Limits...
Cadmium induces transcription independently of intracellular calcium mobilization.

Tvermoes BE, Bird GS, Freedman JH - PLoS ONE (2011)

Bottom Line: It has been proposed that cadmium activates transcription by altering intracellular calcium concentration ([Ca(2+)](i)) and disrupting calcium-mediated intracellular signaling processes.In HEK293 constitutively expressing YC3.60, this calcium sensor was found to be insensitive to cadmium.Exposing HEK293::YC3.60 cells to non-cytotoxic cadmium concentrations was sufficient to induce transcription of cadmium-responsive genes but did not affect [Ca(2+)](i) mobilization or increase steady-state mRNA levels of calcium-responsive genes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.

ABSTRACT

Background: Exposure to cadmium is associated with human pathologies and altered gene expression. The molecular mechanisms by which cadmium affects transcription remain unclear. It has been proposed that cadmium activates transcription by altering intracellular calcium concentration ([Ca(2+)](i)) and disrupting calcium-mediated intracellular signaling processes. This hypothesis is based on several studies that may be technically problematic; including the use of BAPTA chelators, BAPTA-based fluorescent sensors, and cytotoxic concentrations of metal.

Methodology/principal finding: In the present report, the effects of cadmium on [Ca(2+)](i) under non-cytotoxic and cytotoxic conditions was monitored using the protein-based calcium sensor yellow cameleon (YC3.60), which was stably expressed in HEK293 cells. In HEK293 constitutively expressing YC3.60, this calcium sensor was found to be insensitive to cadmium. Exposing HEK293::YC3.60 cells to non-cytotoxic cadmium concentrations was sufficient to induce transcription of cadmium-responsive genes but did not affect [Ca(2+)](i) mobilization or increase steady-state mRNA levels of calcium-responsive genes. In contrast, exposure to cytotoxic concentrations of cadmium significantly reduced intracellular calcium stores and altered calcium-responsive gene expression.

Conclusions/significance: These data indicate that at low levels, cadmium induces transcription independently of intracellular calcium mobilization. The results also support a model whereby cytotoxic levels of cadmium activate calcium-responsive transcription as a general response to metal-induced intracellular damage and not via a specific mechanism. Thus, the modulation of intracellular calcium may not be a primary mechanism by which cadmium regulates transcription.

Show MeSH

Related in: MedlinePlus

panel A, Traces represent [Ca2+]i measured in control HEK293::YC3.60 cells (black line) or cells following exposure to 1 µM cadmium for 4 hr (red line).The traces are representative of typical responses observed in at least three independent experiments. panel B, Means of the peak values in thapsigargin response following exposure to 1 µM cadmium for 4 hr (gray bar) or non-cadmium treated (black bar). Data were expressed as the mean ± SEM and were analyzed by an unpaired t-test. There were no significant differences between control and the cadmium exposed groups. panel C, Traces represent [Ca2+]i measured in HEK 293::YC3.60 cells under control conditions (black line) or following a 4 h exposure to 1 µM cadmium (red line). Following incubation with cadmium, cells were treated with an ionomycin-BAPTA solution in calcium-free HBSS. The traces were representative of typical responses observed in at least three independent experiments. panel D, Mean peak values of ionomycin responses in HEK293::YC3.60 cells following exposure to 0, 1, 3, 10, and 30 µM cadmium for 4 h (black bar) or 24 h (gray bar). Data were expressed as the mean ± SEM and were analyzed by one-way ANOVA followed by Dunnett's post-test. Asterisks (*) indicate significant difference (p<0.001) between control and cadmium exposed groups.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3111418&req=5

pone-0020542-g004: panel A, Traces represent [Ca2+]i measured in control HEK293::YC3.60 cells (black line) or cells following exposure to 1 µM cadmium for 4 hr (red line).The traces are representative of typical responses observed in at least three independent experiments. panel B, Means of the peak values in thapsigargin response following exposure to 1 µM cadmium for 4 hr (gray bar) or non-cadmium treated (black bar). Data were expressed as the mean ± SEM and were analyzed by an unpaired t-test. There were no significant differences between control and the cadmium exposed groups. panel C, Traces represent [Ca2+]i measured in HEK 293::YC3.60 cells under control conditions (black line) or following a 4 h exposure to 1 µM cadmium (red line). Following incubation with cadmium, cells were treated with an ionomycin-BAPTA solution in calcium-free HBSS. The traces were representative of typical responses observed in at least three independent experiments. panel D, Mean peak values of ionomycin responses in HEK293::YC3.60 cells following exposure to 0, 1, 3, 10, and 30 µM cadmium for 4 h (black bar) or 24 h (gray bar). Data were expressed as the mean ± SEM and were analyzed by one-way ANOVA followed by Dunnett's post-test. Asterisks (*) indicate significant difference (p<0.001) between control and cadmium exposed groups.

Mentions: It has been proposed that cadmium activates transcription by depleting ER calcium stores to alter [Ca2+]i [34]. To determine if non-cytotoxic concentrations of cadmium had an effect on calcium homeostasis or ER calcium stores, HEK293::YC3.60 cells were treated with the SERCA pump inhibitor thapsigargin prior to cadmium addition. Treating HEK293::YC3.60 cells with thapsigargin resulted in a typical biphasic change in [Ca2+]i, which represented depletion of ER calcium stores and the re-entry of calcium across the plasma membrane by the activation of store-operated calcium entry (SOCE) (Fig. 4A) [35], [36]. The biphasic calcium response induced by thapsigargin allowed several aspects of calcium homeostasis to be investigated: (i) thapsigargin-mediated inhibition of SERCA pump activity; (ii) proper operation of plasma membrane calcium-ATPases; and (iii) the activation mechanism and permeation properties of SOCE [37]. To determine if cadmium exposure altered the activity of these homeostatic processes, the thapsigargin-induced biphasic calcium response in cells exposed to 1 µM cadmium for 4 h was compared to control cells. The biphasic calcium response in HEK293::YC3.60 cells exposed to 1 µM cadmium was not significantly different from control cells (Fig. 4B). This indicated that exposure to cadmium at a concentration sufficient to induce transcription did not disrupt calcium homeostasis.


Cadmium induces transcription independently of intracellular calcium mobilization.

Tvermoes BE, Bird GS, Freedman JH - PLoS ONE (2011)

panel A, Traces represent [Ca2+]i measured in control HEK293::YC3.60 cells (black line) or cells following exposure to 1 µM cadmium for 4 hr (red line).The traces are representative of typical responses observed in at least three independent experiments. panel B, Means of the peak values in thapsigargin response following exposure to 1 µM cadmium for 4 hr (gray bar) or non-cadmium treated (black bar). Data were expressed as the mean ± SEM and were analyzed by an unpaired t-test. There were no significant differences between control and the cadmium exposed groups. panel C, Traces represent [Ca2+]i measured in HEK 293::YC3.60 cells under control conditions (black line) or following a 4 h exposure to 1 µM cadmium (red line). Following incubation with cadmium, cells were treated with an ionomycin-BAPTA solution in calcium-free HBSS. The traces were representative of typical responses observed in at least three independent experiments. panel D, Mean peak values of ionomycin responses in HEK293::YC3.60 cells following exposure to 0, 1, 3, 10, and 30 µM cadmium for 4 h (black bar) or 24 h (gray bar). Data were expressed as the mean ± SEM and were analyzed by one-way ANOVA followed by Dunnett's post-test. Asterisks (*) indicate significant difference (p<0.001) between control and cadmium exposed groups.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111418&req=5

pone-0020542-g004: panel A, Traces represent [Ca2+]i measured in control HEK293::YC3.60 cells (black line) or cells following exposure to 1 µM cadmium for 4 hr (red line).The traces are representative of typical responses observed in at least three independent experiments. panel B, Means of the peak values in thapsigargin response following exposure to 1 µM cadmium for 4 hr (gray bar) or non-cadmium treated (black bar). Data were expressed as the mean ± SEM and were analyzed by an unpaired t-test. There were no significant differences between control and the cadmium exposed groups. panel C, Traces represent [Ca2+]i measured in HEK 293::YC3.60 cells under control conditions (black line) or following a 4 h exposure to 1 µM cadmium (red line). Following incubation with cadmium, cells were treated with an ionomycin-BAPTA solution in calcium-free HBSS. The traces were representative of typical responses observed in at least three independent experiments. panel D, Mean peak values of ionomycin responses in HEK293::YC3.60 cells following exposure to 0, 1, 3, 10, and 30 µM cadmium for 4 h (black bar) or 24 h (gray bar). Data were expressed as the mean ± SEM and were analyzed by one-way ANOVA followed by Dunnett's post-test. Asterisks (*) indicate significant difference (p<0.001) between control and cadmium exposed groups.
Mentions: It has been proposed that cadmium activates transcription by depleting ER calcium stores to alter [Ca2+]i [34]. To determine if non-cytotoxic concentrations of cadmium had an effect on calcium homeostasis or ER calcium stores, HEK293::YC3.60 cells were treated with the SERCA pump inhibitor thapsigargin prior to cadmium addition. Treating HEK293::YC3.60 cells with thapsigargin resulted in a typical biphasic change in [Ca2+]i, which represented depletion of ER calcium stores and the re-entry of calcium across the plasma membrane by the activation of store-operated calcium entry (SOCE) (Fig. 4A) [35], [36]. The biphasic calcium response induced by thapsigargin allowed several aspects of calcium homeostasis to be investigated: (i) thapsigargin-mediated inhibition of SERCA pump activity; (ii) proper operation of plasma membrane calcium-ATPases; and (iii) the activation mechanism and permeation properties of SOCE [37]. To determine if cadmium exposure altered the activity of these homeostatic processes, the thapsigargin-induced biphasic calcium response in cells exposed to 1 µM cadmium for 4 h was compared to control cells. The biphasic calcium response in HEK293::YC3.60 cells exposed to 1 µM cadmium was not significantly different from control cells (Fig. 4B). This indicated that exposure to cadmium at a concentration sufficient to induce transcription did not disrupt calcium homeostasis.

Bottom Line: It has been proposed that cadmium activates transcription by altering intracellular calcium concentration ([Ca(2+)](i)) and disrupting calcium-mediated intracellular signaling processes.In HEK293 constitutively expressing YC3.60, this calcium sensor was found to be insensitive to cadmium.Exposing HEK293::YC3.60 cells to non-cytotoxic cadmium concentrations was sufficient to induce transcription of cadmium-responsive genes but did not affect [Ca(2+)](i) mobilization or increase steady-state mRNA levels of calcium-responsive genes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.

ABSTRACT

Background: Exposure to cadmium is associated with human pathologies and altered gene expression. The molecular mechanisms by which cadmium affects transcription remain unclear. It has been proposed that cadmium activates transcription by altering intracellular calcium concentration ([Ca(2+)](i)) and disrupting calcium-mediated intracellular signaling processes. This hypothesis is based on several studies that may be technically problematic; including the use of BAPTA chelators, BAPTA-based fluorescent sensors, and cytotoxic concentrations of metal.

Methodology/principal finding: In the present report, the effects of cadmium on [Ca(2+)](i) under non-cytotoxic and cytotoxic conditions was monitored using the protein-based calcium sensor yellow cameleon (YC3.60), which was stably expressed in HEK293 cells. In HEK293 constitutively expressing YC3.60, this calcium sensor was found to be insensitive to cadmium. Exposing HEK293::YC3.60 cells to non-cytotoxic cadmium concentrations was sufficient to induce transcription of cadmium-responsive genes but did not affect [Ca(2+)](i) mobilization or increase steady-state mRNA levels of calcium-responsive genes. In contrast, exposure to cytotoxic concentrations of cadmium significantly reduced intracellular calcium stores and altered calcium-responsive gene expression.

Conclusions/significance: These data indicate that at low levels, cadmium induces transcription independently of intracellular calcium mobilization. The results also support a model whereby cytotoxic levels of cadmium activate calcium-responsive transcription as a general response to metal-induced intracellular damage and not via a specific mechanism. Thus, the modulation of intracellular calcium may not be a primary mechanism by which cadmium regulates transcription.

Show MeSH
Related in: MedlinePlus