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GDNF stimulates the proliferation of cultured mouse immature Sertoli cells via its receptor subunit NCAM and ERK1/2 signaling pathway.

Yang Y, Han C - BMC Cell Biol. (2010)

Bottom Line: In the present study, we have reported that the proliferation of cultured ISCs was significantly enhanced by GDNF.The receptor subunits GFRα1 and NCAM but not RET were expressed in ISCs, and the stimulatory effect of GDNF on the proliferation of ISCs was significantly reduced by anti-NCAM antibody blocking or siRNA that specifically targets NCAM mRNA.Additionally, the ERK1/2 inhibitor, PD98059, completely abolished the mitogenic effect of GDNF on ISCs.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.

ABSTRACT

Background: The proliferation and final density of Sertoli cells in the testis are regulated by hormones and local factors. Glial cell line-derived neurotrophic factor (GDNF), a distantly related member of the transforming growth factor-β superfamily, and its receptor subunits GDNF family receptor alpha 1 (GFRα1), RET tyrosine kinase, and neural cell adhesion molecule (NCAM) have been reported to be expressed in the testis and involved in the regulation of proliferation of immature Sertoli cells (ISCs). However, the expression patterns of these receptor subunits and the downstream signaling pathways have not been addressed in ISCs.

Results: In the present study, we have reported that the proliferation of cultured ISCs was significantly enhanced by GDNF. The receptor subunits GFRα1 and NCAM but not RET were expressed in ISCs, and the stimulatory effect of GDNF on the proliferation of ISCs was significantly reduced by anti-NCAM antibody blocking or siRNA that specifically targets NCAM mRNA. Additionally, the ERK1/2 inhibitor, PD98059, completely abolished the mitogenic effect of GDNF on ISCs.

Conclusions: GDNF stimulates the proliferation of ISCs via its receptor subunit NCAM and the consequent activation of the ERK1/2 signaling pathway.

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Phosphorylation of ERK1/2 but not AKT in ISCs is upregulated by GDNF. (A) Time-course of ERK1/2 phosphorylation levels in cultured ISCs stimulated with GDNF and the first bar (0) is cells without GDNF treatment. Total ERK1/2 was the internal control in the semi-quantitative densitometry analysis (right panel). (B) Time course of AKT phosphorylation levels in ISCs stimulated with GDNF, with total AKT as an internal control in the semi-quantitative densitometry analysis (right panel). (C) Time course of ERK1/2 phosphorylation level in PD98059 pre-treated (10 μM) ISCs stimulated with GDNF and the bar (control) is cells without GDNF or PD98059 treatment, with total ERK1/2 as the internal control in the semi-quantitative densitometry analysis (right panel). (D) Time course of AKT phosphorylation levels in PD98059 pre-treated (10 μM) ISCs stimulated with GDNF, with total AKT as the internal control in the semi-quantitative densitometry analysis (right panel). The data were presented as means ± SD from three independent experiments. Statistically significant differences (p < 0.05) among groups are indicated by an asterisk.
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Figure 4: Phosphorylation of ERK1/2 but not AKT in ISCs is upregulated by GDNF. (A) Time-course of ERK1/2 phosphorylation levels in cultured ISCs stimulated with GDNF and the first bar (0) is cells without GDNF treatment. Total ERK1/2 was the internal control in the semi-quantitative densitometry analysis (right panel). (B) Time course of AKT phosphorylation levels in ISCs stimulated with GDNF, with total AKT as an internal control in the semi-quantitative densitometry analysis (right panel). (C) Time course of ERK1/2 phosphorylation level in PD98059 pre-treated (10 μM) ISCs stimulated with GDNF and the bar (control) is cells without GDNF or PD98059 treatment, with total ERK1/2 as the internal control in the semi-quantitative densitometry analysis (right panel). (D) Time course of AKT phosphorylation levels in PD98059 pre-treated (10 μM) ISCs stimulated with GDNF, with total AKT as the internal control in the semi-quantitative densitometry analysis (right panel). The data were presented as means ± SD from three independent experiments. Statistically significant differences (p < 0.05) among groups are indicated by an asterisk.

Mentions: It has been reported that GDNF plays an essential role in regulating the self-renewal of SSCs by activating the AKT and ERK1/2 signaling pathways [30,31]. To identify the signaling pathways activated by GDNF in ISCs, we first examined the phosphorylation levels of ERK1/2 and AKT in ISCs with and without GDNF treatment by Western blotting assays. The results showed that the phosphorylation level of ERK1/2 was significantly up-regulated 5 min post-GDNF stimulation and reached its highest levels after 30 min (Figure 4A). Notably, the increase of GDNF-induced ERK1/2 phosphorylation was completely blocked by pre-treatment of ISCs with the ERK1/2 inhibitor, PD98059 (10 μM) for 45 min (Figure 4C). The basal levels of Erk1/2 phosphorylation were also down-regulated by PD98059 treatment compared with the control group (Figure 4C). In contrast, GDNF stimulation (Figure 4B) or PD98059 pre-treatment and GDNF stimulation (Figure 4D) did not influence the phosphorylation level of AKT in ISCs. More importantly, PD98059 pre-treatment completely abolished GDNF stimulated proliferation of ISCs compared with the GDNF treatment group (Figures 5A-E).


GDNF stimulates the proliferation of cultured mouse immature Sertoli cells via its receptor subunit NCAM and ERK1/2 signaling pathway.

Yang Y, Han C - BMC Cell Biol. (2010)

Phosphorylation of ERK1/2 but not AKT in ISCs is upregulated by GDNF. (A) Time-course of ERK1/2 phosphorylation levels in cultured ISCs stimulated with GDNF and the first bar (0) is cells without GDNF treatment. Total ERK1/2 was the internal control in the semi-quantitative densitometry analysis (right panel). (B) Time course of AKT phosphorylation levels in ISCs stimulated with GDNF, with total AKT as an internal control in the semi-quantitative densitometry analysis (right panel). (C) Time course of ERK1/2 phosphorylation level in PD98059 pre-treated (10 μM) ISCs stimulated with GDNF and the bar (control) is cells without GDNF or PD98059 treatment, with total ERK1/2 as the internal control in the semi-quantitative densitometry analysis (right panel). (D) Time course of AKT phosphorylation levels in PD98059 pre-treated (10 μM) ISCs stimulated with GDNF, with total AKT as the internal control in the semi-quantitative densitometry analysis (right panel). The data were presented as means ± SD from three independent experiments. Statistically significant differences (p < 0.05) among groups are indicated by an asterisk.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Phosphorylation of ERK1/2 but not AKT in ISCs is upregulated by GDNF. (A) Time-course of ERK1/2 phosphorylation levels in cultured ISCs stimulated with GDNF and the first bar (0) is cells without GDNF treatment. Total ERK1/2 was the internal control in the semi-quantitative densitometry analysis (right panel). (B) Time course of AKT phosphorylation levels in ISCs stimulated with GDNF, with total AKT as an internal control in the semi-quantitative densitometry analysis (right panel). (C) Time course of ERK1/2 phosphorylation level in PD98059 pre-treated (10 μM) ISCs stimulated with GDNF and the bar (control) is cells without GDNF or PD98059 treatment, with total ERK1/2 as the internal control in the semi-quantitative densitometry analysis (right panel). (D) Time course of AKT phosphorylation levels in PD98059 pre-treated (10 μM) ISCs stimulated with GDNF, with total AKT as the internal control in the semi-quantitative densitometry analysis (right panel). The data were presented as means ± SD from three independent experiments. Statistically significant differences (p < 0.05) among groups are indicated by an asterisk.
Mentions: It has been reported that GDNF plays an essential role in regulating the self-renewal of SSCs by activating the AKT and ERK1/2 signaling pathways [30,31]. To identify the signaling pathways activated by GDNF in ISCs, we first examined the phosphorylation levels of ERK1/2 and AKT in ISCs with and without GDNF treatment by Western blotting assays. The results showed that the phosphorylation level of ERK1/2 was significantly up-regulated 5 min post-GDNF stimulation and reached its highest levels after 30 min (Figure 4A). Notably, the increase of GDNF-induced ERK1/2 phosphorylation was completely blocked by pre-treatment of ISCs with the ERK1/2 inhibitor, PD98059 (10 μM) for 45 min (Figure 4C). The basal levels of Erk1/2 phosphorylation were also down-regulated by PD98059 treatment compared with the control group (Figure 4C). In contrast, GDNF stimulation (Figure 4B) or PD98059 pre-treatment and GDNF stimulation (Figure 4D) did not influence the phosphorylation level of AKT in ISCs. More importantly, PD98059 pre-treatment completely abolished GDNF stimulated proliferation of ISCs compared with the GDNF treatment group (Figures 5A-E).

Bottom Line: In the present study, we have reported that the proliferation of cultured ISCs was significantly enhanced by GDNF.The receptor subunits GFRα1 and NCAM but not RET were expressed in ISCs, and the stimulatory effect of GDNF on the proliferation of ISCs was significantly reduced by anti-NCAM antibody blocking or siRNA that specifically targets NCAM mRNA.Additionally, the ERK1/2 inhibitor, PD98059, completely abolished the mitogenic effect of GDNF on ISCs.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.

ABSTRACT

Background: The proliferation and final density of Sertoli cells in the testis are regulated by hormones and local factors. Glial cell line-derived neurotrophic factor (GDNF), a distantly related member of the transforming growth factor-β superfamily, and its receptor subunits GDNF family receptor alpha 1 (GFRα1), RET tyrosine kinase, and neural cell adhesion molecule (NCAM) have been reported to be expressed in the testis and involved in the regulation of proliferation of immature Sertoli cells (ISCs). However, the expression patterns of these receptor subunits and the downstream signaling pathways have not been addressed in ISCs.

Results: In the present study, we have reported that the proliferation of cultured ISCs was significantly enhanced by GDNF. The receptor subunits GFRα1 and NCAM but not RET were expressed in ISCs, and the stimulatory effect of GDNF on the proliferation of ISCs was significantly reduced by anti-NCAM antibody blocking or siRNA that specifically targets NCAM mRNA. Additionally, the ERK1/2 inhibitor, PD98059, completely abolished the mitogenic effect of GDNF on ISCs.

Conclusions: GDNF stimulates the proliferation of ISCs via its receptor subunit NCAM and the consequent activation of the ERK1/2 signaling pathway.

Show MeSH
Related in: MedlinePlus