DBI in Steroid Neurosteroid Synthesis

In search of a cytoplasmic steroidogenesis-stimulating factor(s), a protein of 8,2 kDa molecular size was isolated from bovine adrenals shown to stimulate transport of cholesterol into mitochondria and transport from the outer to the inner membrane (95). This 8,2 kDa protein was shown to be identical to DBI, except the loss of two amino acids (Gly-Ile) from the carboxy terminus (96), and to have a long-half life (97). We examined the effect of isolated 10 kDa DBI on mitochondria from adrenocortical and Leydig cells (94). Dose-response curves indicated that a threefold stimulation is obtained with low concentrations (0.1-1 ^M) of DBI, whereas higher concentrations have lower stimulatory effect on PREG formation. The stimulation obtained was similar to those reported for the 8.2 kDa des-(Gly-Ile)-DBI on bovine adrenocortical mitochondria (95,98). Moreover, similar results were obtained using purified rat and bovine testis DBI (18). In order to exclude the possibility that the stimulatory effect of DBI was owing to the a-helical structure of the protein, we used as control ^-endorphin, which also possesses a-helical structures. ^-endorphin did not affect the mitochondrial steroid synthesis (94).

As previously noted, high concentrations of DBI (10 ^M) gave lower stimulation of steroid synthesis than 100 nM DBI. When DBI was added in combination with a maximally stimulating concentration of Ro5-4864 (100 nM), the stimulatory effect of Ro5-4864 was abolished, suggesting that in C6-2B glial cells DBI may act as a partial agonist of PBR (99).

We then showed that the amino-acid sequence 17-50 of the DBI bears the biological activity because the triacontatetraneuropeptide (TTN, DBI[17-50]) specifically stimulated mitochondrial steroidogenesis with a potency and efficacy similar to that of DBI (94). TTN, together with other DBI peptide fragments were also found in adrenal and testis extracts, and we noted that DBI could be processed in vitro by mitochondria. Binding studies on mitochondria also indicated that TTN binds specifically to PBR (93).

DBI and DBI processing products were also found to be present in brain and C6-2B glioma cell extract. DBI stimulated PREG formation by twofold in mitochondrial fractions from C6-2B glioma cells and rat brain (55; Table 2). In addition to DBI, the DBI peptide fragments DBI[17-50] and DBI[39-75] were found to be biological active in in vitro assays (55,82,84), whereas conflicting data has been presented for the fragment octadecaneuropeptide DBI[33-50] (55,82).

Taking into account the findings that

1. hCG increases PBR ligand binding (57);

2. DBI stimulates mitochondrial steroid formation acting via PBR (61,94); and

3. DBI is preferentially localized in the periphery of mitochondria (100), the possibility is raised that trophic hormones, by altering PBR, increase PBR interaction with DBI; PBR-DBI interaction triggers steroidogenesis.

In order to determine the in situ role of DBI in steroidogenesis, we suppressed cell DBI levels using antisense oligodeoxynucleotides. In order to overcome the commonly encountered oligodeoxynucleotide-uptake problems, we took advantage of the ability of steroidogenic cells to utilize exogenous cholesterol via the lipoprotein endocy-totic pathway (101). Thus, we constructed cholesterol-linked phosphorothioate oligodeoxynucleotides (CHOL-ODNs) complementary to either the sense or the antisense strand of the 24 nucleotides encoding mouse DBI, 9 bases immediately 5' to the initiation codon ATG and 12 downstream the ATG codon. Treating MA-10 cells with CHOL-ODN antisense to DBI resulted in a dose-dependent reduction of DBI levels. In contrast, CHOL-ODN sense to DBI did not affect its expression. Saturating amounts of hCG increased MA-10 progesterone production by 150-fold. The addition of increasing concentrations of CHOL-ODNs sense to DBI or of a nonrelated sequence did not reduce the MA-10 response to hCG. In contrast, a twofold increase in the amount of steroids produced was observed owing to the cholesterol linked to the ODN, liberated in the cells and used as substrate for steroid synthesis. However, in the presence of CHOL-ODN antisense to DBI, in amounts shown to reduce DBI levels, MA-10 cells lost their ability to respond to hCG. In these studies the hCG-stimulated cAMP levels and P-450scc activity were not affected by the CHOL-ODNs used (101).

Using similar technology we also decreased DBI levels in the R2C Leydig cells (61). DBI-depleted R2C cells did not produce steroids, suggesting that DBI plays a vital role both in the acute stimulation of steroidogenesis by trophic hormones and in the constitutive steroid synthesis. Because we showed that DBI is not the long-sought labile factor, and that the site of hormone action is in the mitochondrion, we propose that hormones, by altering PBR, increase its interaction with DBI, which, in turn, triggers steroidogenesis.

Although PBR drug ligands did not have any direct effect on P450scc activity examined in mitoplasts, DBI induced a twofold stimulation of PREG synthesis. Evidence has been previously discussed that indicates that the outer mitochondrial membrane PBR mediates the effects of PBR ligands and DBI on intact mitochondria. However, the observation that DBI stimulates PREG production by inner mitochondrial membranes implies that this protein can also act via an additional PBR-independent mechanism. Further evidence which indicates the DBI acts directly on P450scc was then provided by observations in an in vitro reconstituted enzyme system (102,103), where DBI stimulated the production of PREG, suggesting that the non-PBR mechanism involved in steroido-

genesis may result from direct activation of P450scc, or alternatively an indirect mechanism that may act via increasing the availability of cholesterol or by altering the rate of reduction of P450scc.

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