Localization of De Novo Sterologenesis in Mammalian Skin

Abstract

Previous studies have demonstrated that the skin is an important site of de novo sterol synthesis and that there is a sex difference in cutaneous sterologenesis with male animals synthesizing more sterols than females. The aim of the present study was to localize the major sites of sterol synthesis within the skin and to determine which of these sites accounted for the sex differences in sterologenesis. In male and female rats whose dermal and epidermal layers are separated by dithiothreitol treatment, the dermis is the major site of skin sterologenesis (males 86% of total, females 82% of total). Moreover, the sex difference in skin sterol synthesis is quantitatively localized primarily within the dermal layer (approximately 2.5-fold greater in the dermis of males). Sterol synthesis is also increased in the epidermis of males. To rule out the possibility that sebaceous gland production accounted for our observations, we treated animals with isotretinoin (13-cis-retinoic acid), a drug that suppresses sebaceous gland sebum production. Sterol synthesis in the skin of both male and female rats is not significantly altered by isotretinoin administration and the sex difference in skin sterologenesis is unaffected. To further localize the sites of sterol synthesis within the skin, studies of hairless mice whose skin was split by DTT were initiated. In hairless mice, DTT separates the epidermis into upper (stratum corneum and granulosum) and lower (basal and spinous cells) strata. The basal layer was separated from the dermis by gentle scraping. As in rats, the dermis is the chief site of sterol synthesis in the skin. In addition, the lower layer of the epidermis (basal and spinous cells) is also a very active site of sterologenesis, accounting for 20% of total skin nonsaponifiable lipid synthesis. The upper epidermis accounted for only a small portion of total skin synthesis. It is highly likely that the bulk of cutaneous sterol synthesis occurs in the pilosebaceous epithelium.

Biphasic Regulation of HMG-CoA Reductase Expression and Activity during Wound Healing and Its Functional Role in the Control of Keratinocyte Angiogenic and Proliferative Responses

“…These lipids consist mainly of cholesterol, fatty acids, and ceramides (2). Thus, it is not remarkable that skin tissue appears as an active site of cholesterol biosynthesis in rodents (3) and also in primates (4). More importantly, the epidermis in particular accounts for most of the cutaneous cholesterol synthetic activity and must be considered as a prime tissue of cholesterol biosynthesis (3).…”

Expression and regulation of GPAT isoforms in cultured human keratinocytes and rodent epidermis

“…For day 17 and 18 rats, to obtain suffi cient material for RNA analysis, epidermis was pooled from two to three fetuses for each data point. The upper [SC/stratum granulosum (SG)]/stratum spinosum (SS) and lower (stratum basale, SB) epidermal fractions were obtained by incubating epidermis from hairless mice in 10 mM DTT/PBS at 37°C, as previously described in detail ( 22 ).…”

“…To determine the localization of GPAT1, 3, and 4 in intact mouse epidermis, upper (SC/SG/SS) and lower epidermal (SB) fractions were isolated using DTT, as reported previously ( 22,29 ). To verify the purity of our epidermal preparations, we fi rst measured the mRNA levels of two marker genes, ABCA12 (highly expressed in the upper epidermis) and aquaporin 3 (highly expressed in the basal layer).…”

Expression and regulation of 1-acyl-sn-glycerol- 3-phosphate acyltransferases in the epidermis

“…To prepare epidermis from hairless mouse, flank skin was excised and epidermis was separated from dermis by incubating in 10 mM EDTA in Ca 2 ϩ -and Mg 2 ϩ -free phosphate-buffered saline (pH 7.4) at 37 Њ C for 45 min. In another group of mice, the full-thickness skin was incubated in 10 mM DTT in PBS for 40 min at 37 Њ C, and epidermal fractions (lower and upper layers) were prepared (24,25). The separation occurs above the basal layer; thus, the lower layer comprises stratum basale and the upper layer comprises stratum granulosum, stratum spinosum, and stratum corneum.…”

“…To determine the localization of mAGPAT isoforms, we next measured mAGPAT mRNA levels in the lower versus upper layers of epidermis. Using the DTT method (24,25), we separated fractions of lower (stratum basale) and upper (stratum spinosum, stratum granulosum, and stratum corneum) epidermis. To obtain sufficient signals for mAGPAT 1 and 2, PCR conditions were optimized and 32 cycles, instead of 25, were used for PCR amplification.…”