Mediator of DNA damage checkpoint 1 (MDC1) contributes to high NaCl-induced activation of the osmoprotective transcription factor TonEBP/OREBP

Abstract

Background: Hypertonicity, such as induced by high NaCl, increases the activity of the transcription factor TonEBP/OREBP whose target genes increase osmoprotective organic osmolytes and heat shock proteins.

Methodology: We used mass spectrometry to analyze proteins that coimmunoprecipitate with TonEBP/OREBP in order to identify ones that might contribute to its high NaCl-induced activation.

Principal findings: We identified 20 unique peptides from Mediator of DNA Damage Checkpoint 1 (MDC1) with high probability. The identification was confirmed by Western analysis. We used small interfering RNA knockdown of MDC1 to characterize its osmotic function. Knocking down MDC1 reduces high NaCl-induced increases in TonEBP/OREBP transcriptional and transactivating activity, but has no significant effect on its nuclear localization. We confirm six previously known phosphorylation sites in MDC1, but do not find evidence that high NaCl increases phosphorylation of MDC1. It is suggestive that MDC1 acts as a DNA damage response protein since hypertonicity reversibly increases DNA breaks, and other DNA damage response proteins, like ATM, also associate with TonEBP/OREBP and contribute to its activation by hypertonicity.

Conclusions/significance: MDC1 associates with TonEBP/OREBP and contributes to high NaCl-induced increase of that factor's transcriptional activity.

Figure 1. Identification of MDC1 in immunoprecipitates of TonEBP/OREBP-1-547-V5 by mass spectrometry.

MS² spectra of four MDC1 peptides. The arrows indicate ions that are site determining for phosphorylation.

Figure 2. Confirmation by Western analysis of identification of MDC1 in immunoprecipitates of TonEBP/OREBP-1-547-V5.

“Input” is the protein extract before immunoprecipitation. A. Osmolality bathing HEK 293 cells stably transfected with TonEBP/OREBP-1-547-V5 or empty vector-V5 (EV) was raised from 300 to 500 mosmol/kg by adding NaCl for 1, 3 and 6 h. Proteins were immunoprecipitated with rabbit IgG or anti-V5 antibodies and immunoblotted with anti-V5 or anti-MDC1 antibody. B. Osmolality bathing HEK 293 cells stably transfected with TonEBP/OREBP-1-547-V5 or empty vector-V5 was raised from 300 to 500 mosmol/kg by adding NaCl for 2 h. Proteins were immunoprecipitated from nuclear lysates with rabbit IgG or anti-MDC1 and immunoblotted with anti-MDC1 or anti-V5 antibody. C. 100 µg/ml ethidium bromide was added during immunoprecipitation with anti-MDC1 from nuclear extracts of the stably transfected HEK293 cells expressing TonEBP/OREBP-1-547-V5.

Figure 3. Effect of high NaCl on MDC1 abundance and distribution.

HEK293 cells were exposed to media of the indicated total osmolalities (NaCl varied) for 2 h. A. High NaCl increases the abundance of MDC1 in the soluble fraction and decreases it in the chromatin-bound fraction. (n = 3, *P<0.05, t test). B. High NaCl decreases total MDC1 abundance (n = 3, *P<0.05, t test). C. After 2 h of exposure to high NaCl medium osmolality was decreased to 300 mosmol/kg for 30 min. MDC1 returns to the chromatin-bound fraction within that time (representative of 2 experiments).

Figure 4. Effect on TonEBP/OREBP transcriptional and transactivating activities of knocking down MDC1 expression.

A. HEK293 cells stably expressing an ORE-X reporter were transiently transfected with 25 nM of siRNA against MDC1 or control siRNA for 48 hours. Then, the osmolality of medium was changed to 500 mosm/kg by adding NaCl or kept at 300 mosmol/kg, and reporter activity was measured 24 h later. Knock down of MDC1 is shown in the upper panel. ORE-X reporter activity is relative to “control” at 300 mosmol/kg. B. Control for specificity for ORE. Same as (A) except using an IL2 min reporter (no ORE-X DNA element). C. Same as (A) except using HEK293 stably expressing the binary GAL4dbd TAD reporter and measuring luciferase activity 16 hours after adjusting osmolality. D. Control for specificity for TonEBP/OREBP TAD. Same as (C) except using HEK293 cells stably transfected with Gal4 DBD (no TAD). Mean ±SEM, *, P<0.01, n = 3.

Figure 5

A. Lack of effect of siRNA-mediated knockdown of MDC1 on nuclear localization of TonEBP/OREBP. HEK 293 cells were transiently transfected with 25 nM MDC1 siRNA or control siRNA. 48 h later the osmolality of medium was changed to 200 or 500 mosmol/kg or kept at 300 mosmol/kg for 1 h. Nuclear and cytoplasmic extracts were prepared. TonEBP/OREBP nuclear/cytoplasmic ratio was calculated from its abundance in nuclear and cytoplasmic extracts. Antibodies to BRG1 and aldose reductase serve as controls for nuclear and cytoplasmic fractionation, respectively. B. High NaCl decreases the nuclear localization of MDC1. Osmolality was changed to 200 or 500 mosmol/kg or kept at 300 mosmol/kg for 1 h. Nuclear and cytoplasmic extracts were prepared. MDC1 nuclear/cytoplasmic ratio was calculated from its abundance in nuclear and cytoplasmic extracts. C. Lack of effect of siRNA-mediated knockdown of MDC1 on phosphorylation of ATM on Ser1981. As in (A), except that abundance of ATM was measured in whole cell extracts by Western analysis using a non-phosphospecific antibody and its phosphorylation on Ser1981 was measured using phosphospecific anti-ATM 1981S-P antibody. Results are presented as the ratio of phosphorylated to non phosphorylated ATM. (* P<.05, compared to 300 mosmol).