LGI3 is secreted and binds to ADAM22 via TRIF-dependent NF-κB pathway in response to LPS in human keratinocytes.

Recently, we reported that HaCaT human keratinocytes secreted leucine-rich repeat LGI family member 3 (LGI3) protein after exposure to ultraviolet B (UVB) irradiation. In the present study, we aimed to determine whether LGI3 is also released in response to stimulation by lipopolysaccharides (LPS), membrane components of gram-negative bacteria. Our results showed that LGI3 was indeed secreted by LPS-stimulated HaCaT cells. We also found that LPS potently stimulated the induction of cycloxygenase-2 (COX-2), which is involved in the inflammatory response. In addition, LPS-induced LGI3 secretion and COX-2 expression were blocked by NS-398, a selective COX-2 inhibitor. Moreover, LPS activated nuclear factor-κB (NF-κB) via a TRIF-dependent pathway, and activated NF-κB led to LGI3 production in HaCaT cells. For the first time, we predicted the LGI3 promoter sequence and demonstrated that NF-κB bound to the LGI3 gene promoter region. LPS treatment also increased the expression of a disintegrin and metalloproteinase domain-containing protein 22 (ADAM22), a candidate LGI3 receptor. Furthermore, co-immunoprecipitation, flow cytometry, and immunocytochemistry revealed that LGI3 associated with ADAM22 in LPS-treated keratinocytes. Thus, ADAM22 may be an LGI3 receptor in human keratinocytes. Taken together, these data suggest that the TRIF-dependent pathway is a novel regulator of LGI3 secretion in response to LPS stimulation in HaCaT cells and that keratinocyte-derived LGI3 interacts with ADAM22 and mediates LPS-induced inflammation.

LGI3 promotes human keratinocyte differentiation via the Akt pathway.

Leucine-rich repeat LGI family member 3 (LGI3), a member of the LGI family, is a secreted protein that is expressed not only in the brain and adipose tissues, but also in various skin cells. We previously reported that LGI3 was secreted after exposure to ultraviolet B and promoted the migration of HaCaT human keratinocytes. In the present study, we investigated whether LGI3 influences the differentiation of keratinocytes. The results show that the expression of involucrin, a keratinocyte differentiation marker, was reduced in tissue from LGI3-knockout mice. Those results indicate that LGI3 plays an important role in keratinocyte differentiation. Therefore, we treated HaCaT cells with LGI3 to examine its effect on keratinocyte differentiation. Protein levels of various differentiation markers were enhanced by treatment with LGI3. Furthermore, expression of differentiation markers was inhibited when keratinocytes were transfected with an siRNA for LGI3. LGI3 strongly activated Akt, whereas it had no apparent effect on extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, or the c-Jun N-terminal kinase. A specific inhibitor of phosphoinositide 3-kinase, LY294002, reduced LGI3-induced expression of differentiation markers in HaCaT cells. Taken together, these results suggest that LGI3 promotes keratinocyte differentiation and could be used as a therapeutic agent to recover skin barrier function in epidermal barrier disruption.

Laminin peptide YIGSR enhances epidermal development of skin equivalents.

Since the use of animal experimentation is restricted with regard to cosmetic materials, alternative in vitro models such as skin equivalents (SEs) are needed. Laminin is one of the major non-collagenous glycoproteins. The pentapeptide YIGSR (Tyr-Ile-Gly-Ser-Arg) is a functional motif of laminin that binds to the laminin receptor. In the present study, we examined whether YIGSR could improve the reconstruction of SEs. YIGSR has no effects on monolayer cell proliferation of CCD25-Sk fibroblasts or HaCaT keratinocytes. Interestingly, YIGSR decreased TGF-ß1 levels, although it promoted type Ι collagen synthesis in CCD25-Sk cells. In HaCaT cells, YIGSR decreased the expression of involucrin and loricrin, which are differentiation markers. Furthermore, YIGSR increased levels of proliferating cell nuclear antigen (PCNA), p63, and integrin α6, and decreased involucrin in SE models. In addition, two models containing YIGSR (mixed with dermal equivalents or added into media) did not show any differences in expression levels of PCNA, p63, integrin α6, and involucrin. Therefore, YIGSR is a useful agent for reconstruction of SEs, independent of its method of application. These results indicate that YIGSR stimulates epidermal proliferation and basement membrane formation while inhibiting keratinocyte differentiation of SEs. Taken together, these results indicate that YIGSR promotes the reconstruction of SEs, potentially via decreased TGF-ß1 levels and consequent inhibition of epidermal differentiation.

Avian Collagen Is Useful for the Construction of Skin Equivalents.

As a result of restrictions on animal experimentation, improved skin equivalents (SEs) are needed as alternative test models. This work investigated the effects of avian collagen on the construction of SEs, and to the best of our knowledge is the first study to do so. Hematoxylin and eosin and immunohistochemical staining were used to analyze the SEs. In models containing avian collagen as a dermal equivalent (DE) ingredient, fibroblast proliferation increased by about 60% relative to the control model. Immunohistochemical staining showed that the expression of proliferating cell nuclear antigen (PCNA) and p63 increased in the avian collagen models, while the expression of involucrin, integrin α6, and integrin ß1 remained unchanged. Next, DEs were cryopreserved to allow the easier creation of SEs. Keratinocytes were seeded on thawed DEs, and SEs were constructed. Avian collagen increased the viability of DEs relative to the control. Furthermore, avian collagen increased the expression of PCNA and p63 in keratinocytes on thawed DEs. The results indicate that DEs containing avian collagen can be thawed as needed after cryopreservation. Avian collagen can improve the construction of SEs and be used as part of a dermal kit for SE construction.

Leucine-rich glioma inactivated 3 and tumor necrosis factor-α regulate mutually through NF-κB.

Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein member of LGI family. We previously reported that LGI3 increased in obese adipose tissues and suppressed adipogenesis through its receptor, ADAM23. We proposed that LGI3 may be a pro-inflammatory adipokine secreted predominantly by preadipocytes and macrophages. In this study, we showed that LGI3 and tumor necrosis factor-α (TNF-α) upregulated each other in 3T3-L1 cells. Treatment of 3T3-L1 preadipocytes with LGI3 protein increased TNF-α mRNA and protein. LGI3 treatment led to NF-κB activation and binding to an NF-κB binding site (-523 to -514) in TNF-α promoter. TNF-α treatment increased mRNA and protein expression of LGI3 and ADAM23. TNF-α increased NF-κB binding to a predicted binding site (-40 to -31) in LGI3 promoter. High fat diet-fed mice showed that LGI3 and TNF-α were increased and colocalized in adipose tissue inflammation. Taken together, these results suggested that mutual upregulation of LGI3 and TNF-α may play a role in adipose tissue inflammation in obesity.

The DNA methylation inhibitor 5-azacytidine decreases melanin synthesis by inhibiting CREB phosphorylation.

Here we examined the effects of a DNA methylation inhibitor, 5-azacytidine, on melanogenesis in Mel-Ab cells. We found that 5-azacytidine decreased the melanin content and tyrosinase activity in these cells in a dose-dependent manner; importantly, 5-azacytidine was not cytotoxic at the concentrations used in these experiments. On the other hand, 5-azacytidine did not affect tyrosinase activity in a cell-free system, indicating that 5-azacytidine is not a direct tyrosinase inhibitor. Instead, 5-azacytidine decreased the protein levels of microphthalmia-associated transcription factor (MITF) and tyrosinase. Thus, we investigated the effects of 5-azacytidine on signal transduction pathways related to melanogenesis. However, 5-azacytidine did not have any effect on either Akt or glycogen synthase kinase 3ß (GSK3ß) phosphorylation. The phosphorylation of cAMP response element-binding protein (CREB) is well known to regulate MITF expression, thereby also regulating tyrosinase expression. We found that 5-azacytidine decreased the phosphorylation of CREB. Therefore, we propose that 5-azacytidine may decrease melanin synthesis by downregulating MITF and tyrosinase via CREB inactivation.

Methyl gallate from Acer barbinerve decreases melanin synthesis in Mel-Ab cells.

Methyl gallate (MG) was isolated from the bark of Acer barbinerve, which has traditionally been used in Oriental medicine. In the present study, we examined the effects of MG on melanin synthesis in Mel-Ab melanocyte cells. MG decreased melanin pigmentation in a concentration-dependent manner, but did not directly inhibit tyrosinase activity. Further analysis showed that MG had no effect on extracellular signal-regulated kinase (ERK) activation, but induced phosphorylation of glycogen synthase kinase (GSK)3ß, which is known to increase ß-catenin accumulation. Accordingly, the ß-catenin level was increased by MG. However, a specific GSK3ß inhibitor did not rescue the MG-induced inhibition of melanogenesis. Additionally, MG decreased the protein expression of microphthalmia-associated transcription factor (MITF) and tyrosinase, which regulate melanin synthesis. Based on these results, we conclude that MG inhibits melanogenesis by decreasing the expression of MITF and tyrosinase.

ERK Activation by Fucoidan Leads to Inhibition of Melanogenesis in Mel-Ab Cells.

Fucoidan, a fucose-rich sulfated polysaccharide derived from brown seaweed in the class Phaeophyceae, has been widely studied for its possible health benefits. However, the potential of fucoidan as a possible treatment for hyperpigmentation is not fully understood. This study investigated the effects of fucoidan on melanogenesis and related signaling pathways using Mel-Ab cells. Fucoidan significantly decreased melanin content. While fucoidan treatment decreased tyrosinase activity, it did not do so directly. Western blot analysis indicated that fucoidan downregulated microphthalmia-associated transcription factor and reduced tyrosinase protein expression. Further investigation showed that fucoidan activated the extracellular signal-regulated kinase (ERK) pathway, suggesting a possible mechanism for the inhibition of melanin synthesis. Treatment with PD98059, a specific ERK inhibitor, resulted in the recovery of melanin production. Taken together, these findings suggest that fucoidan inhibits melanogenesis via ERK phosphorylation.

Leucine-rich glioma inactivated 3 is a melanogenic cytokine in human skin.

Recently, we demonstrated that leucine-rich glioma inactivated 3 (LGI3) is expressed in human skin. However, the effects of LGI3 on melanocytes remain unknown. The present study demonstrated that LGI3 can serve to stimulate melanogenesis without affecting cell viability. To determine the effects of LGI3 on melanin synthesis, normal human melanocytes and Mel-Ab cells were treated with recombinant LGI3 and melanin content was measured. Our results showed that LGI3 promoted melanin synthesis in both cell types. Moreover, upregulation of microphthalmia-associated transcription factor (MITF) and tyrosinase was observed at both the mRNA and protein levels via RT-PCR and Western blotting, respectively. Furthermore, immunohistochemical staining showed that the expression of LGI3 increased in the basal layer of melasma skin samples, whereas it decreased slightly in vitiligo samples. These results suggest that LGI3 may play a role as a melanogenic cytokine in human skin.

Docosahexaenoic acid inhibits melanin synthesis in murine melanoma cells in vitro through increasing tyrosinase degradation.

AIM: To investigate the effects of docosahexaenoic acid (DHA) on melanin synthesis and related regulatory mechanisms. METHODS: B16F10 mouse melanoma cells were exposed to DHA for 3 d, and melanin content and tyrosinase activity were measured. Western blot analysis was used to analyze the protein levels in DHA-mediated signal transduction pathways. RESULTS: DHA (1-25 µmol/L) did not affect the viability of B16F10 cells, but decreased α-MSH-induced melanin synthesis in a concentration-dependent manner. DHA concentration-dependently reduced tyrosinase activity in the cells, but did not affect mushroom tyrosinase activity in a cell-free system. Furthermore, DHA treatment significantly reduced tyrosinase level without affecting microphthalmia-associated transcription factor (MITF) in the cells. DHA did not activate ERK and Akt in the cells. Pretreatment with the proteasome inhibitor MG132 (80 nmol/L) abolished DHA-induced tyrosinase reduction. CONCLUSION: DHA inhibits melanogenesis in B16F10 cells in vitro through increasing tyrosinase degradation. The results suggest that DHA may be a potential agent for treatment of hyperpigmentary disorders of skin.

Hypopigmentary effects of ethyl P-methoxycinnamate isolated from Kaempferia galanga.

We isolated crystals from the chloroform fraction of an ethanol extract of Kaempferia galanga and identified it as ethyl p-methoxycinnamate through nuclear magnetic resonance analysis. In the present study, we found that ethyl p-methoxycinnamate significantly decreased melanin synthesis in B16F10 murine melanoma cells stimulated with α-melanocyte stimulating hormone (α-MSH). In a cell-free system, however, ethyl p-methoxycinnamate did not directly inhibit tyrosinase, the rate-limiting enzyme of melanogenesis. Instead, it inhibited tyrosinase activity in B16F10 cells in a dose-dependent manner. Furthermore, Western blot analysis showed that ethyl p-methoxycinnamate decreased microphthalmia-associated transcription factor and tyrosinase levels in α-MSH-stimulated B16F10 cells. These results indicate that the pigment-inhibitory effect of ethyl p-methoxycinnamate results from downregulation of tyrosinase. Ethyl p-methoxycinnamate isolated from K. galanga could be developed as a skin whitening agent to treat hyperpigmentary disorders.

Myriocin, a serine palmitoyltransferase inhibitor, increases melanin synthesis in Mel-Ab cells and a skin equivalent model.

The purpose of this study was to investigate effects of myriocin, an inhibitor of serine palmitoyltransferase, on melanogenesis. It was found that myriocin increased melanin synthesis in a concentration-dependent manner. Moreover, myriocin up-regulated microphthalmia-associated transcription factor (MITF) and tyrosinase expression via phosphorylation of CREB, but it did not directly activate tyrosinase, a rate-limiting melanogenic enzyme. Furthermore, we demonstrated increased melanin synthesis with myriocin on a pigmented skin equivalent model established using Cervi cornus Colla (deer antler glue). One and 5 microM of myriocin darkened the color of the skin equivalent. These results suggest that myriocin may have potential effects for the treatment of hypopigmentary skin diseases like vitiligo or for sunless tanning.

Fucoidan promotes the reconstruction of skin equivalents.

In this study we investigated the effects of fucoidan on the proliferation of fibroblasts and the reconstruction of a skin equivalent (SE). Fucoidan significantly stimulated the proliferation of CCD-25Sk human fibroblasts and Western blot analysis demonstrated that fucoidan markedly increased the expression of cyclin D1 and decreased the expression of p27. Fucoidan was used to reconstruct SE. Immunohistochemical staining showed that the addition of fucoidan to dermal equivalents increased expression of proliferating cell nuclear antigen (PCNA) and p63. In addition, expression of α6-integrin was significantly increased by fucoidan, whereas expression of ß1-integrin, type 1 collagen, elastin, fibronectin did not markedly change. These results suggest that fucoidan has positive effects on epidermal reconstruction and will therefore be beneficial in the reconstruction of SE.

KHG26792 Inhibits Melanin Synthesis in Mel-Ab Cells and a Skin Equivalent Model.

The purpose of this study is to characterize the effects of KHG26792 (3-(naphthalen-2-yl(propoxy) methyl)azetidine hydrochloride), a potential skin whitening agent, on melanin synthesis and identify the underlying mechanism of action. Our data showed that KHG26792 significantly reduced melanin synthesis in a dose-dependent manner. Additionally, KHG26792 downregulated microphthalmia-associated transcription factor (MITF) and tyrosinase, the rate-limiting enzyme in melanogenesis, although tyrosinase was not inhibited directly. KHG26792 activated extracellular signal-regulated kinase (ERK), whereas an ERK pathway inhibitor, PD98059, rescued KHG26792-induced hypopigmentation. These results suggest that KHG26792 decreases melanin production via ERK activation. Moreover, the hypopigmentary effects of KHG26792 were confirmed in a pigmented skin equivalent model using Cervi cornus Colla (deer antler glue), in which the color of the pigmented artificial skin became lighter after treatment with KHG26792. In summary, our findings suggest that KHG26792 is a novel skin whitening agent.

Leucine-rich glioma inactivated 3 regulates adipogenesis through ADAM23.

Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein and a member of LGI/epitempin family. We previously showed that LGI3 was highly expressed in brain and played regulatory roles in neuronal exocytosis and differentiation. Besides the nervous system, LGI3 was shown to be expressed in diverse tissues. In this study, we found that LGI3 and its receptor candidate ADAM23 were expressed in adipose tissues and 3T3-L1 cells. 3T3-L1 preadipocytes secreted a 60-kDa protein, a major secreted form of LGI3, which declined with adipocyte differentiation. LGI3 was also expressed in adipose tissue macrophages in the ob/ob mice and in macrophage cell line. The 60-kDa LGI3 protein was selectively increased in the ob/ob adipose tissues comparing with the lean mice. Pull-down experiments, coimmunoprecipitation and immunocytochemistry indicated that LGI3 associated with ADAM23 in adipose tissues and 3T3-L1 cells. Knockdown of LGI3 or ADAM23 by siRNA increased adipogenesis in 3T3-L1 cells. Treatment with LGI3 protein did not affect preadipocyte proliferation but attenuated adipogenesis and this effect was reversed by siRNA-mediated knockdown of ADAM23. Taken together, we propose that LGI3 may be a candidate adipokine that is perturbed in obesity and suppresses adipogenesis through its receptor, ADAM23.

Leucine-rich glioma inactivated 3 associates negatively with adiponectin.

Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein member of LGI/epitempin family. We previously reported that LGI3 was expressed in adipose tissues and suppressed adipogenesis through its receptor, ADAM23. We proposed that LGI3 may be a candidate adipokine with pro-inflammatory activity. To investigate the role of LGI3 in adipose tissues, we analyzed cytokine profile in LGI3 knockout mice. Protein array analysis showed that adiponectin was significantly increased in adipose tissues and plasma of LGI3 knockout mice. SiRNA-mediated knockdown of LGI3 increased adiponectin in 3T3-L1 preadipocytes. Treatment of differentiating 3T3-L1 cells with LGI3 protein decreased adiponectin in a dose-dependent manner. High fat diet (HFD)-fed mice showed expression of LGI3 in adipose tissue macrophages in addition to adipocytes that expressed LGI3 in both normal chow-fed and HFD-fed mice. The 60-kDa LGI3 was selectively increased in adipose tissues of HFD mice in which adiponectin was downregulated. Taken together, these results suggested that LGI3 may participate in adipose tissue homeostasis by negatively regulating adiponectin.

Ceramide PC102 inhibits melanin synthesis via proteasomal degradation of microphthalmia-associated transcription factor and tyrosinase.

A few types of ceramide are reported to decrease melanin synthesis. In the present study, we examined the effects of an artificial ceramide analog, PC102, on melanogenesis using a spontaneously immortalized melanocyte cell line (Mel-Ab). PC102 is currently used as a moisturizing additive in a variety of cosmetics. Our data showed that PC102 inhibited melanin production and tyrosinase activity in a dose-dependent manner, but did not directly affect tyrosinase activity. Microphthalmia-associated transcription factor (MITF), tyrosinase, and ß-catenin protein levels decreased after 48 h of PC102 treatment. In contrast, PC102 did not decrease MITF, tyrosinase, and ß-catenin mRNA levels. Therefore, we investigated whether the decrease in MITF and tyrosinase by PC102 is due to proteasomal degradation. MG132, a proteasomal inhibitor, completely abolished tyrosinase downregulation due to PC102 and partially reduced the downregulation of MITF and ß-catenin due to PC102. Moreover, MG132 abrogated the inhibition of melanin synthesis by PC102. Taken together, our data suggest that PC102 may inhibit melanin synthesis through MITF and tyrosinase degradation.

Leucine-rich glioma inactivated 3 promotes HaCaT keratinocyte migration.

Our finding that human skin expresses leucine-rich glioma inactivated 3 (LGI3) raises the question of the function of this cytokine in keratinocytes. We have shown that LGI3 stimulates human HaCaT keratinocyte migration without affecting viability or proliferation. Western blot analysis showed that LGI3 induced focal adhesion kinase activation, Akt phosphorylation, and glycogen synthase kinase 3ß (GSK3ß) phosphorylation in these cells. Using the scratch wound assay and a modified Boyden chamber, we found that LY294002, a selective phosphatidylinositol 3-kinase inhibitor, and LiCl, a selective GSK3ß inhibitor, abolished LGI3-induced cell migration. We tested ß-catenin levels after LGI3 treatment because the Akt-GSK3ß pathway regulates ß-catenin accumulation, and ß-catenin promotes cell migration. LGI3 treatment increased ß-catenin protein and nuclear localization, whereas LY294002 prevented LGI3-induced focal adhesion kinase and Akt activation as well as ß-catenin accumulation. Overall, these data suggest that LGI3 stimulates HaCaT cell migration following ß-catenin accumulation through the Akt pathway.

Okadaic acid suppresses melanogenesis via proteasomal degradation of tyrosinase.

Okadaic acid is a C38 fatty acid derivative that is known to specifically inhibit the activity of protein phosphatase 2A (PP2A). Previously, we reported that inhibition of PP2A by okadaic acid elicited extracellular signal-regulated kinase (ERK) activation, and that PP2A may be involved in melanogenesis. However, the effects of okadaic acid on melanogenesis have not been completely evaluated. In the present study, we investigated the molecular mechanisms involved in okadaic acid modulation of melanin synthesis in a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Treatment with okadaic acid inhibited melanin production in a dose-dependent manner. Moreover, okadaic acid led to a decrease in tyrosinase protein levels without altering mRNA expression. Therefore, we investigated whether the decreased level of tyrosinase by okadaic acid was related to proteasomal degradation of tyrosinase. We found that MG132, a proteasome inhibitor, almost completely abolished both the downregulation of tyrosinase levels and the inhibition of melanin synthesis by okadaic acid. Taken together, our data indicate that okadaic acid inhibits melanin synthesis via proteasomal degradation of tyrosinase.

Ultraviolet B-induced LGI3 secretion protects human keratinocytes.

Leucine-rich glioma inactivated 3 (LGI3) is known to be expressed mainly in the brain. However, the expression and physiological roles of LGI3 in skin cells remain unknown. In this study, it was found for the first time that LGI3 is expressed mostly by normal human keratinocytes. Furthermore, ELISA analysis showed that HaCaT human keratinocytes increased LGI3 secretion after exposure to ultraviolet B (UVB) in a time- and dose-dependent manner. We next investigated the possible role of LGI3 in keratinocytes. LGI3 (50 ng/ml) increased survival of HaCaT cells by 20% after UVB irradiation (150 mJ/cm(2) ). It was also found that LGI3 stimulates the phosphorylation of Akt, which is involved in the cell survival-signalling cascade. Furthermore, LGI3 led to the phosphorylation of MDM2 and subsequent p53 degradation. Taken together, the data suggest that LGI3 may regulate p53 levels and that keratinocyte-derived LGI3 may act as a novel cytokine for skin homoeostasis.