Relationship between heat shock protein 70 expression and life span in Daphnia

Abstract

The longevity of an organism is directly related to its ability to effectively cope with cellular stress. Heat shock response (HSR) protects the cells against accumulation of damaged proteins after exposure to elevated temperatures and also in aging cells. To understand the role of Hsp70 in regulating life span of Daphnia, we examined the expression of Hsp70 in two ecotypes that exhibit strikingly different life spans. Daphnia pulicaria, the long lived ecotype, showed a robust Hsp70 induction as compared to the shorter lived Daphnia pulex. Interestingly, the short-lived D. pulex isolates showed no induction of Hsp70 at the mid point in their life span. In contrast to this, the long-lived D. pulicaria continued to induce Hsp70 expression at an equivalent age. We further show that the Hsp70 expression was induced at transcriptional level in response to heat shock. The transcription factor responsible for Hsp70 induction, heat shock factor-1 (HSF-1), although present in aged organisms did not exhibit DNA-binding capability. Thus, the decline of Hsp70 induction in old organisms could be attributed to a decline in HSF-1's DNA-binding activity. These results for the first time, present a molecular analysis of the relationship between HSR and life span in Daphnia.

Keywords: Aging; Daphnia; HSF-1; Heat shock; Hsp70; Longevity.

Fig. 1. Induction of Hsp70 in D. pulicaria in response to heat shock

A. Western blot: D. pulicaria (lake XVI-clone11) were subjected to heat shock at indicated temperatures for 30 minutes and allowed to recover for 4h. Western Blot analysis was performed using 50 μg of total protein using anti-Hsp70 antibody. C lanes indicate control samples with no heat shock treatment. The blot was re-probed with anti-β actin and anti-α tubulin antibodies to ensure equal amounts of protein were loaded in each lane. The positions of molecular weight markers are as indicated on the left. The arrows indicate bands corresponding to Hsp70, β-actin, and α-tubulin. The antibody used in each panel is indicated at the bottom of the blot. B: Quantification of western blot data: The chemifluorescent band intensities were quantified using STORM phosphorimager and the averages from 4 independent biological replicates is represented as a bar graph and the error bars represent standard deviations. Fold-changes are calculated with respect to signals in control lanes. The black bars represent Hsp70, the white bars represent β-actin, and the grey bars represent α-tubulin bands. As shown above bars, *, * *, #, and # # symbols indicate P values that indicate a significant difference (0.0019, 0.0009, 0.0008, and 0.0007 resp.). There was no significant difference observed in signals from β-actin or α-tubulin bands in various lanes with significant values being < 0.01.

Fig. 1. Induction of Hsp70 in D. pulicaria in response to heat shock

A. Western blot: D. pulicaria (lake XVI-clone11) were subjected to heat shock at indicated temperatures for 30 minutes and allowed to recover for 4h. Western Blot analysis was performed using 50 μg of total protein using anti-Hsp70 antibody. C lanes indicate control samples with no heat shock treatment. The blot was re-probed with anti-β actin and anti-α tubulin antibodies to ensure equal amounts of protein were loaded in each lane. The positions of molecular weight markers are as indicated on the left. The arrows indicate bands corresponding to Hsp70, β-actin, and α-tubulin. The antibody used in each panel is indicated at the bottom of the blot. B: Quantification of western blot data: The chemifluorescent band intensities were quantified using STORM phosphorimager and the averages from 4 independent biological replicates is represented as a bar graph and the error bars represent standard deviations. Fold-changes are calculated with respect to signals in control lanes. The black bars represent Hsp70, the white bars represent β-actin, and the grey bars represent α-tubulin bands. As shown above bars, *, * *, #, and # # symbols indicate P values that indicate a significant difference (0.0019, 0.0009, 0.0008, and 0.0007 resp.). There was no significant difference observed in signals from β-actin or α-tubulin bands in various lanes with significant values being < 0.01.

Fig. 2

A. Comparison of HSR in D. pulex and D. pulicaria. One week old D. pulex (TCO) and D. pulicaria (lake XVI-clone11) were subjected to heat shock at 32° C for 30 minutes, extracts were prepared after 4h or 6h recovery period. Western Blot analysis was performed using 50 μg of total protein with anti-Hsp70 antibody. The source of the extract and recovery periods after heat shock are as indicated above each lane. C indicates control extract from organisms that were not subjected to heat shock. HeLa extract from heat-shocked cells was used as a positive control for Hsp70. The blot was re-probed with anti-β actin antibody to ensure equal loading. B. Comparison of HSR in young versus old D. pulicaria. One and five week old D. pulicaria (lake XVI-clone11) were subjected to heat shock at 32° C for 30 minutes, allowed to recover for indicated time periods, and protein was extracted. Western Blot analysis was performed using 50 μg of total protein with anti-Hsp70 antibody. Lanes C: non-heat shocked control samples, 4h: after 4h recovery period, and 6h: after 6h recovery period. The age of Daphnia is indicated below the lanes. Blot was re-probed with anti-β actin antibody to ensure even loading. C. Quantification of western blot data in Fig. 2 A: The chemifluorescent band intensities were quantified using STORM phosphorimager and the averages from 5 independent biological replicates is represented as a bar graph and the error bars represent standard deviations. Fold-changes are calculated with respect to signals in control lanes and all Hsp70 signals were normalized to band intensities of β-actin in each lane. The black bars represent D. pulex, and the white bars represent D. pulicaria. As shown above bars, *, * *, #, and # # symbols indicate P values that indicate a significant difference (0.0009, 0.0007, 0.0018, and 0.0008 resp.). There was no significant difference observed in signals for β-actin in various lanes with significant values being < 0.01. D. Quantification of western blot data in Fig. 2 B: The chemifluorescent band intensities were quantified using STORM phosphorimager and the averages from 3 independent biological replicates is represented as a bar graph and the error bars represent standard deviations. Fold-changes are calculated with respect to signals in control lanes and all Hsp70 signals were normalized to band intensities of β-actin in each lane. The black bars represent samples from 1 wk old D. pulicaria, and the white bars represent samples from 5 wk old D. pulicaria. As shown above bars, *, #, * *, and # # symbols indicate P values that indicate a significant difference (0.0013, 0.0027, 0.0018, and 0.0038 resp.). There was no significant difference observed in signals for β-actin in various lanes with significant values being < 0.01.

Fig. 2

A. Comparison of HSR in D. pulex and D. pulicaria. One week old D. pulex (TCO) and D. pulicaria (lake XVI-clone11) were subjected to heat shock at 32° C for 30 minutes, extracts were prepared after 4h or 6h recovery period. Western Blot analysis was performed using 50 μg of total protein with anti-Hsp70 antibody. The source of the extract and recovery periods after heat shock are as indicated above each lane. C indicates control extract from organisms that were not subjected to heat shock. HeLa extract from heat-shocked cells was used as a positive control for Hsp70. The blot was re-probed with anti-β actin antibody to ensure equal loading. B. Comparison of HSR in young versus old D. pulicaria. One and five week old D. pulicaria (lake XVI-clone11) were subjected to heat shock at 32° C for 30 minutes, allowed to recover for indicated time periods, and protein was extracted. Western Blot analysis was performed using 50 μg of total protein with anti-Hsp70 antibody. Lanes C: non-heat shocked control samples, 4h: after 4h recovery period, and 6h: after 6h recovery period. The age of Daphnia is indicated below the lanes. Blot was re-probed with anti-β actin antibody to ensure even loading. C. Quantification of western blot data in Fig. 2 A: The chemifluorescent band intensities were quantified using STORM phosphorimager and the averages from 5 independent biological replicates is represented as a bar graph and the error bars represent standard deviations. Fold-changes are calculated with respect to signals in control lanes and all Hsp70 signals were normalized to band intensities of β-actin in each lane. The black bars represent D. pulex, and the white bars represent D. pulicaria. As shown above bars, *, * *, #, and # # symbols indicate P values that indicate a significant difference (0.0009, 0.0007, 0.0018, and 0.0008 resp.). There was no significant difference observed in signals for β-actin in various lanes with significant values being < 0.01. D. Quantification of western blot data in Fig. 2 B: The chemifluorescent band intensities were quantified using STORM phosphorimager and the averages from 3 independent biological replicates is represented as a bar graph and the error bars represent standard deviations. Fold-changes are calculated with respect to signals in control lanes and all Hsp70 signals were normalized to band intensities of β-actin in each lane. The black bars represent samples from 1 wk old D. pulicaria, and the white bars represent samples from 5 wk old D. pulicaria. As shown above bars, *, #, * *, and # # symbols indicate P values that indicate a significant difference (0.0013, 0.0027, 0.0018, and 0.0038 resp.). There was no significant difference observed in signals for β-actin in various lanes with significant values being < 0.01.

Fig. 3. Comparison of HSR at different ages in two isolates each of D. pulex and D. pulicaria

A. HSR in D. pulex isolate TCO. B: HSR in D. pulex isolate RW20. C. HSR in D. pulicaria isolate 3L2-1. D. HSR in D. pulicaria isolate lake XVI-clone 11. Daphnia were subjected to heat shock at 32° C for 30 minutes, allowed to recover for indicated time periods, and protein was extracted. Western Blot analysis was performed using 50 μg of total protein with anti-Hsp70 antibody. The recovery periods after heat shock are as indicated above the lanes and the age of the Daphnia are indicated in weeks below the panels. Blots were re-probed with anti-α tubulin antibody to ensure even loading. E-F. Quantification of western blot data in Figs. 3 A-D: The chemifluorescent band intensities were quantified using STORM phosphorimager and the averages from several independent biological replicates (3 replicates for pulex ecotypes and 4 replicates for pulicaria ecotypes) is represented as bar graphs and the error bars represent standard deviations. Fold-changes are calculated with respect to signals in control lanes and all Hsp70 signals were normalized to band intensities of β-actin for each lane. The black bars represent control samples, the white bars represent samples after 4 h recovery, and grey bars represent samples after 6 h recovery. As shown above bars, a, d, g, h, j and k labels indicate P values that show a significant difference (0.0010, 0.0018, 0.0014, 0.0008, 0.0021, and 0.0038 resp.) compared to controls. The P values indicated by labels b, c, e, f, i, and l exhibited no significant difference (0.034, 0.045, 0.086, 0.052, 0.11, and 0.21) compared to controls.There was no significant difference observed in signals for β-actin in various lanes with significant values being < 0.01.

Fig. 3. Comparison of HSR at different ages in two isolates each of D. pulex and D. pulicaria

A. HSR in D. pulex isolate TCO. B: HSR in D. pulex isolate RW20. C. HSR in D. pulicaria isolate 3L2-1. D. HSR in D. pulicaria isolate lake XVI-clone 11. Daphnia were subjected to heat shock at 32° C for 30 minutes, allowed to recover for indicated time periods, and protein was extracted. Western Blot analysis was performed using 50 μg of total protein with anti-Hsp70 antibody. The recovery periods after heat shock are as indicated above the lanes and the age of the Daphnia are indicated in weeks below the panels. Blots were re-probed with anti-α tubulin antibody to ensure even loading. E-F. Quantification of western blot data in Figs. 3 A-D: The chemifluorescent band intensities were quantified using STORM phosphorimager and the averages from several independent biological replicates (3 replicates for pulex ecotypes and 4 replicates for pulicaria ecotypes) is represented as bar graphs and the error bars represent standard deviations. Fold-changes are calculated with respect to signals in control lanes and all Hsp70 signals were normalized to band intensities of β-actin for each lane. The black bars represent control samples, the white bars represent samples after 4 h recovery, and grey bars represent samples after 6 h recovery. As shown above bars, a, d, g, h, j and k labels indicate P values that show a significant difference (0.0010, 0.0018, 0.0014, 0.0008, 0.0021, and 0.0038 resp.) compared to controls. The P values indicated by labels b, c, e, f, i, and l exhibited no significant difference (0.034, 0.045, 0.086, 0.052, 0.11, and 0.21) compared to controls.There was no significant difference observed in signals for β-actin in various lanes with significant values being < 0.01.

Fig. 4. Sequence alignment of Daphnia Hsp70

D. pulex Hsp70 protein sequence is aligned with Hsp70 sequence from other organisms. Gray shaded boxes indicate identity or similarity. Red boxes indicate residues that are involved in nucleotide binding while blue boxes indicate residues that are involved in substrate binding. Gaps (designated as dashes) were entered for the best alignment. C. elegans: Caenorhabditis elegans. D. mel: Drosophila melanogaster. H. sapiens: Homo sapiens. M. musculus: Mus musculus.

Fig. 5. Heat shock leads to an increase in Hsp70 mRNA levels in D. pulicaria

A. RT-PCR analysis. Total RNA was isolated from 1 week-old D. pulicaria (lake XVI-clone 11) either after heat shock or non-heat shock (control) conditions. Hsp70 mRNA levels were analyzed via reverse-transcriptase PCR and visualized on a 1% agarose gel. β-actin mRNA levels were utilized as a control for normalization. The labels above the lanes indicate control or heat-shocked samples and the length of recovery periods after heat shock. B: qRT-PCR analysis. qRT-PCR analysis was performed to examine the fold-change in expression between non-heat shocked and heat shocked samples from one week old D. pulicaria (lake XVI-clone 11). Data represents the average from nine replicate experiments from three different RNA isolations. C: without heat shock, HS-4h: 4h recovery after heat shock, and HS-6h: 6h recovery after heat shock. All results are normalized to β-actin. Error bars indicate standard deviation calculated from the nine replicates. Statistical analysis was performed to calculate P values and the symbol * represents P value (0.0012) that indicated significant difference compared to control and the symbol # represents P value (0.032) that indicates no significant difference compared to control values.

Fig. 6. Activation of HSF-1 in response to heat shock at different ages in D. pulicaria

A. Western blot analysis. D. pulicaria (lake XVI-clone 11) were subjected to heat shock at 32° C for 30 minutes, allowed to recover for indicated time periods, and protein was extracted. Western Blot analysis was performed using 50 μg of total protein with anti-HSF-1 antibody. The recovery periods after heat shock are as indicated above the lanes and the age of the Daphnia are indicated in weeks below the panels. Blots were re-probed with anti-β actin antibody to ensure even loading. Arrowhead marked “ui” indicates HSF-1 position from non-heat shocked samples, and an arrowhead marked “i” indicates upward mobility shift of HSF-1 in heat shocked samples. B. Electrophoretic mobility shift analysis. EMSA was performed using nuclear extracts prepared from D. pulicaria (lake XVI-clone 11) with (HS) or without heat shock (C) at different ages as indicated. 5 μg of nuclear extracts were incubated with P³²-labeled HSE probe. Above each lane, the age of the Daphnia in weeks, addition of a competitor (HSE: specific, Oct1: nonspecific), or addition of an antibody (HSF-1) is as indicated. The ‘-’ lane indicates probe alone lane without any added nuclear extract. Arrows indicate the position of HSF-1 containing complex, and arrowhead indicates position of complex after antibody super-shift. Specific and non-specific non-radioactive competitor oligonucleotides were used in 50-fold molar excess to confirm the specificity of the bound complex.