Leucine-rich Glioma Inactivated 3 Research Articles

The Suppressive Effect of Leucine-Rich Glioma Inactivated 3 (LGI3) Peptide on Impaired Skin Barrier Function in a Murine Model Atopic Dermatitis.

This study aimed to restore the skin barrier function from atopic dermatitis (AD) via treatment with leucine-rich glioma inactivated 3 (LGI3) peptide. Male NC/Nga mice (7 weeks, 20 g) were randomly allocated into three groups (control, AD, and LGI3 group). After induction of AD skin lesions with Dermatophagoides farinae ointment, mice were treated with LGI3. The clinical score of AD was the highest and the dorsal skin thickness was the thickest in the AD group. In contrast, LGI3 treatment improved the clinical score and the dorsal skin thickness compared to the AD model. LGI3 treatment suppressed histopathological thickness of the epithelial cell layer of the dorsal skin. LGI3 treatment could indirectly reduce mast cell infiltration through restoring the barrier function of the skin. Additionally, the filaggrin expression was increased in immunohistochemical evaluation. In conclusion, the ameliorating effect and maintaining skin barrier homeostasis in the AD murine model treated with LGI3 could be attributed to complete re-epithelialization of keratinocytes. Hence, LGI3 might be considered as a new potential therapeutic target for restoring skin barrier function in AD.

Leucine-rich glioma inactivated 3: a novel keratinocyte-derived melanogenic cytokine in vitiligo patients

AbstractBackground: In-vitro studies showed that Leucine-rich glioma inactivated 3 (LGI3) is a keratinocyte-derived cytokine that stimulates melanin synthesis and is increased after ultra violet B (UVB) irradiation. So, we postulated that LGI3 may be involved in vitiligo aetiopathogenesis and may participate in narrow band ultra violet B (NB-UVB) induced pigmentation in vitiligo.Objectives: To assess this hypothesis, lesional LGI3 immunohistochemical expression of vitiligo patients before and after NB-UVB phototherapy was studied, and its correlation with repigmentation was evaluated.Methods: Forty vitiligo patients and 20 age, sex, and skin phenotype-matched controls were enrolled. Patients were treated with NB-UVB thrice weekly for 12 weeks. VASI score was evaluated before and after NB-UVB sessions. For vitiligo patients, baseline LGI3 immunohistochemical staining was estimated, and compared to that of controls and to its post-treatment data in those patients. Results: Baseline LGI3 immunohistochemical studied parameters (expression, intensity, percentage and H score) were significantly lower in vitiligo cases than controls (p=0.003, 0.013, 0.001 and 0.001 respectively). After 12 weeks of NB-UVB phototherapy, these LGI3 immunohistochemical parameters were up-regulated and became comparable to that of controls (p >0.05 for all). There was a significant positive correlation between the improvement of both VASI score and LGI3 H score mean values (r=-0.349 , p=0.027).Study limitations: Small number of investigated subjects.Conclusion: Decreased LGI3 protein may play an active role in vitiligo pathogenesis and its up-regulation after NB-UVB phototherapy, may actively participate in NB-UVB photo-induced melanogenesis.

Leucine-rich glioma inactivated 3: Integrative analyses reveal its potential prognostic role in cancer.

Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein in vertebrates, which belongs to the LGI family. In our previous study, LGI3 was found to be expressed in brain, adipose tissues and the skin, where it functions as a multifunctional cytokine. In the present study, we used bioinformatic tools to perform data mining, phylogenetics and prognostic association analysis to investigate the prognostic role of LGI3 in cancers. The sequences of LGI3 orthologues were analyzed from various species, and it was found that LGI3 was highly conserved in mammals and that the subsets of amino acid residues were phylogenetically coevolved in four major clusters. Single nucleotide polymorphisms (SNPs) of the human LGI3 gene included 228 functionally relevant variants (missense, nonsense and frameshift) in a total of 1,042 SNPs. Four missense SNPs had a global minor allele frequency ≥0.001. Somatic mutations in cancer with functional relevance were found in various types of cancer, including uterine, stomach and lung cancer. In addition, five amino acid residues with cancer mutations were shown to be coevolved in the vertebrate phylogeny, suggesting their importance in protein dysfunctions in cancer. One conserved amino acid and three SNPs were found to be mutated in stomach cancer and melanoma. Analysis of expression microarray data demonstrated that the expression of LGI3 was significantly associated with the prognosis of brain, colorectal and lung cancer. Taken together, these results suggested that the genetic variations and expression levels of LGI3 have potential value in cancer prognosis.

Leucine-rich glioma inactivated 3: integrative analyses support its prognostic role in glioma.

BackgroundLeucine-rich glioma inactivated 3 (LGI3) is a secreted protein member of LGI family. We previously reported that LGI3 was expressed in brain, adipose tissues and skin, where it played roles as a multifunctional cytokine. We postulated that LGI3 may be involved in cytokine network in cancers.AimThis study aimed to analyze differentially expressed genes in glioma tissues and glioma cohort data to investigate the prognostic role of LGI3 and its receptors.Materials and methodsExpression microarray data from Gene Expression Omnibus and glioma cohort data were analyzed using bioinformatic tools for statistical analysis, protein–protein interactions, functional enrichment and pathway analyses and prognostic association analysis.ResultsWe found that LGI3 and its receptors, ADAM22 and ADAM23, were significantly downregulated in glioma tissues. Eleven upregulated genes and two downregulated genes in glioma tissues were found to be the previously reported LGI3-regulated genes. Protein–protein interaction network analysis showed that 85% of the LGI3-regulated and glioma-altered genes formed a cluster of interaction network. Functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed the association of these genes with hypoxia responses, p53 and Akt signaling and various cancer-related pathways including glioma. Analysis of expression microarray data of glioma cohorts demonstrated that low expression levels of LGI3, ADAM22 and ADAM23 were significantly associated with poor prognosis of glioma.ConclusionThese results propose that LGI3 and its receptors may play a prognostic role in glioma.

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.

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.

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 3β (GSK3β) 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 GSK3β inhibitor, abolished LGI3-induced cell migration. We tested β-catenin levels after LGI3 treatment because the Akt-GSK3β pathway regulates β-catenin accumulation, and β-catenin promotes cell migration. LGI3 treatment increased β-catenin protein and nuclear localization, whereas LY294002 prevented LGI3-induced focal adhesion kinase and Akt activation as well as β-catenin accumulation. Overall, these data suggest that LGI3 stimulates HaCaT cell migration following β-catenin accumulation through the Akt pathway.

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.

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 (50ng/ml) increased survival of HaCaT cells by 20% after UVB irradiation (150mJ/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.

LGI3 prevents UVB‐induced cell death of human keratinocytes

Leucine‐rich glioma inactivated 3 (LGI3), a member of LGI/epitempin (EPTP) family, is known to be mainly expressed in brain. However, the expression and physiological roles of LGI3 in skin cells remain unknown. In the present study, it was found for the first time that LGI3 is expressed in human skin. LGI3 was mostly expressed by normal human keratinocytes, although normal human fibroblasts and melanocytes also produced LGI3. Furthermore, ELISA and Western blot analysis showed that HaCaT human keratinocytes secreted LGI3 protein after exposure to UVB in a time‐ and dose‐dependent manner. We next investigated the possible role of LGI3 in keratinocytes. Our results showed that LGI3 protected HaCaT cells from UVB‐induced cell death. It was also found that LGI3 potently stimulates the phosphorylation of Akt, which is involved in the cell survival‐signaling cascade. Furthermore, LGI3 led to the phosphorylation of MDM2 and subsequent p53 degradation. Taken together, these data suggest that LGI3 may regulate p53 levels, and that keratinocyte‐derived LGI3 may act as a novel cytokine for skin homeostasis. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011‐0026587).

LGI3 increases human keratinocyte migration

We recently found that that leucine‐rich glioma inactivated 3 (LGI3) is expressed in human skin. However, the physiological roles of LGI3 in human keratinocytes remain unknown. In the present study, it was demonstrated that LGI3 acts as a stimulator for human HaCaT keratinocyte migration without affecting cell viability and proliferation. Western blot analysis showed that LGI3 induced FAK activation, Akt phosphorylation, and GSK‐3β phosphorylation in HaCaT cells. Further study demonstrated that LY294002, a selective PI3K inhibitor and LiCl, a selective GSK‐ 3β inhibitor abrogated the increased cell migration induced by LGI3. Next, we examined β‐catenin levels after LGI3 treatment, because the Akt‐GSK‐3β pathway is known to regulate accumulation of β‐catenin, which is an important mediator of cell migration. LGI3 treatment increased β‐catenin protein level and nuclear localization, whereas LY294002 prevented LGI3‐induced FAK and Akt activation as well as β‐catenin accumulation. Taken together, it is suggested that LGI3 stimulates HaCaT cell migration via the accumulation of β‐catenin through the Akt pathway. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011‐0026587).

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.

LGI3 interacts with flotillin-1 to mediate APP trafficking and exosome formation

We recently showed that leucine-rich glioma inactivated 3 (LGI3) mediates the internalization of beta-amyloid protein and transferrin, a well-known marker for clathrin-dependent endocytosis, in neural cells. These findings strongly suggest that LGI3 is involved in the endocytosis system in the brain; however, the precise function of LGI3 remains unclear. Here, we show that LGI3 interacts with flotillin-1 (Flo1), and RNA interference analysis shows that LGI3 stabilized Flo1, and Flo1 also stabilized LGI3 vice versa. Moreover, the downregulation of the LGI3/Flo1 complex altered beta-amyloid precursor protein trafficking directly to late endosomes and disrupted exosome formation, suggesting that LGI3 is involved not only in endocytosis but also in another intracellular transport system through binding with its co-factor such as Flo1.

Leucine-Rich Glioma Inactivated 3 Induces Neurite Outgrowth Through Akt and Focal Adhesion Kinase

Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein that belongs to LGI/epitempin family. LGI3 is highly expressed in brain in a transcriptionally and developmentally regulated manner. Here we found that LGI3 induced neurite outgrowth in Neuro-2a cells and dorsal root ganglia explants. LGI3 treatment or overexpression increased neurite outgrowth and knockdown of LGI3 by siRNA had opposite effect. LGI3 treatment increased phosphorylation of Akt and a 125-kDa protein. Immunoprecipitation identified the 125-kDa protein as focal adhesion kinase (FAK). LGI3 overexpression increased phospho-Akt, phospho-FAK and FAK protein. Inhibition of Akt activation by PI3 kinase inhibitor attenuated LGI3-induced FAK phosphorylation and neurite outgrowth. Taken together, we propose that LGI3 is a neuritogenic factor whose signaling pathway involves Akt-mediated FAK activation.

Leucine-rich glioma inactivated 3 associates with syntaxin 1

Leucine-rich glioma inactivated 3 (LGI3) is a member of LGI/epitempin (EPTP) family. The biological function of LGI3 and its association with disease are not known. We previously reported that mouse LGI3 was highly expressed in brain in a developmentally and transcriptionally regulated manner. In this study, we identified syntaxin 1, a SNARE component in exocytosis, as a candidate functional target of LGI3. Western blot analysis of mouse brain extract with LGI3 antibodies detected multiple protein forms (75-, 60-, 35- and 25-kDa). Proteomic analysis, pull-down and coimmunoprecipitation experiments identified syntaxin 1 as an LGI3-associated protein. LGI3 colocalized with syntaxin 1 in processes of cortical neurons with punctate synaptic pattern and was enriched in synaptosomal fraction. Coimmunoprecipitation showed that LGI3-syntaxin 1 complex did not contain other SNARE components, SNAP25 and VAMP2. Recombinant LGI3 attenuated Ca 2+-evoked glutamate release from digitonin-permeabilized synaptosomes and transfection of PC12 cells with LGI3 decreased K +-induced secretion of human growth hormone. Thus, LGI3 may play a regulatory role in neuronal exocytosis via its interaction with syntaxin 1.

Leucine-rich glioma inactivated 3 is involved in amyloid β peptide uptake by astrocytes and endocytosis itself

We earlier showed that leucine-rich glioma inactivated 3 (LGI3) colocalizes with amyloid beta peptide (Abeta) taken up by astrocytes both in vitro and in vivo, and that LGI3 accumulated with endocytosis-associated proteins in aged monkey brains. In this study, we confirmed that LGI3 localizes to the endocytic pathway and found that its accumulation is caused by endocytic perturbation. Most notably, RNA interference experiments demonstrated that the downregulation of LGI3 clearly inhibited Abeta uptake by cultured rat astrocytes, moreover, transferrin uptake by both astrocytes and neuronal cells. Together with our earlier findings, our results suggest that LGI3 is involved in Abeta uptake by astrocytes and even endocytosis itself.

Mouse LGI3 gene: Expression in brain and promoter analysis

Leucine-rich glioma inactivated 3 ( LGI3) is a member of LGI/epitempin family of which the first member, LGI1/epitempin, was shown to be mutated in glioma and autosomal dominant lateral temporal epilepsy. Similar to LGI1, LGI3 is expressed predominantly in brain and its function is unknown. In this study, we examined the expression of mouse LGI3 ( mLGI3) in adult and developing brain and analyzed the 5′-upstream transcriptional regulatory regions of mLGI3 gene. In situ hybridization showed that mLGI3 was expressed in widespread areas with selective regional variation in adult brain. In developing brain, mLGI3 mRNA was expressed at low level during embryo stages and markedly increased in broad areas after birth. Analysis of the 5′- and 3′-ends of mLGI3 mRNA identified a single transcription start site and two alternative 3′-ends. Luciferase reporter analysis using Neuro-2a cells and electrophoretic mobility shift assays identified a neuronal restrictive silencer element (NRSE; − 2573∼− 2553) and a phorbol ester-sensitive AP-2 element with repressor activity (− 44∼− 33) among multiple positive and negative regulatory regions. Since NRSE and AP-2 are implicated in neuron-specific gene expression and developmental regulation of many genes in brain, respectively, these results suggested that NRSE and AP-2 might play important roles in regulation of mLGI3 expression in brain.