The sea cucumber, Holothuria glaberrima, has the capacity to regenerate its internal organs. Intestinal regeneration is accomplished by the thickening of the mesenteric border and the invasion of this thickening by mucosal epithelium from the esophagus and the cloaca. Extracellular matrix (ECM) remodeling has been associated with morphogenetic events during embryonic development and regeneration. We have used immunohistochemical techniques against ECM components to show that differential changes occur in the ECM during early regeneration. Labeling of fibrous collagenous components and muscle-related laminin disappear from the regenerating intestine and mesentery, while fibronectin labeling and 4G7 (an echinoderm ECM component) are continuously present. Western blots confirm a decrease in fibrous collagen content during the first 2 weeks of regeneration. We have also identified five 1,10-phenanthroline-sensitive bands in collagen gelatin zymographs. The gelatinolytic activities of these bands are enhanced during early stages of regeneration, suggesting that the metalloprotease activity is associated with ECM remodeling. Inhibition of MMPs in vivo with 1,10-phenanthroline, p-aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamate or N-CBZ-Pro-Leu-Gly hydroxamate produces a reversible inhibition of intestinal regeneration and ECM remodeling. Our results show that significant changes in ECM content occur during intestine regeneration in the sea cucumber and that the onset of these changes is correlated to the proteolytic activities of MMPs.
BackgroundDetermining the type and source of cells involved in regenerative processes has been one of the most important goals of researchers in the field of regeneration biology. We have previously used several cellular markers to characterize the cells involved in the regeneration of the intestine in the sea cucumber Holothuria glaberrima.ResultsWe have now obtained a monoclonal antibody that labels the mesothelium; the outer layer of the gut wall composed of peritoneocytes and myocytes. Using this antibody we studied the role of this tissue layer in the early stages of intestinal regeneration. We have now shown that the mesothelial cells of the mesentery, specifically the muscle component, undergo dedifferentiation from very early on in the regeneration process. Cell proliferation, on the other hand, increases much later, and mainly takes place in the mesothelium or coelomic epithelium of the regenerating intestinal rudiment. Moreover, we have found that the formation of the intestinal rudiment involves a novel regenerative mechanism where epithelial cells ingress into the connective tissue and acquire mesenchymal phenotypes.ConclusionsOur results strongly suggest that the dedifferentiating mesothelium provides the initial source of cells for the formation of the intestinal rudiment. At later stages, cell proliferation supplies additional cells necessary for the increase in size of the regenerate. Our data also shows that the mechanism of epithelial to mesenchymal transition provides many of the connective tissue cells found in the regenerating intestine. These results present some new and important information as to the cellular basis of organ regeneration and in particular to the process of regeneration of visceral organs.
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.
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