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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.


The influence of BMP6 on the function of human Sertoli cells.(A) Western blots demonstrated ZO1, OCLN, SCF, GDNF, AR and AMH proteins in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. ACTB and GAPDH served as loading controls of proteins. Full length blots of ZO1, OCLN and GDNF were presented in Supplementary Figure B. (B) The relative expression of ZO1, OCLN, SCF, GDNF, AR and AMH 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 control and BMP6 siRNA-treated group. (C,D) ELISA showed SCF secretion by BMP6 siRNA-1 (C) and BMP6 (D) in human Sertoli cells. *Indicated statistically significant differences (p < 0.05) between the control siRNA and BMP6 siRNA-1, or the control and BMP6-treated group.
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f5: The influence of BMP6 on the function of human Sertoli cells.(A) Western blots demonstrated ZO1, OCLN, SCF, GDNF, AR and AMH proteins in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. ACTB and GAPDH served as loading controls of proteins. Full length blots of ZO1, OCLN and GDNF were presented in Supplementary Figure B. (B) The relative expression of ZO1, OCLN, SCF, GDNF, AR and AMH 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 control and BMP6 siRNA-treated group. (C,D) ELISA showed SCF secretion by BMP6 siRNA-1 (C) and BMP6 (D) in human Sertoli cells. *Indicated statistically significant differences (p < 0.05) between the control siRNA and BMP6 siRNA-1, or the control and BMP6-treated group.

Mentions: We next utilized small RNAs to investigate the effect of endogenous BMP6 on the function of human Sertoli cells. ZO1 and OCLN are the indispensable components of tight junction23. Western blots revealed that the level of ZO1 protein was reduced by 22.9% ± 3.2% at 48 hours after transfection of BMP6 siRNA-1 (Fig. 5A,B), while OCLN and AMH levels were not significantly changed (Fig. 5A,B). Sertoli cells can secrete some important growth factors, including GDNF and SCF, which regulate the self-renewal and differentiation of spermatogonial stem cells. Western blots showed that the production of SCF, GDNF and AR (androgen receptor) was reduced in human Sertoli cells at 48 hours after transfection of BMP6 siRNA-1 (Fig. 5A,B). We further measured SCF secretion in human Sertoli cells using ELISA, and the expression of SCF was significantly reduced by 17.7 ± 3.3 μg/ml/106cells by BMP6 siRNA-1 compared to the control siRNA at 48 hours after transfection (Fig. 5C). In contrast, the secretion of SCF was increased to 81.6 ± 9.0 μg/ml/106cells by BMP6 (Fig. 5D) at 72 hours, compared with control group (61.5 ± 3.0 μg/ml/106cells). Taken together, these data implicate that BMP6 stimulates the function of human Sertoli cells.


BMP6 Regulates Proliferation and Apoptosis of Human Sertoli Cells Via Smad2/3 and Cyclin D1 Pathway and DACH1 and TFAP2A Activation
The influence of BMP6 on the function of human Sertoli cells.(A) Western blots demonstrated ZO1, OCLN, SCF, GDNF, AR and AMH proteins in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. ACTB and GAPDH served as loading controls of proteins. Full length blots of ZO1, OCLN and GDNF were presented in Supplementary Figure B. (B) The relative expression of ZO1, OCLN, SCF, GDNF, AR and AMH 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 control and BMP6 siRNA-treated group. (C,D) ELISA showed SCF secretion by BMP6 siRNA-1 (C) and BMP6 (D) in human Sertoli cells. *Indicated statistically significant differences (p < 0.05) between the control siRNA and BMP6 siRNA-1, or the control and BMP6-treated group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5384448&req=5

f5: The influence of BMP6 on the function of human Sertoli cells.(A) Western blots demonstrated ZO1, OCLN, SCF, GDNF, AR and AMH proteins in human Sertoli cells at 48 hours after transfection with control siRNA or with BMP6 siRNA-1. ACTB and GAPDH served as loading controls of proteins. Full length blots of ZO1, OCLN and GDNF were presented in Supplementary Figure B. (B) The relative expression of ZO1, OCLN, SCF, GDNF, AR and AMH 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 control and BMP6 siRNA-treated group. (C,D) ELISA showed SCF secretion by BMP6 siRNA-1 (C) and BMP6 (D) in human Sertoli cells. *Indicated statistically significant differences (p < 0.05) between the control siRNA and BMP6 siRNA-1, or the control and BMP6-treated group.
Mentions: We next utilized small RNAs to investigate the effect of endogenous BMP6 on the function of human Sertoli cells. ZO1 and OCLN are the indispensable components of tight junction23. Western blots revealed that the level of ZO1 protein was reduced by 22.9% ± 3.2% at 48 hours after transfection of BMP6 siRNA-1 (Fig. 5A,B), while OCLN and AMH levels were not significantly changed (Fig. 5A,B). Sertoli cells can secrete some important growth factors, including GDNF and SCF, which regulate the self-renewal and differentiation of spermatogonial stem cells. Western blots showed that the production of SCF, GDNF and AR (androgen receptor) was reduced in human Sertoli cells at 48 hours after transfection of BMP6 siRNA-1 (Fig. 5A,B). We further measured SCF secretion in human Sertoli cells using ELISA, and the expression of SCF was significantly reduced by 17.7 ± 3.3 μg/ml/106cells by BMP6 siRNA-1 compared to the control siRNA at 48 hours after transfection (Fig. 5C). In contrast, the secretion of SCF was increased to 81.6 ± 9.0 μg/ml/106cells by BMP6 (Fig. 5D) at 72 hours, compared with control group (61.5 ± 3.0 μg/ml/106cells). Taken together, these data implicate that BMP6 stimulates the function of 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.