Cytochrome P450scc-dependent metabolism of 7-dehydrocholesterol in placenta and epidermal keratinocytes

Abstract

The discovery that 7-dehydrocholesterol (7DHC) is an excellent substrate for cytochrome P450scc (CYP11A1) opens up new possibilities in biochemistry. To elucidate its biological significance we tested ex vivo P450scc-dependent metabolism of 7DHC by tissues expressing high and low levels of P450scc activity, placenta and epidermal keratinocytes, respectively. Incubation of human placenta fragments with 7DHC led to its conversion to 7-dehydropregnenolone (7DHP), which was inhibited by dl-aminoglutethimide, and stimulated by forskolin. Final proof for P450scc involvement was provided in isolated placental mitochondria where production of 7DHP was almost completely inhibited by 22R-hydroxycholesterol. 7DHC was metabolized by placental mitochondria at a faster rate than exogenous cholesterol, under both limiting and saturating conditions of substrate transport, consistent with higher catalytic efficiency (k(cat)/K(m)) with 7DHC as substrate than with cholesterol. Ex vivo experiments showed five 5,7-dienal intermediates with MS spectra of dihydroxy and mono-hydroxy-7DHC and retention time corresponding to 20,22(OH)(2)7DHC and 22(OH)7DHC. The chemical structure of 20,22(OH)(2)7DHC was defined by NMR. 7DHP was further metabolized by either placental fragments or placental microsomes to 7-dehydroprogesterone as defined by UV, MS and NMR, and to an additional product with a 5,7-dienal structure and MS corresponding to hydroxy-7DHP. Furthermore, epidermal keratinocytes transformed either exogenous or endogenous 7DHC to 7DHP. 7DHP inhibited keratinocytes proliferation, while the product of its pholytic transformation, pregcalciferol, lost this capability. In conclusion, tissues expressing P450scc can metabolize 7DHC to biologically active 7DHP with 22(OH)7DHC and 20,22(OH)(2)7DHC serving as intermediates, and with further metabolism to 7-dehydroprogesterone and (OH)7DHP.

Figure 1

Production of reaction intermediates by purified human P450scc incubated with 7DHC. Human cytochrome P450scc was incubated with 200 µM 7DHC in buffer containing 0.9% 2-hydroxypropyl-β-cyclodextrin, 0.4 µM adrenodoxin reductase and 50 µM NADPH in the absence (control, A) or presence (test, B) of 15 µM adrenodoxin, for 5 min at 37°C. Products were separated by HPLC as described in the Materials and Methods. The inset in B is an expansion of the region of the chromatogram showing products additional to 7DHP.

Figure 2

Metabolism of hydroxy-7DHC products of P450 action on 7DHC, by purified human P450scc. Human P450scc was incubated for 20 min with vesicles containing hydroxy-7DHC products at a ratio of 0.025 mol sterol/mol phospholipid. Substrates and products were separated by HPLC as described in the Materials and Methods. The hydroxy-7DHC derivatives tested for further metabolism by P450scc are labelled according to Fig. 1: A, product 2 (P2); B, product 3 (P3); C, product 5 (P5). No metabolites were seen in control incubations where adrenodoxin was omitted, not shown but similar to Fig. 1A.

Figure 3

MASS and NMR spectra of 20,22(OH)₂7DHC. (A) MASS; (B)1D Proton; (C) ¹H-¹³C HSQC; (D) ¹H-¹³C HMBC; (E) ¹H-¹H TOCSY; (F) ¹H-¹H COSY. Full spectra are shown in Supplemental Figures (Fig. S1-6).

Figure 4

HPLC chromatograms showing metabolism of 7DHC to 7DHP by placental mitochondria. TEST, mitochondria were incubated with 200 µM 7DHC and 5 µM N-62 StAR protein for 2 h at 37°C. CONTROL, the reaction was stopped at zero time, prior to starting the reaction with isocitrate. TEST + 22R-hydroxycholesterol, 100 µM 22R-hydroxycholesterol was included in the Test incubation.

Figure 5

Time courses for metabolism of exogenous 7DHP and cholesterol by placental mitochondria. Mitochondria were incubated with either 200 µM 7DHC or 200 µM [4-¹⁴C]cholesterol (Chol) in the presence or absence of N-62 StAR protein. 7DHP production from 7DHC was measured by HPLC at 280 nm and [4-¹⁴C]pregnenolone production from [4-¹⁴C]cholesterol was measured by scintillation counting following TLC separation.

Figure 6

Detection of 7DHP in human placenta fragments incubated with 7DHC for 20 h. The production of 7DHP was determined by both LC/MS-TIC (A) and LC/MS-SIM (C) and was absent in the control incubation without substrate (B). The arrow shows the retention time corresponding to 7DHP standard.

Figure 7

Transformation of 7DHC to 7DHP in placentas is dependent on substrate concentration (A), incubation time (B), the amount of tissues in the incubation mixture (C). Production of 7DHP by placental fragments was monitored by LC/MS-SIM. Lower right panels in A and C and inset in B show mean values in relation to 7DHC concentration, incubation time and amount of tissue, respectively. The incubation time for the amount of tissue (A) and substrate concentration (C) are 20 h.

Figure 8

Production of 7DHP is inhibited by 200 µM DL-aminoglutethimide (AGT) (A, B) and stimulated by 100 µM forskolin (C,D). LC/MS-SIM analysis shows relative concentrations of 7DHP in placentas incubated with 7DHC only (A and C) or 7DHC plus AGT (B) or plus forskolin (D). The inset shows means value −/+SEM (n=3). The incubation time is 20 h.

Figure 9

Identification of 5,7-dienal intermediates of 7DHC metabolism to 7DHP. A, Placenta incubated with 1 mM 7DHC for 24 h. B. Boiled placenta incubated with 1 mM 7DHC for 24 h with an inset showing total consumption of 7DHC. C. UV and mass spectra of the four metabolites detected.

Figure 10

Placental microsomes metabolize 7DHP. Placental microsomes were incubated with 100 µM 7DHP in the presence of NAD⁺ for 10 min and products analyzed by reverse phase HPLC. A, Test reaction; B, control reaction carried out as for the test except that 16 µM cyanoketone was present; C, spectrum of the major product recorded after HPLC purification, removal of HPLC solvent (a few h at room temperature under a gentle stream of nitrogen) and dissolving in ethanol.

Figure 11

MASS (A) and NMR (B-D) spectra of 7-dehydroprogesterone produce d by placental microsomes. A, MS identify the molecular ion of 335 [M + Na]⁺ consistent with real mass (MW=312) of 7-dehydroprogesterone. B, 1H NMR; C, 1H-1H COSY and D, 1H-1H TOCSY spectra.

Figure 12

Ex-utero transformation of 7DHP to 7-dehydroprogestrone (A) and to monohydroxy-7DHP tentatively assigned as 20(OH)7DHP (D) by placental fragments. A and D, Placenta incubated with 1 mM 7DHP for 20 h. B and E, Boiled placenta incubated with 1 mM 7DHP for 20 h. C and F, Placenta incubated without substrate for 20 h. The chromatogram was monitored at 240nm for 7-dehydroprogesterone (A-C) and 280nm for 20(OH)7DHP (D-F). RP-HPLC was performed using Waters C18 column (250 × 4.6 mm, 5µm particle size). Elution was carried out with a gradient of methanol in water (50%–100%) at a flow rate 0.35 ml/min (30 min), followed by a wash with 100% methanol (10 min). Other conditions are described in materials and methods.

Figure 13

Endogenous metabolism of 7DHC to 7DHP in epidermal keratinocytes and biological activity of 7DHP. A. Endogenous production of 22(OH)7DHC, 20,22(OH)₂7DHC and 7DHP in pig skin cells. Epidermal keratinocytes isolated from pig skin were incubated ex- vivo for 20 h, extracted with methylene chloride and extracts analyzed by HPLC and LCMS as described before (Slominski et al., 2012a). Inserts show analysis by LCMS with APCI in the MRM mode for 7DHP (MRM: m/z = 315 → 297), and in the SIM mode for 20,22(OH)₂7DHC (SIM: m/z = 417) and 22(OH)7DHC (SIM: m/z = 401). B. 7DHP show antiproliferative activity against HaCaT keratinocytes, while the product of its phototransformation, pregnacalciferol (pD), has little activity. C. Scheme showing UVB induced conversion of 7DHP to pD.

Figure 14

Proposed pathways of 7DHC and 7DHP metabolism in placenta (lower panel) and skin (upper panel).