Nrf2 In Keratinocytes Research Articles

Alteration of reactive oxygen species master transcription factor Nrf2 in keratinocytes exposed to monoclonal pathogenic antibody AK23 against desmoglein-3 in pemphigus vulgaris

Keratinocytes in mucosal and skin tissues maintain tissue integrity via desmosomes and desmoglein-3 (Dsg3). Pemphigus Vulgaris (PV) is a life-threatening autoimmune blistering disease characterized by autoantibodies against Dsg3, disrupting desmosomes. Nuclear factor erythroid 2–related factor 2 (Nrf2) regulates oxidative stress responses crucial for skin tissue protection. Although the pathogenesis of PV is known, the detailed molecular events remain unclear. This study investigates changes in Nrf2 expression in keratinocytes following pathogenic anti-Dsg3 antibody AK23 exposure, using dose- and time-dependent studies employing immunofluorescence analysis. N/TERT keratinocytes were cultured in keratinocytes serum-free medium and treated with AK23 at varying doses (5 µg/mL,40µg/mL,75µg/mL) and durations (2, 6, 24 h). Immunofluorescence staining was performed to assess the expression of Nrf2 and Dsg3. All fluorescent images were analyzed using ImageJ software. A dose-dependent increase in Dsg3 was noted following AK23 treatment, while Nrf2 expression and subcellular localization varied. Time-course analyses showed decreased Nrf2 at 24 h and increased Dsg3 levels. Early time-point (2 and 6 h) variations were evident in Nrf2 levels. This study highlights the impact of AK23 on Nrf2 expression, potentially disrupting Nrf2-mediated cytoprotection and implicating oxidative stress (ROS generation) in PV pathogenesis. Further investigation is necessary to validate the findings.

592-P: Epidermal Nrf2 Orchestrates Tissue Regeneration through Direction Regulation of Ccl2

Diabetic ulcers are costly chronic wounds and are the leading cause of lower-extremity amputations. Dysfunctional redox balance underlies a key part of diabetic wound repair pathology that results from misregulation of master antioxidant gene regulator Nrf2. While Nrf2 regulation is relevant for wound repair, how Nrf2 onsets a regenerative response is unclear. Using in-depth analyses, we uncover Nrf2 expression in keratinocytes to be vital for physiologic wound repair. In vivo spatio-temporal analysis shows deregulated nuclear translocation of Nrf2 in diabetic wound keratinocytes (WT: 96.97%±3.6%; Db: 22.50%±16.4%; p<.005), which correlates with reduced activation of many antioxidant gene targets (p<.005). To elucidate the role of Nrf2 in the epidermis, we utilized double transgenic mice to conditionally delete Nrf2 in keratinocytes (K14 CreER; Nrf2fl/fl; cKO) in vivo. Importantly, we find loss of epidermal Nrf2 results in a significant wound repair delay that reminisces delays observed in diabetic mice (WT: 16.0±0d; Db: 32±0d; cKO: 33±2d; p<.0005). RNA-seq of cKO wound keratinocytes reveals Nrf2 regulates epidermal release of extrinsic cues that affect non-autologous responses that are critical for regeneration, most notably immune cell guidance (p<.05). The magnitude of this signaling defect is shown through failure of cKO wounds to traffic monocyte/macrophages to the injury site (WT: 130.0±15.0cells/area; cKO 35.0±2.6cells/area; p<.05). This defect results from reduced expression of Ccl2 in cKO wounds, which we find to possess a Nrf2-binding motif that exhibits dynamic Nrf2 binding upon wounding. Induced expression of Nrf2 in primary keratinocytes results in Ccl2 upregulation, and its application is sufficient for restoring physiologic wound regeneration in cKO and diabetic wounds (p<.0001). We provide evidence describing an essential role of Nrf2 in regulating critical wound healing events and provide the basis for continued investigation on its therapeutic value. Disclosure A.P. Villarreal Ponce: None. M. Tiruneh-Worku: None. J. Lee: None. J.A. David: None. C. Guerrero-Juarez: None. K. Dammeyer: None. J. Kuhn: None. J. Kwong: None. D. Ceradini: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-16-ACE-08 to D.C.)

Nrf2 in keratinocytes protects against skin fibrosis via regulating epidermal lesion and inflammatory response

Nuclear factor-E2-related factor 2 (Nrf2) is a master transcription factor in antioxidant response, protecting against oxidative damage and various diseases. Previous studies suggest that Nrf2 is suppressed in fibrotic skin and Nrf2 agonists represent a therapeutic strategy, which is mainly attributed to Nrf2 function in fibroblasts. However, constitutive activation of Nrf2 may endow cells with proliferation and survival advantage, facilitating skin tumorigenesis. Non-invasive and mild modulation of Nrf2 via topical application may be helpful. Keratinocytes, which are essential for epidermal formation and function maintenance, have been shown to modulate differentiation of fibroblasts in different stages of fibrosis. In this respect, the role of Nrf2 in keratinocytes in skin fibrosis remains elusive. In the present study, bleomycin (BLM)-induced skin fibrosis model was applied to keratinocyte-specific Nrf2 knockout (Nrf2(K)-KO) mice generated with Keratin 14-Cre/loxp system. BLM treatment significantly suppressed Nrf2 expression in the epidermis. Nrf2 deficiency in keratinocytes exacerbated BLM-induced skin fibrosis according to dermal thickness, and immunostaining of collagen and α-SMA. One-dose BLM treatment led to the emergence of apoptotic cells in the epidermis and an elevated number of macrophages and neutrophils in the dermis, which was aggravated by Nrf2 deficiency, as indicated by TUNEL staining, and expression of F4/80 and Ly6G. In line with in vivo evidence, NRF2 silencing in HaCaT cells significantly decreased cell survival rate in response to BLM due to suppressed expression of antioxidative genes and increased intracellular levels of reactive oxygen species (ROS). The mRNA levels of chemokines and cytokines that are capable of recruiting macrophages and neutrophils, including Mcp-1, Il-6 and Il-8, were increased by Nrf2 deficiency in primary mouse keratinocytes. Moreover, bardoxolone methyl (CDDO-Me), a potent Nrf2 activator, ameliorated BLM-induced skin fibrosis after topical administration. These findings indicate that Nrf2 in keratinocytes protects against skin fibrosis via regulating cell resistance to apoptosis and expression of cytokines and chemokines. The restoration of Nrf2 through topical application might be a potential pharmacologic approach to combat skin fibrosis.

1312 Nrf2 activation enhances the healing of cutaneous wounds through the activation of hair follicle stem cells

The transcription factor Nrf2 is a key regulator of the cellular stress response through the regulation of antioxidant enzymes, cytoprotective proteins and various transporters. Strong genetic activation of Nrf2 in keratinocytes leads to a pilosebaceous phenotype, characterized by hyperplasia of the sebaceous glands and infundibula, hyperkeratosis, and seborrhea, implicating Nrf2 in several other key processes in the epidermis. Here we show that Nrf2 activation in keratinocytes promotes the proliferation and expansion of the junctional zone (JZ) and upper isthmus (UI) hair follicle stem cells, while bulge stem cells are only mildly affected. This was observed to be functionally important for wound repair, since Nrf2 activation also led to the faster closure of excisional wounds through the formation of a longer and larger wound epithelium. This however, was neither due to changes in proliferation or apoptosis of keratinocytes in the wound epithelium, nor their migration as measured in vitro. Instead, an increased number and proliferation of follicular JZ and UI stem cells were observed peripheral to the wound. An enhancement of wound healing in Nrf2 transgenic mice following tape stripping of the epidermis revealed a functional link between the Nrf2-mediated expansion of hair follicles stem cells and accelerated re-epithelialization. The effect of Nrf2 activation on JZ and UI stem cells resulted from the Nrf2-mediated up-regulation of the EGF family member Epigen and subsequent EGF receptor activation. These results suggest pharmacological Nrf2 activation as a promising approach for the enhancement of wound healing through expansion of hair follicle stem cell pools.

Nrf2 in keratinocytes modulates UVB-induced DNA damage and apoptosis in melanocytes through MAPK signaling

Responses of melanocytes (MC) to ultraviolet (UV) irradiation can be influenced by their neighbouring keratinocytes (KC). We investigated the role of Nrf2 in regulating paracrine effects of KC on UVB-induced MC responses through phosphorylation of MAPKs in association with oxidative stress in primary human MC cocultured with primary human KC using a transwell co-culture system and small-interfering RNA-mediated silencing of Nrf2 (siNrf2). The mechanisms by which Nrf2 modulated paracrine factors including α-melanocyte-stimulating hormone (α-MSH) and paracrine effects of KC on UVB-mediated apoptosis were also assessed. Our findings showed that co-culture of MC with siNrf2-transfected KC enhanced UVB-mediated cyclobutane pyrimidine dimer (CPD) formation, apoptosis and oxidant formation, together with phosphorylation of ERK, JNK and p38 in MC. Treatment of MC with conditioned medium (CM) from Nrf2-depleted KC also increased UVB-mediated MC damage, suggesting that KC modulated UVB-mediated MC responses via paracrine effects. Additionally, depletion of Nrf2 in KC suppressed UVB-induced α-MSH levels as early as 30min post-irradiation, although pretreatment with N-acetylcysteine (NAC) elevated its levels in CM from siNrf2-transfected KC. Furthermore, NAC reversed the effect of CM from Nrf2-depleted KC on UVB-induced apoptosis and inflammatory response in MC. Our study demonstrates for the first time that KC provided a rescue effect on UVB-mediated MC damage, although depletion of Nrf2 in KC reversed its protective effects on MC in a paracrine fashion in association with elevation of ROS levels and activation of MAPK pathways in MC. Nrf2 may indirectly regulate the paracrine effects of KC probably by affecting levels of the paracrine factor α-MSH via a ROS-dependent mechanism.

672 Palladium and platinum nanoparticles activate AHR and NRF2 in keratinocytes— Implications in vitiligo therapy

672 Palladium and platinum nanoparticles activate AHR and NRF2 in keratinocytes— Implications in vitiligo therapy

65 - Nrf2 in Keratinocytes Modulates UVB-Induced DNA Damage and Apoptosis in Melanocytes through ROS-Dependent Mechanism

Responses of melanocytes (MC) to ultraviolet (UV) irradiation can be regulated by their neighbouring keratinocytes (KC). Aims We investigated the role of Nrf2 in regulating paracrine effects of KC on UVB-induced MC responses through phosphorylation of MAPKs in association with oxidative stress in primary human MC co-cultured with primary human KC using a trans-well co-culture system and small-interfering RNA-mediated silencing of Nrf2 (siNrf2). The mechanisms by which Nrf2 modulated paracrine factors including α-melanocyte-stimulating hormone (α-MSH) and paracrine effects of KC on UVB-mediated apoptosis were also assessed. Results Co-culture of MC with siNrf2-transfected KC enhanced UVB-mediated cyclobutane pyrimidine dimer (CPD) formation, apoptosis and oxidant formation in correlation with phosphorylation of ERK, JNK and p38 in MC. MC damage induced by UVB was increased in MC treated with conditioned medium (CM) derived from Nrf2-depleted KC, suggesting that KC modulated UVB-mediated MC responses via paracrine effects. Additionally, at 30 min post-irradiation, depletion of Nrf2 in KC suppressed UVB-induced α-MSH levels, although pre-treatment with N-acetylcysteine (NAC) elevated its levels in CM from siNrf2-transfected KC. Furthermore, NAC reversed the effect of CM from Nrf2-depleted KC on UVB-induced apoptosis in MC. Conclusion Our study demonstrates for the first time that KC provided a rescue effect on UVB-mediated MC responses, although depletion of Nrf2 in KC reversed its protective effects on MC in a paracrine fashion in association with elevation of reactive oxygen species (ROS) levels and activation of MAPK pathways in MC. Nrf2 may indirectly regulate paracrine effect of KC probably by affecting levels of the paracrine factor α-MSH via a ROS-dependent mechanism.

P-17 - UVB-induced DNA damage and apoptosis in melanocytes: modulation by Nrf2 in keratinocytes

P-17 - UVB-induced DNA damage and apoptosis in melanocytes: modulation by Nrf2 in keratinocytes

Autocrine and Paracrine Regulation of Keratinocyte Proliferation through a Novel Nrf2-IL-36γ Pathway.

The Nrf2 transcription factor is well known for its cytoprotective functions through regulation of genes involved in the detoxification of reactive oxygen species or toxic compounds. Therefore, activation of Nrf2 is a promising strategy for the protection of tissues from various types of insults and for cancer prevention. However, recent studies revealed a proinflammatory activity of activated Nrf2 and a stimulating effect on epithelial cell proliferation, but the underlying mechanisms of action and the responsible target genes are largely unknown. Using a combination of gene expression profiling, chromatin immunoprecipitation, and targeted proteomics via selected reaction monitoring, we show that the gene encoding the proinflammatory cytokine IL-36γ is a novel direct target of Nrf2 in keratinocytes and hepatocytes in vitro and in vivo. As a consequence, upregulation of IL-36γ expression occurred upon genetic or pharmacological activation of Nrf2 in the epidermis and in the normal and regenerating liver. Functional in vitro studies demonstrate that IL-36γ directly stimulates proliferation of keratinocytes. In particular, it induces expression of keratinocyte mitogens in fibroblasts, suggesting that the Nrf2-IL-36γ axis promotes keratinocyte proliferation through a double paracrine loop. These results provide mechanistic insight into Nrf2 action in the control of inflammation and cell proliferation through regulation of a proinflammatory cytokine with a key function in various inflammatory diseases.

Nrf2 Activation Promotes Keratinocyte Survival during Early Skin Carcinogenesis via Metabolic Alterations.

Pharmacologic activation of the transcription factor NRF2 has been suggested to offer a strategy for cancer prevention. In this study, we present evidence from murine tumorigenesis experiments suggesting there may be limitations to this possibility, based on tumorigenic effects of Nrf2 in murine keratinocytes that have not been described previously. In this setting, Nrf2 expression conferred metabolic alterations in keratinocytes that were protumorigenic in nature, affecting enzymes involved in glutathione biosynthesis or in the oxidative pentose phosphate pathway and other NADPH-producing enzymes. Under stress conditions, coordinate increases in NADPH, purine, and glutathione levels promoted the survival of keratinocytes harboring oncogenic mutations, thereby promoting tumor development. The protumorigenic activity of Nrf2 in keratinocytes was particularly significant in a mouse model of skin tumorigenesis that did not rely upon chemical carcinogenesis. In exploring the clinical relevance of our findings, we confirm that NRF2 and protumorigenic NRF2 target genes were activated in some actinic keratoses, the major precancerous lesion in human skin. Overall, our results reveal an unexpected tumor-promoting activity of activated NRF2 during early phases of skin tumorigenesis.

Vitamin C Compound Mixtures Prevent Ozone-Induced Oxidative Damage in Human Keratinocytes as Initial Assessment of Pollution Protection.

IntroductionOne of the main functions of cutaneous tissues is to protect our body from the outdoor insults. Ozone (O3) is among the most toxic stressors to which we are continuously exposed and because of its critical location, the skin is one of the most susceptible tissues to the oxidative damaging effect of O3. O3 is not able to penetrate the skin, and although it is not a radical per se, the damage is mainly a consequence of its ability to induce oxidative stress via the formation of lipid peroxidation products.Aim of StudyIn this study we investigated the protective effect of defined “antioxidant” mixtures against O3 induced oxidative stress damage in human keratinocytes and understand their underlying mechanism of action.ResultsResults showed that the mixtures tested were able to protect human keratinocytes from O3-induced cytotoxicity, inhibition of cellular proliferation, decrease the formation of HNE protein adducts, ROS, and carbonyls levels. Furthermore, we have observed the decreased activation of the redox sensitive transcription factor NF-kB, which is involved in transcribing pro-inflammatory cytokines and therefore constitutes one of the main players associated with O3 induced skin inflammation. Cells exposed to O3 demonstrated a dose dependent increase in p65 subunit nuclear expression as a marker of NF-kB activation, while pre-treatment with the mixtures abolished NF-kB nuclear translocation. In addition, a significant activation of Nrf2 in keratinocytes treated with the mixtures was also observed.ConclusionOverall this study was able to demonstrate a protective effect of the tested compounds versus O3-induced cell damage in human keratinocytes. Pre-treatment with the tested compounds significantly reduced the oxidative damage induced by O3 exposure and this protective effect was correlated to the abolishment of NF-kB nuclear translocation, as well as activation of Nrf2 nuclear translocation activating the downstream defence enzymes involved in cellular detoxification process.

Activation of Nrf2 in keratinocytes causes chloracne (MADISH)‐like skin disease in mice

The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula. These abnormalities caused dilatation of infundibula, hair loss, and cyst development upon aging. Upregulation of epigen, secretory leukocyte peptidase inhibitor (Slpi), and small proline-rich protein 2d (Sprr2d) in hair follicles was identified as the likely cause of infundibular acanthosis, hyperkeratosis, and cyst formation. These alterations were highly reminiscent to the phenotype of chloracne/“metabolizing acquired dioxin-induced skin hamartomas” (MADISH) patients. Indeed, SLPI, SPRR2, and epigen were strongly expressed in cysts of MADISH patients and upregulated by dioxin in human keratinocytes in an NRF2-dependent manner. These results identify novel Nrf2 activities in the pilosebaceous unit and point to a role of NRF2 in MADISH pathogenesis.

Nrf2 links epidermal barrier function with antioxidant defense

The skin provides an efficient permeability barrier and protects from microbial invasion and oxidative stress. Here, we show that these essential functions are linked through the Nrf2 transcription factor. To test the hypothesis that activation of Nrf2 provides skin protection under stress conditions, we determined the consequences of pharmacological or genetic activation of Nrf2 in keratinocytes. Surprisingly, mice with enhanced Nrf2 activity in keratinocytes developed epidermal thickening, hyperkeratosis and inflammation resembling lamellar ichthyosis. This resulted from upregulation of the cornified envelope proteins small proline-rich proteins (Sprr) 2d and 2h and of secretory leukocyte peptidase inhibitor (Slpi), which we identified as novel Nrf2 targets in keratinocytes. Since Sprrs are potent scavengers of reactive oxygen species and since Slpi has antimicrobial activities, their upregulation contributes to Nrf2's protective function. However, it also caused corneocyte fragility and impaired desquamation, followed by alterations in the epidermal lipid barrier, inflammation and overexpression of mitogens that induced keratinocyte hyperproliferation. These results identify an unexpected role of Nrf2 in epidermal barrier function, which needs to be considered for pharmacological use of Nrf2 activators.

Electrophilic Chemicals but not UV Irradiation or Reactive Oxygen Species Activate Nrf2 in Keratinocytes In Vitro and In Vivo

The NF-E2-related factor 2 (Nrf2) transcription factor is a potent inducer of cytoprotective genes, which encode--among others--enzymes that detoxify reactive oxygen species (ROS). As we demonstrated a crucial role of Nrf2 in the prevention of skin carcinogenesis, it is of interest to identify Nrf2-activating factors in keratinocytes. For this purpose, keratinocytes from mice transgenic for an Nrf2-responsive reporter gene were analyzed. Electrophilic compounds activated the reporter in keratinocytes, and induced nuclear translocation of Nrf2 and the expression of known Nrf2 target genes. This is biologically relevant, as we show that Nrf2-mediated gene expression protects keratinocytes from the toxicity of these substances. By contrast, hydrogen peroxide, glucose oxidase, UVA, and UVB irradiation had no effect, although these treatments strongly increased the levels of intracellular ROS. To verify these results in vivo, transgenic reporter mice with and without functional Nrf2 alleles were topically treated with electrophilic chemicals or irradiated with UVB. Electrophiles but not UVB activated the reporter in an Nrf2-dependent manner. These results provide the basis for the identification of novel Nrf2 activators in keratinocytes with therapeutic potential for skin tumor prevention.