertoli cells, and ELISA assay showed an increase of SCF by BMP6 and a reduction by BMP6 siRNAs. Notably, Smad2/3 phosphorylation and cyclin D1 were enhanced by BMP6 and decreased by BMP6 siRNAs in human Sertoli cells. The levels of DACH1 and TFAP2A were increased by BMP6 and reduced by BMP6 siRNAs, and the growth of human Sertoli cells was inhibited by these siRNAs. Collectively, these results suggest that BMP6 regulates the proliferation and apoptosis of human Sertoli cells via activating the Smad2/3/cyclin D1 and DACH1 and TFAP2A pathway.

No MeSH data available.


BMP6 activated Smad2/3 and cyclin D1 pathway in human Sertoli cells.(A) Western blots showed the change of phos-Smad2/3 in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. Smad2/3 was used as a loading control of total proteins. Full length blots of phos-Smad2/3 and ACTB were presented in Supplementary Figure C. (B–D) Western blots revealed the expression of phosphorylation of Smad1/5/8 (B), phosphorylation of ERK1/2 (C), and phosphorylation of AKT (D) in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. Smad1/5/8, ERK2, and AKT served as loading controls for total proteins, respectively. Full length blots of phos-ERK1/2 and ERK were presented in Supplementary Figure D. (E) Western blots showed the expression of cyclin A, cyclin B1, cyclin D1, CDK2 and cyclin E in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. ACTB and GAPDH served as a loading control of total proteins. Full length blots of cyclin D1, CDK2, cyclin E and ACTB were presented in Supplementary Figure E. (F) The relative expression of phos-Smad2/3, phos-Smad1/5/8, phos-AKT, phos-ERK1/2, cyclin A, cyclin B1, cyclin D1, CDK2, and cyclin E in human Sertoli cells at 48 hours after transfection with BMP6 siRNA-1 or control siRNA after normalization to the signals of their respect loading control. *indicated statistically significant differences (p < 0.05) between the control siRNA and BMP6 siRNA-1-treated group. (G–I) Western blots demonstrated the levels of phos-Smad2/3 and cyclin D1 by BMP6 and BSA (control) in human Sertoli cells.
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f6: BMP6 activated Smad2/3 and cyclin D1 pathway in human Sertoli cells.(A) Western blots showed the change of phos-Smad2/3 in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. Smad2/3 was used as a loading control of total proteins. Full length blots of phos-Smad2/3 and ACTB were presented in Supplementary Figure C. (B–D) Western blots revealed the expression of phosphorylation of Smad1/5/8 (B), phosphorylation of ERK1/2 (C), and phosphorylation of AKT (D) in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. Smad1/5/8, ERK2, and AKT served as loading controls for total proteins, respectively. Full length blots of phos-ERK1/2 and ERK were presented in Supplementary Figure D. (E) Western blots showed the expression of cyclin A, cyclin B1, cyclin D1, CDK2 and cyclin E in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. ACTB and GAPDH served as a loading control of total proteins. Full length blots of cyclin D1, CDK2, cyclin E and ACTB were presented in Supplementary Figure E. (F) The relative expression of phos-Smad2/3, phos-Smad1/5/8, phos-AKT, phos-ERK1/2, cyclin A, cyclin B1, cyclin D1, CDK2, and cyclin E in human Sertoli cells at 48 hours after transfection with BMP6 siRNA-1 or control siRNA after normalization to the signals of their respect loading control. *indicated statistically significant differences (p < 0.05) between the control siRNA and BMP6 siRNA-1-treated group. (G–I) Western blots demonstrated the levels of phos-Smad2/3 and cyclin D1 by BMP6 and BSA (control) in human Sertoli cells.

Mentions: We further explored the signaling pathways and transcription factors activated by BMP6 in human Sertoli cells. To this end, we detected the expression of phos-Smad2/3, phos-Smad1/5/8, phos-AKT and phos-ERK1/2. Western blots revealed that phos-Smad2/3 was reduced by 25.0% ± 2.5% in human Sertoli cells after transfection of BMP6 siRNA-1 compared with control siRNA (Fig. 6A and F). No significant changes were observed in phos-Smad1/5/8, phos-AKT, or phos-ERK1/2 in human Sertoli cells after transfection of BMP6 siRNA-1 (Fig. 6B–D and F). Furthermore, we checked the expression of various cell cycle progression proteins, including cyclin A, B1, D1, E and CDK2, to address which cell cycle proteins were affected by endogenous BMP6. Western blots demonstrated that the level of cyclin D1 was reduced by 41.1% ± 3.2% in human Sertoli cells transfected with BMP6 siRNA-1 compared with control siRNA at 48 hours (Fig. 6E,F). In contrast, no obvious changes were seen in the levels of cyclin A, B1, E, and CDK2 by BMP6 siRNA-1 compared to control siRNA (Fig. 6E,F). Additionally, Western blots revealed that BMP6 increased the level of phos-Smad2/3 by 70.9% ± 4.9% at 30 min and cyclin D1 at 24 hours by 56.3% ± 3.2% in human Sertoli cells (Fig. 6G–I). Therefore, BMP6 activates Smad2/3 pathway and enhances the level of cyclin D1 in human Sertoli cells.


BMP6 Regulates Proliferation and Apoptosis of Human Sertoli Cells Via Smad2/3 and Cyclin D1 Pathway and DACH1 and TFAP2A Activation
BMP6 activated Smad2/3 and cyclin D1 pathway in human Sertoli cells.(A) Western blots showed the change of phos-Smad2/3 in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. Smad2/3 was used as a loading control of total proteins. Full length blots of phos-Smad2/3 and ACTB were presented in Supplementary Figure C. (B–D) Western blots revealed the expression of phosphorylation of Smad1/5/8 (B), phosphorylation of ERK1/2 (C), and phosphorylation of AKT (D) in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. Smad1/5/8, ERK2, and AKT served as loading controls for total proteins, respectively. Full length blots of phos-ERK1/2 and ERK were presented in Supplementary Figure D. (E) Western blots showed the expression of cyclin A, cyclin B1, cyclin D1, CDK2 and cyclin E in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. ACTB and GAPDH served as a loading control of total proteins. Full length blots of cyclin D1, CDK2, cyclin E and ACTB were presented in Supplementary Figure E. (F) The relative expression of phos-Smad2/3, phos-Smad1/5/8, phos-AKT, phos-ERK1/2, cyclin A, cyclin B1, cyclin D1, CDK2, and cyclin E in human Sertoli cells at 48 hours after transfection with BMP6 siRNA-1 or control siRNA after normalization to the signals of their respect loading control. *indicated statistically significant differences (p < 0.05) between the control siRNA and BMP6 siRNA-1-treated group. (G–I) Western blots demonstrated the levels of phos-Smad2/3 and cyclin D1 by BMP6 and BSA (control) in human Sertoli cells.
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f6: BMP6 activated Smad2/3 and cyclin D1 pathway in human Sertoli cells.(A) Western blots showed the change of phos-Smad2/3 in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. Smad2/3 was used as a loading control of total proteins. Full length blots of phos-Smad2/3 and ACTB were presented in Supplementary Figure C. (B–D) Western blots revealed the expression of phosphorylation of Smad1/5/8 (B), phosphorylation of ERK1/2 (C), and phosphorylation of AKT (D) in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. Smad1/5/8, ERK2, and AKT served as loading controls for total proteins, respectively. Full length blots of phos-ERK1/2 and ERK were presented in Supplementary Figure D. (E) Western blots showed the expression of cyclin A, cyclin B1, cyclin D1, CDK2 and cyclin E in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. ACTB and GAPDH served as a loading control of total proteins. Full length blots of cyclin D1, CDK2, cyclin E and ACTB were presented in Supplementary Figure E. (F) The relative expression of phos-Smad2/3, phos-Smad1/5/8, phos-AKT, phos-ERK1/2, cyclin A, cyclin B1, cyclin D1, CDK2, and cyclin E in human Sertoli cells at 48 hours after transfection with BMP6 siRNA-1 or control siRNA after normalization to the signals of their respect loading control. *indicated statistically significant differences (p < 0.05) between the control siRNA and BMP6 siRNA-1-treated group. (G–I) Western blots demonstrated the levels of phos-Smad2/3 and cyclin D1 by BMP6 and BSA (control) in human Sertoli cells.
Mentions: We further explored the signaling pathways and transcription factors activated by BMP6 in human Sertoli cells. To this end, we detected the expression of phos-Smad2/3, phos-Smad1/5/8, phos-AKT and phos-ERK1/2. Western blots revealed that phos-Smad2/3 was reduced by 25.0% ± 2.5% in human Sertoli cells after transfection of BMP6 siRNA-1 compared with control siRNA (Fig. 6A and F). No significant changes were observed in phos-Smad1/5/8, phos-AKT, or phos-ERK1/2 in human Sertoli cells after transfection of BMP6 siRNA-1 (Fig. 6B–D and F). Furthermore, we checked the expression of various cell cycle progression proteins, including cyclin A, B1, D1, E and CDK2, to address which cell cycle proteins were affected by endogenous BMP6. Western blots demonstrated that the level of cyclin D1 was reduced by 41.1% ± 3.2% in human Sertoli cells transfected with BMP6 siRNA-1 compared with control siRNA at 48 hours (Fig. 6E,F). In contrast, no obvious changes were seen in the levels of cyclin A, B1, E, and CDK2 by BMP6 siRNA-1 compared to control siRNA (Fig. 6E,F). Additionally, Western blots revealed that BMP6 increased the level of phos-Smad2/3 by 70.9% ± 4.9% at 30 min and cyclin D1 at 24 hours by 56.3% ± 3.2% in human Sertoli cells (Fig. 6G–I). Therefore, BMP6 activates Smad2/3 pathway and enhances the level of cyclin D1 in human Sertoli cells.

View Article: PubMed Central - PubMed

ABSTRACT

Sertoli cells are essential for regulating normal spermatogenesis. However, the mechanisms underlying human Sertoli cell development remain largely elusive. Here we examined the function and signaling pathways of BMP6 in regulating human Sertoli cells. RT-PCR, immunocytochemistry and Western blots revealed that BMP6 and its multiple receptors were expressed in human Sertoli cells. CCK-8 and EDU assays showed that BMP6 promoted the proliferation of Sertoli cells. Conversely, BMP6 siRNAs inhibited the division of these cells. Annexin V/PI assay indicated that BMP6 reduced the apoptosis in human Sertoli cells, whereas BMP6 knockdown assumed reverse effects. BMP6 enhanced the expression levels of ZO1, SCF, GDNF and AR in human Sertoli cells, and ELISA assay showed an increase of SCF by BMP6 and a reduction by BMP6 siRNAs. Notably, Smad2/3 phosphorylation and cyclin D1 were enhanced by BMP6 and decreased by BMP6 siRNAs in human Sertoli cells. The levels of DACH1 and TFAP2A were increased by BMP6 and reduced by BMP6 siRNAs, and the growth of human Sertoli cells was inhibited by these siRNAs. Collectively, these results suggest that BMP6 regulates the proliferation and apoptosis of human Sertoli cells via activating the Smad2/3/cyclin D1 and DACH1 and TFAP2A pathway.

No MeSH data available.