Epidermal Cell Line Research Articles

Enhancing efficacy and safety of azelaic acid via encapsulation in cyclodextrin nanosponges: development, characterization and evaluation

Azelaic acid (AA), a promising agent for acne and hyperpigmentation disorders, is associated with side effects viz. rashes, skin irritation, dryness, burning and stinging. Its poor solubility also pose challenge in the development of suitable formulation. Therefore, this research was aimed to design cyclodextrin nanosponges (CDNS) of AA to address above-mentioned challenges. Herein, the fabrication of CDNS by melt method was demonstrated employing β-cyclodextrin (β-CD) as polymer and diphenyl carbonate (DPC) as a cross-linker. AA was loaded in CDNS via lyophilization and appropriately characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray powder diffraction and nuclear magnetic resonance. For morphological evaluation, field emission scanning electron and transmission electron microscopy were also performed. The particle size of AANS was in nanorange, with acceptable zeta potential, low polydispersity index and delayed release. The safety of the nanoformulation was assessed using Human Epidermal Keratinocyte cell lines. Further, molecular docking studies for AA was carried out. In vitro antibacterial, antioxidant and antityrosinase assay were also conducted for prepared nanoformulation. The results of all the studies performed revealed that encapsulation of AA in nanosponges led to improvement in efficacy of drug in terms of solubility, release and safety, with adequate antimicrobial, antioxidant and antityrosinase activity.

EGF Conjugation Improves Safety and Uptake Efficacy of Titanium Dioxide Nanoparticles.

Titanium dioxide nanoparticles (TiO2 NPs) have a strong potential for cancer therapeutic and bioimaging applications such as photodynamic therapy (PDT) and photodynamic diagnosis (PDD). Our previous results have shown that TiO2 NPs have a low cellular uptake and can induce cell proliferation. This suggests that TiO2 NPs could increase the risk of tumor overgrowth while being used for PDD and PDT. To solve this problem, we constructed epidermal growth factor-ligated polyethylene glycol-coated TiO2 NPs (EGF-TiO2 PEG NPs). In this work, we studied the effect of EGF conjugation on the cellular uptake of TiO2 PEG NPs. Then, we investigated the effect of both non-conjugated and EGF-TiO2 PEG NPs on the A431 epidermal cancer cell line, proliferation and growth via the investigation of EGFR localization and expression. Our results indicated that TiO2 PEG NPs induced EGFRs aggregation on the A431 cells surface and induced cell proliferation. In addition, EGF-TiO2 PEG NPs induced the internalization of EGFRs inside of cells with increased cellular NPs uptake and decreased cellular proliferation compared to TiO2 PEG NPs-treated cells. These findings suggest that EGF conjugation can increase the efficacy of TiO2 PEG NPs for biomedical applications such as PDD and PDT with decreased risk of tumor overgrowth.

Identification and accumulation of phenolic compounds in the leaves and bark of Salix alba (L.) and their biological potential.

The study examines the phenolic compounds in hydromethanolic extracts of Salix alba (L.) leaves and bark as well as their antioxidant activity and cytotoxic potential. UPLC-PDA-Q/TOF-MS analysis showed a total of 29 phenolic compounds in leaves and 34 in bark. Total phenolic compound content was 5575.96 mg/100 g of dry weight (DW) in leaves and 2330.31 mg/100 g DW in bark. The compounds were identified as derivatives of phenolic acids (seven in leaves and five in bark), flavanols and procyanidins (eight in leaves and 26 in bark) and flavonols (14 in leaves and three in bark). Both extracts exhibited strong antioxidant potential, assessed by radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), but the bark extract was even stronger than the ascorbic acid used as a standard. The cytotoxicity of both extracts was evaluated against human skin fibroblasts and human epidermal keratinocytes cell lines using the Presto Blue cell viability assay. The keratinocytes were more resistant to tested extracts than fibroblasts. The leaf and bark extracts at concentrations which exhibited antioxidant activity were also not toxic against the keratinocyte cell line. Thus, S. alba extracts, especially the leaf extract, offer promise as a nontoxic natural antioxidant, in cosmetic products or herbal medicines, and as a source of bioactive secondary metabolites.

49P Role of microRNAs in a panel of human cervical cancer cell lines

Genetic and epigenetic changes have been shown to be crucial regulators in cervical cancer progression. However, the mechanisms and role of miRNA dysregulation in cervical carcinogenesis are still not clear. We aimed to evaluate four miRNAs (miR-130a-3p, miR-205-5p, miR-4531, and miR-381-3p) and the regulation of their targets in human cervical cancer (CC) cell lines. We used a panel of CC cell lines including five commercial cell lines (HeLa, SiHa, CasKi, C4-I, and C-33A) and one primary cell line (HCB-514) established in our laboratory. Primary Epidermal Keratinocytes cell line (HaCaT) was used as control. The differential expression level of miRNAs was assessed by quantitative PCR analysis. Gene expression profile analysis was performed using nCounter PanCancer Pathways panel (NanoString Technologies®) to compare HCB-514 and HaCaT cell lines. The interaction networks of the miRNA-targets were constructed by the Cytoscape program and REACTOME FI plug-in with the CC category. We found that miR-130a-3p was overexpressed in all CC cell lines and miR-205-5p was upregulated in CasKi, C4-IA, C-33A and HCB-514, both with a ≤2 fold-change. However, miR-4531 showed no difference between expression levels in CC cell lines compared to HaCaT cells. On the other hand, miR-381-3p was downregulated only in the HCB-514 cell line. We identified 14 differentially expressed genes: eight downregulated (WNT10A, ITGA2, BMP7, DLL1, PPP2R2B, SOX9, BMP4, and DUSP6) and six upregulated (LTBP1, TMPRSS2, RASAL1, PAX8, IL6R, and TLRB2) in the HCB-514 cell line compared to the HaCaT cell line. In the miRNA-target interaction analysis, we verified that WNT10A can be being silenced by upregulation of miR-130a-3p and miR-205-5p. This study revealed that miR-130a-3p and miR-205-5p are upregulated in CC cell lines. WNT expression was predicted as a specific target of both upregulated miRNAs (miR-130a-3p and miR-205-5p). These results may be useful to better understand CC progression and the role of these microRNAs in this process.

In Vitro Photodynamic Therapy of Mononuclear and Dinuclear Iridium(III) Bis(terpyridine) Complexes

Three mononuclear or dinuclear bis(terpyridine) (tpy) iridium(III) complexes bearing pyren-1-yl (pyr) group(s) were synthesized. Their photophysical properties in water and in vitro photodynamic therapy (PDT) effects toward the human lung epithelial cancer cell line A549 and the human epidermal skin cancer cell line A431 were investigated to evaluate the effects of dinuclear versus mononuclear complexes and the impact of the oligoether substituent at the ligand. All complexes possessed pyr-tpy ligand-associated charge transfer (1CT)/1π,π* absorption bands at 350-550 nm, with the dinuclear complex Ir3 showing the much enhanced absorptivity of this band. These complexes exhibited dual emission upon excitation at >430 nm in most cases, with the emitting states being ascribed to 1ILCT (intraligand charge transfer) and 3π,π*/3CT states, respectively. All complexes exhibited relatively weak to moderate cytotoxicity in the dark but high photocytotoxicity upon broadband visible light irradiation. Among them, the dinuclear complex Ir3 showed the highest intracellular reactive oxygen species (ROS) generation and PDT efficiency compared to its mononuclear counterpart Ir1. Introducing an oligoether substituent on one of the tpy ligands in Ir2 also improved its intracellular ROS generation and PDT efficacy compared to those induced by Ir1. Ir3 induced both mitochondrial dysfunction and lysosomal damage upon light activation toward both cell lines, whereas Ir1 and Ir2 caused both mitochondrial dysfunction and lysosomal damage in A431 cells but only lysosomal damage in A549 cells. The dominant cell death pathway induced by Ir1-Ir3 PDT is apoptosis.

Reversible cross-tolerance to platelet-activating factor signaling by bacterial toxins

Bacterial toxins signaling through Toll-like receptors (TLRs) are implicated in the pathogenesis of many inflammatory diseases. Among the toxins, lipopolysaccharide (LPS) exerts its action via TLR-4 while lipoteichoic acid (LTA) and bacterial lipoproteins such as Braun lipoprotein (BLP) or its synthetic analogue Pam3CSK4 act through TLR-2. Part of the TLR mediated pathogenicity is believed to stem from endogenously biosynthesized platelet-activating factor (PAF)- a potent inflammatory phospholipid acting through PAF-receptor (PAF-R). However, the role of PAF in inflammatory diseases like endotoxemia is controversial. In order to test the direct contribution of PAF in TLR-mediated pathogenicity, we intraperitoneally injected PAF to Wistar albino mice in the presence or absence of bacterial toxins. Intraperitoneal injection of PAF (5 μg/mouse) causes sudden death of mice, that can be delayed by simultaneously or pre-treating the animals with high doses of bacterial toxins- a phenomenon known as endotoxin cross-tolerance. The bacterial toxins- induced tolerance to PAF can be reversed by increasing the concentration of PAF suggesting the reversibility of cross-tolerance. We did similar experiments using human platelets that express both canonical PAF-R and TLRs. Although bacterial toxins did not induce human platelet aggregation, they inhibited PAF-induced platelet aggregation in a reversible manner. Using rabbit platelets that are ultrasensitive to PAF, we found bacterial toxins (LPS and LTA) and Pam3CSK4 causing rabbit platelet aggregation via PAF-R dependent way. The physical interaction of PAF-R and bacterial toxins is also demonstrated in a human epidermal cell line having stable PAF-R expression. Thus, we suggest the possibility of direct physical interaction of bacterial toxins with PAF-R leading to cross-tolerance.

Ester derivatives of phyllohydroquinone effectively deliver the active form of vitamin K1 topically, owing to their non-photosensitivity

Topical application of phylloquinone (PK) is beneficial to the skin; however, its topical use is limited in Europe owing to its photosensitive properties such as photodegradation and phototoxicity. We evaluated the suitability of ester derivatives of phyllohydroquinone (PKH), the active form of PK, for topical application to overcome the abovementioned problems of PK. We used the PKH derivatives PKH-1,4-bis-N,N-dimethylglycinate hydrochloride (PKH-DMG) and PKH-1,4-bis-hemisuccinate (PKH-SUC) for our studies. Photostability was determined by measuring the residual concentration after irradiation with artificial sunlight and multi-wavelength light. Phototoxicity after ultraviolet A (UVA) irradiation was assessed by measuring drug-induced singlet oxygen and intracellular reactive oxygen species (ROS) generation, and cell viability of a human epidermal keratinocyte cell line (HaCaT). Delivery of PKH into HaCaT cells was assessed by measuring PK epoxide (PKO) levels. The PKH derivatives showed higher photostability than PK. After UVA irradiation, PK induced high singlet oxygen levels and intracellular ROS generation, and reduced cell viability, whereas the PKH derivatives showed no effects. The PKH derivatives increased intracellular PKO levels. AUCPKO(0-72 h) values after PKH-DMG and PKH-SUC treatments were 0.741- and 22.9-fold higher than that after PK treatment, respectively. In conclusion, PKH derivatives act as PKH prodrugs and are suitable for topical application without the need for special protection from light.

Mechanisms of Resorcinol Antagonism of Benzo[a]pyrene-Induced Damage to Human Keratinocytes.

Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon and ubiquitous environmental toxin with known harmful effects to human health. Abnormal phenotypes of keratinocytes are closely associated with their exposure to B[a]P. Resorcinol is a component of argan oil with reported anticancer activities, but its mechanism of action and potential effect on B[a]P damage to the skin is unknown. In this study, we investigated the effects of resorcinol on B[a]P-induced abnormal keratinocyte biology and its mechanisms of action in human epidermal keratinocyte cell line HaCaT. Resorcinol suppressed aryl hydrocarbon receptor (AhR) activity as evidenced by the inhibition of B[a]P-induced xenobiotic response element (XRE)-reporter activation and cytochrome P450 1A1 (CYP1A1) expression. In addition, resorcinol attenuated B[a]P-induced nuclear translocation of AhR, and production of ROS and pro-inflammatory cytokines. We also found that resorcinol increased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activity. Antioxidant response element (ARE)-reporter activity and expression of ARE-dependent genes NAD(P)H dehydrogenase [quinone] 1 (NQO1), heme oxygenase-1 (HO-1) were increased by resorcinol. Consistently, resorcinol treatment induced nuclear localization of Nrf2 as seen by Western analysis. Knockdown of Nrf2 attenuated the resorcinol effects on ARE signaling, but knockdown of AhR did not affect resorcinol activation of Nrf2. This suggests that activation of antioxidant activity by resorcinol is not mediated by AhR. These results indicate that resorcinol is protective against effects of B[a]P exposure. The mechanism of action of resorcinol is inhibition of AhR and activation of Nrf2-mediated antioxidant signaling. Our findings suggest that resorcinol may have potential as a protective agent against B[a]P-containing pollutants.

Surface modified nanoliposome formulations provide sustained release for 5-FU and increase cytotoxicity on A431 cell line

Malignant melanoma is a type of skin cancer with high risk of metastasis. 5-Fluorouracil is commonly used for treatment of skin cancer, however its penetration through the skin is found to be insufficient in some cases. Therefore, we optimized its pharmacokinetics by fabricating 5- Fluorouracil-loaded nanoliposome formulations modified with Poly-L-lysine coating. 5-Fluorouracil-loaded nanoliposome formulations were prepared using dipalmitoylphosphatidylcholine, dicethylphosphate and cholesterol having encapsulation efficiency of 45 ± 9.61%. The particle size, zeta potential, polydispersity index and encapsulation rate of the prepared formulation was found to be 237.9 ± 0.986 nm, 41.4 ± 1.060 mV, 0.233 ± 0.019 and 88.2 ± 7.85%, respectively. Surface characterization, molecular structure and thermal property illumination of the formulations were performed alongside stability studies. The In-vitro release of 5-FU from Lipo-FU6 and PLL-1 formulations was investigated by dialysis membrane method. Within the first 12 hours, the percentage release of 5-FU from Lipo-FU6 and PLL-1 formulations was observed to be 47.17% and 20.84%, respectively. Moreover, the cytotoxicity study on A431 epidermal carcinoma cell lines has revealed that 5-FU-loaded formulations were toxic to cells unlike the 5-FU free formulations. In conclusion, PLL coated nanoliposome formulations showed a potential to be an effective option for further combined drug/gene therapy applications.

MiR-143 acts as an inhibitor of migration and proliferation as well as an inducer of apoptosis in melanoma cancer cells in vitro.

Melanoma is a serious form of skin cancers begins in the melanocyte. Micro-RNAs are small noncoding RNA with 19 to 25 nucleotides in length involves in the regulation of a wide range of biological processes. MicroRNAs are affected by an aberrant epigenetic alteration in the tumors that may lead to their dysregulation and formation of cancer. Recently, dysregulation of numerous microRNAs has been reported in different types of cancer. The present study focused on the role of miR-143 in carcinogenesis of melanoma cancer. Here, we evaluated the expression level of miR-143 in three melanoma cell lines in comparison with the normal human epidermal melanocyte cell line. Then, miR-143 gene plasmid transfected into the WM115 cell line, for having the lowest expression of miR-143. In addition, the effect of miR-143 transfection on mRNA and protein levels of metastasis-related genes was performed along with MTT assay, wound healing assay, and flow cytometry. The results showed that mRNA and protein expression levels of metastasis-related genes including MMP-9, E-cadherin, Vimentin, and CXCR4 have been reduced following transfection of miR-143. Moreover, the results of the scratch test showed that miR-143 re-expression inhibited cell migration. Also, the role of miR-143 in the induction of apoptosis and inhibition of proliferation by flow cytometry and MTT was confirmed. As a result, the present study showed that miR-143 was involved in metastatic and apoptotic pathways, suggesting that miR-143 acts as a tumor-suppressor microRNA in melanoma cancer.

195 N/TERTs replicate primary keratinocytes in barrier formation and viral infectivity

Atopic dermatitis (AD) is a skin disease characterized by an impaired epidermal barrier and increased susceptibility to cutaneous viral infections. We have previously observed that barrier disruption (caused by our tight junction disrupting peptide [TJDP®]), enhances vaccinia virus (VV) infection of primary human foreskin keratinocytes (PHFK). To evaluate the importance of AD-relevant tight junction proteins in epidermal viral infections, we sought to identify an epidermal cell line that we could genetically manipulate. We tested N/TERT cells (immortalized PHFK) to determine whether they faithfully recapitulated primary cells in both our barrier and VV infection models. We found that N/TERTs developed a robust barrier, as measured by transepithelial electrical resistance (TEER), with peak values of 300–400 ohms/cm2 observed 4-5 days after treatment with high Ca+2 (1.8mM) containing media. After exposure to our TJDP®, N/TERTs exhibited a substantial reduction in barrier compared to media controls (e.g. TEER reduction: 46% ± 13 and 68% ± 11 at Days 2 and 3, respectively; n=3). We previously observed that PHFK are more susceptible to VV when treated with TJDP® during differentiation. With N/TERTs, we observed an increase in VV susceptibility as measured by % change in plaque number (179% ± 64 at Day 2; n=3) and % of the monolayer infected (27% ± 24 vs 42% ± 20 at Day 2 for media compared to TJDP® treated samples, n=3). These observations suggest that N/TERTs behave similarly to PHFK in both our infection and barrier experiments. Future studies will focus on determining how reduction in AD-relevant TJ barrier proteins (Claudin 1, 4, 23, occludin, and zonula occludens 1) using the CRISPR/Cas9 system impacts the susceptibility of N/TERTs to viral infections (VV and herpes simplex virus).

Adenovirus-Mediated LAMA3 Transduction Enhances Hemidesmosome Formation and Periodontal Reattachment during Wound Healing

A robust dento-epithelial junction prevents external pathogenic factors from entering connective tissue and could be crucial for periodontal reattachment after periodontal surgery. The junctional epithelium (JE) is attached to the tooth surface through the hemidesmosome (HD) and internal basal lamina, where the primary component is laminin-332. Destruction of the JE leads to the loss of periodontal attachment. Traditional treatments are effective in eliminating local inflammation of the gingiva; however, few directly promote periodontal reattachment and HD formation. Here, we designed a gene-therapy strategy using the adenovirus-mediated human laminin-332 α3 chain (LAMA3) gene (Ad-LAMA3) transduced into a human-immortalized epidermal cell line (HaCaT) to study the formation of HD in vitro. Ad-LAMA3 promoted early adhesion and fast migration of HaCaT cells and increased expression of LAMA3 and type XVII collagen (BP180) significantly. Furthermore, HaCaT cells could facilitate formation of mature HDs after LAMA3 overexpression. In vivo experiments demonstrated that the JE transduced with Ad-LAMA3 could increase expression of LAMA3 and BP180 and “biological sealing” between the tooth and gingival epithelium. These results suggested that adenovirus-mediated LAMA3 transduction is a novel therapeutic strategy that promotes the stability and integration of the JE around the tooth during wound healing.

Pre-mRNA processing factor 3 enhances the progression of keratinocyte-derived cutaneous squamous cell carcinoma by regulating the JAK2/STAT3 pathway

The precise role of pre-mRNA processing factors (PRPs) in human tumorigenesis has not been yet explored. The object of the present study was to explore the effects of PRP3 in a common metastatic skin cancer, keratinocyte-derived cutaneous squamous cell carcinoma (cSCCs). RT-qPCR and western blotting were conducted to measure the expression levels of PRP3 in various cSCC cell lines and cSCC tissues. A benign epidermal keratinocyte cell line was transfected with a eukaryotic expression plasmid to overexpress PRP3. In addition, the endogenous expression level of PRP3 in cSCC cells was silenced using a short hairpin RNA method, and the role of PRP3 on cell proliferation and migration was examined by Cell Counting Kit-8, colony formation, wound healing assay and Transwell assays following knockdown in cSCC cells, and overexpression in keratinovcyte cells. Elevated levels of PRP3 mRNA and protein were noted in cSCC cell lines or cSCC tissues compared with actinic keratosis (AK) or benign epidermal keratinocyte cell line, respectively. Upregulation of PRP3 expression was found to be associated with poor clinical outcomes in patients with cSCCs. The upregulation of PRP3 promoted cell viability, metastasis and the activity of the JAK2/STAT3 pathway in epidermal keratinocyte cells. Interestingly, loss of PRP3 had no obvious impact on cell viability and migration in benign epidermal keratinocyte cells. Functionally, the inhibition of the JAK2/STAT3 pathway reversed the increased cell viability and migration of cSCC cells induced by PRP3. Taken together, the present observations indicated that PRP3 served as a tumor active factor in cSCCs by targeting the JAK2/STAT3 pathway. Moreover, it is implied that impeding the PRP3 activity may selectively constrain cancer cell growth and migration with limited effect on normal skin cells.

Design, Implementation, and Validation of a Piezoelectric Device to Study the Effects of Dynamic Mechanical Stimulation on Cell Proliferation, Migration and Morphology.

Cell functions and behavior are regulated not only by soluble (biochemical) signals but also by biophysical and mechanical cues within the cells’ microenvironment. Thanks to the dynamical and complex cell machinery, cells are genuine and effective mechanotransducers translating mechanical stimuli into biochemical signals, which eventually alter multiple aspects of their own homeostasis. Given the dominant and classic biochemical-based views to explain biological processes, it could be challenging to elucidate the key role that mechanical parameters such as vibration, frequency, and force play in biology. Gaining a better understanding of how mechanical stimuli (and their mechanical parameters associated) affect biological outcomes relies partially on the availability of experimental tools that may allow researchers to alter mechanically the cell’s microenvironment and observe cell responses. Here, we introduce a new device to study in vitro responses of cells to dynamic mechanical stimulation using a piezoelectric membrane. Using this device, we can flexibly change the parameters of the dynamic mechanical stimulation (frequency, amplitude, and duration of the stimuli), which increases the possibility to study the cell behavior under different mechanical excitations. We report on the design and implementation of such device and the characterization of its dynamic mechanical properties. By using this device, we have performed a preliminary study on the effect of dynamic mechanical stimulation in a cell monolayer of an epidermal cell line (HaCaT) studying the effects of 1 Hz and 80 Hz excitation frequencies (in the dynamic stimuli) on HaCaT cell migration, proliferation, and morphology. Our preliminary results indicate that the response of HaCaT is dependent on the frequency of stimulation. The device is economic, easily replicated in other laboratories and can support research for a better understanding of mechanisms mediating cellular mechanotransduction.

The Desmoglein‐1 Transmembrane Domain Drives Lipid Raft Association and Desmosome Assembly

The epidermis is a barrier to the outside world, but mutations in genes encoding desmosomal proteins can inhibit this function, causing human diseases of skin fragility. Severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome is a severe homozygous recessive disorder of skin fragility caused by mutations in the gene encoding desmoglein 1 (DSG1), a component of desmosomes. Desmosomes are junctional complexes that function in mechanical stress resistance across tissues. In these junctions, desmosomal cadherins such as DSG1 interact across extracellular spaces and with intracellular adaptor proteins to link cytoskeletal elements of adjacent cells. Both assembly and disassembly of desmosomes have been shown to rely on lipid raft membrane microdomains, highly ordered cholesterol and sphingolipid‐enriched regions of the plasma membrane important in signaling and trafficking. Our current model predicts that raft association promotes clustering of desmosomal proteins to drive assembly and mediate adhesion. However, the mechanisms of raft association and desmosome assembly are not fully understood. We created a DSG‐null A431 cell line to examine the relationship between DSG raft association and desmosomal adhesion. A431s are an immortal human epidermal carcinoma cell line commonly used in desmosomal studies that express a single DSG species, DSG2. The absence of DSG2 in these cells was confirmed by western blot and immunofluorescence. Further characterization of these DSG‐null A431 cells revealed mislocalized desmoplakin, a marker of mature desmosomes, and a lack of strong cell‐cell adhesion. Exogenous stable expression of GFP‐tagged wildtype DSG1 (DSG1WT‐GFP) rescued both desmosome formation and function, suggesting that these DSG‐null cells would be suitable for probing the relationship between DSG raft association and desmosome assembly and function. Recently, we discovered a novel, heterozygous mutation in the transmembrane domain (TMD)‐encoding region of DSG1 that causes a glycine to arginine substitution and SAM syndrome. DSG1SAM is a lipid raft targeting mutant suggesting an important role for the TMD in raft association. Stable expression of DSG1SAM‐GFP could not rescue desmosome formation or function in the DSG‐null cells, suggesting that a loss of raft association also impacts desmosome assembly and adhesive function. Intriguingly, a chimeric DSG1 protein containing the TMD from E‐cadherin (DSG1Ecad‐GFP), a non‐raft associating adhesive protein, only partly rescued desmosome formation and function. Furthermore, these results correlated with the predicted lipid raft affinity of each TMD, suggesting that desmosome assembly and function is dependent on raft association. We are currently analyzing a panel of GFP‐tagged DSG1TMD variants with differential raft targeting affinities and assessing their ability to support desmosome assembly and function. In conclusion, the TMD targets desmogleins to lipid rafts and modulates desmoglein adhesive function.Support or Funding InformationNIH R01AR050501, NIH R01AR048266

Expression of Concern: “Cadmium Induces Intracellular Ca2+- and H2O2-Dependent Apoptosis through JNK- and p53-Mediated Pathways in Skin Epidermal Cell line”

Expression of Concern: “Cadmium Induces Intracellular Ca2+- and H2O2-Dependent Apoptosis through JNK- and p53-Mediated Pathways in Skin Epidermal Cell line”

Blockade of ROCK inhibits migration of human primary keratinocytes and malignant epithelial skin cells by regulating actomyosin contractility

Actomyosin contractility, crucial for several physiological processes including migration, is controlled by the phosphorylation of myosin light chain (MLC). Rho-associated protein kinase (ROCK) and Myosin light chain kinase (MLCK) are predominant kinases that phosphorylate MLC. However, the distinct roles of these kinases in regulating actomyosin contractility and their subsequent impact on the migration of healthy and malignant skin cells is poorly understood. We observed that blockade of ROCK in healthy primary keratinocytes (HPKs) and epidermal carcinoma cell line (A-431 cells) resulted in loss of migration, contractility, focal adhesions, stress fibres, and changes in morphology due to reduction in phosphorylated MLC levels. In contrast, blockade of MLCK reduced migration, contractile dynamics, focal adhesions and phosphorylated MLC levels of HPKs alone and had no effect on A-431 cells due to the negligible MLCK expression. Using genetically modified A-431 cells expressing phosphomimetic mutant of p-MLC, we show that ROCK dependent phosphorylated MLC controls the migration, focal adhesion, stress fibre organization and the morphology of the cells. In conclusion, our data indicate that ROCK is the major kinase of MLC phosphorylation in both HPKs and A-431 cells, and regulates the contractility and migration of healthy as well as malignant skin epithelial cells.

Targeting Angiogenesis by Blocking the ATM-SerRS-VEGFA Pathway for UV-Induced Skin Photodamage and Melanoma Growth.

Retinoic acid (RA) has been widely used to protect skin from photo damage and skin carcinomas caused by solar ultraviolet (UV) irradiation, yet the mechanism remains elusive. Here, we report that all-trans retinoic acid (tRA) can directly induce the expression of a newly identified potent anti-angiogenic factor, seryl tRNA synthetase (SerRS), whose angiostatic role can, however, be inhibited by UV-activated ataxia telangiectasia mutated (ATM) kinase. In both a human epidermal cell line, HaCaT, and a mouse melanoma B16F10 cell line, we found that tRA could activate SerRS transcription through binding with the SerRS promoter. However, UV irradiation induced activation of ATM-phosphorylated SerRS, leading to the inactivation of SerRS as a transcriptional repressor of vascular endothelial growth factor A (VEGFA), which dampened the effect of tRA. When combined with ATM inhibitor KU-55933, tRA showed a greatly enhanced efficiency in inhibiting VEGFA expression and a much better protection of mouse skin from photo damage. Also, we found the combination greatly inhibited tumor angiogenesis and growth in mouse melanoma xenograft in vivo. Taken together, tRA combined with an ATM inhibitor can greatly enhance the anti-angiogenic activity of SerRS under UV irradiation and could be a better strategy in protecting skin from angiogenesis-associated skin damage and melanoma caused by UV radiation.

3,6-Anhydro-L-galactose increases hyaluronic acid production via the EGFR and AMPKα signaling pathway in HaCaT keratinocytes

BackgroundHyaluronic acid (HA) is an important factor in skin hydration maintenance. In mammalian keratinocytes, hyaluronan synthase 2 (HAS2) is a critical enzyme in HA production. Therefore, the promotion of HAS2 expression in keratinocytes may be a strategy for maintaining skin moisture. ObjectiveThe aim was to determine the skin hydration effect and regulatory mechanisms of 3,6-anhydro-L-galactose (L-AHG), a main component of red macroalgal carbohydrates in human keratinocytes. MethodsL-AHG was applied to an immortalized human epidermal keratinocyte cell line (HaCaT cells). HA production, HAS2 protein and mRNA levels, and the activation of the signaling pathways involved in HAS2 expression were measured. HA levels were also evaluated for three dimensional (3D) reconstructed human skin. ResultsOur results suggest that L-AHG upregulates HA production and may enhance HAS2 expression by activating EGFR-mediated ERK, PI3K/Akt, and STAT3 signaling pathways. We confirmed that L-AHG activated the AMPKα signaling pathway which in turn could regulate HAS2 expression in HaCaT cells. The effects of L-AHG on HA production were observed in the 3D reconstructed human skin model. ConclusionOur results suggest that L-AHG may enhance skin moisture retention by increasing HA synthesis in human epidermal keratinocytes.

The steroid hormone 20-hydroxyecdysone induces phosphorylation and aggregation of stromal interacting molecule 1 for store-operated calcium entry

Oligomerization of stromal interacting molecule 1 (STIM1) promotes store-operated calcium entry (SOCE); however, the mechanism of STIM1 aggregation is unclear. Here, using the lepidopteran insect and agricultural pest cotton bollworm (Helicoverpa armigera) as a model and immunoblotting, RT-qPCR, RNA interference (RNAi), and ChIP assays, we found that the steroid hormone 20-hydroxyecdysone (20E) up-regulates STIM1 expression via G protein-coupled receptors (GPCRs) and the 20E nuclear receptor (EcRB1). We also identified an ecdysone-response element (EcRE) in the 5'-upstream region of the STIM1 gene and also noted that STIM1 is located in the larval midgut during metamorphosis. STIM1 knockdown in larvae delayed pupation time, prevented midgut remodeling, and decreased 20E-induced gene transcription. STIM1 knockdown in a H. armigera epidermal cell line, HaEpi, repressed 20E-induced calcium ion influx and apoptosis. Moreover, 20E-induced STIM1 clustering to puncta and translocation toward the cell membrane. Inhibitors of GPCRs, phospholipase C (PLC), and inositol trisphosphate receptor (IP3R) repressed 20E-induced STIM1 phosphorylation, and we found that two GPCRs are involved in 20E-induced STIM1 phosphorylation. 20E-induced STIM1 phosphorylation on Ser-485 through protein kinase C (PKC), and we observed that Ser-485 phosphorylation is critical for STIM1 clustering, interaction with calcium release-activated calcium channel modulator 1 (Orai1), calcium ion influx, and 20E-induced apoptosis. These results suggest that 20E up-regulates STIM1 phosphorylation for aggregation via GPCRs, followed by interaction with Orai1 to induce SOCE, thereby promoting apoptosis in the midgut during insect metamorphosis.