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BACKGROUND: The African clawed frog, Xenopus laevis, is capable of enduring seasonal bouts of severe dehydration stress resulting from transcriptional regulation that facilitates a pro-survival response. Previous studies have shown that dehydration increases antioxidant gene expression in this amphibian. As FoxO4 is known to regulate several antioxidant genes, we sought to understand how differential phosphorylation and environmental factors (urea, temperature) may contribute to its transcriptional regulation during dehydration exposure.
METHODS: Immunoblotting was used to quantify relative amount of total FoxO4, of phosphorylated FoxO4, and of the factors in the Ras-Ral pathway that regulate FoxO4 activity in X. laevis skeletal muscle during dehydration. DNA-protein interaction (DPI)-ELISA was used to measure transcription factor-binding to their consensus sequences in the promoters of target genes. Environmental DPI-ELISA was used to assess the effect of the cellular environment on transcription factor binding.
RESULTS: FoxO4 protein levels do not change during dehydration, but FoxO4-binding to DNA increases with higher dehydration. The Ras-Ral pathway does not appear to be involved in regulating FoxO4 during dehydration, but Akt-mediated FoxO4 phosphorylation at Ser-193 decreases during high dehydration exposure, which is indicative of increased FoxO4 activity. Further assessment indicated that FoxO4-DNA binding affinity is drastically affected by environmental changes in urea and temperature.
CONCLUSION: FoxO4 plays an important role during dehydration stress in X. laevis, and its activity could be regulated through Akt-mediated phosphorylation, and changes in temperature or urea.
GENERAL SIGNIFICANCE: Dehydration triggers regulatory mechanisms of transcription by inducing differential phosphorylation and changes to urea in X. laevis.
Fig. 1. Changes in Ras, RalA, and Ralbp1 total protein levels in the skeletal muscle of X. laevis during dehydration stress. Ras, RalA, and Ralbp1 total protein levels were visualized at three sampling points: control, medium dehydration, and high dehydration. Western blots and Coomassie total protein loading controls representative of the results are shown for the three sampling points. Also shown are histograms with mean standardized band densities (±S.E.M., n = 4 independent protein isolations from different animals). Data was analyzed using a one-way analysis of variance with a post hoc Tukey's test (p < 0.05); for each parameter measured, values that are not statistically different from each other share the same letter notation.
Fig. 2. Changes in total FoxO4, as well as phosphorylated FoxO4 Thr451 (T451), Ser197 (S197), and S193 protein levels in the skeletal muscle of X. laevis during dehydration stress. Total FoxO4 as well as p-FoxO4 T451, S197, and S193 protein levels were visualized at three sampling points: control, medium dehydration, and high dehydration. Western blots and Coomassie total protein loading controls representative of the results are shown for the three sampling points. Also shown are histograms with mean standardized band densities (±S.E.M., n = 4 independent protein isolations from different animals). Data was analyzed using a one-way analysis of variance with a post hoc Tukey's test (p < 0.05); for each parameter measured, values that are not statistically different from each other share the same letter notation.
Fig. 3. Changes in binding of the transcription factors FoxO4 and MyoG to DNA-binding elements designed for their respective consensus sequences in the skeletal muscle of X. laevis during dehydration stress. DNA-Protein Interaction (DPI)-ELISA absorbance readings were corrected by subtraction of negative controls containing no protein and values were expressed relative to EC. Histograms show mean relative values ± S.E.M., n = 4 independent biological replicates for each of the three experimental conditions. Data was analyzed using a one-way analysis of variance with a post hoc Tukey's test (p < 0.05); for each parameter measured, values that are not statistically different from each other share the same letter notation.
Fig. 4. The effect of adding free urea on the transcription factor-DNA binding affinity of FoxO4. Transcription factor-DNA binding was measured during the control and high dehydration sampling points with no urea added (control), 100 mM urea, 200 mM urea, 300 mM urea, and 600 mM urea added. Modified DPI-ELISA absorbance readings were corrected by subtraction of negative control containing no protein, and values were expressed relative to the control (no urea added) for the control and high dehydration sampling points. Histograms show mean relative values ± S.E.M., n = 4 independent biological replicates for both experimental conditions. Data was analyzed using a one-way analysis of variance with a post hoc Tukey's test (p < 0.05); for each parameter measured, values that are not statistically different from each other share the same letter notation.
Fig. 5. The effect of adding free urea on the transcription factor-DNA binding affinity of MyoG. Transcription factor-DNA binding was measured during the control and high dehydration sampling points with no urea added (control), 100 mM urea, 300 mM urea, and 600 mM urea added. Modified DPI-ELISA absorbance readings were corrected by subtraction of negative control containing no protein, and values were expressed relative to the control (no urea added) for the control and high dehydration sampling points. Histograms show mean relative values ± S.E.M., n = 4 independent biological replicates for both experimental conditions. Data was analyzed using a one-way analysis of variance with a post hoc Tukey's test (p < 0.05); for each parameter measured, values that are not statistically different from each other share the same letter notation.
Fig. 6. The effects of adjusting temperature on the transcription factor-DNA binding affinities of FoxO4 and MyoG. Transcription factor-DNA binding was measured at 37 and 21 °C (room temperature) at the control and high dehydration sampling points for FoxO4 and MyoG. Modified DPI-ELISA absorbance readings were corrected by subtraction of negative controls containing no protein, and values were expressed relative to 37 °C for FoxO4 and MyoG at both sampling points. Histograms show mean relative values ± S.E.M., n = 4 independent biological replicates for both experimental conditions. Data was analyzed using unpaired t-tests (p < 0.05); to test for the effect of temperature change on each target at each sampling point, *indicates a significant difference from 37 °C.
