Adapalene suppresses sebum accumulation via the inhibition of triacylglycerol biosynthesis and perilipin expression in differentiated hamster sebocytes in vitro.

BACKGROUND: Acne is a chronic inflammatory disease in sebaceous glands and pilosebaceous units where excess sebum production and follicular hyperkeratinization are observed. Adapalene, which exerts comedolytic and anti-inflammatory effects, is used for the topical treatment of mild to moderate acne. OBJECTIVE: We examined the effect of adapalene on sebum production and accumulation in sebaceous gland cells (sebocytes). METHODS: The regulation of sebum production was examined by oil red O and nile red staining and the measurement of triacylglycerols (TGs) in differentiated hamster sebocytes. The gene expression and production of diacylglycerol acyltransferase-1 (DGAT-1) and perilipin 1 (PLIN1) were analyzed using real-time PCR and Western blotting, respectively. RESULTS: Adapalene suppressed sebum accumulation as lipid droplets in spontaneously and insulin-differentiated hamster sebocytes. The TG production, and the gene expression and production of DGAT-1, a rate-limiting enzyme of TG biosynthesis, were dose-dependently inhibited by adapalene in insulin-, 5α-dihydrotestosterone- or a peroxisome proliferator activating receptor γ agonist, troglitazone-differentiated hamster sebocytes. In addition, the inhibition of TG production by adapalene interfered with antagonists against nuclear retinoic acid and retinoid X receptors (CD2665 and UVI3006, respectively) in the differentiated sebocytes. Furthermore, the production of PLIN1, a lipid storage droplet protein, was transcriptionally inhibited by adapalene in the differentiated sebocytes. CONCLUSIONS: These results suggest that adapalene exerts an inhibitory action for sebum accumulation due to the suppression of TG and PLIN1 production in differentiated hamster sebocytes. Furthermore, these findings may contribute to a novel understanding of the molecular mechanisms of adapalene for acne treatment and prevention.

Differentiated hamster sebocytes exhibit apoptosis-resistant phenotype by the augmentation of intracellular calcium level in vitro.

Sebaceous glands play important roles in the maintenance of the skin barrier function by secreting sebum onto the skin surface. In our study, we demonstrated that differentiated hamster sebocytes (DHS) exhibited apoptosis resistance and the loss of Ca(2+) influx against a calcium ionophore, A23187 treatment, which induced both apoptosis and Ca(2+) influx in undifferentiated hamster sebocytes (unDHS). The Fluo-3-related signal of intracellular Ca(2+) in the DHS was higher than that in unDHS and was sustained even though there was a depletion of Ca(2+) from the culture medium. Furthermore, the intracellular Ca(2+) chelator, BAPTA-AM, was found to decrease the Ca(2+) signal in the DHS, which induced apoptosis. Thus, these results provide novel evidence that the cell differentiation-dependent increase in store-operated Ca(2+) release is associated with apoptosis resistance in the DHS. Moreover, these findings should accelerate the understanding of the mechanisms of sebogenesis and/or sebum production and secretion under physiological conditions.

Identification and characterization of ABCB1-mediated and non-apoptotic sebum secretion in differentiated hamster sebocytes.

Sebaceous glands secrete sebum onto the skin surface in a holocrine manner and as such a thin lipid layer is formed as a physiological barrier. In the present study, extracellular level of triacylglycerols (TG), a major sebum component, as well as intracellular TG accumulation was augmented in insulin-differentiated hamster sebocytes (DHS). The DHS exhibited phosphatidylserine exposure in an apoptosis-independent manner. In addition, intracellular ATP level and membrane-transporter activity using a substrate, Rhodamine 123, were highly detectable in the DHS rather than in the undifferentiated hamster sebocytes. A membrane-transporter activating reagent, 2'(3')-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP), enhanced transporter activity, extracellular TG level, and phosphatidylserine exposure in the DHS. Both transporter activity and TG secretion were suppressed by R-verapamil, a potent membrane-transporter inhibitor, in the BzATP-treated and untreated DHS. Furthermore, the gene expression and production of ATP-binding cassette subfamily B member 1 (ABCB1) were augmented in the DHS. ABCB1 was also detectable in sebaceous glands in the skin of hamsters. Moreover, the cell-differentiation- and BzATP-augmented transporter activity and TG secretion were dose-dependently inhibited by adding not only an ABCB1 antibody but also a selective inhibitor of ABCB1, PSC833. Thus, these results provide novel evidence that ABCB1 is involved in sebum secretion in the DHS, which is associated with non-apoptotic phosphatidylserine exposure and the increased level of intracellular ATP. These findings should accelerate the understanding of sebum secretion occurring in a holocrine-independent manner in sebaceous glands, and may contribute to the development of therapies for sebaceous gland disorders such as acne, seborrhea, and xerosis.

Augmentation of gene expression and production of promatrix metalloproteinase 2 by Propionibacterium acnes-derived factors in hamster sebocytes and dermal fibroblasts: a possible mechanism for acne scarring.

Aberrant extracellular matrix (ECM) remodeling in sebaceous glands and pilosebaceous units in the skin is associated with scar formation under acne conditions. To investigate the involvement of Propionibacterium acnes (P. acnes), a Gram-positive anaerobic microbial species, in ECM remodeling in sebaceous glands and pilosebaceous units, we examined the effects of P. acnes culture media, formalin-fixed P. acnes, and peptidoglycan (PGN) from Gram-positive bacteria walls on the production of promatrix metalloproteinase 2 (proMMP-2)/progelatinase A in hamster sebocytes and dermal fibroblasts. When hamster sebocytes (1.8×10(5) cells) and dermal fibroblasts (1×10(5) cells) were treated with P. acnes culture media and formalin-fixed P. acnes (corresponding to 1×10(6) and 1×10(7) bacterial cells), the production of proMMP-2 was augmented. In addition, PGN (5-50 µg/ml) dose-dependently augmented the production of proMMP-2 in both cells. Furthermore, the PGN (50 µg/ml)-augmented proMMP-2 production was resulted from an increase of its transcript. In contrast, there were no changes in cell proliferative activity in either the P. acnes or PGN-treated sebocytes and dermal fibroblasts, indicating that the augmented proMMP-2 production was not due to an increase in cell numbers. Therefore, these results provide novel evidence that PGN transcriptionally up-regulates the production of proMMP-2 in hamster sebocytes and dermal fibroblasts. Given an increase in the quantity of Gram-positive bacteria, including P. acnes in acne lesions, the aberrant ECM degradation may progress in sebaceous glands and pilosebaceous units, which is associated with acne scar formation.

Induction of inflammatory reactions by lipopolysaccharide in hamster sebaceous glands and pilosebaceous units in vivo and in vitro.

Lipopolysaccharide (LPS) from Gram-negative bacteria has been reported to exert inflammatory reactions in epidermis, dermis, and sebaceous glands. Here, we demonstrated that the intradermal administration of Escherichia coli-derived LPS, three times a week for 4 weeks, to hamster auricle skin did not influence sebaceous morphology or sebum accumulation in sebaceous glands but in fact induced epidermal thickness. In addition, the administration of LPS, once a day for 2 days, augmented the production of cyclooxygenase 2 (COX-2) in sebaceous glands. Furthermore, LPS increased the production of prostaglandin F(2α) (PGF(2α) ) in hamster sebocytes. Moreover, the production of progelatinase A/promatrix metalloproteinase 2 (proMMP-2) was transcriptionally augmented by LPS and PGF(2α) in hamster sebocytes. Therefore, these results suggest that LPS directly increases inflammation by augmenting COX-2, PGF(2α) , and the PGF(2α) -mediated proMMP-2 production in sebaceous glands as well as epidermal inflammatory events in skin disorders including acne and folliculitis.