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Cadmium and proliferation in human uterine leiomyoma cells: evidence of a role for EGFR/MAPK pathways but not classical estrogen receptor pathways.

Gao X, Yu L, Moore AB, Kissling GE, Waalkes MP, Dixon D - Environ. Health Perspect. (2014)

Bottom Line: Cd mimics the effects of estrogen in the rat uterus, and blood Cd concentrations positively correlate with ER levels in uteri of women with fibroids.Cd did not significantly bind to ERα or ERβ, nor did it show transactivation in both cell types transiently transfected with ERE reporter genes.Additional studies in ht-UtLM cells showed that AG1478, an EGFR inhibitor, abolished Cd-induced phosphorylation of EGFR and MAPK.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathogenesis Group, National Toxicology Program (NTP) Laboratory, Division of the NTP (DNTP).

ABSTRACT

Background: It has been proposed that cadmium (Cd) is an environmental "metalloestrogen" and that its action is mediated via the estrogen receptor (ER). Cd mimics the effects of estrogen in the rat uterus, and blood Cd concentrations positively correlate with ER levels in uteri of women with fibroids.

Objectives: In the present study we explored whether Cd could stimulate proliferation of estrogen-responsive human uterine leiomyoma (ht-UtLM) cells and uterine smooth muscle cells (ht-UtSMCs) through classical interactions with ERα and ERβ, or by nongenomic mechanisms.

Methods: We used estrogen response element (ERE) reporters, phosphorylated receptor tyrosine kinase arrays, Western blot analysis, estrogen binding, and cell proliferation assays to evaluate the effects of Cd on ht-UtLM cells and ht-UtSMCs.

Results: Cd stimulated growth of both cell types at lower concentrations and inhibited growth at higher concentrations (≥ 50 μM). Cd did not significantly bind to ERα or ERβ, nor did it show transactivation in both cell types transiently transfected with ERE reporter genes. However, in both cells types, Cd (0.1 μM and 10 μM) activated p44/42 MAPK (ERK1/2), and a MAPK inhibitor (PD98059) abrogated Cd-induced cell proliferation. Cd in ht-UtLM cells, but not in ht-UtSMCs, activated the growth factor receptors EGFR, HGFR, and VEGF-R1 upstream of MAPK. Additional studies in ht-UtLM cells showed that AG1478, an EGFR inhibitor, abolished Cd-induced phosphorylation of EGFR and MAPK.

Conclusions: Our results show that low concentrations of Cd stimulated cell proliferation in estrogen-responsive uterine cells by nongenomic activation of MAPK, but not through classical ER-mediated pathways.

No MeSH data available.


Related in: MedlinePlus

Effect of PD98059 (PD) on Cd-induced cell proliferation and p44/42 MAPK phosphorylation. Cell proliferation was evaluated in ht-UtLM cells (A) and ht-UtSMCs (B) treated with vehicle (control), Cd (0.1 μM or 10 μM), with 10 μM PD98059 (PD) alone, or Cd in combination with 10 μM PD for 72 hr. The experiments were repeated three times with independent cultures. Absorbance values were determined at a 490 nm wavelength. Data are presented as mean + SE (n = 6). (C) Confocal images of ht-UtLM cells (a,b,c,d) and ht-UtSMCs (e,f,g,h) treated with vehicle (control; a,e), 10 μM Cd (b,f), PD (50 μM; c,g), or Cd plus PD (d,h) for 10 min. Red indicates phospho-p44/42 MAPK, and blue indicates DAPI staining; bar = 50 μm.*p < 0.05 compared with control.
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f3: Effect of PD98059 (PD) on Cd-induced cell proliferation and p44/42 MAPK phosphorylation. Cell proliferation was evaluated in ht-UtLM cells (A) and ht-UtSMCs (B) treated with vehicle (control), Cd (0.1 μM or 10 μM), with 10 μM PD98059 (PD) alone, or Cd in combination with 10 μM PD for 72 hr. The experiments were repeated three times with independent cultures. Absorbance values were determined at a 490 nm wavelength. Data are presented as mean + SE (n = 6). (C) Confocal images of ht-UtLM cells (a,b,c,d) and ht-UtSMCs (e,f,g,h) treated with vehicle (control; a,e), 10 μM Cd (b,f), PD (50 μM; c,g), or Cd plus PD (d,h) for 10 min. Red indicates phospho-p44/42 MAPK, and blue indicates DAPI staining; bar = 50 μm.*p < 0.05 compared with control.

Mentions: We evaluated whether the activation of the p44/42 MAPK pathway plays a role in Cd-induced cell proliferation. By adding a specific ERK inhibitor (PD, 10 μM) prior to Cd treatment (0.1 and 10 μM), Cd-induced cell proliferation was substantially abolished (p < 0.05, vs. Cd alone) in both cell types (Figure 3A,B). As shown in Figure 3C, treatment with 10 μM Cd resulted in robust activation of p44/42 MAPK as indicated by intense red positive signals in ht-UtLM cells and ht-UtSMCs (Figure 3C-b, 3C-f), whereas PD dramatically inhibited phospho-p44/42 MAPK expression (Figure 3C-c and 3C-g). Cd administration in the presence of PD did not result in activation of p44/42 MAPK (Figure 3C-d, 3C-h). Taken together, these data suggest that Cd-induced cell proliferation in ht-UtLM cells and ht-UtSMCs was mediated by activation of p44/42 MAPK.


Cadmium and proliferation in human uterine leiomyoma cells: evidence of a role for EGFR/MAPK pathways but not classical estrogen receptor pathways.

Gao X, Yu L, Moore AB, Kissling GE, Waalkes MP, Dixon D - Environ. Health Perspect. (2014)

Effect of PD98059 (PD) on Cd-induced cell proliferation and p44/42 MAPK phosphorylation. Cell proliferation was evaluated in ht-UtLM cells (A) and ht-UtSMCs (B) treated with vehicle (control), Cd (0.1 μM or 10 μM), with 10 μM PD98059 (PD) alone, or Cd in combination with 10 μM PD for 72 hr. The experiments were repeated three times with independent cultures. Absorbance values were determined at a 490 nm wavelength. Data are presented as mean + SE (n = 6). (C) Confocal images of ht-UtLM cells (a,b,c,d) and ht-UtSMCs (e,f,g,h) treated with vehicle (control; a,e), 10 μM Cd (b,f), PD (50 μM; c,g), or Cd plus PD (d,h) for 10 min. Red indicates phospho-p44/42 MAPK, and blue indicates DAPI staining; bar = 50 μm.*p < 0.05 compared with control.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384203&req=5

f3: Effect of PD98059 (PD) on Cd-induced cell proliferation and p44/42 MAPK phosphorylation. Cell proliferation was evaluated in ht-UtLM cells (A) and ht-UtSMCs (B) treated with vehicle (control), Cd (0.1 μM or 10 μM), with 10 μM PD98059 (PD) alone, or Cd in combination with 10 μM PD for 72 hr. The experiments were repeated three times with independent cultures. Absorbance values were determined at a 490 nm wavelength. Data are presented as mean + SE (n = 6). (C) Confocal images of ht-UtLM cells (a,b,c,d) and ht-UtSMCs (e,f,g,h) treated with vehicle (control; a,e), 10 μM Cd (b,f), PD (50 μM; c,g), or Cd plus PD (d,h) for 10 min. Red indicates phospho-p44/42 MAPK, and blue indicates DAPI staining; bar = 50 μm.*p < 0.05 compared with control.
Mentions: We evaluated whether the activation of the p44/42 MAPK pathway plays a role in Cd-induced cell proliferation. By adding a specific ERK inhibitor (PD, 10 μM) prior to Cd treatment (0.1 and 10 μM), Cd-induced cell proliferation was substantially abolished (p < 0.05, vs. Cd alone) in both cell types (Figure 3A,B). As shown in Figure 3C, treatment with 10 μM Cd resulted in robust activation of p44/42 MAPK as indicated by intense red positive signals in ht-UtLM cells and ht-UtSMCs (Figure 3C-b, 3C-f), whereas PD dramatically inhibited phospho-p44/42 MAPK expression (Figure 3C-c and 3C-g). Cd administration in the presence of PD did not result in activation of p44/42 MAPK (Figure 3C-d, 3C-h). Taken together, these data suggest that Cd-induced cell proliferation in ht-UtLM cells and ht-UtSMCs was mediated by activation of p44/42 MAPK.

Bottom Line: Cd mimics the effects of estrogen in the rat uterus, and blood Cd concentrations positively correlate with ER levels in uteri of women with fibroids.Cd did not significantly bind to ERα or ERβ, nor did it show transactivation in both cell types transiently transfected with ERE reporter genes.Additional studies in ht-UtLM cells showed that AG1478, an EGFR inhibitor, abolished Cd-induced phosphorylation of EGFR and MAPK.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathogenesis Group, National Toxicology Program (NTP) Laboratory, Division of the NTP (DNTP).

ABSTRACT

Background: It has been proposed that cadmium (Cd) is an environmental "metalloestrogen" and that its action is mediated via the estrogen receptor (ER). Cd mimics the effects of estrogen in the rat uterus, and blood Cd concentrations positively correlate with ER levels in uteri of women with fibroids.

Objectives: In the present study we explored whether Cd could stimulate proliferation of estrogen-responsive human uterine leiomyoma (ht-UtLM) cells and uterine smooth muscle cells (ht-UtSMCs) through classical interactions with ERα and ERβ, or by nongenomic mechanisms.

Methods: We used estrogen response element (ERE) reporters, phosphorylated receptor tyrosine kinase arrays, Western blot analysis, estrogen binding, and cell proliferation assays to evaluate the effects of Cd on ht-UtLM cells and ht-UtSMCs.

Results: Cd stimulated growth of both cell types at lower concentrations and inhibited growth at higher concentrations (≥ 50 μM). Cd did not significantly bind to ERα or ERβ, nor did it show transactivation in both cell types transiently transfected with ERE reporter genes. However, in both cells types, Cd (0.1 μM and 10 μM) activated p44/42 MAPK (ERK1/2), and a MAPK inhibitor (PD98059) abrogated Cd-induced cell proliferation. Cd in ht-UtLM cells, but not in ht-UtSMCs, activated the growth factor receptors EGFR, HGFR, and VEGF-R1 upstream of MAPK. Additional studies in ht-UtLM cells showed that AG1478, an EGFR inhibitor, abolished Cd-induced phosphorylation of EGFR and MAPK.

Conclusions: Our results show that low concentrations of Cd stimulated cell proliferation in estrogen-responsive uterine cells by nongenomic activation of MAPK, but not through classical ER-mediated pathways.

No MeSH data available.


Related in: MedlinePlus