Related Experiment Videos
Cadmium-induced expression of immediate early genes in LLC-PK1 cells
1Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts, USA.
Abstract:
To identify molecular mechanisms underlying renal cell damage by cadmium, the effect of this heavy metal on the level of immediate early genes (IEGs) transcripts in LLC-PK1 cells was studied. Cadmium chloride (CdCl2) induced the expression of four IEGs examined, but with differing time courses. The level of c-fos mRNA peaked at 30 minutes, and then decreased. The levels of c-jun and c-myc transcripts reached a maximum at one hour, and remained elevated up to four hours. Egr-1 mRNA level peaked at one hour, and returned to the control level by three hours. Experiments with cycloheximide and actinomycin D showed, respectively, that induction of IEGs by cadmium occurred in a protein synthesis-independent and transcriptional activation-dependent manner. Cadmium induction of c-fos mRNA was reduced markedly by the intracellular calcium chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)-ester (BAPTA/AM), and was decreased partially by a protein kinase C (PKC) inhibitor, 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine (H-7). These data indicate that IEG induction by cadmium requires intracellular calcium mobilization and occurs in part by a PKC-dependent pathway. Exposure of LLC-PK1 cells to CdCl2 (20 microM for 1 to 24 hr) resulted loss of cell viability and DNA fragmentation, which was indicative of apoptosis.
Insights
Cadmium chloride exposure increases immediate early genes (IEGs) in kidney cells, indicating a role for intracellular calcium and protein kinase C (PKC) in cadmium-induced renal damage and apoptosis.
Area of Science:
- Toxicology
- Molecular Biology
- Cell Biology
Background:
- Cadmium is a toxic heavy metal contributing to renal cell damage.
- Immediate early genes (IEGs) are rapidly activated by cellular stimuli.
- Understanding IEG regulation by cadmium is crucial for elucidating kidney injury mechanisms.
Purpose of the Study:
- To investigate the molecular mechanisms of renal cell damage induced by cadmium.
- To determine the effect of cadmium on immediate early genes (IEGs) transcripts in LLC-PK1 cells.
Main Methods:
- LLC-PK1 cells were exposed to cadmium chloride (CdCl2).
- Expression levels of c-fos, c-jun, c-myc, and Egr-1 mRNA were quantified.
- Experiments utilized cycloheximide, actinomycin D, a calcium chelator (BAPTA/AM), and a PKC inhibitor (H-7).
- Cell viability and DNA fragmentation were assessed.
Main Results:
- Cadmium chloride induced the expression of four IEGs with distinct time courses.
- IEG induction by cadmium was protein synthesis-independent but transcriptionally dependent.
- Cadmium-induced c-fos mRNA expression was reduced by BAPTA/AM and partially by H-7.
- Cadmium exposure led to loss of cell viability and DNA fragmentation, indicative of apoptosis.
Conclusions:
- Cadmium-induced IEG expression in renal cells requires intracellular calcium mobilization.
- The process involves, in part, a protein kinase C (PKC)-dependent pathway.
- Cadmium exposure triggers apoptosis in LLC-PK1 cells.