Regulation of de novo ceramide synthesis: the role of dihydroceramide desaturase and transcriptional factors NFATC and Hand2 in the hypoxic mouse heart

Abstract

We have previously shown that ceramide, a proapoptotic molecule decreases in the mouse heart as it adapts to hypoxia. We have also shown that its precursor, dihydroceramide, accumulates with hypoxia. This implicates the enzyme dihydroceramide desaturase (DHC-DS), which converts dihydroceramide to ceramide, in a potential regulatory checkpoint in cardiomyocytes. We hypothesised that the regulation of de novo ceramide synthesis plays an important role in the cardiomyocyte adaptation to hypoxia. We used an established mouse model to induce acute and chronic hypoxia. Cardiac tissues were extracted and quantitative real-time polymerase chain reaction (qRT-PCR) was used to evaluate the expression levels of DHC-DS. Electrophoretic Mobility Shift Assays (EMSAs) and qRT-PCR were used to evaluate the activity and expression levels of an array of transcription factors that might regulate DEGS1 gene expression. We demonstrated that DEGS1 mRNA levels decrease with time in hypoxic mice concurrent with the decrease in HAND2 transcripts. Interestingly, the DEGS1 promoter harbors overlapping sites for Hand2 and Nuclear Factor of Activated T-cells (NFATC) transcription factors. We have demonstrated a physical interaction between NFATC1 and the E-Box proteins with EMSA and coimmunoprecipitation assays. The regulation of de novo ceramide synthesis in response to hypoxia and this newly described interaction between E-box and NFATC transcription factors will pave the way to identify new pathways in the adaptation of the cardiomyocyte to stress. The elucidation of these pathways will in the long-term provide insights into potential targets for novel therapeutic regimens.

FIG. 1.

LASS genes regulation of de novo ceramide synthesis in the hypoxic heart. Comparison of the mean normalized expression (MNE) of the different ceramide synthetase enzymes (LASS1 to LASS6) between normal and hypoxic heart tissues at different ages (1 day, 1, 4, and 8 weeks). The results are the mean of two independent RNA extractions followed by qPCR done on five samples from each group of mice (normal and hypoxic at 1 day, 1, 4, and 8 weeks). *p<0.05. LV, left ventricle; RV, right ventricle.

FIG. 2.

DEGS1, Hand2, and activated calcineurin (PPP3CA) expression in the hypoxic heart. Comparison of the MNE of the following different transcripts between control and hypoxic left and right ventricles: DEGS1, PPP3CA, Hand2. The results are the mean of two independent RNA extractions followed by qPCR done on five samples from each group of mice (normal and hypoxic at 1 day, 1, 4, and 8 weeks). *p<0.05. LV, left ventricle; RV, right ventricle.

FIG. 3.

Cardiac-enriched transcription factors and NPPA (Atrial natriuretic factor, ANF) expression. Comparison of the MNE of GATA4, GATA5, HIF-1a and ANF (NPPA) between normal and hypoxic heart tissues at different ages (1 day, 1 week, 4 weeks, and 8 weeks). The results are the mean of two independent RNA extractions followed by qPCR done on 5 samples from each group of mice (normal and hypoxic at 1 day, 1, 4, and 8 weeks). *p<0.05. LV, left ventricle; RV, right ventricle.

FIG. 4.

NFATC and Hand2 regulate the DEGS1 promoter. (A) The mouse DEGS1 promoter (1500 bp upstream of the transcription initiation site) harbors multiple binding sites for GATA (underlined) and bHLH proteins in addition to two juxtaposed binding sites for bHLH and NFAT (bold). Capital letters are for the transcript and small letters for the 5′ upstream region. (B) NFAT and bHLH protein hypothetical interaction using Hex4.5. The Rel domain of NFATC1 (blue) and the bHLH domain of Pan and Hand2 (red) were used and results showed a favorable exothermic association with a docking energy of −500 KJ/mol for HAND2 and −546 KJ/mol for Pan. (C) Co-immunoprecipitation of Flag-tagged Hand2 and NFATC1 proteins showing a strong interaction between both transcription factors. Western blots with the Flag antibody (left panel) showing 15% input of protein extracts used for the co-IP. Immunoprecipitation was done using an anti-Hand2 antibody and immunoblotted using the anti-Flag antibody. (D) Gel shift analysis with whole cell protein extracts from AD293 cells overexpressing Hand2, NFATC1, and Pan showed that all Pan, NFATC1, and Pan/Hand2 can bind specifically the E-box/NFAT-binding site on the DEGS1 promoter. They can also form a stable ternary complex (*). Control refers to mock transfected cells. (E) A functional interaction between NFATC1 and Hand2. Luciferase assays shows that both proteins interact together to synergistically activate the DEGS1 promoter (+ and ++ refers to increasing doses of the plasmid being used). *p<0.05. bHLH, basic-helix-loop-helix; NFATC, Nuclear Factor of Activated T-cells. Color images available online at www.liebertpub.com/dna

FIG. 4.

NFATC and Hand2 regulate the DEGS1 promoter. (A) The mouse DEGS1 promoter (1500 bp upstream of the transcription initiation site) harbors multiple binding sites for GATA (underlined) and bHLH proteins in addition to two juxtaposed binding sites for bHLH and NFAT (bold). Capital letters are for the transcript and small letters for the 5′ upstream region. (B) NFAT and bHLH protein hypothetical interaction using Hex4.5. The Rel domain of NFATC1 (blue) and the bHLH domain of Pan and Hand2 (red) were used and results showed a favorable exothermic association with a docking energy of −500 KJ/mol for HAND2 and −546 KJ/mol for Pan. (C) Co-immunoprecipitation of Flag-tagged Hand2 and NFATC1 proteins showing a strong interaction between both transcription factors. Western blots with the Flag antibody (left panel) showing 15% input of protein extracts used for the co-IP. Immunoprecipitation was done using an anti-Hand2 antibody and immunoblotted using the anti-Flag antibody. (D) Gel shift analysis with whole cell protein extracts from AD293 cells overexpressing Hand2, NFATC1, and Pan showed that all Pan, NFATC1, and Pan/Hand2 can bind specifically the E-box/NFAT-binding site on the DEGS1 promoter. They can also form a stable ternary complex (*). Control refers to mock transfected cells. (E) A functional interaction between NFATC1 and Hand2. Luciferase assays shows that both proteins interact together to synergistically activate the DEGS1 promoter (+ and ++ refers to increasing doses of the plasmid being used). *p<0.05. bHLH, basic-helix-loop-helix; NFATC, Nuclear Factor of Activated T-cells. Color images available online at www.liebertpub.com/dna

FIG. 5.

DEGS1 upregulation by the coexpression of NFATC1 and Hand2. AD293 cells were transiently transfected with, mock empty vector, Flag-NFATC1, and HA-Hand2. Whole cell extracts were prepared 48 h post-transfection and were validated to express tagged NFATC1 and Hand2 proteins by Western blots (A). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate the expression of DEGS1 in the transfected cells using ACTB as a reference housekeeping gene (B).