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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.


Related in: MedlinePlus

Transfection of BMP6 siRNAs and the influence of BMP6 knockdown on the proliferation and apoptosis of human Sertoli cells.(A) The transfection with FAM-labeled siRNA at 6 hours showed the transfection efficiency of BMP6 siRNAs. Scale bars in A = 10 μm. (B) Real-time PCR showed the transcription of BMP6 in human Sertoli cells at 24 hours after transfection with BMP6 siRNAs and control siRNA. (C) Real-time PCR displayed ACTB transcript in human Sertoli cells at 24 hours after transfection with BMP6 siRNA-1 and -3, control siRNA, lipofectamine 2000, or no transfection. (D) Real-time PCR revealed the expression of BMP2, BMP4, BMP9, and BMP15 in human Sertoli cells treated with BMP6 siRNA-1 and the control siRNA. (E,F) Western blots demonstrated the BMP6 protein expression in human Sertoli cells at 48 hours after transfection with control siRNA or BMP6 siRNA-1 or BMP6 siRNA-3. ACTB served as a loading control of proteins. (G) CCK-8 assay showed the growth curve of human Sertoli cells after transfection of control siRNA or BMP6 siRNA-1 for 1 to 5 days. (H,I) EDU incorporation assay showed the EDU-positive cells in human Sertoli cells after transfection of control siRNA or BMP6 siRNA-1. Cell nuclei were counterstained with Hoechst 33342. The percentages of EDU-positive cells were counted out of 500 total cells from three independent experiments. Scale bars in H = 10 μm. (J–L) Annexin V-APC/PI and flow cytometry analysis revealed apoptosis in human Sertoli cells at 48 hours after transfection of control siRNA (J) or BMP6 siRNA-1 (K). *Denoted statistically significant differences (p < 0.05) between the control and BMP6 siRNA-1-treated group.
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f4: Transfection of BMP6 siRNAs and the influence of BMP6 knockdown on the proliferation and apoptosis of human Sertoli cells.(A) The transfection with FAM-labeled siRNA at 6 hours showed the transfection efficiency of BMP6 siRNAs. Scale bars in A = 10 μm. (B) Real-time PCR showed the transcription of BMP6 in human Sertoli cells at 24 hours after transfection with BMP6 siRNAs and control siRNA. (C) Real-time PCR displayed ACTB transcript in human Sertoli cells at 24 hours after transfection with BMP6 siRNA-1 and -3, control siRNA, lipofectamine 2000, or no transfection. (D) Real-time PCR revealed the expression of BMP2, BMP4, BMP9, and BMP15 in human Sertoli cells treated with BMP6 siRNA-1 and the control siRNA. (E,F) Western blots demonstrated the BMP6 protein expression in human Sertoli cells at 48 hours after transfection with control siRNA or BMP6 siRNA-1 or BMP6 siRNA-3. ACTB served as a loading control of proteins. (G) CCK-8 assay showed the growth curve of human Sertoli cells after transfection of control siRNA or BMP6 siRNA-1 for 1 to 5 days. (H,I) EDU incorporation assay showed the EDU-positive cells in human Sertoli cells after transfection of control siRNA or BMP6 siRNA-1. Cell nuclei were counterstained with Hoechst 33342. The percentages of EDU-positive cells were counted out of 500 total cells from three independent experiments. Scale bars in H = 10 μm. (J–L) Annexin V-APC/PI and flow cytometry analysis revealed apoptosis in human Sertoli cells at 48 hours after transfection of control siRNA (J) or BMP6 siRNA-1 (K). *Denoted statistically significant differences (p < 0.05) between the control and BMP6 siRNA-1-treated group.

Mentions: In addition, we utilized small RNAs to elucidate the effect of endogenous BMP6 on the growth of human Sertoli cells. To improve the efficiency of BMP6 knockdown, we designed four pairs of BMP6 siRNAs targeting different regions of BMP6 mRNA. FAM-labeled siRNA represented the transfection efficiency. At 6 hours after transfection, the transfection efficiency of siRNA in human Sertoli cells was over 80% (Fig. 4A). The viability of human Sertoli cells after transfecting BMP6 siRNAs was about 98%, as assessed by trypan blue exclusion assay (data not shown). Quantitative real-time PCR revealed that BMP6 siRNA-1, -2, -3, and-4 apparently decreased BMP6 mRNA in Sertoli cells at 24 hours after transfection (Fig. 4B), while the interfering effect of BMP6 siRNA-1 was the most prominent (The knockdown rates of BMP6 siRNAs were shown in Table S3). Real-time PCR showed that no obvious difference existed in the transcription of ACTB of human Sertoli cells after transfection with BMP6 siRNAs, control siRNA, lipofectamine 2000, or no transfection (Fig. 4C), thus confirming that ACTB could be used as a good control. Notably, there was no significant change of BMP2, BMP4, BMP9, and BMP15 transcripts by BMP6 siRNA-1 and the control siRNA (Fig. 4D), thus verifying the specific silencing of BMP6 siRNA-1 in human Sertoli cells. Western blots revealed that BMP6 siRNA-1 reduced the expression of BMP6 protein by 300.0% ± 27.7% at 48 hours after transfection (Fig. 4E,F). Therefore, we chose BMP6 siRNA-1 to investigate the effect of BMP6 knockdown on the proliferation in human Sertoli cells. CCK-8 assay was executed from 24 hours to 120 hours after transfection of BMP6 siRNA-1 in human Sertoli cells, and BMP6 siRNA-1 evidently reduced the proliferation in human Sertoli cells at 48 hours to 120 hours (Fig. 4G). Similarly, the percentage of EDU-positive cells was reduced in human Sertoli cells with BMP6 siRNA-1 treatment compared with the control siRNA (55.4% ± 3.9% vs. 43.8% ± 4.2%, n = 5) at 48 hours after transfection (Fig. 4H,I). Additionally, we found that the apoptosis of Sertoli cells was increased by 10.7% ± 2.6% after BMP6 siRNA-1 transfection at 48 hours (Fig. 4J–L). Collectively, these data indicate that BMP6 stimulates DNA synthesis and the proliferation and inhibits the apoptosis 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
Transfection of BMP6 siRNAs and the influence of BMP6 knockdown on the proliferation and apoptosis of human Sertoli cells.(A) The transfection with FAM-labeled siRNA at 6 hours showed the transfection efficiency of BMP6 siRNAs. Scale bars in A = 10 μm. (B) Real-time PCR showed the transcription of BMP6 in human Sertoli cells at 24 hours after transfection with BMP6 siRNAs and control siRNA. (C) Real-time PCR displayed ACTB transcript in human Sertoli cells at 24 hours after transfection with BMP6 siRNA-1 and -3, control siRNA, lipofectamine 2000, or no transfection. (D) Real-time PCR revealed the expression of BMP2, BMP4, BMP9, and BMP15 in human Sertoli cells treated with BMP6 siRNA-1 and the control siRNA. (E,F) Western blots demonstrated the BMP6 protein expression in human Sertoli cells at 48 hours after transfection with control siRNA or BMP6 siRNA-1 or BMP6 siRNA-3. ACTB served as a loading control of proteins. (G) CCK-8 assay showed the growth curve of human Sertoli cells after transfection of control siRNA or BMP6 siRNA-1 for 1 to 5 days. (H,I) EDU incorporation assay showed the EDU-positive cells in human Sertoli cells after transfection of control siRNA or BMP6 siRNA-1. Cell nuclei were counterstained with Hoechst 33342. The percentages of EDU-positive cells were counted out of 500 total cells from three independent experiments. Scale bars in H = 10 μm. (J–L) Annexin V-APC/PI and flow cytometry analysis revealed apoptosis in human Sertoli cells at 48 hours after transfection of control siRNA (J) or BMP6 siRNA-1 (K). *Denoted statistically significant differences (p < 0.05) between the control and BMP6 siRNA-1-treated group.
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f4: Transfection of BMP6 siRNAs and the influence of BMP6 knockdown on the proliferation and apoptosis of human Sertoli cells.(A) The transfection with FAM-labeled siRNA at 6 hours showed the transfection efficiency of BMP6 siRNAs. Scale bars in A = 10 μm. (B) Real-time PCR showed the transcription of BMP6 in human Sertoli cells at 24 hours after transfection with BMP6 siRNAs and control siRNA. (C) Real-time PCR displayed ACTB transcript in human Sertoli cells at 24 hours after transfection with BMP6 siRNA-1 and -3, control siRNA, lipofectamine 2000, or no transfection. (D) Real-time PCR revealed the expression of BMP2, BMP4, BMP9, and BMP15 in human Sertoli cells treated with BMP6 siRNA-1 and the control siRNA. (E,F) Western blots demonstrated the BMP6 protein expression in human Sertoli cells at 48 hours after transfection with control siRNA or BMP6 siRNA-1 or BMP6 siRNA-3. ACTB served as a loading control of proteins. (G) CCK-8 assay showed the growth curve of human Sertoli cells after transfection of control siRNA or BMP6 siRNA-1 for 1 to 5 days. (H,I) EDU incorporation assay showed the EDU-positive cells in human Sertoli cells after transfection of control siRNA or BMP6 siRNA-1. Cell nuclei were counterstained with Hoechst 33342. The percentages of EDU-positive cells were counted out of 500 total cells from three independent experiments. Scale bars in H = 10 μm. (J–L) Annexin V-APC/PI and flow cytometry analysis revealed apoptosis in human Sertoli cells at 48 hours after transfection of control siRNA (J) or BMP6 siRNA-1 (K). *Denoted statistically significant differences (p < 0.05) between the control and BMP6 siRNA-1-treated group.
Mentions: In addition, we utilized small RNAs to elucidate the effect of endogenous BMP6 on the growth of human Sertoli cells. To improve the efficiency of BMP6 knockdown, we designed four pairs of BMP6 siRNAs targeting different regions of BMP6 mRNA. FAM-labeled siRNA represented the transfection efficiency. At 6 hours after transfection, the transfection efficiency of siRNA in human Sertoli cells was over 80% (Fig. 4A). The viability of human Sertoli cells after transfecting BMP6 siRNAs was about 98%, as assessed by trypan blue exclusion assay (data not shown). Quantitative real-time PCR revealed that BMP6 siRNA-1, -2, -3, and-4 apparently decreased BMP6 mRNA in Sertoli cells at 24 hours after transfection (Fig. 4B), while the interfering effect of BMP6 siRNA-1 was the most prominent (The knockdown rates of BMP6 siRNAs were shown in Table S3). Real-time PCR showed that no obvious difference existed in the transcription of ACTB of human Sertoli cells after transfection with BMP6 siRNAs, control siRNA, lipofectamine 2000, or no transfection (Fig. 4C), thus confirming that ACTB could be used as a good control. Notably, there was no significant change of BMP2, BMP4, BMP9, and BMP15 transcripts by BMP6 siRNA-1 and the control siRNA (Fig. 4D), thus verifying the specific silencing of BMP6 siRNA-1 in human Sertoli cells. Western blots revealed that BMP6 siRNA-1 reduced the expression of BMP6 protein by 300.0% ± 27.7% at 48 hours after transfection (Fig. 4E,F). Therefore, we chose BMP6 siRNA-1 to investigate the effect of BMP6 knockdown on the proliferation in human Sertoli cells. CCK-8 assay was executed from 24 hours to 120 hours after transfection of BMP6 siRNA-1 in human Sertoli cells, and BMP6 siRNA-1 evidently reduced the proliferation in human Sertoli cells at 48 hours to 120 hours (Fig. 4G). Similarly, the percentage of EDU-positive cells was reduced in human Sertoli cells with BMP6 siRNA-1 treatment compared with the control siRNA (55.4% ± 3.9% vs. 43.8% ± 4.2%, n = 5) at 48 hours after transfection (Fig. 4H,I). Additionally, we found that the apoptosis of Sertoli cells was increased by 10.7% ± 2.6% after BMP6 siRNA-1 transfection at 48 hours (Fig. 4J–L). Collectively, these data indicate that BMP6 stimulates DNA synthesis and the proliferation and inhibits the apoptosis 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.


Related in: MedlinePlus