La castration induit des changements de l’expression et de la distribution des récepteurs dans l’épididyme chez le rat. Implications dans la écrétion de la cathespine D

CARVELLI L; AGUILERA AC; BANNOUD N; MORALES CR; SOSA MA

Aguas de Sao Pedro

Conferencia; Fifth International Conference on the Epididymis.; 2010

Introduction: The mammalian epididymis is involved in the maturation of gametes and its integrity and functionality depends on steroid hormones. The epididymal epithelium secretes proteins into the lumen, which could be involved in sperm maturation. In the epididymis of mammals, the secretion is enriched in acid hydrolases, an unusual fact, because these enzymes are normally confined to the lysosomal environment. In most cell types, the normal distribution of lysosomal enzymes is mediated by receptors that recognize mannose-6-phosphate (MPRs). Two forms of these receptors have been described so far, the cation-dependent (CD-MPR) and cation-independent (CI-MPR). In some cell types, the CD-MPR participates in the secretion of lysosomal enzymes, while the CI-MPR is involved in endocytosis of ligands. From previous results,we observed an increase in the expression and secretion of procathepsin D in rats deprived of androgens (castration), indicating that these events could be regulated by steroid hormones. Furthermore, it is known that the gene encoding Cathepsin D contains an element of response to estrogen, indicating that these hormones regulate the expression of the enzyme. Although procathepsin D is carried by the MPRs in most cell types, alternative routes have been proposed for transport to lysosomes. For example, in some cell types, procathepsin D complexes with prosaposin (a soluble lysosomal protein), which are transported to lysosomes or released into the extracellular medium through the sortilin receptor. Aims: From this evidence, we wonder if the transport and secretion of procatepsin D are mediated by CD-MPR or sortilin or both in epididymal cells and whether this mechanism is regulated by estrogenic hormones. We proposed to study the incidence of Sortilin and CD-MPR in the secretion of procathepsin D in epididymal cells subjected to hormonal changes. Methods: A cell line derived from rat epididymis (RCE-1, generously provided by Dr. D. Cyr), was used in the experiments. The cells were grown in D-MEM-F12 medium. They were then subjected to treatment with estradiol or with its antagonist, tamoxifen. In some experiments, hormone treatment was combined with NH4Cl. After 48 hr of treatment, the media were collected and cells were homogenized to study the expression of proteins. Co-immunoprecipitation assays were performed to detect prosaposin in the extracellular medium and to observe if this protein formed complexes with procathepsin D. Results and Discussion: We observed that procathepsin D and prosaposin are complexed in the extracellular medium and these complexes are increased in the RCE-1 cells treated with estradiol. In addition, treatment with estradiol induced an increase in the expression and secretion of cathepsin D. Moreover, NH4Cl caused a reduction in the secretion of procathepsin D and an increased intracellular retention of the enzyme. This retention could be justified by the increased expression of sortilin due to estradiol, which is powered by the acidotropic amine. All this suggests that increased secretion of the enzyme due to estradiol is not "by default". and that alternative routes for the transport of cathepsin D (sortilin mediated) would be involved, which are regulated by estrogens. Although the expression of CD-MPR was not affected by estradiol, its involvement in intracellular trafficking of the enzyme in RCE-1 cells can not be ruled out. Unexpectedly, NH4Cl caused a greater secretion of prosaposin in RCE-1 cells, and probably this is explained by an alteration in the turnover of this protein. Based on these results, we decided to directly investigate the incidence of sortilin on transport and secretion of procathepsin D in epididymal cells. For this, we designed an experimental model on RCE-1 cells, with silenced sortilin-gene, by using siRNA. This knock down resulted in an increase in the expression of CD-MPR and prosaposin, and a redistribution of both proteins. However, procathepsin D showed no major changes in intracellular distribution, suggesting that the enzyme could be transported either by sortilin or CD-MPR. In conclusion, this study shows that some proteins of epididymal cells could be transported by alternate routes which are regulated by hormones. Finantial Support: this work was supported by a Grant (06/J280) from SECyT (UNCuyo). Key words: epididymis, lysosomal enzymes, receptors, hormones. Corresponding author: Dr. MASosa – IHEM-CONICET, ICB-(UNCuyo) 5500-Mendoza (Argentina) E-mail: msosa@fcm.uncu.edu.ar