Limits...
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.

Show MeSH

Related in: MedlinePlus

The proliferation stimulating effect of GDNF on ISCs is mediated by NCAM. (A-C) BrdU-positive ISCs in response to GDNF stimulation with cells pre-treated with non-specific IgG (A) and NCAM polyclonal antibody (B) and the quantitative comparison (C). (D-G) NCAM mRNA (D, E) and protein (F, G) levels in ISCs and TM4 cells were significantly reduced by NCAM-specific siRNA but not by the negative control siRNA when compared with control groups (1, normal culture control; 2, negative control siRNA; 3, NCAM siRNA). G3PDH and β-actin served as the loading controls for RNA and protein respectively. Expression values of NCAM mRNA (E) or protein (G) were normalized against G3PDH or β-actin signals, respectively. (H-L) BrdU-positive ISCs with negative control siRNA (H) and NCAM siRNA groups (I) stimulated with GDNF and in normal culture controls (J) and the NCAM siRNA group (K) not treated with GDNF, as well as the quantitative comparison among all groups (L). The data is presented as means ± SD from three independent experiments. Statistically significant differences (p < 0.05) among groups are indicated by * or ** (p < 0.01). Scale bar indicates 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2967512&req=5

Figure 3: The proliferation stimulating effect of GDNF on ISCs is mediated by NCAM. (A-C) BrdU-positive ISCs in response to GDNF stimulation with cells pre-treated with non-specific IgG (A) and NCAM polyclonal antibody (B) and the quantitative comparison (C). (D-G) NCAM mRNA (D, E) and protein (F, G) levels in ISCs and TM4 cells were significantly reduced by NCAM-specific siRNA but not by the negative control siRNA when compared with control groups (1, normal culture control; 2, negative control siRNA; 3, NCAM siRNA). G3PDH and β-actin served as the loading controls for RNA and protein respectively. Expression values of NCAM mRNA (E) or protein (G) were normalized against G3PDH or β-actin signals, respectively. (H-L) BrdU-positive ISCs with negative control siRNA (H) and NCAM siRNA groups (I) stimulated with GDNF and in normal culture controls (J) and the NCAM siRNA group (K) not treated with GDNF, as well as the quantitative comparison among all groups (L). The data is presented as means ± SD from three independent experiments. Statistically significant differences (p < 0.05) among groups are indicated by * or ** (p < 0.01). Scale bar indicates 10 μm.

Mentions: Cultured ISCs were pre-treated with a polyclonal human NCAM antibody that was raised against the N-terminal 300 amino acids, and then treated with GDNF and pulse-labeled with BrdU. After immunocytochemical staining, the numbers of BrdU-positive cells and DAPI stained nuclei were counted. As shown in Figures 3A-C, the percentage of the BrdU positive cells in the NCAM antibody-treated group was significantly lower than that in the non-specific IgG treated group. Next, we knocked down the expression of NCAM by siRNAs that specifically targeted the NCAM mRNA, and tested whether the proliferation stimulation effect of GDNF on ISCs could also be abolished. As shown by Figures 3D-G, NCAM mRNA and protein expression in both ISCs and TM4 cells were significantly reduced in the NCAM siRNA transfected group compared with the negative control siRNA group. Importantly, the proliferation stimulating effect of GDNF on ISCs was significantly reduced in the NCAM siRNA transfected group compared with the negative control siRNA group (Figures 3H-L).


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)

The proliferation stimulating effect of GDNF on ISCs is mediated by NCAM. (A-C) BrdU-positive ISCs in response to GDNF stimulation with cells pre-treated with non-specific IgG (A) and NCAM polyclonal antibody (B) and the quantitative comparison (C). (D-G) NCAM mRNA (D, E) and protein (F, G) levels in ISCs and TM4 cells were significantly reduced by NCAM-specific siRNA but not by the negative control siRNA when compared with control groups (1, normal culture control; 2, negative control siRNA; 3, NCAM siRNA). G3PDH and β-actin served as the loading controls for RNA and protein respectively. Expression values of NCAM mRNA (E) or protein (G) were normalized against G3PDH or β-actin signals, respectively. (H-L) BrdU-positive ISCs with negative control siRNA (H) and NCAM siRNA groups (I) stimulated with GDNF and in normal culture controls (J) and the NCAM siRNA group (K) not treated with GDNF, as well as the quantitative comparison among all groups (L). The data is presented as means ± SD from three independent experiments. Statistically significant differences (p < 0.05) among groups are indicated by * or ** (p < 0.01). Scale bar indicates 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2967512&req=5

Figure 3: The proliferation stimulating effect of GDNF on ISCs is mediated by NCAM. (A-C) BrdU-positive ISCs in response to GDNF stimulation with cells pre-treated with non-specific IgG (A) and NCAM polyclonal antibody (B) and the quantitative comparison (C). (D-G) NCAM mRNA (D, E) and protein (F, G) levels in ISCs and TM4 cells were significantly reduced by NCAM-specific siRNA but not by the negative control siRNA when compared with control groups (1, normal culture control; 2, negative control siRNA; 3, NCAM siRNA). G3PDH and β-actin served as the loading controls for RNA and protein respectively. Expression values of NCAM mRNA (E) or protein (G) were normalized against G3PDH or β-actin signals, respectively. (H-L) BrdU-positive ISCs with negative control siRNA (H) and NCAM siRNA groups (I) stimulated with GDNF and in normal culture controls (J) and the NCAM siRNA group (K) not treated with GDNF, as well as the quantitative comparison among all groups (L). The data is presented as means ± SD from three independent experiments. Statistically significant differences (p < 0.05) among groups are indicated by * or ** (p < 0.01). Scale bar indicates 10 μm.
Mentions: Cultured ISCs were pre-treated with a polyclonal human NCAM antibody that was raised against the N-terminal 300 amino acids, and then treated with GDNF and pulse-labeled with BrdU. After immunocytochemical staining, the numbers of BrdU-positive cells and DAPI stained nuclei were counted. As shown in Figures 3A-C, the percentage of the BrdU positive cells in the NCAM antibody-treated group was significantly lower than that in the non-specific IgG treated group. Next, we knocked down the expression of NCAM by siRNAs that specifically targeted the NCAM mRNA, and tested whether the proliferation stimulation effect of GDNF on ISCs could also be abolished. As shown by Figures 3D-G, NCAM mRNA and protein expression in both ISCs and TM4 cells were significantly reduced in the NCAM siRNA transfected group compared with the negative control siRNA group. Importantly, the proliferation stimulating effect of GDNF on ISCs was significantly reduced in the NCAM siRNA transfected group compared with the negative control siRNA group (Figures 3H-L).

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