Most newly synthesized peroxisomal proteins are imported in a receptor-mediated fashion, depending on the interaction of a peroxisomal targeting signal (PTS) with its cognate targeting receptor Pex5 or Pex7 located in the cytoplasm. Apart from this classic mechanism, heterologous protein complexes that have been proposed more than a decade ago are also to be imported into peroxisomes. However, it remains still unclear if this so-called piggyback import is of physiological relevance in mammals. Here, we show that Cu/Zn superoxide dismutase 1 (SOD1), an enzyme without an endogenous PTS, is targeted to peroxisomes using its physiological interaction partner 'copper chaperone of SOD1' (CCS) as a shuttle. Both proteins have been identified as peroxisomal constituents by 2D-liquid chromatography mass spectrometry of isolated rat liver peroxisomes. Yet, while a major fraction of CCS was imported into peroxisomes in a PTS1-dependent fashion in CHO cells, overexpressed SOD1 remained in the cytoplasm. However, increasing the concentrations of both CCS and SOD1 led to an enrichment of SOD1 in peroxisomes. In contrast, CCS-mediated SOD1 import into peroxisomes was abolished by deletion of the SOD domain of CCS, which is required for heterodimer formation. SOD1/CCS co-import is the first demonstration of a physiologically relevant piggyback import into mammalian peroxisomes.
Instructive influences of fetal mesenchyme were examined in heterotypic tissue recombinants consisting of urogenital sinus mesenchyme (UGM) from male and female rats and distal ductal tips from adult rat prostate. Tissues were grown under the renal capsule of male hosts for periods up to 28 days. Resultant growths exhibited typical prostate histology. Expression of lobe-specific proteins for the ventral (prostatic steroid binding protein [PSBP]) lateral (seminal vesicle secretion II [SVS II]), and dorsal prostate (secretory transglutaminase [TGase]) were examined by immunocytochemistry. Male or female UGM combined with terminal segments of the ventral or dorsal prostate and immunolabeled with antibodies to lobe-specific proteins demonstrated expression of all three secretory products. The pattern of staining was consistent with a compound inductive response from the UGM. Unique to this study was our ability to use a defined mesenchymal tissue (female ventral mesenchymal pad [VMP]). This tissue is specifically associated with ductal branching morphogenesis and cytodifferentiation of the ventral prostate. Distal ductal tips from the dorsal lobe of the adult male prostate when recombined with female VMP and grown in vivo exhibited transformation of secretory phenotype, and the epithelium expressed mRNAs for PSBP. Immunocytochemistry of serial sections did not demonstrate labeling for TGase in the new epithelial growth. Ultrastructural analysis of the heterotypic recombinants indicated that the epithelium had similar characteristics to those of normal ventral prostate. Early stages of the mesenchymal-epithelial interactions resulted in dedifferentiation of the adult epithelium to solid cords of stratified cells. These findings illustrate the potent instructive capacity of a defined fetal UGM to influence development and cytodifferentiation of adult prostate epithelium.
Macrophage migration inhibitory factor (MIF), described originally as a product of activated T lymphocytes, recently has been found to be released by monocytes/macrophages and the anterior pituitary gland. Immunohistochemical studies of the adult rat testis using an affinity-purified polyclonal antimurine MIF antibody demonstrated strong staining for MIF in Leydig cells and their putative precursors. Peritubular myoid cells and the seminiferous epithelium were negative for MIF staining; however, a weak reaction around the heads of elongated spermatids also was observed. The expression of MIF messenger RNA and protein in whole rat testis was demonstrated by Northern blot and Western blot analyses, respectively. Both MIF messenger RNA and protein immunoreactivity in Leydig cells was observed in testes obtained from long term hypophysectomized rats. Significant concentrations of intracellular MIF were detected in lysates of the TM3 Leydig cell line (7.23 +/- 2.6 pg/microgram protein), and testicular interstitial fluid contained 14.7 +/- 1.6 ng/ml MIF protein, as measured by MIF-specific enzyme-linked immunosorbent assay. To gain insight into the possible biological role of MIF in the testis, cultures of adult rat seminiferous tubules and purified Leydig cells were incubated together with recombinant murine MIF (rMIF). Neither rMIF (50 ng/ml) nor a neutralizing anti-MIF antiserum was found to affect basal or LH-stimulated Leydig cell steroidogenesis in vitro. However, a dose-dependent decrease in the secretion of inhibin by the seminiferous tubules was observed at rMIF concentrations ranging from 10-100 ng/ml. Taken together, these data indicate that Leydig cells produce MIF in vivo and suggest an important regulatory role for this newly discovered mediator of testicular function.
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