Normal Epidermal Keratinocytes Research Articles

Unraveling the Mechanisms Involved in the Beneficial Effects of Magnesium Treatment on Skin Wound Healing.

The skin wound healing process consists of hemostatic, inflammatory, proliferative, and maturation phases, with a complex cellular response by multiple cell types in the epidermis, dermis, and immune system. Magnesium is a mineral essential for life, and although magnesium treatment promotes cutaneous wound healing, the molecular mechanism and timing of action of the healing process are unknown. This study, using human epidermal-derived HaCaT cells and human normal epidermal keratinocyte cells, was performed to investigate the mechanism involved in the effect of magnesium on wound healing. The expression levels of epidermal differentiation-promoting factors were reduced by MgCl2, suggesting an inhibitory effect on epidermal differentiation in the remodeling stage of the late wound healing process. On the other hand, MgCl2 treatment increased the expression of matrix metalloproteinase-7 (MMP7), a cell migration-promoting factor, and enhanced cell migration via the MEK/ERK pathway activation. The enhancement of cell migration by MgCl2 was inhibited by MMP7 knockdown, suggesting that MgCl2 enhances cell migration which is mediated by increased MMP7 expression. Our results revealed that MgCl2 inhibits epidermal differentiation but promotes cell migration, suggesting that applying magnesium to the early wound healing process could be beneficial.

Expression and Function of VEGFRs in Normal Epidermis and Psoriatic Lesional Keratinocytes.

The study aimed to explore the expression and function of VEGFRs in normal epidermis and keratinocytes of psoriatic lesions. In this study, the expression and role of VEGFRs in keratinocytes were examined using examples from psoriatic and healthy individuals. The experiment was completed by immunofluorescence analysis, reverse transcription polymerase chain reaction, Western blot, and real-time quantitative RT-PCR after the skin of nonlesional, adjacent, and lesional skin was excised. Observations indicated that in non-lesional psoriatic areas and adjacent lesional areas of the skin of psoriasis patients, the fluorescent signals of VEGFR-1 and VEGFR-2 were strongly labelled with keratinocytes, and in psoriatic lesions, keratinocytes were present throughout the entire thickness of the epidermis, with the exception of the stratum corneum. The distribution of VEGFR-3 in psoriatic nonlesional and adjacent lesional skin was consistent with that in normal epidermis, whereas all layers of the epidermis of psoriatic lesions expressed VEGFR-3. The mRNA expression levels of VEGFR-1,2,3 steadily increased from the normal epidermis to the psoriatic nonlesional, adjacent lesional, and perilesional areas, with the lesional epidermis' keratinocytes exhibiting the greatest levels of mRNA expression. Ca ions upregulate VEGFR-1,2,3 mRNA and protein expression in keratinocytes of nonlesional areas of psoriasis. VEGFRs protein expression and cortical IOD values of psoriatic and normal population cells showed a positive correlation. Hence, in comparison to normal epidermal keratinocytes, psoriatic lesional regions' keratinocytes considerably enhanced their expression of VEGFR-1,2,3 mRNA and protein. The overexpression of VEGFR-1,2,3 in psoriatic lesions may be encouraged by VEGF and Ca þ ions.

Topical Ozone Accelerates Diabetic Wound Healing by Promoting Re-Epithelialization through the Activation of IGF1R–EGFR Signaling

Ozonated oil increases the healing of chronic diabetic wounds, but the underlying mechanisms remain unclear. We investigated the effect of topical ozonated oil on wound healing in mice with diabetes with diet-induced obesity and further elucidated the role of EGFR and IGF1R signaling in diabetic wound healing. We found that topical ozonated oil accelerated wound healing; increased phosphorylation of IGF1R, EGFR, and VEGFR; and improved vascularization at the wound leading edge in mice with diabetes with diet-induced obesity. Exposure of normal epidermal keratinocytes to ozonated medium (20 μM for 2 hours daily) increased cell proliferation and migration distance by increasing phosphorylation of IGF1R and EGFR and downstream phosphoinositide 3-kinase, protein kinase B, and extracellular signal-regulated kinase. These findings shed light on the mechanism for topical ozone action in chronic wounds and support its potential therapeutic application.

Methanolic extract of Kigelia africana and wound healing: an in vitro study.

Kigelia africana (Lam.) Benth. (Bignoniaceae) syn. Kigelia pinnata (Jacq. DC) is a tropical plant that is native to tropical Africa. The aim of this study was to determine if a methanolic extract prepared from Kigelia africana (KAE) can promote wound healing in treated human normal epidermal keratinocyte (HaCaT) cells and human normal foreskin fibroblast cell line (BJ) cells compared with untreated cells. Experimental steps included: the methanolic extraction of the leaf and fruit of the Kigelia africana plant; the preparation of HaCaT and BJ cell lines; cell culture with a stable tetrazolium salt-based proliferation assay; and the evaluation of the wound healing effect of KAE (2μg/ml) in BJ and HaCaT cells. The phytochemical contents of KAE were determined using liquid chromatography quadrupole time-of-flight mass spectrometry. The following molecules were identified as being present in the KAE, among others: cholesterol sulfate; lignoceric acid; embelin; isostearic acid; linoleic acid; dioctyl phthalate; arg-pro-thr; 15-methyl-15(S)-PGE1; sucrose; benzododecinium (Ajatin); and 9-Octadecenamide (oleamide). KAE effected faster wound healing in treated cells compared with untreated cells for both cell lines. HaCaT cells that had been mechanically injured and treated with KAE healed completely in 48 hours compared with 72 hours for untreated HaCaT cells. Treated BJ cells healed completely in 72 hours compared with 96 hours for untreated BJ cells. Concentrations of KAE up to 300μg/ml had a very low cytotoxic effect on treated BJ and HaCaT cells. The experimental data in this study support the potential of KAE-based wound healing treatment to accelerate wound healing.

Bioactivity and molecular docking of lactones isolated from Centaurea pseudosinaica Czerep

Two cytotoxic sesquiterpene lactones, 17-epichlorohyssopifolin A (1) and chlorjanerin (2), and a monoterpene lactone, loliolide (3) were isolated from Centaurea pseudosinaica. The cytotoxicity of the total extract and terpenoids 1–3 were evaluated against three human cancer cells (HepG2, PC-3, and HT-29), along with the human normal primary epidermal keratinocytes (HEKa) cells. With IC50 values ranging between 0.6 ± 0.04 and 5.0 ± 0.61 μg/mL against HepG2; 0.2 ± 0.01 and 11.9 ± 1.31 μg/mL against PC-3, and 0.04 ± 0.013 and 8.9 ± 0.97 μg/mL against HT-29, the total extract, and lactones 1–3 demonstrated cytotoxic effects. Compound 1 displayed the strongest impact on all cancer cells and a slightly safe effect on the normal cells HEKa. Compound 1 caused accumulation of HepG2 and HT-29 cells in G1 phase as displayed cell cycle analysis. On the other hand, the cell distributions were increased in the S phase in PC-3 cells. Furthermore, 1 caused apoptosis in PC-3 and HePG2 cells with 91.50%, and 79.72 %, respectively. A higher fraction of necrotic cells was observed in HT-29 cells amounting to 23.60%. These results suggested that the promising cytotoxicity exhibited by 1 is brought by the apoptosis induction in the cancer cells, which were evaluated. As the compounds showed antiproliferative effect against the HT-29 cells, the docking simulation was performed aiming at determining how they would interact with the EGFR enzyme, whose PDB: 4I23 is considered one of the two distinct wild types of EGFR enzymes. The antibacterial activity results revealed that 3 showed the most remarkable antibacterial effects, especially against the examined Gram-positive bacteria. The total extract exhibited potent activity against all examined bacteria. The total extract showed a potent antifungal effect against two Candida and two Aspergillus pathogens. The antioxidant activity revealed the potency of the total extract and 3 as antioxidant candidates. The obtained results refer to the importance of Centaurea pseudosinaica as a source of potent antiproliferative agents and the whole plant as an antipathogenic and antioxidant agent.

The Role of the Integrated Stress Response in the Biologic Response of Keratinocytes to UVB Exposure

Background and Hypothesis:Ultraviolet B (UVB) irradiation from the sun can disrupt normal epidermal keratinocyte function, leading to the development of skin cancer. The integrated stress response (ISR) is a conserved cellular mechanism against environmental stressors, such as UV irradiation, through the phosphorylation of the alpha subunit of the initiation factor eIF2 (eIF2a-P). Previously, we demonstrated that the ISR kinase GCN2 plays a role in the response of keratinocytes exposed to UV irradiation. However, the role of other components of the ISR in UV-induced stress has not been well elucidated. We hypothesize that in addition to GCN2, other components of ISR such as PERK, GCN1, and ZAK1 are involved in apoptotic pathways induced by prolonged UVB exposure. Methods:Gene-specific knockouts (KO) were generated using CRISPR/Cas9 and validated through immunoblotting. Wild-type (WT) and KO NTERT keratinocytes were irradiated with UVB at 0, 100, 200, and 400J/m2. Six hours post-UVB, cell photomicrographs were taken at 0 and 400J/m2. Measurements for apoptosis were then performed by measuring enzymatic levels of caspase-3. Results:Measurements of caspase-3 following UVB exposure on WT NTERT, PERK-KO, and GCN2-KO showed no difference between the groups. GCN1-KO had significantly greater caspase-3 activity at 400J/m2, whereas ZAK1-KO had significantly reduced caspase-3 activity at the same concentration. GCN2/PERK double-KO had significantly increased caspase-3 activity at 400 J/m2 compared to the individual KO cells. Conclusion and Potential Impact:Collectively, our data suggests that the ISR may be involved in the apoptotic responses that ensue in keratinocytes exposed to UVB. How GCN2 and PERK could co-interact with GCN1 or ZAK1 to facilitate apoptotic fate in keratinocytes still warrants further research. Nevertheless, elucidating the role of the ISR in normal keratinocyte response to UV irradiation will allow us to define novel therapeutic targets for the treatment or prevention of UV-related skin malignancies.

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

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

Differential Regulation of Integrin α5 and β4 in Normal and Psoriatic Epidermal Keratinocytes

Differential Regulation of Integrin α5 and β4 in Normal and Psoriatic Epidermal Keratinocytes

Role of TRPV4 in matrix stiffness-induced expression of EMT-specific LncRNA.

Long non-coding RNAs (LncRNAs) are long (> 200 bases), non-coding, single-stranded RNAs that have emerged as major regulators of gene expression, cell differentiation, development, and oncogenesis. In view of the fact that matrix stiffness plays a role in cellular functions associated with these processes, it is important to ask what role matrix stiffness plays in regulating expression of LncRNAs. In this report, we show that (i) matrix stiffness causes differential expression of epithelial-mesenchymal transition (EMT)-related LncRNAs and mRNAs in primary mouse normal epidermal keratinocytes, (ii) differential expression of EMT-related LncRNAs and mRNAs occurs in response to combined stimulation of transforming growth factor β1 and matrix stiffness, and (iii) transient receptor potential (TRP) channel of the vanilloid subfamily, TRPV4, a matrix stiffness-sensitive ion channel, plays a role in differential expression of EMT-related LncRNAs and mRNAs in response to combined stimulation by TGFβ1 and matrix stiffness. These data identify TRPV4 as a candidate plasma membrane mechanosensor that transmits matrix-sensing signals essential to LncRNA expression. Our results also show that we have established and validated an assay system capable of discovering novel LncRNAs and mRNAs sensitive to matrix stiffening.

Enzymes C1r and C1s may show progression of a type of skin cancer called keratinocyte‐derived cutaneous squamous cell carcinoma

The incidence of skin cancers is increasing worldwide. The epidermis is the surface layer of the skin and the main cell type is the keratinocyte. Keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic (meaning it can spread) skin cancer and it is responsible for 20% of skin cancer related deaths. The main risk factor for cSCC includes long term exposure to sun ultraviolet radiation. The progression of cSCC is as follows: it starts in a premalignant form (not yet cancerous) called actinic keratosis (AK), then becomes locally restricted cSCC, then cSCC in situ (cSCCIS), and eventually becomes invasive and metastatic cSCC. This research, from Finland, aimed to find biomarkers (chemicals in the body which act as signs of a disease) which could predict the progression and aggressiveness of cSCC. The authors found significantly elevated levels of mRNA (messenger molecules) for enzymes called C1r and C1s (which play a role in our immune system), in cSCC cells and tumors compared to normal epidermal keratinocytes and normal skin, respectively. Abundant expression of C1r and C1s by tumor cells was detected in invasive cSCCs and in an aggressive form of cSCC (recessive dystrophic epidermolysis bullosa-associated cSCC), whereas the expression of C1r and C1s was lower in cSCCIS, actinic keratosis, and normal skin tissue samples. The roles of C1r and C1s in cSCC cells were investigated by knocking down (reducing) the expression of C1r and C1s, and through complex methods, this was shown to potentially suppress growth of cSCC tumours. These results provide evidence that tumor cell-derived C1r and C1s play a part in the progression of cSCC and identify them as biomarkers and possible treatment targets in cSCC. This is a summary of the study: Tumour-cell-derived complement components C1r and C1s promote growth of cutaneous squamous cell carcinoma

Fibroblast growth factor receptor promotes progression of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte-derived invasive and metastatic tumor of the skin. It is the second-most commonly diagnosed form of skin cancer striking 200 000 Americans annually. Further, in organ transplant patients, there is a 65- to 100-fold increased incidence of cSCC compared to the general population. Excision of cSCC of the head and neck results in significant facial disfigurement. Therefore, increased understanding of the mechanisms involved in the pathogeneses of cSCC could identify means to prevent, inhibit, and reverse this process. In our previous studies, inhibition of fibroblast growth factor receptor (FGFR) significantly decreased ultraviolet B-induced epidermal hyperplasia and hyperproliferation in SKH-1 mice, suggesting an important role for FGFR signaling in skin cancer development. However, the role of FGFR signaling in the progression of cSCC is not yet elucidated. Analysis of the expression of FGFR in cSCC cells and normal epidermal keratinocytes revealed protein overexpression and increased FGFR2 activation in cSCC cells compared to normal keratinocytes. Further, tumor cell-specific overexpression of FGFR2 was detected in human cSCCs, whereas the expression of FGFR2 was low in premalignant lesions and normal skin. Pretreatment with the pan-FGFR inhibitor; AZD4547 significantly decreased cSCC cell-cycle traverse, proliferation, migration, and motility. Interestingly, AZD4547 also significantly downregulated mammalian target of rapamycin complex 1 and AKT activation in cSCC cells, suggesting an important role of these signaling pathways in FGFR-mediated effects. To further bolster the in vitro studies, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice with SCC12A tumor xenografts treated with AZD4547 (15 mg/kg/bw, twice weekly oral gavage) exhibited significantly decreased tumor volume compared to the vehicle-only treatment group. The current studies provide mechanistic evidence for the role of FGFR and selectively FGFR2 in the early progression of cSCC and identifies FGFR as a putative therapeutic target in the treatment of skin cancer.

THE CYTOSKELETAL ALTERATION MODULATES CELL INVASIVENESS OF OSCC CELLS THROUGH RHOA-YAP SIGNALING IN STROMAL fiBROBLASTS

Objectives Cancer-associated fibroblasts (CAFs) are most abundant stromal cells among tumor microenvironment that participate in carcinogenesis. This study aimed to investigate the mechanism of cytoskeletal alteration of CAFs and its role in carcinogenesis of oral squamous cell carcinoma (OSCC). Findings We first evaluated if immortalized normal fibroblasts(hTERT-hNOFs) can be substituted for CAFs. hTERT- hNOFs co-cultured with OSCC cells showedmyofibroblastic and senescent phenotypeslike CAFs.Next, we observed the cytoskeletal alteration in hTERT-hNOFs co-cultured with OSCC cells, including enlarged cellular size, distinct F-actin assembly (stress fibers). To further understand the mechanisms, we identified the expression of RhoGT- Pase gene family. Among them, RhoA was significantly increased. These results were confirmed by RhoA-ROCK inhibitor(Y27632). In spite of fibroblasts grown with OSCC cells, Y27632 reduced cell size and stress fibers. Furthermore,YAP distribution, as a downstream transcriptional factor of RhoA, was examined. YAP was mainly localized at nucleus in hTERT-hNOF co-cultured with OSCC cells, unlike hTERT-hNOFs co-cultured with HEK(human normal epidermal keratinocyte). To further verify if RhoA and cytoskeletal change modulate YAP distribution, Actin polymerization inhibitor(Lat.A) and Y27632 were used. As results, the inhibitors interrupted nuclear YAP localization, suggesting that YAP can be regulated by RhoA-induced cytoskeletal alteration. Lastly, we examined if nuclear YAP localization of fibroblasts exacerbates OSCC progression. YAPS127A mutant fibroblasts, maintained in nuclear YAP, were generated. As results, YAPS127A showed cytoskeletal rearrangement, such as increased gel contractility and matrix stiffness, and thereby enhanced the invasiveness of OSCC cells. Conclusions The alteration of tumor microenvironment, such as cytoskeletal change and matrix remodeling via RhoA-YAP in CAFs, modulates OSCC progression. These understandings will provide the novel approaches for CAFs-based OSCC therapy.

158 Triggering immortalized keratinocyte growth arrest by protein kinase C delta activation

Normal epidermal keratinocyte (KC) differentiation is marked by irreversible cell cycle exit and a step-wise transition through the suprabasal layers of the epidermis. In contrast, KCs in both actinic keratosis and squamous cell carcinomas (SCCs) continue to proliferate in the suprabasal compartment, indicating a defect in their growth arrest signaling. Activation of multiple protein kinase C (PKC) isoforms have been associated with KC cell cycle withdrawal. To study the involvement of PKC in KC growth arrest, we evaluated the effects of the PKC agonist 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on normal human epidermal keratinocytes (NHEKs) and HaCaT cells, an immortalized, non-tumorigenic human keratinocyte cell line. Treating NHEKs with 10 nM TPA for as little as 15 minutes induced complete growth arrest over a 7 day period while untreated KCs underwent a 93-fold increase in cell number (p<0.005). 10 nM TPA treatment of HaCaT cells did not induce any growth arrest, indicating that immortalized KCs have defective PKC-dependent growth arrest signaling. To test if PKCδ activation was able to enforce growth arrest in HaCaT cells, we transduced HaCaT cells with retroviruses encoding either EGFP or a PKCδ-EGFP fusion and sorted EGFP positive cells by flow cytometry. HaCaT-EGFP cells behaved similar to HaCaTs and were not growth arrested by TPA treatment. In contrast, HaCaT-PKCδ-EGFP treated with TPA increased in cell number only 3-fold compared to a 15-fold increase in untreated HaCaT-PKCδ-EGFP over a 4 day growth assay. Activation of PKCδ in HaCaT-PKCδ-EGFP cells was confirmed by observing TPA-induced translocation of PKCδ-EGFP to the plasma membrane and increased S299 autophosphorylation. TPA-induced growth inhibition in HaCaT-PKCδ-EGFP cells was due to arrest in G1 and G2/M, not apoptosis. Global phosphoproteomics are being employed to identify PKCδ signaling pathways that can overcome the defective growth arrest in immortalized keratinocytes and may have therapeutic benefit for the treatment of actinic keratosis and SCCs.

Preparation and investigation of in vitro cytotoxic activity of pH-sensitive liposomes with sanguinarine

Sanguinarine is a benzophenanthridine alkaloid of plant origin showing antitumour, anti-inflammatory and a number of other activities. To increase the effectiveness of its therapeutic effect, sanguinarine was introduced into the pH-sensitive liposomes by the remote loading method using ammonium citrate. The average diameter of the obtained liposomes was equal to 144.8 ± 2.3 nm; zeta potential was equal to -16.3 ± 1.5 mV, the effectiveness of sanguinarine inclusion in liposomes was 68.7%. The release of sanguinarine from liposomes was prolonged; the lowest level of release was observed in a medium with a pH of 7.4, the highest at pH 5.5, which may be useful in preventing the release of the active substance in the blood stream and, conversely, in activating its release in the tumour growth zone. The cytotoxic activity of liposomal sanguinarine against cells of human squamous carcinoma A431 (IC50 9.5 μM) was significantly higher than its activity against the normal epidermal keratinocytes (NHEK) (IC50 25.1 μM). The drug also caused an effective dose-dependent induction of apoptosis in tumour cells of A431, while not affecting normal cells. Thus, liposomal sanguinarine can be considered as a promising antitumour agent acting on tumour cells by inducing apoptosis and exhibiting low cytotoxicity to normal human cells.

TRPV4 regulates matrix stiffness and TGFβ1-induced epithelial-mesenchymal transition.

Substrate stiffness (or rigidity) of the extracellular matrix has important functions in numerous pathophysiological processes including fibrosis. Emerging data support a role for both a mechanical signal, for example, matrix stiffness, and a biochemical signal, for example, transforming growth factor β1 (TGFβ1), in epithelial‐mesenchymal transition (EMT), a process critically involved in fibrosis. Here, we report evidence showing that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive channel, is the likely mediator of EMT in response to both TGFβ1 and matrix stiffness. Specifically, we found that: (a) genetic ablation or pharmacological inhibition of TRPV4 blocked matrix stiffness and TGFβ1‐induced EMT in normal mouse primary epidermal keratinocytes (NMEKs) as determined by changes in morphology, adhesion, migration and alterations of expression of EMT markers including E‐cadherin, N‐cadherin (NCAD) and α‐smooth muscle actin (α‐SMA), and (b) TRPV4 deficiency prevented matrix stiffness‐induced EMT in NMEKs over a pathophysiological range. Intriguingly, TRPV4 deletion in mice suppressed expression of mesenchymal markers, NCAD and α‐SMA, in a bleomycin‐induced murine skin fibrosis model. Mechanistically, we found that: (a) TRPV4 was essential for the nuclear translocation of YAP/TAZ (yes‐associated protein/transcriptional coactivator with PDZ‐binding motif) in response to matrix stiffness and TGFβ1, (b) TRPV4 deletion inhibited both matrix stiffness‐ and TGFβ1‐induced expression of YAP/TAZ proteins and (c) TRPV4 deletion abrogated both matrix stiffness‐ and TGFβ1‐induced activation of AKT, but not Smad2/3, suggesting a mechanism by which TRPV4 activity regulates EMT in NMEKs. Altogether, these data identify a novel role for TRPV4 in regulating EMT.

Effects of insulin-like growth factor binding protein 3 on apoptosis of cutaneous squamous cell carcinoma cells.

BackgroundCutaneous squamous cell carcinoma (CSCC) is the second most common carcinoma worldwide. Clinical treatment for patients with CSCC remains non-ideal. Insulin-like growth factor binding protein 3 (IGFBP3), a member of the insulin-like growth (IGF) system, participates in several biological processes, including cellular proliferation and apoptosis. Here, we explored the functional role of IGFBP3 in apoptosis and proliferation of A431 cells, a human CSCC cell line.Materials and methodsDifferential expression analysis, immunohistochemistry, immunoblotting, TUNEL assay, and CCK8 assay techniques were used to investigate the IGFBP3 expression levels in both A431 cells and CSCC tissue surgically obtained from humans as well as to explore the functional role of IGFBP3 in the apoptosis and proliferation of A431 cells.ResultsBy using normal epidermal keratinocytes for comparison, we identified the top 10 ranked differentially upregulated genes expressed in human cutaneous squamous cell carcinoma cell lines. Among these 10 genes, IGFBP3 was ranked number 1. By using immunohistochemistry, we found that the expression level of IGFBP3 was significantly elevated in CSCC tissue compared with that in normal human skin tissue. Knockdown of IGFBP3 in A431 cells by transfection with IGFBP3-specific siRNA markedly altered the expression of proteins that contribute to apoptosis via mitochondrial pathways, significantly suppressing the expression of Bax and active caspase-3, while significantly increasing B-cell lymphoma-2 expression. TUNEL assay confirmed the effect of knockdown of IGFBP3 on the apoptosis as well. In addition, knockdown of IGFBP3 inhibited the proliferation of A431 cells.ConclusionIGFBP3 is overexpressed in both CSCC cell lines and tissue. Knockdown of IGFBP3 enhanced the apoptosis via a mitochondrial pathway and inhibited the proliferation of A431 cells. These findings indicate that IGFBP3 may be a biomarker and a potential therapeutic target for CSCC.

Transcription and microRNA Profiling of Cultured Human Tympanic Membrane Epidermal Keratinocytes.

The human tympanic membrane (TM) has a thin outer epidermal layer which plays an important role in TM homeostasis and ear health. The specialised cells of the TM epidermis have a different physiology compared to normal skin epidermal keratinocytes, displaying a dynamic and constitutive migration that maintains a clear TM surface and assists in regeneration. Here, we characterise and compare molecular phenotypes in keratinocyte cultures from TM and normal skin. TM keratinocytes were isolated by enzymatic digestion and cultured in vitro. We compared global mRNA and microRNA expression of the cultured cells with that of human epidermal keratinocyte cultures. Genes with either relatively higher or lower expression were analysed further using the biostatistical tools g:Profiler and Ingenuity Pathway Analysis. Approximately 500 genes were found differentially expressed. Gene ontology enrichment and Ingenuity analyses identified cellular migration and closely related biological processes to be the most significant functions of the genes highly expressed in the TM keratinocytes. The genes of low expression showed a marked difference in homeobox (HOX) genes of clusters A and C, giving the TM keratinocytes a strikingly low HOX gene expression profile. An in vitro scratch wound assay showed a more individualised cell movement in cells from the tympanic membrane than normal epidermal keratinocytes. We identified 10 microRNAs with differential expression, several of which can also be linked to regulation of cell migration and expression of HOX genes. Our data provides clues to understanding the specific physiological properties of TM keratinocytes, including candidate genes for constitutive migration, and may thus help focus further research.

TRPV4 channels regulates matrix stiffness and TGFβ1‐induced epithelial‐mesenchymal transition

Epithelial‐mesenchymal transition (EMT) has important functions in cellular processes including development, wound healing, oncogenesis, and fibrosis. Emerging data support a role for both a mechanical signal, and a biochemical signal, e.g., transforming growth factor β1 (TGFβ1), in EMT. Here, we report evidence showing that transient receptor potential vanilloid 4 (TRPV4), calcium‐permeable channel and member of TRP superfamily, is the likely mediator of EMT in response to both TGFβ1 and matrix stiffness. Specifically, we found that: i) genetic ablation of TRPV4 blocked TGFβ1‐induced EMT in normal mouse primary epidermal keratinocytes (NMEKs) as determined by changes in morphology and alterations of expression of EMT markers including E‐cadherin (ECAD), N‐cadherin (NCAD), and α‐smooth muscle actin (α‐SMA), and ii) TRPV4 deficiency prevented matrix stiffness‐induced EMT in NMEKs over a pathophysiological range. Intriguingly, TRPV4 deletion in mice suppressed expression of mesenchymal markers, NCAD and α‐SMA, in a bleomycin‐induced murine skin fibrosis model. We found an increased co‐localization of TRPV4 with NCAD, and decreased co‐localization of TRPV4 with epithelial marker ECAD in skin tissues of bleomycin‐treated wild‐type mice compared to saline controls. Mechanistically, we found that: i) TRPV4 was essential for the nuclear translocation of YAP/TAZ (Yes‐associated protein/transcriptional coactivator with PDZ‐binding motif) in response to matrix stiffness and TGFβ1, ii) TRPV4 deletion inhibited both matrix stiffness‐ and TGFβ1‐induced expression of YAP/TAZ proteins, and iii) TRPV4 deletion abrogated both matrix stiffness‐ and TGFβ1‐induced activation of AKT, but not Smad2/3, suggesting a mechanism by which TRPV4 activity regulates EMT in NMEKs. Altogether, these data identify a novel role for TRPV4 in regulating EMT, and thus suggest that therapeutic targeting of TRPV4 may provide a selective approach to ameliorate the development of skin fibrosis and oncogenesis.Support or Funding InformationAHA (13SDG17310007), Startup grant from University of Maryland, NIH (1R01EB024556‐01), and NSF (CMMI‐1662776) grants to SOR.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Green Tea Root Is a Potential Natural Surfactant and Is Protective against the Detrimental Stimulant PM2.5 in Human Normal Epidermal Keratinocytes

Green tea (Camellia sinensis L.) has been widely explored for its medicinal applications. However, most of the studies had targeted the green tea leaf, while other parts remained unexplored. In this study, protective effect of green tea root extract on Normal Human Epidermal Keratinocytes (NHEKs) against the damage induced by an external stimulant (PM2.5) was confirmed. Thirty-year-old green tea root samples were collected from Amorepacific’s Dolsongi tea field and green tea root extract was prepared with 70% ethanol. Total crude saponin content in green tea root extract was 54%, which is much higher than that in ginseng extract. Our results suggest that green tea root extract can be used as a natural surfactant in cosmetics. For evaluating its protective effect against the damage induced by PM2.5, IL-36G was used as a biomarker. IL-36G mRNA expression level increased remarkable upon PM2.5 treatment in NHEKs. Moreover, IL-36G was recently reported to be expressed in psoriasis lesions. Results showed significant decrease of IL-36G expression by treatment of green tea root extract. In conclusion, thirty-year-old green tea root extract can be used as a natural surfactant with a high saponin content and may have protective effect against the damage induced by PM2.5.

Reduced SMAD2/3 activation independently predicts increased depth of human cutaneous squamous cell carcinoma.

The incidence of cutaneous squamous cell carcinoma (cSCC) is rising. Whilst the majority are cured surgically, aggressive metastatic cSCC carry a poor prognosis. Inactivating mutations in transforming growth factor beta (TGF-β) receptors have been identified amongst genetic drivers of sporadic tumours and murine models of cSCC, suggesting a tumour suppressor function for TGF-β in normal skin. However, paradoxically, TGF-β acts as a tumour promoter in some murine model systems. Few studies have analysed the role of TGF-β/activin signalling in human normal skin, hyper-proliferative skin disorders and cSCC. Antibodies recognising phospho-SMAD proteins which are activated during canonical TGF-β/activin signalling were validated for use in immunohistochemistry. A tissue microarray comprising FFPE lesional and perilesional tissue from human primary invasive cSCC (n=238), cSCC in-situ (n=2) and keratocanthoma (n=9) were analysed in comparison with tissues from normal human scalp (n=10). Phosphorylated SMAD2 and SMAD3 were detected in normal interfollicular epidermal keratinocytes and were also highly localised to inner root sheath, matrix cells and Keratin 15 positive cells. Lesional cSCC tissue had significantly reduced activated SMAD2/3 compared to perilesional tissue, consistent with a tumour suppressor role for SMAD2/3 activators in cSCC. Increased cSCC tumour thickness inversely correlated with the presence of phospho-SMADs in tumour tissue suggesting that a reduction in canonical TGF-β/activin signalling may be associated with disease progression.