A novel pathway for sequential transformation of 7‐dehydrocholesterol and expression of the P450scc system in mammalian skin

Abstract

Following up on our previous findings that the skin possesses steroidogenic activity from progesterone, we now show widespread cutaneous expression of the full cytochrome P450 side-chain cleavage (P450scc) system required for the intracellular catalytic production of pregnenolone, i.e. the genes and proteins for P450scc enzyme, adrenodoxin, adrenodoxin reductase and MLN64. Functionality of the system was confirmed in mitochondria from skin cells. Moreover, purified mammalian P450scc enzyme and, most importantly, mitochondria isolated from placenta and adrenals produced robust transformation of 7-dehydrocholesterol (7-DHC; precursor to cholesterol and vitamin D3) to 7-dehydropregnenolone (7-DHP). Product identity was confirmed by comparison with the chemically synthesized standard and chromatographic, MS and NMR analyses. Reaction kinetics for the conversion of 7-DHC into 7-DHP were similar to those for cholesterol conversion into pregnenolone. Thus, 7-DHC can form 7-DHP through P450scc side-chain cleavage, which may serve as a substrate for further conversions into hydroxy derivatives through existing steroidogenic enzymes. In the skin, 5,7-steroidal dienes (7-DHP and its hydroxy derivatives), whether synthesized locally or delivered by the circulation, may undergo UVB-induced intramolecular rearrangements to vitamin D3-like derivatives. This novel pathway has the potential to generate a variety of molecules depending on local steroidogenic activity and access to UVB.

Differential expression of HPA axis homolog in the skin

“…This suggests that mammalian skin cells are biochemically equipped for cholesterol transport as the basis for steroidogenesis. This was confirmed when isolated mitochondria from skin cells were shown to be able to transform cholesterol into pregnenolone and progesterone, albeit at low efficiency (1% of placenta) (Slominski et al, 2004c). Cultured skin cells (squamous cell carcinoma and melanoma) transformed 22R-hydroxycholesterol into pregnenolone.…”

“…It is now clear that cytochrome P450scc can also efficiently use other substrates that include 7-DHC, ergosterol, and vitamins D2 and D3, to produce steroidal 5, 7-dienes or hydroxyderivatives of vitamin D or ergosterol (Guryev et al, 2003;Slominski et al, 2006a;Slominski et al, 2005c;Slominski et al, 2004c). This is pertinent since mammalian skin expresses the CYP11A1 gene and the actual P450scc protein (Slominski et al, 2004c). This expression was detected in a wide assortment of mouse and human skin samples, subcutaneous adipose tissue, and in normal, immortalized and malignant epidermal and dermal cell lines.…”

“…This expression was detected in a wide assortment of mouse and human skin samples, subcutaneous adipose tissue, and in normal, immortalized and malignant epidermal and dermal cell lines. Moreover, an alternatively spliced CYP11A1 was detected in keratinocytes, squamous cell carcinoma and melanoma cells (GeneBank # AY603498) (Slominski et al, 2004c). The genes for components of the P450scc system, adrenodoxin (FDX1) and adrenodoxin reductase (FDXR), are also expressed in human and mouse skin and cultured skin cells, with the resulting proteins being recognized by specific adrenodoxin and adrenodoxin reductase antibodies, respectively (Slominski et al, 2004c).…”

“…Using specific antibodies that recognize a common epitope for both MLN64 and StAR (Bose et al, 2000), we detected the expected protein with MW of 55 kDa (Slominski et al, 2004c) corresponding to MLN64 (Uribe et al, 2003) in human and rodent skin samples. Additional immunoreactive proteins of lower MW (37 kDa and 18 kDa) could represent products of MLN64 processing and/or the full-length StAR protein (Bose et al, 2000;Stocco, 2000;Watari et al, 1997).…”

“…Cultured skin cells (squamous cell carcinoma and melanoma) transformed 22R-hydroxycholesterol into pregnenolone. Pregnenolone was only observed in the presence of 8 μM cyanoketone (a 3βHSD inhibitor); in its absence pregnenolone was rapidly metabolized, most likely via the delta-4 pathway (Slominski et al, 2004c). P450scc, adrenodoxin reductase, and steroidogenic factor 1 (SF-1) have also been detected in immortalized sebocytes and sebaceous gland, and the corresponding antigens have been detected in situ in human epidermis, hair follicle and sebaceous gland (Thiboutot et al, 2003).…”