Promote Keratinocyte Differentiation Research Articles

Topical histone deacetylase inhibitor remetinostat improves IMQ-induced psoriatic dermatitis via suppressing dendritic cell maturation and keratinocyte differentiation and inflammation

Psoriasis is a chronic inflammatory skin disease characterized by excessive proliferation of keratinocytes and infiltration of immune cells. Although psoriasis has entered the era of biological treatment, there is still a need to explore more effective therapeutic targets and drugs due to the presence of resistance and adverse reactions to biologics. Remetinostat, an HDAC inhibitor, can maintain its potency within the skin with minimal systemic effects, making it a promising topical medication for treating psoriasis. But its effectiveness in treating psoriasis has not been evaluated. In this study, the topical application of remetinostat significantly improved psoriasiform inflammation in an imiquimod-induced mice model by inhibiting CD86 expression of CD11C+I-A/I-E+ dendritic cells (DCs) in the skin. Moreover, remetinostat could dampen the maturation and activation of bone marrow-derived DCs in vitro, as well as the expression of psoriasis-related inflammatory mediators by keratinocytes. In addition, remetinostat could promote keratinocyte differentiation without affecting its proliferation. Our findings demonstrate that remetinostat improves psoriasis by inhibiting the maturation and activation of DCs and the differentiation and inflammation of keratinocytes, which may facilitate the potential application of remetinostat in anti-psoriasis therapy.

Promoter Hypermethylation Downregulates MiR-125b-5p and MiR-199b-5p Targeting of ΔNp63, Resulting in PI3K/AKT/mTOR Pathway Activation and Keratinocyte Differentiation.

Normal keratinocyte differentiation is important for epidermal wound healing. ΔNp63 is a major gene regulating epidermal formation and differentiation. We identified miRNAs targeting ΔNp63 and studied the association between the miRNAs and DNA methylation in keratinocyte differentiation. This study aimed to explore the mechanisms regulating ΔNp63 expression during keratinocyte differentiation. Bioinformatics analysis was performed to screen the miRNAs targeting ΔNp63 and uncover potential pathway mechanisms. The differentiation model of keratinocytes was established by CaCl2 treatment. Furthermore, the effects of the miRNA transgenic technique on Δ Np63 and keratinocyte differentiation were studied. In addition, the RNA FISH experiment was conducted to detect the location of different miRNAs. A double luciferase reporter experiment was carried out to verify the potential bindings between the miRNAs and ΔNp63. A rescue experiment was also performed to assess the effects of different miRNAs targeting ΔNp63 on keratinocyte differentiation. We analyzed the methylation patterns of the promoter regions of miRNAs using keratinocytes treated with 5-Aza-2'-deoxycytidine. Finally, we designed a methylation rescue experiment to verify the effects of miRNA promoter methylation on keratinocyte differentiation. Bioinformatics analysis showed that the miR-125b-5p and miR-199b-5p binding to the ΔNp63 3'UTR region decreased during skin development. Moreover, such binding may downregulate the PI3K/AKT/mTOR pathway. The expression levels of CK10, Inv, TG1, ΔNp63, and PI3K/AKT/mTOR were all significantly increased during keratinocyte differentiation. Both miR- 125b-5p and miR-199b-5p were localized in the cytoplasm. Luciferase assay results showed that both miR-125b-5p and miR-199b-5p can bind to the 3'UTR region of ΔNp63. Overexpression of ΔNp63 can significantly counteract the inhibitory effect of miRNA mimics on keratinocyte differentiation. Moreover, the promoter regions of both miR-125b-5p and miR-199b-5p had methylation sites, and the methylation levels in those promoter regions were significantly increased during keratinocyte differentiation. 5-Aza-2'-Deoxycytidine treatment increased the expression of miR- 125b-5p and miR-199b-5p and inhibited the differentiation of keratinocytes. Finally, miRNA inhibitors reversed the inhibitory effects of 5-Aza-2'-deoxycytidine on keratinocyte differentiation. Promoter hypermethylation in miR-125b-5p and miR-199b-5p seem to promote keratinocyte differentiation via upregulation of ΔNp63 expression and the activation of the PI3K/AKT/mTOR pathway. The findings of this study are helpful for future research on skin development and clinical scar-free healing.

MEK inhibition induces expression of differentiation marker Keratin 10 in human keratinocytes

BRAF mutant metastatic melanoma was regularly treated in the past with a BRAF inhibitor (BRAFi) alone or in combination with inhibitors of the mitogen-activated protein kinase kinase (MEKi), which is still a common treatment. This combination therapy strongly reduced the occurrence of keratoacanthomas and squamous cell carcinoma, which was frequently seen when BRAFi was used as monotherapy. Here we addressed the question whether MEK inhibition counteracts squamous cell carcinoma development in part by promoting keratinocyte differentiation. Exposure of human immortalized keratinocytes to different concentrations of MEKi revealed a significant increase in the expression of differentiation-associated keratins K10 and K1 as determined by qRT-PCR and immunofluorescence staining. Taken together, the present study suggests that in a combined treatment of melanoma with BRAFi/MEKi, MEKi reduces the incidence of squamous cell carcinomas by promoting keratinocyte differentiation under combined BRAFi/MEKi treatment in melanoma. This might open further treatment perspectives for skin cancer treatment.

Retromer Chaperones: Potential Therapeutics for Treatment of Skin Disease?

Retromer Chaperones: Potential Therapeutics for Treatment of Skin Disease?

Dual targeting of mTOR/IL-17A and autophagy by fisetin alleviates psoriasis-like skin inflammation.

Psoriasis is a chronic autoimmune inflammatory skin disorder characterized by epidermal hyperplasia and aberrant immune response. In addition to aberrant cytokine production, psoriasis is associated with activation of the Akt/mTOR pathway. mTOR/S6K1 regulates T-lymphocyte activation and migration, keratinocytes proliferation and is upregulated in psoriatic lesions. Several drugs that target Th1/Th17 cytokines or their receptors have been approved for treating psoriasis in humans with variable results necessitating improved therapies. Fisetin, a natural dietary polyphenol with anti-oxidant and anti-proliferative properties, covalently binds mTOR/S6K1. The effects of fisetin on psoriasis and its underlying mechanisms have not been clearly defined. Here, we evaluated the immunomodulatory effects of fisetin on Th1/Th17-cytokine-activated adult human epidermal keratinocytes (HEKa) and anti-CD3/CD28-stimulated inflammatory CD4+ T cells and compared these activities with those of rapamycin (an mTOR inhibitor). Transcriptomic analysis of HEKa revealed 12,713 differentially expressed genes (DEGs) in the fisetin-treated group compared to 7,374 DEGs in the rapamycin-treated group, both individually compared to a cytokine treated group. Gene ontology analysis revealed enriched functional groups related to PI3K/Akt/mTOR signaling pathways, psoriasis, and epidermal development. Using in silico molecular modeling, we observed a high binding affinity of fisetin to IL-17A. In vitro, fisetin significantly inhibited mTOR activity, increased the expression of autophagy markers LC3A/B and Atg5 in HEKa cells and suppressed the secretion of IL-17A by activated CD4+ T lymphocytes or T lymphocytes co-cultured with HEKa. Topical administration of fisetin in an imiquimod (IMQ)-induced mouse psoriasis model exhibited a better effect than rapamycin in reducing psoriasis-like inflammation and Akt/mTOR phosphorylation and promoting keratinocyte differentiation and autophagy in mice skin lesions. Fisetin also significantly inhibited T-lymphocytes and F4/80+ macrophage infiltration into skin. We conclude that fisetin potently inhibits IL-17A and the Akt/mTOR pathway and promotes keratinocyte differentiation and autophagy to alleviate IMQ-induced psoriasis-like disease in mice. Altogether, our findings suggest fisetin as a potential treatment for psoriasis and possibly other inflammatory skin diseases.

Involvement of hypoxia-inducible factor activity in inevitable air-exposure treatment upon differentiation in a three-dimensional keratinocyte culture.

Formation of the human skin epidermis can be reproduced by a three-dimensional (3D) keratinocyte culture system, in which air-exposure is inevitable upon initiation of differentiation. In the continuous submerged culture without air-exposure, even with a differentiation-compatible medium, several keratinocyte-specific proteins were not induced resulting in the formation of aberrant epidermal layers. To clarify the mechanism by whichair-exposure promotes keratinocyte differentiation, we performed a comparative analysis on biological properties between submerged and air-liquid interphase culture systems. By transcriptomic analysis, hypoxia-inducible factor (HIF)-related genes appeared to significantly change in these cultured cells. In submerged culture, the transcriptional activity of HIF on its canonical response element was enhanced, while air-exposure treatment drastically reduced the transcriptional activity despite the high HIF protein level. Regulating HIF activity through reagents and genetic manipulation revealed that the reduced but retained HIF-transcriptional activity was essentially involved in differentiation. Furthermore, we showed, for the first time, that artificial supplementation of oxygen in the submerged culture system could restore keratinocyte differentiation as observed in the air-exposed culture. Thus, we mechanistically evaluated how HIF regulates the air-exposure-dependent differentiation of keratinocytes in a 3D culture system.

434 Molecular events in the epidermis upon aryl hydrocarbon receptor targeting: AHR-TFAP2A axis drives epidermal keratinocyte differentiation

The aryl hydrocarbon receptor (AHR) is an environmental sensor and ligand-activated transcription factor that is involved in epithelial homeostasis and barrier development of the skin. Through genome-wide transcriptomic and epigenomic analyses of human primary keratinocytes upon AHR activation using TCDD we identified early and late AHR responsive genes. The early responsive genes were enriched for canonical transcription factors known to promote keratinocyte differentiation, barrier development, and host defense, such as Transcription Factor AP-2α (TFAP2A).

428 RORα promotes keratinocyte differentiation by reducing SOX9 stability

Retinoic acid receptor-related orphan receptors α (RORα) is a transcription factor essential for keratinocyte differentiation. The transcriptional network of RORα and how it regulates various keratinocyte functions remain understudied. We have identified Sex Determining Region Y-Box9 (SOX9) as a critical downstream target gene of RORα in human keratinocytes. SOX9 is an epithelial-to-mesenchymal transition (EMT)-inducing transcription factor involved in embryonic development and cancer metastasis.

Natural Compounds Tapinarof and Galactomyces Ferment Filtrate Downregulate IL-33 Expression via the AHR/IL-37 Axis in Human Keratinocytes.

Interleukin (IL)-37 suppresses systemic and local inflammation. It is expressed in the epidermis, the external layer of the skin, and is decreased in inflammatory skin diseases including atopic dermatitis (AD) and psoriasis. Therefore, an agent applied topically on the skin that can increase IL-37 could be promising for treating AD and psoriasis; however, the mechanism regulating IL-37 remains largely unknown. Given that IL-37 expression is induced in differentiated keratinocytes, a major component of the epidermis, and that activation of aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, promotes keratinocyte differentiation, we hypothesized that AHR might be involved in the IL-37 expression in human keratinocytes. We analyzed normal epidermal human keratinocytes (NHEKs) treated with tapinarof and Galactomyces ferment filtrate (GFF), which are potent AHR modulators. We found that tapinarof and GFF upregulated IL-37 in NHEKs, which was canceled by the knockdown of AHR using siRNA transfection, indicating that AHR mediates IL-37 expression in NHEKs. Furthermore, we found that the knockdown of IL-37 resulted in the upregulation of IL-33, an alarmin cytokine with crucial roles in the pathogenesis of AD and psoriasis. These findings suggest that IL-37 negatively regulates IL-33 expression in NHEKs. Finally, we examined whether tapinarof and GFF treatment modulates IL-33 expression in NHEKs. Such treatment inhibited IL-33 expression, which was partially reversed by the knockdown of either AHR or IL-37. Taken together, our findings provide the first evidence that tapinarof and GFF could have potential to prevent IL-33-overexpressing disorders such as AD and psoriasis via the AHR/IL-37 axis.

Shotokuseki Extract Promotes Keratinocyte Differentiation Even at a Low Calcium Concentration

The switch between keratinocyte proliferation and differentiation is regulated by extracellular calcium levels, requiring high concentrations (>1 mol/L) of extracellular calcium to induce differentiation. The Shotokuseki extract (SE) contains various ions such as calcium, but its effect on keratinocytes is unknown. This study focused on calcium-induced differentiation of keratinocytes and investigated the effects of simultaneous application of calcium and other ions on keratinocyte differentiation. The expression of differentiation markers increased when SE was added to a keratinocyte culture but not when only calcium was added at the same concentration present in SE. The calcium concentration in SE was found to be too low (0.01 mol/L) to induce differentiation of keratinocytes. In addition, the application of SE increased intracellular calcium concentration compared with calcium solution alone. Therefore, the induction of keratinocyte differentiation by SE is not calcium-dependent, or SE may alter the calcium sensitivity of keratinocytes. In our study, we found that simultaneous application of multiple ions and/or the application of trace ions may alter calcium sensitivity and the epidermal cell response. The function of ion transporters associated with these ions and the response of cells to ions depends largely on the balance among various ions and the function of trace ions.

Effect of galactosylceramide on stratum corneum intercellular lipid synthesis in a three-dimensional cultured human epidermis model

Intercellular lipids in the stratum corneum (SC), such as ceramide (CER), free fatty acid (FFA), and cholesterol (CHOL), contribute to the formation of stable lamellar structures in the SC, making them important for skin barrier function. β-Galactosylceramide (GalCer) is a glycosphingolipid that is used in some cosmetics and quasi-drugs in anticipation of a moisturizing effect. GalCer promotes keratinocyte differentiation and increases CER production by increasing β-glucocerebrosidase (β-GCase) activity. However, few reports have described the mechanism of these effects, and detailed studies on the role of GalCer in intercellular lipid production in the SC have not been conducted. This study investigated the effect of GalCer on the metabolism and production of intercellular lipids in the SC in a three-dimensional cultured epidermis model. After reacting GalCer with a homogenate solution of three-dimensional cultured epidermis, GalCer was hardly metabolized. Treatment of the three-dimensional cultured epidermis with GalCer increased the expression of genes involved in the β-GCase metabolic pathway and promoted CER production. In addition, GalCer treatment reduced the expression of FFA metabolism-related genes as well as palmitic acid levels. In addition, transepidermal water loss, which is a barrier index, was reduced by GalCer treatment. These findings suggested that GalCer, which is hardly metabolized, affects the production of intercellular lipids in the SC and improves skin barrier function.

Glycerol Improves Skin Lesion Development in the Imiquimod Mouse Model of Psoriasis: Experimental Confirmation of Anecdotal Reports from Patients with Psoriasis.

Glycerol is used in many skin care products because it improves skin function. Anecdotal reports by patients on the National Psoriasis Foundation website also suggest that glycerol may be helpful for the treatment of psoriasis, although to date no experimental data confirm this idea. Glycerol entry into epidermal keratinocytes is facilitated by aquaglyceroporins like aquaporin-3 (AQP3), and its conversion to phosphatidylglycerol, a lipid messenger that promotes keratinocyte differentiation, requires the lipid-metabolizing enzyme phospholipase-D2 (PLD2). To evaluate whether glycerol inhibits inflammation and psoriasiform lesion development in the imiquimod (IMQ)-induced mouse model of psoriasis, glycerol’s effect on psoriasiform skin lesions was determined in IMQ-treated wild-type and PLD2 knockout mice, with glycerol provided either in drinking water or applied topically. Psoriasis area and severity index, ear thickness and ear biopsy weight, epidermal thickness, and inflammatory markers were quantified. Topical and oral glycerol ameliorated psoriasiform lesion development in wild-type mice. Topical glycerol appeared to act as an emollient to induce beneficial effects, since even in PLD2 knockout mice topical glycerol application improved skin lesions. In contrast, the beneficial effects of oral glycerol required PLD2, with no improvement in psoriasiform lesions observed in PLD2 knockout mice. Our findings suggest that the ability of oral glycerol to improve psoriasiform lesions requires its PLD2-mediated conversion to phosphatidylglycerol, consistent with our previous report that phosphatidylglycerol itself improves psoriasiform lesions in this model. Our data also support anecdotal evidence that glycerol can ameliorate psoriasis symptoms and therefore might be a useful therapy alone or in conjunction with other treatments.

054 Phenformin promotes keratinocyte differentiation via the calcineurin/NFAT pathway

Phenformin is a drug in the biguanide classify that was previously used to treat type 2 diabetes. We have reported the anti-tumor activities of phenformin to enhance the efficacy of BRAF-MEK-ERK pathway inhibition and to inhibit myeloid-derived suppressor cells in various melanoma models. Here we demonstrate that phenformin suppresses tumor growth and promotes keratinocyte differentiation in the DMBA/TPA two stage skin carcinogenesis mouse model. Moreover, phenformin enhances the suspension-induced differentiation of mouse and human keratinocytes. Mechanistically, phenformin induces the nuclear translocation of NFATc1 in keratinocytes in an AMPK-dependent manner. Pharmacological or genetic inhibition of calcineurin/NFAT signaling reverses the effects of phenformin on keratinocyte differentiation. Taken together, our study reveals an anti-tumor activity of phenformin to promote keratinocyte differentiation that warrants future translational efforts to repurpose phenformin for the treatment of cutaneous squamous cell carcinomas.

The long noncoding RNA MALAT1 suppresses miR-211 to confer protection from ultraviolet-mediated DNA damage in vitiligo epidermis by upregulating sirtuin 1.

The absence of melanocytes poses a challenge for long-term tissue homeostasis in vitiligo. Surprisingly, while individuals with Fitzpatrick phototypes I-II (low melanin content) have a higher incidence of melanoma and nonmelanoma skin cancer, people with vitiligo are at a decreased risk for the same. To understand the molecular mechanisms that protect vitiligo skin from ultraviolet (UV)-induced DNA damage by (i) characterizing differentially expressed microRNAs in lesional vs. nonlesional epidermis and (ii) identifying their upstream regulators and downstream gene targets. Genome-wide microRNA profiling of nonlesional and lesional epidermis was performed on five individuals with stable nonsegmental vitiligo using next-generation RNA sequencing. The relevance of the upstream regulator and downstream target gene of the most differentially expressed microRNA was studied. Our study found sirtuin1 (SIRT1), an NAD-dependent deacetylase, to be a direct target of miR-211 - the most significantly downregulated microRNA in lesional epidermis. Inhibition of SIRT1 with EX-527 downregulated keratin 10 and involucrin, suggesting that SIRT1 promotes keratinocyte differentiation. Overexpression of miR-211 mimic led to a significant increase in γ-H2AX positivity and cyclobutane pyrimidine dimer (CPD) formation, hallmarks of UVB-mediated DNA damage. These effects could be ameliorated by the addition of resveratrol, a SIRT1 activator. Furthermore, a long noncoding RNA, MALAT1, was identified as a negative upstream regulator of miR-211. Overexpression of MALAT1 resulted in increased expression of SIRT1 and a concomitant removal of UVB-induced CPDs in primary keratinocytes. These findings establish a novel MALAT1-miR-211-SIRT1 signalling axis that potentially confers protection to the 'amelanotic' keratinocytes in vitiligo.

Phenformin: AMP(K)ed for Potential Repurposing

The results in the article by Zhou etal. (2020) demonstrate that the antidiabetic drug phenformin inhibits skin tumor growth and promotes keratinocyte differentiation, and an underlying mechanism is also defined. In this commentary, additional potential mechanisms through which phenformin may exert its antitumorigenic effect are described. Thus, the proposed repurposing of phenformin to treat skin cancer has merit.

Phenformin Promotes Keratinocyte Differentiation via the Calcineurin/NFAT Pathway

Phenformin is a drug in the biguanide class that was previously used to treat type 2 diabetes. We have reported the antitumor activities of phenformin to enhance the efficacy of BRAF-MAPK kinase-extracellular signal-regulated kinase pathway inhibition and to inhibit myeloid-derived suppressor cells in various melanoma models. Here we demonstrate that phenformin suppresses tumor growth and promotes keratinocyte differentiation in the 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate two-stage skin carcinogenesis mouse model. Moreover, phenformin enhances the suspension-induced differentiation of mouse and human keratinocytes. Mechanistically, phenformin induces the nuclear translocation of NFATc1 in keratinocytes in an AMPK-dependent manner. Pharmacologic or genetic inhibition of calcineurin and NFAT signaling reverses the effects of phenformin on keratinocyte differentiation. Taken together, our study reveals an antitumor activity of phenformin to promote keratinocyte differentiation that warrants future translational efforts to repurpose phenformin for the treatment of cutaneous squamous cell carcinomas.

Desmoglein 1 Regulates Invadopodia by Suppressing EGFR/Erk Signaling in an Erbin-Dependent Manner.

Loss of the desmosomal cell-cell adhesion molecule, Desmoglein 1 (Dsg1), has been reported as an indicator of poor prognosis in head and neck squamous cell carcinomas (HNSCC) overexpressing epidermal growth factor receptor (EGFR). It has been well established that EGFR signaling promotes the formation of invadopodia, actin-based protrusions formed by cancer cells to facilitate invasion and metastasis, by activating pathways leading to actin polymerization and ultimately matrix degradation. We previously showed that Dsg1 downregulates EGFR/Erk signaling by interacting with the ErbB2-binding protein Erbin (ErbB2 Interacting Protein) to promote keratinocyte differentiation. Here, we provide evidence that restoring Dsg1 expression in cells derived from HNSCC suppresses invasion by decreasing the number of invadopodia and matrix degradation. Moreover, Dsg1 requires Erbin to downregulate EGFR/Erk signaling and to fully suppress invadopodia formation. Our findings indicate a novel role for Dsg1 in the regulation of invadopodia signaling and provide potential new targets for development of therapies to prevent invadopodia formation and therefore cancer invasion and metastasis. IMPLICATIONS: Our work exposes a new pathway by which a desmosomal cadherin called Dsg1, which is lost early in head and neck cancer progression, suppresses cancer cell invadopodia formation by scaffolding ErbB2 Interacting Protein and consequent attenuation of EGF/Erk signaling.

The Type IV Collagenase Inhibitor, BiPS, Alters Keratin Expression in Sulfur Mustard Exposed Mouse Skin

Sulfur mustard (SM) is a potent chemical vesicant targeting many tissues including skin. The SM induced cutaneous toxicity is associated with edema, epidermal erosions, inflammation, and prolonged wound repair. Earlier studies have shown that overexpression of matrix metalloproteinase 9 (MMP9) degrades extracellular matrix proteins and leads to the separation of epidermis from dermis in skin exposed to SM. We have found that MMP9, a type IV collagenase, disrupts collagen IV (Col IV) a major component of the basement membrane (BM) zone in SM induced blisters. Col IV in the BM network has recently been reported to be a regulator of epidermal differentiation and keratin expression during wound repair. The type IV collagen (Col IV) BM network may regulate epidermal differentiation and keratin expression during development and wound repair. In this present study, we evaluated the MMP9 inhibitor BiPS [N‐hydroxy‐3‐phenyl‐2‐(4‐phenylbenzenesulfonamido) propanamide] in an epidermal wound healing model following SM exposure. Time course (24, 72, and 168 hrs post‐SM exposure) and compound evaluation studies (BiPS as a single pretreatment) were performed in a mouse ear vesicant model (MEVM) using histopathological changes, degree of hyperplasia and edema, and expression of type IV collagen, MMP9, keratin 10 (K10, differentiation marker), and keratin 6 (K6, wound‐inducible marker) for comparison. MMP9 mRNA and protein expression increased with time. Dual immunofluorescence studies using Col IV α1 and MMP9 antibodies showed BM zone breakdown of Col IV and apparent MMP9 expression in the epidermis and the adjacent dermal matrix of the SM injured skin. Dual immunofluorescent studies using K6 and K10 showed strong K6 and weak K10 expression in the hyperplastic epidermis. In the compound evaluation study, the BiPS pretreatment group demonstrated significant reduction in edema with 25% reduction of epidermal thickness, and improvement in overall tissue structure by 72 hr post‐SM exposure. mRNA expression of MMP9 was significantly downregulated in the BiPS treated group by 168 hr post exposure. Immunofluorescent studies showed that significant reduction of MMP9 expression in the skin and a continuous expression pattern of Col IV; similar to the unexposed control skin. Dual immunofluorescent studies show reduced K6 and increased K10 expression in hyperplastic epidermis when compared to SM treated samples. These results suggest that BiPS treatment may effectively improve BM integrity by the inhibition of MMP9 in SM exposed skin. This may in turn modulate the epidermal keratin expression and promote keratinocyte differentiation for wound repair. Targeting MMP9 for the restoration of BM may be an effective strategy for countering SM‐induced cutaneous injury.Support or Funding InformationGrant funding supported by ES005022, T32ES007148, and NIAMS U54AR055073.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Epidermal Growth Factor Receptor neddylation is regulated by a desmosomal-COP9 (Constitutive Photomorphogenesis 9) signalosome complex.

Cell junctions are scaffolds that integrate mechanical and chemical signaling. We previously showed that a desmosomal cadherin promotes keratinocyte differentiation in an adhesion-independent manner by dampening Epidermal Growth Factor Receptor (EGFR) activity. Here we identify a potential mechanism by which desmosomes assist the de-neddylating COP9 signalosome (CSN) in attenuating EGFR through an association between the Cops3 subunit of the CSN and desmosomal components, Desmoglein1 (Dsg1) and Desmoplakin (Dp), to promote epidermal differentiation. Silencing CSN or desmosome components shifts the balance of EGFR modifications from ubiquitination to neddylation, inhibiting EGFR dynamics in response to an acute ligand stimulus. A reciprocal relationship between loss of Dsg1 and neddylated EGFR was observed in a carcinoma model, consistent with a role in sustaining EGFR activity during tumor progression. Identification of this previously unrecognized function of the CSN in regulating EGFR neddylation has broad-reaching implications for understanding how homeostasis is achieved in regenerating epithelia.