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ABSTRACT: Liver X receptors (LXRs) are nuclear receptors that act as ligand-activated transcription factors regulating lipid metabolism and inflammation. In the skin, activation of LXRs stimulates differentiation of keratinocytes and augments lipid synthesis in sebocytes. However, the function of LXRs in melanocytes remains largely unknown. We investigated whether LXR activation would affect melanogenesis. In human primary melanocytes, MNT-1, and B16 melanoma cells, TO901317, a synthetic LXR ligand, inhibited melanogenesis. Small interfering RNA (siRNA) experiments revealed the dominant role of LXRβ in TO901317-mediated antimelanogenesis. Enzymatic activities of tyrosinase were unaffected, but the expression of tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2 was suppressed by TO901317. Expressions of microphthalmia-associated transcription factor (MITF), a master transcriptional regulator of melanogenesis, and cAMP-responsive element-binding activation were not affected. It is noteworthy that the degradation of MITF was accelerated by TO901317. Extracellular signal-regulated kinase (ERK) contributed to TO901317-induced antimelanogenesis, which was evidenced by recovery of melanogenesis with ERK inhibitor. Other LXR ligands, 22(R)-hydroxycholesterol (22(R)HC) and GW3965, also activated ERK and suppressed melanogenesis. The intermediary role of Ras was confirmed in TO901317-induced ERK phosphorylation. Finally, antimelanogenic effects of TO901317 were confirmed in vivo in UVB-tanning model in brown guinea pigs, providing a previously unreported line of evidence that LXRs may be important targets for antimelanogenesis.Journal of Investigative Dermatology advance online publication, 6 December 2012; doi:10.1038/jid.2012.409.
Journal of Investigative Dermatology 12/2012; · 6.31 Impact Factor
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ABSTRACT: A rapid, sensitive and specific hydrophilic interaction liquid chromatography coupled to tandem mass spectrometric (HILIC-MS/MS) method for the simultaneous determination of pyroglutamic acid, cis- and trans-urocanic acid in human skin stratum corneum (SC) were developed and validated. This method was carried out without derivatization or addition of ion-pair additives in mobile phase. The analytes were extracted by PBS buffer solution and analyzed using an electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode. Chromatographic separation was performed on an AQUITY UPLC amide column using gradient elution with the mobile phase of water and acetonitrile. The standard curves were linear over the concentration range of 1.0-250 ng/mL with a correlation coefficient higher than 0.999 with an LLOQ of 0.5 ng/mL. The lower limits of detection (LLOD) of these analytes were lower than 0.2 ng/mL. The intra- and inter-day precisions were measured to be below 7.7% and accuracies were within the range of 94.3-102.6%. The validated method was successfully applied to determine the level of pyroglutamic acid and cis-/trans-urocanic acid in the SC samples from forearm and forehead region of 19 human volunteers.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 04/2012; 897:55-63. · 2.78 Impact Factor
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ABSTRACT: PAC-14028 ((E)-N-((R)-1-(3,5-difluoro-4-methanesulfonylamino-phenyl)-ethyl)-3-(2-propyl-6-trifluoromethyl-pyridine-3-yl)-acrylamide) is a novel and potent transient receptor potential vanilloid type I (TRPV1) antagonist. We developed and validated a rapid, sensitive and selective liquid chromatography/tandem mass spectrometric method for determination of PAC-14028 in rat and minipig plasma. After protein precipitation PAC-14028 and internal standard (methylated analog, PAC-14026) were separated on a Symmetry C(18) column (4.6 mm × 75 mm, 3.5 μm) with an isocratic mobile phase, acetonitrile: water (8:2, v/v) containing 0.2% formic acid and monitored by electrospray positive ionization with multiple reaction monitoring mode (PAC-14028, 492→156; IS, 506→156, m/z). The calibration curve was linear over the range of 1.0-500 ng/ml (r(2)>0.999) and lower limit of quantitation (LLOQ) was 1 ng/ml. The precision and accuracy were within ± 15% and the stability was acceptable during bench-top, auto-sampler, 3 freeze-thaw cycles and 4-week storage in a freezer at -80°C. This method was successfully applied to the intravenous, oral and topical pharmacokinetic studies of PAC-14028 in rats and minipigs, which showed comparable pharmacokinetic parameters (T1/2, 2.1h and 3.8h; F%, 52.7% and 64.2% for rats and minipigs, respectively). Percutaneous absorption of PAC-14028 was negligible after topical application (F% 0.2-1.7%).
Journal of pharmaceutical and biomedical analysis 11/2011; 61:8-14. · 2.45 Impact Factor
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Journal of Dermatological Science 11/2008; 53(2):150-2. · 3.72 Impact Factor
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ABSTRACT: Alpha-ketoglutarate is a key intermediate in the Krebs cycle, and a rate-limiting cofactor of prolyl-4-hydroxylase. It also has a potent effect on increasing the proline pool during collagen production, but the details underlying the boosting effect on collagen production by alpha-ketoglutarate remain as yet unreported. To investigate the effects of alpha-ketoglutarate on procollagen production and wrinkle formation, we conducted experiments in cultured human dermal fibroblasts and UVB-irradiated hairless mice. Based on ELISA measurements, alpha-ketoglutarate (10 microM) stimulated procollagen production in fibroblasts by 25.6+/-4.6% compared to vehicle (dH(2)O)-treated control cells. Also, we demonstrated that alpha-ketoglutarate increased activities of prolidase, which is known to play an important role in collagen metabolism, in fibroblasts and N-benzyloxycarbonyl-L-proline (Cbz-Pro), prolidase inhibitor, inhibited procollagen synthesis by alpha-ketoglutarate in fibroblasts. To determine the effect of topically applied alpha-ketoglutarate on wrinkle formation, alpha-ketoglutarate (1%) and vehicle (70% propylene glycol, 30% ethanol) were applied on the dorsal skin of UVB-induced hairless mice for twelve weeks. We found that alpha-ketoglutarate decreased wrinkle formation upon long-term topical application. These results suggest that alpha-ketoglutarate diminishes UVB-induced wrinkle formation by increasing collagen production, through a pathway that involves prolidase activation. Therefore, application of alpha-ketoglutarate may represent an effective anti-wrinkle agent for the cosmetic field.
Biological & Pharmaceutical Bulletin 09/2007; 30(8):1395-9. · 1.66 Impact Factor
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ABSTRACT: To investigate the effects of topically applied 17beta-estradiol on the expression of extracellular matrix proteins in aged human skin, 17beta-estradiol (0.01%) and its vehicle (70% propylene glycol, 30% ethanol) were applied to aged (68-82 y, eight females and five males) human buttock skin under occlusion for 2 wk (three times per week). Topical 17beta-estradiol was found to increase the expression of type 1 procollagen mRNA and protein significantly in human aged skin in vivo. In addition, metalloproteinase (MMP-1 protein levels were reduced by topical 17beta-estradiol. The expressions of TGF-beta1, TGF-beta type II receptor, and Sma and Mad related (Smad)3 were increased by topical 17 beta-estradiol in aged human skin, and TGF-beta1 neutralizing antibody inhibited 17beta-estradiol-induced procollagen synthesis in cultured fibroblasts. We also found that the expressions of tropoelastin and fibrillin-1 mRNA and protein, and elastic fibers in aged skin were also increased by topical 17beta-estradiol. Topical 17beta-estradiol also increased keratinocyte proliferation and the epidermal thickness in aged human skin. We also observed the same effects of topical 17beta-estradiol in young skin. In conclusion, our results suggest that topical 17beta-estradiol treatment may improve the cutaneous function of aged human skin by improving the connective tissue and increasing epidermal thickness.
Journal of Investigative Dermatology 07/2005; 124(6):1149-61. · 6.31 Impact Factor
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ABSTRACT: The methanolic extract of the fruits of Torilis japonica showed a potent inhibition against 5 alpha-reductase activity in vitro. Bioassay-guided fractionation of the methanol extract of the fruits followed by repeated silica gel chromatography led to the isolation of an active principle and its structure was identified as torilin on the basis of spectroscopic data. Torilin (IC50 = 31.7 +/- 4.23 microM) showed a stronger inhibition of 5 alpha-reductase than alpha-linolenic acid (IC50 = 160.3 +/- 24.62 microM) but was weaker than finasteride. (IC50 = 0.38 +/- 0.06 microM). Simple guaiane-type compounds, such as (-)-guaiol and guaiazulene showed weak inhibitory effects on the 5 alpha-reductase activity with IC50 values of f 81.6 microM and 100.8 microM, respectively, while azulene was not active. These results suggest that both degrees of unsaturation and the side-chain in the guaiane skeleton are important for the manifestation of 5 alpha-reductase inhibition.
Planta Medica 06/2003; 69(5):459-61. · 2.15 Impact Factor
