Related Experiment Videos
Immediate early gene and HSP70 expression in hyperosmotic stress in MDCK cells
D M Cohen1, J C Wasserman, S R Gullans
1Renal Division, Brigham and Women's Hospital, Boston, Massachusetts.
Abstract:
The early genetic response to hyperosmotic stress remains to be elucidated in eukaryotes. We observed that hyperosmotic NaCl in Madin-Darby canine kidney cells increased levels of mRNA encoding the immediate early gene (IEG) transcription factors Egr-1 and c-fos at 2 h of treatment by two- and threefold, respectively. Sham treatment and hyperosmotic glycerol, and ineffective osmole, had no effect. Hyperosmotic NaCl, but not glycerol, also increased the mRNA level of the stress protein HSP70 by four- to fivefold at 2, 6, and 24 h. These changes occurred despite inhibition of total RNA transcription rate and DNA synthesis rate by NaCl. Neither NaCl nor glycerol treatment manifested significant cytotoxicity. NaCl, and to a lesser extent glycerol, suppressed protein synthesis, a phenomenon previously correlated with IEG superinduction. Therefore, hyperosmotic stimuli with different physiological effects result in differential expression of IEGs and the stress protein HSP70; induction of the former may govern the ensuing program of gene expression that culminates in the osmolyte response, while the latter may serve as a temporizing protective measure.
Insights
Hyperosmotic NaCl rapidly increases immediate early gene (IEG) mRNA levels in kidney cells, despite inhibiting RNA and DNA synthesis. This response may initiate the cell
Area of Science:
- Molecular Biology
- Cellular Stress Response
- Eukaryotic Gene Expression
Background:
- The early genetic mechanisms underlying eukaryotic responses to hyperosmotic stress are not fully understood.
- Immediate early genes (IEGs) are rapidly activated by various stimuli and play crucial roles in cellular signaling.
- Understanding the differential gene expression in response to varying osmotic stimuli is key to elucidating cellular adaptation.
Purpose of the Study:
- To investigate the early genetic transcriptional response of Madin-Darby canine kidney (MDCK) cells to hyperosmotic sodium chloride (NaCl) stress.
- To determine the differential expression of immediate early genes (IEGs) and stress proteins under hyperosmotic conditions.
- To explore the relationship between protein synthesis inhibition and IEG superinduction during osmotic stress.
Main Methods:
- Treatment of MDCK cells with hyperosmotic NaCl or glycerol.
- Quantitative analysis of mRNA levels for immediate early genes (Egr-1, c-fos) and the stress protein HSP70 using RT-PCR.
- Assessment of total RNA transcription rate, DNA synthesis rate, protein synthesis, and cytotoxicity.
Main Results:
- Hyperosmotic NaCl significantly increased mRNA levels of Egr-1 and c-fos within 2 hours, while glycerol had no effect.
- NaCl treatment elevated HSP70 mRNA levels at 2, 6, and 24 hours, an effect not observed with glycerol.
- NaCl inhibited RNA and DNA synthesis rates, and both NaCl and glycerol suppressed protein synthesis, correlating with IEG superinduction.
Conclusions:
- Hyperosmotic stimuli with differing physiological impacts induce distinct patterns of IEG and HSP70 expression.
- IEG induction by hyperosmotic stress likely orchestrates the cellular osmolyte response program.
- HSP70 induction may act as a temporary protective mechanism against osmotic damage.