Characterization of proteolytic activities during intestinal regeneration of the sea cucumber, Holothuria glaberrima

Abstract

Proteolysis carried out by different proteases control cellular processes during development and regeneration. Here we investigated the function of the proteasome and other proteases in the process of intestinal regeneration using as a model the sea cucumber Holothuria glaberrima. This echinoderm possesses the ability to regenerate its viscera after a process of evisceration. Enzymatic activity assays showed that intestinal extracts at different stages of regeneration possessed chymotrypsin-like activity. This activity was inhibited by i) MG132, a reversible inhibitor of chymotrypsin and peptidylglutamyl peptidase hydrolase (PGPH) activities of the proteasome, ii) E64d, a permeable inhibitor of cysteine proteases and iii) TPCK, a serine chymotrypsin inhibitor, but not by epoxomicin, an irreversible and potent inhibitor of all enzymatic activities of the proteasome. To elucidate the role which these proteases might play during intestinal regeneration, we carried out in vivo experiments injecting MG132, E64d and TPCK into regenerating animals. The results showed effects on the size of the regenerating intestine, cell proliferation and collagen degradation. These findings suggest that proteolysis by several proteases is important in the regulation of intestinal regeneration in H. glaberrima.

Fig. 1. Enzymatic activity toward the substrate SLLVY-AMC (10μM) in crude extracts of different stages of intestinal regeneration

Aliquots of intestinal extracts were incubated a 37°C for 15 min and the enzymatic activity was measured in a final volume of 2 ml during 15 min. (A) The cumulative cleavage of SLLVY-AMC at different time points (min) is represented. (B) Summary histogram of the results. Each bar represents the mean fluorescence units/mg proteins at 15 min of assay for each stage of intestinal regeneration ± SE. *Different from normal animals and 3dpe, p<0.05, ONE WAY ANOVA and Tukey's test.

Fig. 2. Effect of proteasome inhibitors on SLLVY-AMC cleavage. Intestinal extracts were prepared as described in Materials and Methods (crude extracts)

Aliquots of these extracts were incubated with MG132 10 μM (A), Epoxomicin 50 μM (B), E64d 80 μM (C) or TPCK 80 μM (D) for 15 min and the enzymatic activity was measured. Results are expressed as percent of activity compared to vehicle (100%). Each bar represents the mean fluorescence units/mg proteins at 15 min of assay for each stage ± SE. The insert in (B), represents a Western blot showing the presence of α-subunits of proteasomes in the homogenates of regenerating intestines *Different from vehicle. p<0.05, TWO WAY ANOVA and t-test. n=3.

Fig. 3. Effect of epoxomicin in the presence of E64d, on the cleavage of the fluorogenic substrate SLLVY-AMC. Intestinal extracts at 7 dpe were prepared as described in methods (HMW protein fractions)

Aliquots of these extracts were incubated with E64d, epoxomicin or E64d plus epoxomicin for 15 min and the enzymatic activity to the substrate was measured for 15 min. Results are expressed as mean fluorescence units/mg protein at 15 min of assay ± SE. *p<0.05 significantly different. t-test. n=3.

Fig. 4. Gene expression profile analysis of H. glaberrima calpain-5 (A) and calpain-7 (B) during intestinal regeneration

Analysis was performed by conventional RT-PCR and normalized against NADH. A significantly increase in the expression calpain 5 is observed at 3, 7 and 14 dpe compared con normal animals. Bar plot represent the mean of the normalized optical density of the bands for each regeneration stage +/− the SE. n=6 (normal, 3, and 7 dpe) and n=4 (14 dpe) for calpain 7 and n=6 for calpain 5. **Different from normal animals (N). p<0.01, ONE WAY ANOVA and Tukey's test.

Fig. 5. Intracoelomic injection of MG132 delays intestinal regeneration

(A,B) Representative histological sections of DMSO-treated (control) animals (A) and MG132-treated animals (B) showing a significant reduction in the size of the regenerating intestinal primordium. (C) Measurement of the primordium area in animals treated with DMSO and MG132 shows a decrease in the regenerating intestine size of MG132-treated animals when compared with control animals (*p<0.05 t-test). Results represent the mean ± SE of at least ten animals. Scale bar, 100 μm.

Fig. 6. Injections of MG132 and E64d affect cell proliferation in the regenerating intestinal primordia

The percentage of cells that incorporated BrdU in the mesothelial layer of the intestinal primordia (MIP) and mesentery (MM) was determined in controls and animals treated with the inhibitors. Fewer mesothelial cells of animals treated with MG132 (A) and E64d (D) incorporated BrdU when compared with DMSO-treated animals. The photos below each graph are representative histological sections of animal showing a minor amount of Brdu-positive cell in animals treated with MG132 (C) and E64d (F) compared with the respective control animals (B,E). Results represent the mean ± SE of at least seven animals for MG132 and six for E64d and TPCK. *Different from DMSO (control), p<0.05. (A) t-test; (B) ONE WAY ANOVA and Tukey-test. Scale bar, 100 μm.

Fig. 7. Effect of E64d and TPCK injections on collagen expression

(A)Animals treated with E64d and TPCK show a delay in collagen degradation compared with DMSO (control animals). (B) Representative pictures showing the presence of collagen (white color) in animals treated with DMSO, E64d and TPCK. Results represent the mean ± SE of at least five experiments for E64d and TPCK. *Different from DMSO (control). p<0.05 (A) t-test; (B) ONE WAY ANOVA and Tukey-tes.t. Scale bar, 100 μm.

Fig. 8. TUNEL assay in H. glaberrima

Percentage of apoptosis in mesothelial cells of the regenerating primordial of animals treated with MG132, E64d and TPCK. Results are represented as mean ± standard error. n=4 experiments. Not significantly different. ANOVA ONE WAY.