Diazepam binding inhibitor is a paracrine/autocrine regulator of Leydig cell proliferation and steroidogenesis: action via peripheral-type benzodiazepine receptor and independent mechanisms.

Abstract

Previous studies demonstrated that the polypeptide diazepam binding inhibitor (DBI) and its receptor, the peripheral-type benzodiazepine receptor (PBR), are involved in the regulation of steroid biosynthesis and that one site of PBR action resides in mitochondria. In the present investigation, evidence is presented that a functional form of PBR is also present at the cell surface. First, PBR was immunolocalized in the rat testis using biotin-streptavidin peroxidase immunocytochemistry, and results revealed that PBR was present exclusively in the interstitial Leydig cells. Next, the distribution of PBR in MA-10 Leydig cells was further examined using confocal microscopy. MA-10 cells were either fixed and immunostained or fixed/permeabilized and immunostained for PBR, followed by generation of confocal microscope optical sections, three-dimensional reconstructions of these sections, and then generation of vertical confocal sections of the three-dimensional reconstruction. In the fixed/unpermeabilized cells, PBR immunostaining at the cell surface was clearly evident, whereas in the fixed/permeabilized cells, intracellular PBR distribution was more robust. These results suggest that the plasma membrane fraction of the receptor could mediate the action of extracellular PBR ligands on Leydig cell function. Next, we examined whether DBI, the naturally occurring PBR ligand, is secreted by testicular cells and whether it could activate the cell surface PBR. Immunolocalization of DBI demonstrated that it was present in both Leydig and Sertoli cells. Further, using an immunoblot assay, we demonstrated that DBI is present in rat testicular interstitial fluid. Metabolic labeling of cultured immature rat Sertoli cells and MA-10 mouse tumor Leydig cells, followed by immunoprecipitation of the secreted proteins with an anti-DBI antiserum, demonstrated that both Leydig and Sertoli cells secrete DBI and could serve as a cell source for the interstitial fluid DBI. Then, we partially purified the DBI present in conditioned medium and interstitial fluid by reverse phase chromatography and demonstrated it to be bioactive, based on displacement of a radiolabeled benzodiazepine (Ro5-4864)-specific ligand for PBR; pronase treatment of different preparations eliminated all bioactivity. We then examined the effects of DBI on Leydig cell function. DBI added to MA-10 cells affected DNA synthesis and cell growth in a biphasic manner; at low concentrations (1 nM), DBI was mitogenic, increasing [3H]thymidine incorporation and cell numbers by 30-40%, while at high concentrations (1 microM), DBI inhibited cell growth (30-40%). Similar effects on cell growth were obtained using the benzodiazepine Ro5-4864.

Peripheral-type benzodiazepine receptor levels correlate with the ability of human breast cancer MDA-MB-231 cell line to grow in scid mice

“…Our laboratory, as well as others, has previously shown that PBR ligands regulate cell proliferation. [7][8][9][10][11][13][14][15][16][17] The in vivo behavior of the cells, however, differs from their in vitro behavior where no differences in the rates of cell proliferation between the MDA-231 PH and PL is seen. This may be due to a minimum of PBR levels required for cell proliferation.…”

“…5,6 Increased expression of PBR has been found in numerous tumor types and PBR has been shown to regulate the differentiation state and proliferation rates of several cancer cell lines. [7][8][9][10][11][12][13][14][15][16][17][18][19][20] Over the past several years our laboratory has examined the role of PBR in breast cancer using the MDA-231 cell line. While examining MDA-231 cells from various origins we observed variations in their PBR levels.…”

Diazepam-binding inhibitor-derived peptides induce intracellular calcium changes and modulate human neutrophil function

“…Supernatant was recovered and neutrophils were isolated by Ficoll-Paque Plus density-gradient centrifugation as described [21]. Contaminating erythrocytes were eliminated by 5-min hypotonic lysis in distilled water with the following added (g/L): 8.248 NH 4 …”

“…Proteolytic cleavage of DBI results in the production of several biologically active fragments (collectively known as endozepines [2]) that are involved in the regulation of multiple processes, such as mitochondrial steroid biosynthesis in adrenocortical and Leydig cells [3,4], glucose-induced insulin secretion from pancreatic islets [5], and ␥-aminobutyric acid (GABA)-induced inhibition on pituitary melanotrope cells [6]. The biological actions of DBI-derived peptides have been ascribed mainly to their ability to bind central-type benzodiazepine receptors (cBRs) located on the GABA A receptor complex [see ref .…”

Inhibition of hormone-stimulated steroidogenesis in cultured Leydig tumor cells by a cholesterol-linked phosphorothioate oligodeoxynucleotide antisense to diazepam-binding inhibitor.

“…DBI was originally purified from brain by monitoring its ability to displace diazepam from the allosteric modulatory sites for y-taminobutyric acid action on y-aminobutyric acidA receptors (11,13). DBI was found to be present in a variety of tissues (14), to be highly expressed in adrenocortical and testicular Leydig (14)(15)(16) steroidproducing cells, and to have a variety of specialized functions in different tissues (10,11,13,15,(16)(17)(18). We and others (9,10,15,16,19,20) then showed that bovine adrenal DesGly85-Ile86-DBI, rat and bovine brain native DBI, as well as rat testis native DBI stimulate intramitochondrial cholesterol transport and increase pregnenolone formation by isolated adrenocortical, Leydig, or glial-cell mitochondria.…”