Immortalized Keratinocyte Cell Line Research Articles

Angiotensin (1-7) Inhibits Transforming Growth Factor-Β1-Induced Epithelial-Mesenchymal Transition of Human Keratinocyte Hacat Cells in vitro.

Angiotensin (1-7) (Ang-(1-7)) is an emerging component of the renin-angiotensin system (RAS) with effective anti-fibrosis properties and has been shown to interfere with epithelial-mesenchymal transition (EMT) by numerous studies. In recent years, EMT has been proposed as a new therapeutic target for skin fibrotic diseases such as keloids. However, the effect of Ang-(1-7) on EMT in skin is still unclear. Hence, the purpose of this study was to explore the effect of Ang-(1-7) on Transforming growth factor-β1(TGF-β1)-induced EMT of human immortalized keratinocytes HaCaT in vitro. The study involved the use of the human immortalized keratinocyte cell line (HaCaT). The cells were cultured in high-glucose DMEM medium with 10% fetal bovine serum and 1% penicillin-streptomycin. Four groups were created for experimentation: control group (Group C), TGF-β1-treated group (Group T), Ang-(1-7)-treated group (Group A), and a group treated with both TGF-β1 and Ang-(1-7) (Group A + T). Various assays were conducted, including a cell proliferation assay using CCK-8 solution, a scratch wound healing assay to evaluate cell migration, and Western blotting to detect protein expressions related to cell characteristics. Additionally, quantitative real-time polymerase chain reaction (PCR) was performed to analyze epithelial-mesenchymal transition (EMT) related gene expression levels. The study aimed to investigate the effects of TGF-β1 and Ang-(1-7) on HaCaT cells. We found that Ang-(1-7) not only reduced the migration of HaCaT cells induced by TGF-β1 in vitro but also reduced the expression of α-SMA and vimentin, and restored the protein expression of E-cadherin and claudin-1. Mechanistically, Ang-(1-7) inhibits the phosphorylation levels of Smad2 and Smad3 in the TGF-β1 canonical pathway, and suppresses the expression of EMT-related transcription factors (EMT-TFs) such as SNAI2, TWIST1, and ZEB1. Taken together, our findings suggest that Ang-(1-7) inhibits TGF-β1-induced EMT in HaCaT cells in vitro by disrupting the TGF-β1-Smad canonical signaling pathway. These results may be helpful in the treatment of EMT in skin fibrotic diseases such as keloids.

Adipose mesenchymal stem cell-derived exosomes promote skin wound healing in diabetic mice by regulating epidermal autophagy.

Adipose mesenchymal stem cell-derived exosomes (ADSC-Exos) have great potential in the field of tissue repair and regenerative medicine, particularly in cases of refractory diabetic wounds. Interestingly, autophagy plays a role in wound healing, and recent research has demonstrated that exosomes are closely associated with intracellular autophagy in biogenesis and molecular signaling mechanisms. Therefore, this study aimed to investigate whether ADSC-Exos promote the repair of diabetic wounds by regulating autophagy to provide a new method and theoretical basis for the treatment of diabetic wounds. Western blot analysis and autophagy double-labelled adenovirus were used to monitor changes in autophagy flow in human immortalized keratinocyte cell line (HaCaT) cells. ADSC-Exos were generated from ADSC supernatants via ultracentrifugation. The effectiveness of ADSC-Exos on HaCaT cells was assessed using a live-cell imaging system, cell counting kit-8 and cell scratch assays. The cells were treated with the autophagy inhibitor bafilomycin A1 to evaluate the effects of autophagy on cell function. The recovery of diabetic wounds after ADSC-Exo treatment was determined by calculating the healing rates and performing histological analysis. High-throughput transcriptome sequencing was used to analyze changes in mRNA expression after the treatment of HaCaT cells with ADSC-Exos. ADSC-Exos activated autophagy in HaCaT cells, which was inhibited by high glucose levels, and potentiated their cellular functions. Moreover, ADSC-Exos in combination with the autophagy inhibitor bafilomycin A1 showed that autophagy defects further impaired the biological function of epidermal cells under high-glucose conditions and partially weakened the healing effect of ADSC-Exos. Using a diabetes wound model, we found that ADSC-Exos promoted skin wound healing in diabetic mice, as evidenced by increased epidermal autophagy and rapid re-epithelialization. Finally, sequencing results showed that increased expression of autophagy-related genes nicotinamide phosphoribosyltransferase (NAMPT), CD46, vesicle-associated membrane protein 7 (VAMP7), VAMP3 and eukaryotic translation initiation factor 2 subunit alpha (EIF2S1) may contribute to the underlying mechanism of ADSC-Exo action. This study elucidated the molecular mechanism through which ADCS-Exos regulate autophagy in skin epithelial cells, thereby providing a new theoretical basis for the treatment and repair of skin epithelial damage by ADSC-Exos.

Different immortalized keratinocyte cell lines display distinct capabilities to differentiate and reconstitute an epidermis invitro.

Dermatological research relies on the availability of suitable models that most accurately reflect the invivo situation. Primary keratinocytes obtained from skin reduction surgeries are not only limited by availability but have a short lifespan and show donor-specific variations, which hamper the understanding of general mechanisms. The spontaneously immortalized keratinocyte cell line HaCaT displays chromosomal aberrations and is known to differentiate in an abnormal manner. To overcome these issues, we validated different engineered immortalized cell lines created from primary human keratinocytes (NHK) as model systems to study epidermal function. Cell lines either immortalized by the expression of SV40 large T antigen and hTERT (NHK-SV/TERT) or by transduction with HPV E6/E7 (NHK-E6/E7) were analysed for their growth and differentiation behaviour using 2D and 3D culture systems and compared to primary keratinocytes. Both cell lines displayed a robust proliferative behaviour but were still sensitive to contact inhibition. NHK-E6/E7 could be driven into differentiation by Ca2+ switch, while NHK-SV/TERT needed withdrawal from any proliferative signal to initiate a delayed onset of differentiation. In 3D epidermal models both cell lines were able to reconstitute a stratified epidermis and functional epidermal barrier. However, only NHK-E6/E7 showed a degree of epidermal maturation and stratification that was comparable to primary keratinocytes.

CRISPR/Cas9-Mediated Generation of COL7A1-Deficient Keratinocyte Model of Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin fragility and ultimately lethal blistering disease caused by mutations in the COL7A1 gene which is responsible for coding type VII collagen. Investigating the pathological mechanisms and novel candidate therapies for RDEB could be fostered by new cellular models. The aim of this study was to employ CRISPR/Cas9 technology in the development of immortalized COL7A1-deficient keratinocyte cell lines intended for application as a cellular model for RDEB in ex vivo studies. In this experimental study, we used transient transfection to express COL7A1 -targeting guide RNA (gRNA) and Cas9 in HEK001 immortalized keratinocyte cell line followed by enrichment with fluorescent-activated cell sorting (FACS) via GFP expressing cells (GFP+ HEK001). Homogenous single-cell clones were then isolated, genotyped, and evaluated for type VII collagen expression. We performed a scratch assay to confirm the functional effect of COL7A1 knockout. We achieved 46.1% (P<0.001) efficiency of in/del induction in the enriched transfected cell population. Except for 4% of single nucleotide insertions, the remaining in/dels were deletions of different sizes. Out of nine single expanded clones, two homozygous and two heterozygous COL7A1-deficient cell lines were obtained with defined mutation sequences. No off-target effect was detected in the knockout cell lines. Immunostaining and western blot analysis showed lack of type VII collagen (COL7A1) protein expression in these cell lines. We also showed that COL7A1-deficient cells had higher motility compared to their wild-type counterparts. We reported the first isogenic immortalized COL7A1-deficient keratinocyte lines that provide a useful cell culture model to investigate aspects of RDEB biology and potential therapeutic options.

Human epidermis organotypic cultures, a reproducible system recapitulating the epidermis in vitro.

The translatability of research is highly dependent on models that recapitulate human tissues and organs. Here, we describe a procedure for the generation of human epidermis organotypic cultures (HEOCs) from primary keratinocytes isolated from foreskin and adult skin as well as from an immortalized keratinocyte cell line (KerTr). We tested several media conditions to develop a defined HEOC growing and expansion media. We characterized the HEOCs and show that in optimal culture conditions they express the proliferation marker Ki67, the basement membrane protein collagen 17 (col17) and the epidermal differentiation markers keratin 15 (K15), keratin 14 (K14), keratin 5 (K5), keratin 10 (K10), keratin 1 (K1), transglutaminase 1 (TGM1), transglutaminase 3 (TGM3) and filaggrin (FLG). Thus, they recapitulate the human epidermis and are stratified from the basal layer to the stratum corneum. These HEOC can be generated reproducibly on a large scale, making it an invaluable model for screening therapeutic compounds and also for the study of pathologies affecting the epidermis.

Ethylhexadecyldimethylammonium bromide, a quaternary ammonium compound, controls inflammatory response through NRF2 pathway in a human immortalized keratinocyte cell line.

Many everyday products contain quaternary ammonium compounds (QAC) and some of them are known to be skin irritants such as benzalkonium chloride. Others, such as didecyldimethylammonium chloride, have been shown to cause allergic contact dermatitis. Ethylhexadecyldimethylammonium bromide (EHD) is a QAC for which sensitization potential is not clearly known. Therefore, we have studied its mechanism in human keratinocytes (KC), the main cells of the epidermis. We used the well-described human KC cell line KERTr exposed to EHD, cinnamaldehyde (CinA), a well-known skin sensitizer, and a mixture of both. Since chemical sensitizers are known to activate the transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2), leading to cellular detoxification and suppressed proinflammatory cytokines, protein or mRNA expression of NRF2 pathway-related enzymes and pro-inflammatory cytokines were investigated by Western blot and RT-qPCR. The activity of the NRF2 pathway on inflammation was studied by RT-qPCR in NRF2-invalidated KERTr cells. We showed that EHD cannot induce the NRF2 pathway, unlike contact sensitizers like CinA. EHD triggers an inflammatory response by inducing the mRNA expression of pro-inflammatory cytokines such as IL-1β or IL-6. Moreover, mixing EHD and CinA inhibits the effect of CinA on NRF2 expression and mitigates the inflammatory response induced by EHD alone. EHD treatment of KERTr cells in which NRF2 has been invalidated showed an exacerbation of the inflammatory response at the transcriptional level. Hence, EHD may elicit an inflammatory response in KC via the NF-κB pathway, which could lead to irritation when applied to the skin. This inflammation is negatively controlled by the basal activity of the NRF2 pathway.

Auranofin ameliorates psoriasis-like dermatitis in an imiquimod-induced mouse by inhibiting of inflammation and upregulating FA2H expression

Psoriasis vulgaris (PV) is a common inflammatory skin disorder with immune imbalance contributing to its pathology. It has been reported that auranofin, a known anti-rheumatic drug, has the therapeutic effects on inflammatory diseases. However, the effects and underlying mechanisms of auranofin on PV have not been reported. Here, we showed that auranofin significantly attenuated the mixture of five proinflammatory cytokines (termed as M5) induced hyperproliferation and inflammation in HaCaT cells, a kind of human immortalized keratinocyte cell line. Topical administration of auranofin markedly alleviated the imiquimod (IMQ)-induced mouse psoriatic dermatitis both in appearance and in pathology. The psoriasis area and severity index (PASI) score, acanthosis, dermal infiltrated inflammatory cells and Ki-67 expression were improved obviously. The results of RNA-sequencing from both M5-induced HaCaT cells and IMQ-treated mouse dermatitis showed that auranofin could regulate inflammation-related signaling pathway. Meanwhile, we observed that fatty acid 2-hydroxylase (FA2H), a key molecule in lipid metabolism, was significantly increased after auraonfin treatment in both M5-induced HaCaT cells and IMQ-treated mouse skin. Knockdown of FA2H in HaCaT cells remarkably inhibited cell apoptosis, increased cell proliferation and the expression profiles of inflammatory-related genes, whereas FA2H overexpression had opposite effect. In conclusion, auranofin notably alleviated IMQ-induced psoriasis-like skin inflammation, which might be mediated by increasing FA2H expression, suggesting its potential to be a novel strategy for psoriasis treatment.

Cordycepin-induced Keratinocyte Secretome Promotes Skin Cell Regeneration.

Skin regeneration is the intrinsic ability to repair damaged skin tissues to regaining skin well-being. Processes of wound healing, a major part of skin regeneration, involve various types of cells, including keratinocytes and dermal fibroblasts, through their autocrine/paracrine signals. The releasable factors from keratinocytes were reported to influence dermal fibroblasts behavior during wound-healing processes. Here, we developed a strategy to modulate cytokine components and improve the secretome quality of HaCaT cells, a nontumorigenic immortalized keratinocyte cell line, via the treatment of cordycepin, and designated as cordycepin-induced HaCaT secretome (CHS). The bioactivities of CHS were investigated in vitro on human dermal fibroblasts (HDF). The effects of CHS on HDF proliferation, reactive oxygen species-scavenging, cell migration, extracellular matrix production and autophagy activation were investigated by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide cell viability assay, dichloro-dihydro-fluorescein diacetate, the wound-healing assay, reverse transcription polymerase chain reaction and immunofluorescent microscopy. Finally, Proteome Profiler™ Array was used to determine the composition of the secretome. CHS induced fibroblast proliferation/migration, reactive oxygen species-scavenging property, regulation of extracellular matrix synthesis, and autophagy activation. Such enhanced bioactivities of CHS were related to the increase of some key cytokines, including C-X-C motif chemokine ligand 1, interleukin 1 receptor A, interleukin 8, macrophage migration-inhibitory factor, and serpin family E member 1. These findings highlight the implications of cordycepin alteration of the cytokine profile of the HaCaT secretome, which represents a novel biosubstance for the development of wound healing and skin regeneration products.

TNF-α‒Mediated Keratinocyte Expression and Release of Matrix Metalloproteinase 9: Putative Mechanism of Pathogenesis in Stevens‒Johnson Syndrome/Toxic Epidermal Necrolysis

Stevens‒Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are severe cutaneous adverse drug reactions characterized by widespread keratinocyte cell death and epidermal detachment. At present, there is little understanding of how the detachment occurs or how it is abrogated by the TNF-α inhibitor etanercept, an effective SJS/TEN treatment. RNA sequencing was used to identify upregulated transcripts in formalin-fixed paraffin-embedded SJS/TEN skin biopsies. Epidermal matrix metalloproteinase 9 (MMP9) expression was assessed by immunohistochemistry in skin biopsies and cultured human skin explants exposed to serum from patients with cutaneous adverse drug reactions. TNF-α‒induced MMP9 expression and activity and its abrogation by etanercept were determined using the HaCaT immortalized keratinocyte cell line. Epidermal MMP9 expression was significantly higher in SJS/TEN skin (70.6%) than in healthy control skin (0%) (P= 0.0098) and nonbullous skin reactions (10.7%) (P= 0.0002). SJS/TEN serum induced significant MMP9 expression and collagenase activity in healthy skin explants, which was reduced by etanercept. Etanercept was also able to negate the TNF-α‒induced MMP9 expression in the HaCaT cell line. Data suggest that elevated epidermal MMP9 expression and collagenase activity are a putative pathogenic mechanism in SJS/TEN, which is limited by etanercept. Modulation of MMP9 expression and activity represents, to our knowledge, a previously unreported therapeutic target for the treatment of SJS/TEN.

030 The Role of FasL-dependent Signaling in the Pathogenesis of Pemphigus Vulgaris

Pemphigus vulgaris is a chronic, life-altering bullous autoimmune disease with production of antibodies against the desmosomal proteins Desmogleins 3 and 1 (Dsg) causing acantholysis. Ubiquitous antibody deposition but discontinuous disease manifestation indicate the possible role of cofactors. In this study, we focus on FasL as a putative cofactor in PV-related acantholysis. To study cell-death dependent processes during PV, we used the spontaneously immortalized keratinocyte cell line HaCaT stimulated with AK23 (anti-Dsg3 ab) together with FasL as a cofactor.

Interleukin-18 as a severity marker and novel potential therapeutic target for epidermolytic ichthyosis.

Epidermolytic ichthyosis (EI) is a major form of nonsyndromic inherited ichthyosis, characterized by erythroderma, marked hyperkeratosis and scale, bulla and erosion at birth, associated with KRT1/KRT10 mutations. The cytokine and chemokine profiles in EI are poorly understood, and specific treatment options have not been established. To explore novel biomarkers and therapeutic targets in patients with EI. We analysed cytokine levels in serum and skin samples from 10 patients with inherited ichthyosis, including seven patients with EI. Wild-type and mutant KRT1 constructs were established and transfected into HaCaT cells, an immortalized keratinocyte cell line, for in vitro immunoblotting and immunocytochemistry analyses. Multiplex cytokine/chemokine analysis revealed that 10 cytokines/chemokines [interleukin (IL)-1β, IL-4, IL-17A, IL-16, IL-18, IL-1 receptor-α, macrophage colony-stimulating factor, interferon-α2, basic fibroblast growth factor and monocyte chemotactic protein-3] were significantly increased in patients with EI. Furthermore, IL-18 levels were significantly higher in patients with EI [n = 7; 2714.1 (1438.0) pg mL-1] than in healthy controls [n = 11; 218.4 (28.4) pg mL-1, P < 0.01]. Immunohistochemical analyses showed that IL-18 expression was elevated in skin samples from patients with EI. Serum IL-18 levels correlated with the severity of ichthyosis, as measured by the Ichthyosis Scoring System. Immunoblotting analysis revealed that mature IL-18 levels were increased in the supernatant of mutant KRT1 expressing HaCaT cells. Additionally, these cells showed NLRP3 aggregation in the cytoplasm and ASC clustered around mutant keratin aggregations. These findings suggest that mutant keratin might promote the activation of the NLRP3 inflammasome and its downstream caspase-1-mediated IL-18 release in keratinocytes from patients with EI. Our results suggest that serum IL-18 is a severity marker released from the skin of patients with EI. Blockade of IL-18 may be a useful novel therapeutic option for patients with EI.

Salidroside Ameliorates Ultraviolet-Induced Keratinocyte Injury by Inducing SIRT1-Dependent Autophagy.

IntroductionAutophagy is an important process for maintaining intracellular homeostasis and is deregulated in ultraviolet B (UVB)-induced skin injury. Salidroside (SAL) is an active ingredient extracted from Rhodiola rosea, which is a herbal medicine that has shown protection against ultraviolet (UV) radiation. Here, we investigated the functions and mechanisms of SAL on UVB-induced skin cell oxidative damage and autophagy.MethodsHuman immortalized keratinocyte cell line HaCaT was used as a cell model of UV injury. HaCaT cells were exposed to UVB irradiation and then incubated with SAL to investigate cell viability, lactate dehydrogenase (LSD) in culture media, intracellular reactive oxygen species (ROS) level, oxidative stress, autophagy, and regulatory effects on SIRT1 protein.ResultsSAL pretreatment (25, 50 and 100 μM) increased cell viability and inhibited LDH release in UVB-challenged HaCaT cells. SAL (100 μM) significantly reduced intracellular ROS level and suppressed oxidative stress, with increased MDA content and increased SOD activity. In addition, SAL pretreatment enhanced autophagy in UVB-irradiated HaCaT cells, increased protein expressions of Beclin-1 and ATG7, and decreased protein expression of P62. We also found that pretreatment with SAL increased the SIRT1 protein in irradiated HaCaT cells. SAL protected UVB-induced damage in a dependent manner on autophagy and SIRT1, as SAL-induced increase in viability was significantly attenuated by specific autophagy inhibitor Wortmannin (1 μM) or SIRT1 inhibitor EX-527 (100 nM).DiscussionThe present study results speculate that SAL suppresses UVB-induced injury and autophagy by enhancing SIRT1 expression.

Pitfalls in the Application of Dispase-Based Keratinocyte Dissociation Assay for In Vitro Analysis of Pemphigus Vulgaris.

Pemphigus vulgaris (PV) is a chronic, life-altering autoimmune disease due to the production of anti-desmoglein antibodies causing the loss of cell–cell adhesion in keratinocytes (acantholysis) and blister formation in both skin and mucous membranes. The dispase-based keratinocyte dissociation assay (DDA) is the method of choice to examine the pathogenic effect of antibodies and additional co-stimuli on cell adhesion in vitro. Despite its widespread use, there is a high variability of experimental conditions, leading to inconsistent results. In this paper, we identify and discuss pitfalls in the application of DDA, including generation of a monolayer with optimized density, appropriate culturing conditions to obtain said monolayer, application of mechanical stress in a standardized manner, and performing consistent data processing. Importantly, we describe a detailed protocol for a successful and reliable DDA and the respective ideal conditions for three different types of human keratinocytes: (1) primary keratinocytes, (2) the HaCaT spontaneously immortalized keratinocyte cell line, and (3) the recently characterized HaSKpw spontaneously immortalized keratinocyte cell line. Our study provides detailed protocols which guarantee intra- and inter-experimental comparability of DDA.

Royal Jelly Extract Accelerates Keratinocyte Proliferation, and Upregulates Laminin &amp;lt;i&amp;gt;α&amp;lt;/i&amp;gt;3 and Integrin &amp;lt;i&amp;gt;β&amp;lt;/i&amp;gt;1 mRNA Expression, via Akt/mTOR/HIF-1&amp;lt;i&amp;gt;α&amp;lt;/i&amp;gt; Pathway

Background: In the previous decade, various benefits and biological activities of royal jelly, applied in alternative and modern medicine, and cosmetics, have been reported. However, the effects of royal jelly extract (RJ) on keratinocytes have not been fully elucidated. Objective: The primary objectives of this study were to reveal the effects of RJ on keratinocytes and explore the underlying mechanism. Methods: HaCaT cells, an immortal human epidermis-derived keratinocyte cell line, were used in this study. Laminin α3 (LAMA3), integrin β1 (ITGB1), and hypoxia-inducible factor-1α (HIF-1α) mRNA expression levels were determined using real-time PCR. Cell proliferation rate was measured using a bromodeoxyuridine uptake assay. Results: RJ treatment upregulated LAMA3, ITGB1 and HIF-1α mRNA expression, and accelerated HaCaT cell proliferation. Akt and mTOR inhibitors suppressed the RJ-induced HIF-1α expression and cell proliferation. HIF-1α silencing abrogated RJ-induced LAMA3 and ITGB1 mRNA expression and cell proliferation, whereas LAMA3 silencing and antibody-mediated ITGB1 blockade did not affect the effects of RJ. Conclusion: RJ upregulates LAMA3 and ITGB1 mRNA expression levels by HIF-1α expression enhancement. In addition, RJ accelerates keratinocyte proliferation via Akt/mTOR/HIF-1α/NF-κB signaling pathway. These suggest that RJ is beneficial for anti-aging, as a skin care product ingredient.

Catechol functionalized ink system and thrombin-free fibrin gel for fabricating cellular constructs with mechanical support and inner micro channels

The development of 3D bio printing technology has contributed to protocols for the repair and regeneration of tissues in recent years. However, it is still a great challenge to fabricate structures that mimic the complexity of native tissue, including both the biomechanics and microscale internal structure. In this study, a catechol functionalized ink system was developed to produce tough and elastic scaffolds with built-in micro channels that simulate the vascular structure. And a skin model was designed to evaluate the cytocompatibility of the scaffolds. The mechanical support stemmed from the double network based on catechol-hyaluronic acid (HACA) and alginate, the micro channels were generated using sacrificial gelatin. HACA/alginate and gelatin were firstly printed using a 3D extrusion printer. Thrombin-free fibrinogen were then mixed with human dermal fibroblasts and introduced to the printed scaffolds to induce gelation. An immortal human keratinocyte cell line was introduced on top of the cellular construct to mimic the full thickness skin structure. The printed scaffolds demonstrated high elasticity and supported the formation of a double-layered cell-laden skin like structure. The results suggest the 3D printing platform developed here provides a platform for skin regeneration and could be explored further to engineer functional skin tissue by incorporation of other types of cells.

121 Characterization of different immortalized keratinocyte cell lines as models for epidermal differentiation studies

In vitro reconstructed skin models have been widely used to study epidermal physiology. However, these models rely on primary cells that are limited by availability and donor variations. To overcome these issues we validated immortalized cell lines that were generated by the expression of SV40 large T antigen and hTERT (NHEK-SV/TERT) or HPV E6/E7 (NHEK-E6/E7). Both cell lines show indefinite growth up to over 50 passages and displayed increased growth rates compared to primary cells. Primary keratinocytes present a mix of different epidermal cell populations such as keratinocyte stem and cells already committed to differentiation. Immortalized cells did not show distinct populations, arguing that immortalized cells are a more uniform mainly proliferative cell population that needs an exogenous stimulus to commit to differentiation. While NHEK-E6/E7 show a differentiation behavior comparable to primary cells, the highly proliferative NHEK-SV/TERT needed withdrawal of any proliferative stimulus to commit to a delayed onset of differentiation. In 3D epidermal models both cell lines were able to reconstitute a stratified epidermis and formed a tight and functional barrier. Both cell lines responded to inflammatory stimuli leading to an epidermal morphology mimicking Th1 or Th2 type dermatosis. In addition these cell lines are suitable for genome editing using CRISPR/Cas9 technology as different target genes could be successfully knocked out with NHEK-SV/TERT showing a higher transfection efficiency simplifying the isolation of edited single cell clones. Thus, the tested cell lines are suitable substitutes for primary cells and have the potential to serve as suitable models to investigate molecular pathomechanisms and test novel therapeutics during pre-clinical evaluation.

237 Impact of innate immunity on pemphigus vulgaris disease manifestation

Pemphigus vulgaris is a chronic autoimmune blistering disease characterized by antibody production against Desmogleins 3 (Dsg3) and 1 (Dsg1) causing acantholysis. Increased levels of innate cytokines detected in the blister fluid suggest a role of innate immune system activity. To elucidate the discrepancy between binding of pathogenic antibodies and a lack of blister formation, we focused on the role of innate cofactors supporting acantholysis. Here, we used a dispase-based keratinocyte-dissociation assay with a human immortalized keratinocyte cell line (HaCaT) and human primary keratinocytes.

Lycopus lucidus Turcz ameliorates DNCB‑induced atopic dermatitis in BALB/c mice.

Atopic dermatitis (AD) is a chronic inflammatory allergic skin disease, characterized by pruritic and eczematous skin lesions. Lycopus lucidus Turcz (LLT) is a perennial herb that has been reported to have various biological properties, including effects on blood circulation, as well as anti-inflammatory, antioxidant, anti-vascular inflammation and wound-healing effects. However, whether LLT improves dermatitis and the underlying mechanisms has yet to be determined. The aim of the present study was to determine whether LLT can improve 2,4-dinitrochlorobenzene (DNCB)-induced dermatitis and to verify the inhibitory effect of LLT on the expression of chemokines and pro-inflammatory cytokines in the HaCaT immortalized keratinocyte cell line. In addition, the anti-inflammatory function of LLT in RAW264.7 mouse macrophages was investigated. In the DNCB-induced AD mouse model, LLT inhibited infiltration by mast cells, eosinophils and CD8+ cells in the dorsal skin tissue of AD mice, and suppressed the expression of IgE and IL-6 in serum. In addition, LLT inhibited the phosphorylation of ERK and JNK, as well as NF-κB in skin tissue. In the HaCaT cell model induced by TNF-α/IFN-γ, LLT inhibited the expression of thymus and activation-regulated chemokine, granulocyte-macrophage colony-stimulating factor, monocyte chemoattractant protein-1, TNF-α and IL-1β, whilst inhibiting the phosphorylation of NF-κB. In addition, in the lipopolysaccharide-induced RAW 264.7 cell inflammation model, LLT inhibited the expression of TNF-α and IFN-γ, the nuclear translocation of NF-κB and the phosphorylation of ERK and JNK. These results suggested that LLT may be a promising candidate for the treatment of inflammatory dermatitis.

Abstract LB185: BBX and PLK1 feedback mechanisms regulate mitotic entry &amp; exit

Abstract Bobby Sox Homolog (BBX) is a highly conserved member of the high mobility group (HMG) box family of transcription factors, but its function remains largely unknown. We find human papillomavirus positive oropharyngeal squamous cell carcinomas (HPV-OPSCC) are enriched for BBX amplification and single nucleotide variants clustered within the BBX lysine rich domain. We therefore used the immortalized keratinocyte cell line HaCaT to elucidate BBX function. BBX is localized primarily in the nucleus. Cell synchronization at G1-S or G2-M shows BBX mRNA transcription and protein expression are highest at G1-S and rapidly decline at G2-M. Immunofluorescence (IF) shows high nuclear BBX expression during interphase and rapid reduction at mitosis (M). Induced overexpression of BBX decreases cellular proliferation, prolongs M phase, induces senescence, increases frequency of binucleated cells, and increases DNA damage as measured by γH2AX. Knockdown of BBX expression decreases cell proliferation, shortens M phase, and results in several mitotic defects, including lagging chromosomes, micronuclei, multi-polar spindles, and binucleated cells. Because effects of BBX dysregulation mirror those of Polo-like kinase-1 (PLK1), we investigated possible BBX-PLK1 interactions. BBX induction increases, whereas knockdown decreases, PLK1 mRNA and protein expression. BBX strongly activated transcription of the PLK1 promoter in a luciferase reporter assay. In contrast, PLK1 dramatically decreased BBX protein, but not mRNA, expression, and this effect was attenuated by a PLK1 T210 inactivating mutation and by a PLK1 small molecule inhibitor. We found a strong positive correlation between BBX and PLK1 mRNA expression in HNSCC tumors, but not in normal tissues. We conclude that BBX and PLK1 interact in a feedback mechanism to precisely regulate the G2-M transition. Future work will investigate interactions between BBX, HPV oncoproteins, and regulation of the cell cycle. Citation Format: Gaiyun Li, Maura L. Gillison. BBX and PLK1 feedback mechanisms regulate mitotic entry &amp; exit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB185.

Crystal Structure, Spectroscopic Characterization, Antioxidant and Cytotoxic Activity of New Mg(II) and Mn(II)/Na(I) Complexes of Isoferulic Acid.

The Mg(II) and heterometallic Mn(II)/Na(I) complexes of isoferulic acid (3-hydroxy-4-methoxycinnamic acid, IFA) were synthesized and characterized by infrared spectroscopy FT-IR, FT-Raman, electronic absorption spectroscopy UV/VIS, and single-crystal X-ray diffraction. The reaction of MgCl2 with isoferulic acid in the aqueous solutions of NaOH resulted in synthesis of the complex salt of the general formula of [Mg(H2O)6]⋅(C10H9O4)2⋅6H2O. The crystal structure of this compound consists of discrete octahedral [Mg(H2O)6]2+ cations, isoferulic acid anions and solvent water molecules. The hydrated metal cations are arranged among the organic layers. The multiple hydrogen-bonding interactions established between the coordinated and lattice water molecules and the functional groups of the ligand stabilize the 3D architecture of the crystal. The use of MnCl2 instead of MgCl2 led to the formation of the Mn(II)/Na(I) complex of the general formula [Mn3Na2(C10H7O4)8(H2O)8]. The compound is a 3D coordination polymer composed of centrosymmetric pentanuclear subunits. The antioxidant activity of these compounds was evaluated by assays based on different antioxidant mechanisms of action, i.e., with •OH, DPPH• and ABTS•+ radicals as well as CUPRAC (cupric ions reducing power) and lipid peroxidation inhibition assays. The pro-oxidant property of compounds was measured as the rate of oxidation of Trolox. The Mg(II) and Mn(II)/Na(I) complexes with isoferulic acid showed higher antioxidant activity than ligand alone in DPPH (IFA, IC50 = 365.27 μM, Mg(II) IFA IC50 = 153.50 μM, Mn(II)/Na(I) IFA IC50 = 149.00 μM) and CUPRAC assays (IFA 40.92 μM of Trolox, Mg(II) IFA 87.93 μM and Mn(II)/Na(I) IFA 105.85 μM of Trolox; for compounds’ concentration 10 μM). Mg(II) IFA is a better scavenger of •OH than IFA and Mn(II)/Na(I) IFA complex. There was no distinct difference in ABTS•+ and lipid peroxidation assays between isoferulic acid and its Mg(II) complex, while Mn(II)/Na(I) complex showed lower activity than these compounds. The tested complexes displayed only slight antiproliferative activity tested in HaCaT human immortalized keratinocyte cell line within the solubility range. The Mn(II)/Na(I) IFA (16 μM in medium) caused an 87% (±5%) decrease in cell viability, the Mg salt caused a comparable, i.e., 87% (±4%) viability decrease in a concentration of 45 μM, while IFA caused this level of cell activity attenuation (87% ± 5%) at the concentration of 1582 μM (significant at α = 0.05).