Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7alpha-hydroxy dehydroepiandrosterone and 7alpha-hydroxy pregnenolone

Abstract

Steroids produced locally in brain (neurosteroids), including dehydroepiandrosterone (DHEA), influence cognition and behavior. We previously described a novel cytochrome P450, Cyp7b, strongly expressed in rat and mouse brain, particularly in hippocampus. Cyp7b is most similar to steroidogenic P450s and potentially could play a role in neurosteroid metabolism. To examine the catalytic activity of the enzyme mouse Cyp7b cDNA was introduced into a vaccinia virus vector. Extracts from cells infected with the recombinant showed NADPH-dependent conversion of DHEA (Km, 13.6 microM) and pregnenolone (Km, 4.0 microM) to slower migrating forms on thin layer chromatography. The expressed enzyme was less active against 25-hydroxycholesterol, 17beta-estradiol and 5alpha-androstane-3beta,17beta-diol, with low to undetectable activity against progesterone, corticosterone, and testosterone. On gas chromatography and mass spectrometry of the Cyp7b metabolite of DHEA the retention time and fragmentation patterns were identical to those obtained with authentic 7alpha-hydroxy DHEA. The reaction product also comigrated on thin layer chromatography with 7alpha-hydroxy DHEA but not with 7beta-hydroxy DHEA; when [7alpha-3H]pregnenolone was incubated with Cyp7b extracts the extent of release of radioactivity into the medium suggested that hydroxylation was preferentially at the 7alpha position. Brain extracts also efficiently liberated tritium from [7alpha-3H]pregnenolone and converted DHEA to a product with a chromatographic mobility indistinguishable from 7alpha-hydroxy DHEA. We conclude that Cyp7b is a 7alpha-hydroxylase participating in the synthesis, in brain, of neurosteroids 7alpha-hydroxy DHEA, and 7alpha-hydroxy pregnenolone.

Figure 1

TLC (ascending) of steroid transformations in HeLa cells infected with a vaccinia-Cyp7b recombinant (VV-Cyp7b). (a) Products generated by incubating control vaccinia-infected cell extracts (v); or extracts of cells infected by VV expressing Cyp7b (7b), with steroids 25-hydroxycholesterol (25OHC), pregnenolone (Preg), DHEA, progesterone (Prog), corticosterone (B), cortisol (F), testosterone (Test), estradiol (E2), and 5α,androstane-3β,17β-diol (And); lateral arrowheads indicate the origin. (b) Products obtained with no extract control (o), control vaccinia-infected cells (v), VV-Cyp7b infected cells (7b), rat brain extract (Br), and rat liver extract (Li). (c) Products generated by 0, v, and 7b cell preparations (abbreviations as above) using intact live (vaccinia-infected) cells (ic), purified microsomes (m), or by purified microsomes from VV-Cyp7b infected cells incubated in the absence of NADPH (-N) or with 1 or 10 μM clotrimazole (1C, 10C).

Figure 2

Mass spectrometry of the Cyp7b metabolite of DHEA and reference 7HD. Methoxime-trisilyl derivatives of Cyp7b product (a) and reference 7HD (b) were purified by gas chromatography, and mass spectra were determined.

Figure 3

Release of radioactivity from [7α-³H]pregnenolone by Cyp7b and brain extracts. The set of results presented is representative of three independent sets of experiments performed under slightly different conditions but which yielded comparable values. Samples were buffer only (column a); control vaccinia (v) extract (column b); VV-Cyp7b extract (7b) (column c); 10, 20, and 50 μl rat brain (Br) extract (columns d–f); and 10, 20, and 50 μl rat liver (Li) extract (columns g–i). For reaction conditions see Experimental Procedures.

Figure 4

Alternative metabolism of the cholesterol-derived steroids pregnenolone and DHEA to either Cyp7b products or 3β-hydroxysteroid dehydrogenase (3β-HSD) products progesterone and androstenedione (that are not effective substrates for Cyp7b; Table 1). ∗, It is so far unknown whether Cyp17 (steroid 17-hydroxylase/17,20 lyase) catalyzes the conversion of 7HP to 7HD. Cyp11a1, side-chain cleavage enzyme (scc); 7HD, 7α-hydroxy DHEA; 7HP, 7α-hydroxy pregnenolone.