A decrease of mitochondrial ubiquitin ligase increases the secretion of matrix metalloproteinase-1 by dermal fibroblasts through the induction of ER stress.

BACKGROUND: We previously reported that the level of mitochondrial ubiquitin ligase (MITOL) protein in fibroblasts was decreased by UVA and that the knock-down (KD) of MITOL increased the secretion of matrix metalloprotease-1 (MMP-1) by fibroblasts. A recent study reported that MITOL suppresses endoplasmic reticulum (ER) stress by stabilizing the interaction between ER and mitochondria (MT) through the ubiquitination of mitofusin 2. These facts suggest that a decrease of MITOL would increase the secretion of MMP-1 through ER stress, but the detailed mechanism of that process in dermal fibroblasts remains unclear. Thus, this study was conducted to clarify the involvement of ER stress in the oversecretion of MMP-1 induced by the decreased MT quality caused by MITOL-KD. METHODS: MITOL-KD normal human dermal fibroblast (NHDFs) were prepared by treating them with MITOL-small interfering RNA, after which their MMP-1 protein levels were measured. ER stress in NHDFs was evaluated by measuring the mRNA levels of spliced X-box binding protein 1 (sXBP1) and the protein levels of inositol-requiring enzyme 1α (IRE1α). RESULTS: MITOL-KD NHDFs enhanced the secretion of MMP-1 via interleukin-6 (IL-6) elicited by the activation of nuclear factor-kappa B (NF-κB). The secretion of MMP-1 could be abrogated by a neutralizing IL-6 antibody and by JSH23, which is an inhibitor of NF-κB activation. Furthermore, MITOL-KD NHDFs as well as UVA-irradiated NHDFs showed increased ER stress levels. In addition, tunicamycin, which is an inducer of ER stress, also increased MMP-1 secretion. CONCLUSION: These results suggested that the decrease of MITOL caused the oversecretion of MMP-1 via NF-κB-IL-6 signaling through the activation of ER stress in fibroblasts.

Possible Involvement of Dermal Fibroblasts in Modulating Nrf2 Signaling in Epidermal Keratinocytes.

Epidermal keratinocytes protect themselves by cooperating with neighboring cells against internal and external stresses, which leads not only to the maintenance of cell homeostasis but also to the prevention of skin aging. Although it is known that nuclear factor (NF)-E2-related factor 2 (Nrf2) signaling plays a pivotal role in ameliorating oxidative stress and inflammatory responses under stress situations, it is unclear whether Nrf2 signaling in keratinocytes cooperates with neighboring cells such as dermal fibroblasts. Thus, this study was conducted to examine the influence of dermal fibroblasts on Nrf2 signaling in epidermal keratinocytes using a co-culture system. The results show that expression levels of Nrf2-regulated antioxidant factors, such as glutathione and heme oxygenase-1, in HaCaT keratinocytes (HaCaT KCs) are up-regulated in the presence of normal human dermal fibroblasts (NHDFs). In addition, the secretion of pro-inflammatory molecules, including interleukin-1α (IL-1α) and prostaglandin E2 (PGE2), is suppressed in co-cultures of NHDFs and UVB-irradiated HaCaT KCs. Interestingly, the localization of Nrf2 protein in HaCaT KCs was immediately translocated from the cytoplasm to the nucleus after the co-culture with NHDFs. These results suggest the possibility that Nrf2 signaling in keratinocytes is regulated in cooperation with dermal fibroblasts.

Decreased mitochondrial function in UVA-irradiated dermal fibroblasts causes the insufficient formation of type I collagen and fibrillin-1 fibers.

BACKGROUND: Decreases of collagen fibers and the disappearance of oxytalan fibers are typical symptoms of photoaged skin. Although a low quality of mitochondria (MT) in photoaged skin cells has been observed, it is unknown whether the decreased quality of MT is responsible for the insufficient formation of dermal fibers. OBJECTIVE: To identify the role of mitochondrial quality in skin photoaging focusing on the formation of dermal fibers. METHODS: Type I collagen and fibrillin-1 fibers in normal human dermal fibroblasts (NHDFs) were observed by immunostaining. Type I collagen and fibrillin-1 proteins in NHDFs were quantified by ELISA. Mitochondrial quality was evaluated by measuring levels of intracellular ATP and MITOL, which regulate mitochondrial quality. RESULTS: UVA-irradiated NHDFs formed insufficient type I collagen and fibrillin-1 fibers and had a decreased ratio of extracellular versus intracellular levels of those proteins. Although expression levels of motor proteins that transport those proteins intracellularly were not affected by UVA, intracellular ATP levels, which is the driving force of motor proteins, were decreased by UVA along with decreased MITOL protein. Knockdown of MITOL in NHDFs decreased the level of intracellular ATP and caused the insufficient formation of type I collagen and fibrillin-1 fibers due to interfering with the secretion of those proteins. CONCLUSION: These results indicate that a low quality of MT with ATP depletion in dermal fibroblasts caused by irradiation with UVA induces the insufficient formation of type I collagen and fibrillin-1 fibers due to the decreased extracellular secretion of those proteins.

Pyridoxine Has a Potential to Prevent the Appearance of Pigmented Spots: Effects on the Phagocytosis and Differentiation of Keratinocytes.

Pyridoxine (VB6) is a vitamin that is essential to maintain the homeostasis of the human body by contributing to various metabolic reactions. In the skin, although some studies have shown that VB6 is involved in regulating homeostasis through the attenuation of intracellular oxidative stress, there are few reports regarding the effects of VB6 on the prevention or improvement of skin aging. Thus, we conducted this study to determine the potential anti-skin pigmentation effect of VB6 focusing on the phagocytosis of melanosomes (MSs) by keratinocytes. The phagocytosis of MSs by keratinocytes is activated by oxidative stress and is an important factor of skin pigmentation and the eventual appearance of pigmented spots. First, we confirmed the antioxidant property of VB6 that enhanced the expression of several intracellular antioxidants via nuclear erythroid factor 2-related factor 2 (Nrf2). Although the incorporation of fluorescent beads (FBs), which are used as pseudo-MSs, into keratinocytes was increased under higher oxidation conditions caused by UVB and by the depletion of intracellular glutathione, treatment with VB6 suppressed the increased incorporation of FBs into those keratinocytes via Nrf2 activation. Furthermore, VB6 restored the decreased expression of differentiation marker proteins in keratinocytes caused by FB incorporation. Taken together, the results show that VB6 has the potential to prevent the appearance of pigmented spots by suppressing the activation of phagocytosis in keratinocytes caused by oxidative stress, and by restoring the differentiation of keratinocytes disrupted by FB incorporation.

Intracellular oxidative stress induced by calcium influx initiates the activation of phagocytosis in keratinocytes accumulating at S-phase of the cell cycle after UVB irradiation.

BACKGROUND: Phagocytosis is an essential process that maintains cellular homeostasis. In the epidermis, the phagocytosis of melanosomes into keratinocytes is important to protect their DNA against damage from ultraviolet B (UVB) radiation. Furthermore, it is considered that UVB activates the phagocytosis by keratinocytes but the detailed mechanism involved is not fully understood. OBJECTIVE: To clarify the mechanism of UVB-enhanced phagocytosis in keratinocytes, we investigated the relationship between the phagocytic ability of keratinocytes and the cell cycle stage of keratinocytes. METHODS: The phagocytic ability of keratinocytes was evaluated using the incorporation of fluorescent beads after exposure to UVB or oxidative stress. S-phase was evaluated by BrdU incorporation and immunostaining of cyclin D1. Intracellular calcium levels of keratinocytes were measured using the probe Fluo-4AM. RESULTS: The phagocytosis of fluorescent beads into keratinocytes was enhanced by UVB and also by oxidative stress. We found that keratinocytes exposed to UVB or oxidative stress were at S-phase of the cell cycle. Furthermore, keratinocytes synchronized to S-phase showed a higher phagocytic ability according to the increased intracellular ROS level. The UVB-enhanced phagocytosis and entrance into S-phase of keratinocytes was abolished by ascorbic acid, a typical antioxidant. Keratinocytes synchronized to S-phase and exposed to UVB or oxidative stress had increased levels of intracellular calcium and their enhanced phagocytic abilities were diminished by the calcium ion chelator BAPTA-AM. CONCLUSION: Taken together, intracellular oxidative stress induced by intracellular calcium influx mediates the UVB-enhanced phagocytic ability of keratinocytes accumulating at S-phase of the cell cycle.

The role of chaperone complex HSP90-SGT1-RAR1 as the associated machinery for hybrid inviability between Nicotiana gossei Domin and N. tabacum L.

Two cultured cell lines (GTH4 and GTH4S) of a Nicotiana interspecific F1 hybrid (N. gossei × N. tabacum) were comparatively analyzed to find genetic factors related to hybrid inviability. Both cell lines proliferated at 37 °C, but after shifting to 26 °C, GTH4 started to die similar to the F1 hybrid seedlings, whereas GTH4S survived. As cell death requires de novo expression of genes and proteins, we compared expressed protein profiles between the two cell lines, and found that NgSGT1, a cochaperone of the chaperone complex (HSP90-SGT1-RAR1), was expressed in GTH4 but not in GTH4S. Agrobacterium-mediated transient expression of NgSGT1, but not NtSGT1, induced cell death in leaves of N. tabacum, suggesting its possible role in hybrid inviability. Cell death in N. tabacum was also induced by transient expression of NgRAR1, but not NtRAR1. In contrast, transient expression of any parental combinations of three components revealed that NgRAR1 promoted cell death, whereas NtRAR1 suppressed it in N. tabacum. A specific inhibitor of HSP90, geldanamycin, inhibited the progression of hypersensitive response-like cell death in GTH4 and leaf tissue after agroinfiltration. The present study suggested that components of the chaperone complex are involved in the inviability of Nicotiana interspecific hybrid.

Impairment of the autophagy system in repetitively UVA-irradiated fibroblasts.

BACKGROUND: Autophagy is known as an intracellular cleanup system necessary to maintain homeostasis of the skin. Many studies have pointed out the relationship between aging and the inactivation of autophagy function, which suggests that the inactivation of autophagy occurs in aged skin. However, the aging process of the skin is complicated compared with other organs, because the skin is localized at the border between the inside of the body and the environment. Thus, skin aging is strongly affected by environmental factors, and it is well recognized that ultraviolet (UV) radiation is an important environmental factor that promotes skin aging. Therefore, characterizing the autophagic phenotypes induced by environmental factors is important to understand the process of skin aging. METHODS: In order to demonstrate the status of autophagy during environment-induced aging of the skin, we investigated the autophagy profiles of normal human dermal fibroblasts (NHDFs) treated with repetitive UVA irradiation as model fibroblasts in photoaged skin. RESULTS: Repetitively UVA-irradiated NHDFs showed increased numbers of autophagosomes, which coincided with the accumulation of p62 and increased levels of LAMP-1 and lysosomes. The behavior of repetitively UVA-irradiated NHDFs on autophagy was similar to that of NHDFs treated with hydroxychloroquine (HCQ), which is an inhibitor of lysosomal proteinase. CONCLUSION: In summary, these results demonstrate that repetitively UVA-irradiated fibroblasts have reduced autophagy function due to the dysfunction of lysosomes.

3-O-Laurylglyceryl ascorbate improves the development of sensitive skin through the reduction of oxidative stress.

Skin sensitivity is a serious problem for many people, and it can be induced by various factors such as UV irradiation, physical and mental stresses, air pollution, dry air and so on. Skin dryness triggered by UV and dry air is one of the most important causes inducing the development of sensitive skin, and it has been reported that oxidative stress contributes to skin dryness. In this study, we investigated whether treatment with 3-O-laurylglyceryl ascorbate (VC-3LG), which is an amphipathic ascorbic acid derivative, can suppress the development of sensitive skin. The results demonstrate that VC-3LG restores the expression levels of interleukin-1α, nerve growth factor and matrix metalloprotease-9 in the dry skin models of reconstructed human epidermal equivalents (RHEEs) and in H2 O2 -treated keratinocytes. In addition, VC-3LG suppresses the dendrite elongation of nerve cells induced in RHEEs by dry skin conditions and by H2 O2 treatment of keratinocytes. Therefore, we consider that treatment of the skin with VC-3LG is an effective approach to improve the development of sensitive skin.

3-O-Glyceryl-2-O-hexyl Ascorbate Suppresses Melanogenesis through Activation of the Autophagy System.

The formation of skin pigmentation requires multiple steps, namely the activation of melanocytes, the synthesis of melanin, the transport of melanosomes to the tips of melanocyte dendrites and the transfer of melanosomes from melanocytes to surrounding keratinocytes. Recently, we reported that melanosomes accumulate in melanocytes when melanosome transport is disrupted and that they are then degraded by the autophagy system. In this study, we examined whether 3-O-glyceryl-2-O-hexyl ascorbate (VC-HG) suppresses melanogenesis through the activation of autophagy since VC-HG interferes with melanosome transport through the down-regulated expression of MyosinVa and Kinesin. The results demonstrate that VC-HG-treated B16 cells show an activation of autophagy through an increased expression level of Microtubule-associated protein 1 light chain 3 (LC3)-II and a decreased expression level of p62. Furthermore, the decrease of melanin content elicited by VC-HG was partially abolished by hydroxychloroquine or pepstatin A which are inhibitors of autophagy. Taken together, we conclude that VC-HG suppresses melanogenesis by activating the autophagy system.

Structural Requirements of Alkylglyceryl-l-Ascorbic Acid Derivatives for Melanogenesis Inhibitory Activity.

l-Ascorbic acid has multifunctional benefits on skin aesthetics, including inhibition of melanin production, and is widely used in cosmetics. It, however, has low stability and poor skin penetration. We hypothesize that alkylglyceryl-l-ascorbic acid derivatives, highly stable vitamin C-alkylglycerol conjugates, would have similar anti-melanogenic activity with better stability and penetration. We test 28 alkylglyceryl-l-ascorbic acid derivatives (1-28) on theophylline-stimulated B16 melanoma 4A5 cells to determine if they inhibit melanogenesis and establish any structure-function relationships. Although not the most potent inhibitors, 3-O-(2,3-dihydroxypropyl)-2-O-hexyl-l-ascorbic acid (6, IC50 = 81.4 µM) and 2-O-(2,3-dihydroxypropyl)-3-O-hexyl-l-ascorbic acid (20, IC50 = 117 µM) are deemed the best candidate derivatives based on their inhibitory activities and low toxicities. These derivatives are also found to be more stable than l-ascorbic acid and to have favorable characteristics for skin penetration. The following structural requirements for inhibitory activity of alkylglyceryl-l-ascorbic acid derivatives are also determined: (i) alkylation of glyceryl-l-ascorbic acid is essential for inhibitory activity; (ii) the 3-O-alkyl-derivatives (2-14) exhibit stronger inhibitory activity than the corresponding 2-O-alkyl-derivatives (16-28); and (iii) derivatives with longer alkyl chains have stronger inhibitory activities. Mechanistically, our studies suggest that l-ascorbic acid derivatives exert their effects by suppressing the mRNA expression of tyrosinase and tyrosine-related protein-1.

3-O-Glyceryl-2-O-hexyl ascorbate suppresses melanogenesis by interfering with intracellular melanosome transport and suppressing tyrosinase protein synthesis.

BACKGROUND: Ascorbic acid (AsA) has multifunctional benefits on skin beauty, such as the reduction in oxidative stress and the induction of collagen production. Among them, the prevention and improvement of skin pigmentation by AsA is a most important benefit for people. However, it is well known that AsA not only is quite unstable in formulations but it also has a low capability of skin penetration due to its hydrophilic property. In addition, existing water-soluble AsA derivatives that were developed to improve its stability also have low skin penetration. AIM: To investigate the potential of a newly synthesized amphiphilic derivative of AsA, 3-O-Glyceryl-2-O-hexyl ascorbate (VC-HG), which has an added glyceryl group and a hexyl group, on skin beauty focusing on its skin lightening/whitening effects. METHODS: DNA microarray analysis and real-time PCR were used to clarify the effects of VC-HG on melanogenesis using B16 mouse melanoma cells. The effects of VC-HG on melanin synthesis, tyrosinase protein levels, and the inhibition of tyrosinase activity were evaluated. RESULTS: DNA microarray analysis revealed that treatment with VC-HG downregulated the expression of genes encoding tyrosinase and MyosinVa. Further, real-time PCR analysis showed the downregulation of tyrosinase, MyosinVa, Rab27a, and Kinesin mRNAs following VC-HG treatment. In addition, VC-HG caused decreases in tyrosinase protein levels and melanin synthesis. CONCLUSION: We conclude that VC-HG has an impact on skin lightening/whitening by inhibiting tyrosinase protein synthesis and interfering with intracellular melanosome transport.

Disruption of melanosome transport in melanocytes treated with theophylline causes their degradation by autophagy.

Melanosomes containing melanin are transported from the perinuclear area to the tips of dendrites in epidermal melanocytes, and are then transferred to keratinocytes. Thus, skin color is determined by the amount of melanin synthesized in melanocytes and the subsequent dispersion of melanosomes in the epidermis. Therefore, disrupting intracellular melanosome transport in melanocytes is considered an effective approach to regulate skin color. However, the fate of melanosomes that accumulate in melanocytes due to disrupted intracellular transport is unclear. In this study, we disrupted melanosome transport by knockdown of the motor protein MyosinVa. Knock-down of MyosinVa (M-KD) in cells treated with theophylline significantly down-regulated the mRNA and protein expression levels of tyrosinase. Interestingly, intracellular melanin contents in M-KD cells were decreased. Furthermore, M-KD cells showed activation of autophagy through increased expression of Microtubule-associated protein 1 light chain 3 (LC3) -II and decreased expression of p62. The sum of these results indicate that disruption of melanosome transport causes their degradation by autophagy.

Melanogenesis inhibitory activity of a 7-O-9'-linked neolignan from Alpinia galanga fruit.

An aqueous acetone extract from the fruit of Alpinia galanga (Zingiberaceae) demonstrated inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells (IC50=7.3µg/mL). Through bioassay-guided separation of the extract, a new 7-O-9'-linked neolignan, named galanganol D diacetate (1), was isolated along with 16 known compounds including 14 phenylpropanoids (2-15). The structure of 1, including its absolute stereochemistry in the C-7 position, was elucidated by means of extensive NMR analysis and total synthesis. Among the isolates, 1 (IC50=2.5µM), 1'S-1'-acetoxychavicol acetate (2, 5.0µM), and 1'S-1'-acetoxyeugenol acetate (3, 5.6µM) exhibited a relatively potent inhibitory effect without notable cytotoxicity at effective concentrations. The following structural requirements were suggested to enhance the inhibitory activity of phenylpropanoids on melanogenesis: (i) compounds with 4-acetoxy group exhibit higher activity than those with 4-hydroxy group; (ii) 3-methoxy group dose not affect the activity; (iii) acetylation of the 1'-hydroxy moiety enhances the activity; and (iv) phenylpropanoid dimers with the 7-O-9'-linked neolignan skeleton exhibited higher activity than those with the corresponding monomer. Their respective enantiomers [1' (IC50=1.9µM) and 2' (4.5µM)] and racemic mixtures [(±)-1 (2.2µM) and (±)-2 (4.4µM)] were found to exhibit melanogenesis inhibitory activities equivalent to those of the naturally occurring optical active compounds (1 and 2). Furthermore, the active compounds 1-3 inhibited tyrosinase, tyrosine-related protein (TRP)-1, and TRP-2 mRNA expressions, which could be the mechanism of melanogenesis inhibitory activity.

3-O-Laurylglyceryl ascorbate activates the intracellular antioxidant system through the contribution of PPAR-γ and Nrf2.

BACKGROUND: Ascorbic acid (AsA) has multifunctional effects on physiology and aging including the prevention and improvement of skin pigmentation and wrinkles. AsA has scavenging effects against various types of reactive oxygen species (ROS), which are initiators of aging and premature aging of the skin. However, AsA not only has a quite unstable characteristic, but also has low skin penetration. In addition, existing water-soluble AsA derivatives are not effective to improve its penetration of the skin. OBJECTIVE: To investigate the antioxidant effect of a newly synthesized amphipathic derivative of AsA, 3-O-laurylglyceryl ascorbate (VC-3LG), in which a laurylglyceryl group was introduced into AsA. METHODS: Intracellular ROS levels in keratinocytes were evaluated using the 2',7'-Dichlorofluorescein diacetate (DCFHDA) assay. Real-time PCR was used to investigate the mechanism of the antioxidant effect of VC-3LG. RESULTS: Although VC-3LG had less ability to scavenge ROS compared to AsA, it elicited a superior reduction of intracellular ROS levels, with or without extracellular stimuli such as exposure to H2O2 or UVB. The results show that VC-3LG up-regulates the expression of mRNAs encoding peroxisome proliferator activated receptor-γ (PPAR-γ) and nuclear factor E2-related factor 2 (Nrf2), which in turn up-regulate the levels of mRNAs encoding γ-glutamyl cysteine synthase (γ-GCS), heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO1). Furthermore, the Nrf2 mRNA level is down-regulated in siPPAR-γ treated cells, and the effects of VC-3LG on PPAR-γ and Nrf2 mRNA levels are reduced by PPAR-γ knockdown. CONCLUSION: Taken together, we conclude that VC-3LG has an antioxidant effect and scavenges ROS directly as well as stimulating intracellular antioxidants such as GSH through the PPAR-γ and Nrf2 signaling pathway.

Simultaneous quantitative analysis of 12 methoxyflavones with melanogenesis inhibitory activity from the rhizomes of Kaempferia parviflora.

A methanol extract from the rhizomes of Kaempferia parviflora Wall. ex Baker (Zingiberaceae) has shown inhibitory effects against melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells (IC50 = 9.6 µg/mL). Among 25 flavonoids and three acetophenones isolated previously (1-28), several constituents including 5-hydroxy-7,3',4'-trimethoxyflavone (6, IC50 = 8.8 µM), 5,7,3',4'-tetramethoxyflavone (7, 8.6 µM), 5,3'-dihydroxy-3,7,4'-trimethoxyflavone (12, 2.9 µM), and 5-hydroxy-3,7,3',4'-tetramethoxyflavone (13, 3.5 µM) showed inhibitory effects without notable cytotoxicity at the effective concentrations. Compounds 6, 7, 12, and 13 inhibited the expression of tyrosinase, tyrosine-related protein (TRP)-1, and TRP-2 mRNA, which could be the mechanism of their melanogenesis inhibitory activity. In addition, a quantitative analytical method for 12 methoxyflavones (1, 2, 4-11, 13, and 14) in the extract was developed using HPLC. The optimal condition for separation and detection of these constituents were achieved on an ODS column (3 µm particle size, 2.1 mm i.d. × 100 mm) with MeOH-0.1 % aqueous acetic acid solvent systems as the mobile phase, and the detection and quantitation limits of the method were estimated to be 0.08-0.66 ng and 0.22-2.00ng, respectively. The relative standard deviation values of intra- and interday precision were lower than 0.95 and 1.08 %, respectively, overall mean recoveries of all flavonoids were 97.9-102.9 %, and the correlation coefficients of all the calibration curves showed good linearity within the test ranges. For validation of the protocol, extracts of three kinds of the plant's rhizomes collected from different regions in Thailand (Leoi, Phetchabun, and Chiang Mai provinces) were evaluated. The results indicated that the assay was reproducible, precise, and could be readily utilized for the quality evaluation of the plant materials.