Confluent Keratinocytes Research Articles

Recombinant Human Keratinocyte Growth Factor Ameliorates Cancer Treatment-Induced Oral Mucositis on a Chip.

Oral mucositis (OM) is a severe complication of cancer therapies caused by off-target cytotoxicity. Palifermin, which is recombinant human keratinocyte growth factor (KGF), is currently the only mitigating treatment available to a subset of OM patients. This study used a previously established model of oral mucositis on a chip (OM-OC) comprised of a confluent human gingival keratinocytes (GIE) layer attached to a basement membrane-lined subepithelial layer consisting of human gingival fibroblasts (HGF) and human dermal microvascular endothelial cells (HMEC) on a stable collagen I gel. Cisplatin, radiation, and combined treatments are followed by a recovery period in the OM-OC to determine possible cellular and molecular mechanisms of OM under effects of KGF. Cancer treatments affected the keratinocyte layer, causing death and epithelial barrier loss. Both keratinocytes and subepithelial cells died rapidly, as evidenced by propidium iodide staining. In response to radiation exposure, cell death occurred in the apical epithelial layer, predominantly, within 24h. Cisplatin exposure predominantly promoted death of basal epithelial cells within 32-36h. Presence of KGF in OM-OC protected tissues from damage caused by cancer treatments in a dose-dependent manner, being more effective at 10 ng/mL. As verified by F-actin staining and the Alamar Blue assay, KGF contributed to epithelial survival and induced proliferation of GIE and HGF as well as HMEC within 120h. When the expression of eighty inflammatory cytokines is evaluated at OM induction (Day 12) and resolution (Day 18) stages in OM-OC, some cytokines are identified as potential novel therapeutic targets. In comparison with chemoradiation exposure, KGF treatment showed a trend to decrease IL-8 and TNF-a expression at Day 12 and 18, and TGF-β1 at Day 18 in OM-OC. Taken together, these findings support the utility of OM-OC as a platform to model epithelial damage and evaluate molecular mechanisms following OM treatment.

Engineering of Uniform Epidermal Layers via Sacrificial Gelatin Bioink-Assisted 3D Extrusion Bioprinting of Skin.

To reconstruct an ideal full-thickness skin model, basal keratinocytes must be distributed as a confluent monolayer on the dermis. However, the currently available extrusion bioprinting method for the skin is limited when producing an air-exposed cellular monolayer because the cells are encapsulated within a bioink. This is the first study to use sacrificial gelatin-assisted extrusion bioprinting to reproduce a uniform and stratified epidermal layer. Experimental analyses of the rheological properties, printability, cell viability, and initial keratinocyte adhesion shows that the optimal gelatin bioink concentration is 4wt.%. The appropriate thickness of the bioprinted gelatin structure for achieving a confluent keratinocyte layer is determined to be 400µm. The suggested strategy generates a uniform keratinocyte monolayer with tight junctions throughout the central and peripheral regions, whereas manual seeding generates non-uniform cellular aggregates and vacancies. These results influence gene expression, exhibiting a propensity for epidermal differentiation. Finally, the gelatin-assisted keratinocytes are bioprinted onto a dermis composed of gelatin methacryloyl and dermis-derived decellularized extracellular matrix to establish a full-thickness skin model. Thus, this strategy leads to significant improvements in epidermal differentiation/stratification. The findings demonstrate that the gelatin-assisted approach is advantageous for recreating reliable full-thickness skin models with significant consistency for mass production.

ARHGAP29 Regulates Keratinocyte Migration through the RhoA/ROCK Pathway

Genetic variants in Rho GTPase Activating Protein 29 (ARHGAP29) have been associated with non‐syndromic cleft lip and/or palate, one of the most common human birth defects. Orofacial clefting results from the failure of facial processes to fuse during development which can be caused by defects in proliferation, adhesion, and/or migration. ARHGAP29 is widely expressed and preferentially interacts with the small GTPase RhoA, a critical modulator of the actin cytoskeleton required for cell morphology and migration. Therefore, we hypothesized that ARHGAP29 would play a critical role in keratinocyte migration. To test this hypothesis, we knocked down ARHGAP29 in a human keratinocyte cell line using both shRNA and CRISPR technologies. To assess migration, we used an in vitro scratch assay on a confluent keratinocyte monolayer and followed scratch closure over 24 hours. ARHGAP29 knockdown keratinocytes displayed significantly increased cell area and significant delays in scratch closure. By tracing single cells throughout the duration of scratch closure, we found that ARHGAP29 knockdown cells were significantly slower and showed defects in directionality that were rescued with a ROCK inhibitor, Y‐27632, suggesting these migration defects are downstream of the RhoA pathway. Collectively, our results demonstrate that ARHGAP29 regulates keratinocyte morphology and promotes migration speed and directionality through the RhoA/ROCK pathway, providing potential insight into its importance in craniofacial development.

Camphorquinone alters the expression of extracellular proteases in a 3D co-culture model of the oral mucosa

ObjectiveObjective of our investigation was to determine the influence of CQ on the expression of antioxidant proteins and extracellular proteases in a 3D co-culture model (3DCCM) of the oral mucosa and to analyze the distribution and stability of CQ within 3D-CCMs. Methods3D-CCMs consist of confluent keratinocytes (OKF6/TERT2) on cell culture inserts on top of human gingival fibroblasts (HGFs) in collagen. The treatment was carried out by adding CQ to the cell culture inserts at two time points with declining concentrations. Mass spectrometry was used to analyze the CQ concentration above and underneath the OKF6/TERT2-layer. The expression of antioxidant genes was analyzed by qRT-PCR and western blot. The regulation of extracellular proteases from different families was analyzed by qRT-PCR and Proteome Profiler arrays. ResultsGC/MS analysis showed that CQ was evenly distributed within the model. Heme oxygenase-1, NAD(P)H quinone dehydrogenase 1 (NQO1), and superoxide dismutase 1 were induced on the mRNA and protein level in OKF6/TERT2 cells. In HGFs, only the transcription of NQO1 was induced. The transcription of extracellular proteases was increased mainly in OKF6/TERT2 cells 72 h after the initial treatment. The quantity of ten out of 25 analyzed extracellular proteases in the cell culture supernatant above and six underneath the keratinocyte-layer were modulated by CQ. SignificanceDespite its high reactivity, CQ is able to penetrate a dense keratinocyte-layer, presumably across plasma membranes. CQ initially induced the cellular defense machinery against oxidative stress and altered the expression of extracellular proteases. We assume a relationship between both processes.

Scratching Counteracts IL-13 Signaling by Upregulating the Decoy Receptor IL-13Rα2 in Keratinocytes.

The vicious itch–scratch cycle is a cardinal feature of atopic dermatitis (AD), in which IL-13 signaling plays a dominant role. Keratinocytes express two receptors: The heterodimeric IL-4Rα/IL-13Rα1 and IL-13Rα2. The former one transduces a functional IL-13 signal, whereas the latter IL-13Rα2 works as a nonfunctional decoy receptor. To examine whether scratch injury affects the expression of IL-4Rα, IL-13Rα1, and IL-13Rα2, we scratched confluent keratinocyte sheets and examined the expression of three IL-13 receptors using quantitative real-time PCR (qRT-PCR) and immunofluorescence techniques. Scratch injuries significantly upregulated the expression of IL13RA2 in a scratch line number-dependent manner. Scratch-induced IL13RA2 upregulation was synergistically enhanced in the simultaneous presence of IL-13. In contrast, scratch injuries did not alter the expression of IL4R and IL13RA1, even in the presence of IL-13. Scratch-induced IL13RA2 expression was dependent on ERK1/2 and p38 MAPK signals. The expression of IL-13Rα2 protein was indeed augmented in the scratch edge area and was also overexpressed in lichenified lesional AD skin. IL-13 inhibited the expression of involucrin, an important epidermal terminal differentiation molecule. IL-13-mediated downregulation of involucrin was attenuated in IL-13Rα2-overexpressed keratinocytes, confirming the decoy function of IL-13Rα2. Our findings indicate that scratching upregulates the expression of the IL-13 decoy receptor IL-13Rα2 and counteracts IL-13 signaling.

Cyto/chemokine profile of in vitro scratched keratinocyte model: Implications of significant upregulation of CCL20, CXCL8 and IL36G in Koebner phenomenon

BackgroundScratch injury induces Koebner phenomenon in psoriasis. Smoking is also a risk factor for psoriasis. Keratinocytes can produce various psoriasis-related molecules including TNF, IL1 A, IL1B, IL6, IL12B, IL17C, IL23 A, IL36 A, IL36B, IL36 G, CXCL1, CXCL2, CXCL8, CXCL9, CXCL10, CCL20, IFNB, and CAMP. However, the scratch-induced molecular profiling remains elusive. ObjectiveTo profile the induction pattern of above-mentioned psoriasis-related and keratinocyte-derived molecules by scratch injury in the presence or absence of anti-psoriatic drugs or benzo[a]pyrene, a major environmental pollutant of tobacco smoke. MethodsConfluent normal human keratinocytes were scratched and molecules were assayed by qRT-PCR, ELISA and Western blotting with or without drugs and benzo[a]pyrene. ResultsAmong the 18 molecules, the scratch injury on a confluent keratinocyte sheet significantly and selectively upregulated the mRNA expression of four cyto/chemokines, CXCL8, CCL20, IL36G, and TNF, in a scratch-line-number-dependent manner under either low- or high-calcium condition. However, significant protein secretion was only demonstrated for CXCL8 and CCL20. The IL36 G protein was not secreted, but its intracellular level was significantly upregulated by scratch injury, whereas neither the secretion nor the intracellular level of TNF protein was affected by scratch injury. Dexamethasone, but not maxacalcitol nor the phosphodiesterase 4 inhibitor apremilast, partially inhibited the CXCL8 and CCL20 secretion. Benzo[a]pyrene significantly and synergistically enhanced the scratch-induced CCL20 secretion that may explain why smoking is a risk factor for psoriasis. ConclusionCCL20 and to a less extent CXCL8 may play a key role in triggering the Koebner phenomenon after scratch injury to keratinocytes.

Skin renewal activity of non-thermal plasma through the activation of \u03b2-catenin in keratinocytes

For recent years, devices that generate non-thermal plasma (NTP) have been introduced into the field of dermatology. Since NTP has demonstrated strong anti-pathogenic activity with safety of use, NTP was first applied to sterilize the skin surface to aid in the healing of various kinds of skin diseases. However, the effect of NTP on skin regeneration has not yet been fully explored. In this study, the effect of NTP on the growth of keratinocytes was tested using the HaCaT human keratinocyte cell line and HRM2 hairless mice. Treatment with NTP allowed confluent keratinocytes to escape from G1 cell cycle arrest and increased the proportion of cells in S and G2 phases. In particular, NTP treatment immediately dispersed E-cadherin-mediated cell-to-cell interactions, resulting in the translocation of β-catenin to the nucleus and leading to the enhanced transcription of target genes including c-MYC and cyclin D1. Moreover, repeated treatment of the mice with NTP also stimulated epidermal expansion by activating β-catenin in the epidermal cells. The symptoms of cellular DNA damage were not detected after NTP treatment. Taken together, these results demonstrate that NTP may be employed as a new type of skin regenerating device.

Actomyosin contractility provokes contact inhibition in E-cadherin-ligated keratinocytes

Confluence-dependent inhibition of epithelial cell proliferation, termed contact inhibition, is crucial for epithelial homeostasis and organ size control. Here we report that among epithelial cells, keratinocytes, which compose the stratified epithelium in the skin, possess a unique, actomyosin-dependent mechanism for contact inhibition. We have observed that under actomyosin-inhibited conditions, cell-cell contact itself through E-cadherin promotes proliferation of keratinocytes. Actomyosin activity in confluent keratinocytes, however, inhibits nuclear localization of β-catenin and YAP, and causes attenuation of β-catenin- and YAP-driven cell proliferation. Confluent keratinocytes develop E-cadherin-mediated punctate adhesion complexes, to which radial actin cables are connected. Eliminating the actin-to-E-cadherin linkage by depleting α-catenin increases proliferation of confluent keratinocytes. By contrast, enforced activation of RhoA-regulated actomyosin or external application of pulling force to ligated E-cadherin attenuates their proliferation, suggesting that tensile stress at E-cadherin-mediated adhesion complexes inhibits proliferation of confluent keratinocytes. Our results highlight actomyosin contractility as a crucial factor that provokes confluence-dependent inhibition of keratinocyte proliferation.

Drug reaction with eosinophilia and systemic symptoms (DRESS): clinicopathological study of 45 cases.

Drug reaction with eosinophilia and systemic symptoms (DRESS) is a rare and severe adverse drug reaction. Large detailed studies of histopathological features of DRESS are sparse and suggest an association between keratinocyte damage and the severity of visceral involvement. To describe the dermatopathological features in a large series of DRESS and their possible association with clinical features and the severity of the disease. A retrospective analysis of the clinicobiological and dermatopathological features in a monocentric cohort of patients with DRESS. From January 2005 to January 2013, 45 patients were validated as probable or definite cases of DRESS. The median age was 64years (range 3-87). The most frequent clinical and biological features included: fever ≥38.5°C (95%), facial oedema (72%), enlarged lymph nodes (51%), visceral involvement (75%), blood eosinophilia (97%) and atypical lymphocytes (82%). Severe DRESS occurred in 24% and a fatal outcome in 6% of patients. Histopathological analysis showed that no specific histopathological pattern was characteristic for DRESS. However, several changes in different cutaneous compartments were observed in 2 of 3 of cases. Spongiosis (55%) and keratinocyte damage (53%) were the most common epidermal changes. Spongiosis was associated with non-severe DRESS (P=0.041) whereas confluent keratinocyte necrosis correlated with severe DRESS (P=0.011). Vascular changes were frequent (88%). A moderate dermal perivascular lymphocytic infiltrate was invariably present, containing eosinophils, neutrophils and/or atypical lymphocytes in 57% of cases. Epidermal changes are indicative for the severity of DRESS.

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

Topical androgen antagonism promotes cutaneous wound healing without systemic androgen deprivation by blocking β‐catenin nuclear translocation and cross‐talk with TGF‐β signaling in keratinocytes

Orchidectomy in rodents and lower testosterone levels in men are associated with improved cutaneous wound healing. However, due to the adverse effects on skeletal and sexual tissues, systemic androgen blockade is not a viable therapeutic intervention. Accordingly, we tested the hypothesis that topical application of an androgen antagonist would elicit accelerated wound healing without systemic androgen antagonism. Full-thickness cutaneous wounds were created on adult C57BL6/J mice. Daily topical application of androgen receptor antagonist, flutamide, resulted in improved gap closure similar to orchiectomized controls and faster than orchidectomized mice treated with topical testosterone. In vivo data showed that the effects of androgen antagonism on wound closure primarily accelerate keratinocytes migration without effecting wound contraction. Consequently, mechanisms of testosterone action on reepithelialization were investigated in vitro by scratch wounding assays in confluent keratinocytes. Testosterone inhibited keratinocyte migration and this effect was in part mediated through promotion of nuclear translocation of β-catenin and by attenuating transforming growth factor-β (TGF-β) signaling through β-catenin. The link between Wnt and TGF beta signaling was confirmed by blocking β-catenin and by following TGF-β-induced transcription of a luciferase reporter gene. Together, these data show that blockade of β-catenin can, as a potential target for novel therapeutic interventions, accelerate cutaneous wound healing.

HPV16 E5 affects the KGFR/FGFR2b-mediated epithelial growth through alteration of the receptor expression, signaling and endocytic traffic

The E5 oncoprotein of the human papillomavirus type 16 (HPV16 E5) cooperates in cervical carcinogenesis and in epithelial transformation deregulating cell growth, survival and differentiation through the modulation of growth factor receptors. Among the epithelial receptor tyrosine kinases, the keratinocyte growth factor receptor/fibroblast growth factor receptor 2b (KGFR/FGFR2b) is a major paracrine mediator of epithelial homeostasis and appears to have an unique and unusual role in epithelial tissues, exerting a tumor-suppressive function in vitro and in vivo. With the aim to better elucidate the molecular events involved in the pathological activity of 16E5, we investigated if the viral protein would be able to affect the KGFR expression, signaling and turnover by interference with its degradative and recycling endocytic pathways. Quantitative reverse transcriptase-PCR and biochemical approaches on human keratinocytes transfected with 16E5-HA showed that E5 protein is able to induce KGFR down-modulation at both transcript and protein levels. Immunofluorescence microscopy in double-transfected cells expressing both E5 and KGFR revealed that the viral protein alters the receptor endocytic trafficking and triggers its endosomal sorting to the indirect juxtanuclear recycling pathway. The shift from lysosomal degradation to recycling at the plasma membrane correlates with a reduced phosphorylation of the fibroblast growth factor receptor substrate-2α tyrosine 196, the major docking site for Grb2-Cbl complexes responsible for receptor ubiquitination and degradation. 5'-Bromo-deoxyuridine incorporation assay demonstrated that expression of 16E5 induces a decrease in the growth response to the receptor ligands as a consequence of KGFR down-modulation, suggesting that 16E5 might have a role on HPV infection in perturbing the KGFR-mediated physiological behavior of confluent keratinocytes committed to differentiation.

Platelet Lysate Induces In Vitro Wound Healing of Human Keratinocytes Associated with a Strong Proinflammatory Response

Platelet lysates (PL), which are derived from platelets, are cocktails of growth factors and cytokines that can promote tissue regeneration. Until today, most studies have focused on growth factor content of platelets rather than on their potential as a reservoir of mediators and cytokines. Taking advantage of an in vitro scratch assay performed under both normal and inflammatory conditions, in the present work, we report that at physiologic concentrations, PL enhanced wound closure rates of NCTC 2544 human keratinocytes. This effect was clearly detectable 6 h after wounding. Moreover, PL induced a strong cell actin cytoskeletal re-organization that persisted up to 24 h. The accelerated wound closure promoted by PL, in either presence or absence of serum, was associated with a high expression of the inflammatory cytokine interleukin-8. Further, after 24 h PL treatment, confluent keratinocytes also expressed low amounts of interleukin-8 and of the antimicrobial peptide neutrophil gelatinase-associated lipocalin, which dramatically increased under inflammatory conditions. These effects were associated with activation of the inflammatory pathways, p38 mitogen-activated protein kinase, and NF-κB. Our findings support the concept that platelet-derived preparations could accelerate regeneration of difficult-to-heal wounds by triggering an inflammatory cascade and having an antimicrobial role.

The Calcium-Sensing Receptor-Dependent Regulation of Cell–Cell Adhesion and Keratinocyte Differentiation Requires Rho and Filamin A

Extracellular Ca(2+) (Ca(2+)(o)) functioning through the calcium-sensing receptor (CaR) induces E-cadherin-mediated cell-cell adhesion and cellular signals mediating cell differentiation in epidermal keratinocytes. Previous studies indicate that CaR regulates cell-cell adhesion through Fyn/Src tyrosine kinases. In this study, we investigate whether Rho GTPase is a part of the CaR-mediated signaling cascade regulating cell adhesion and differentiation. Suppressing endogenous Rho A expression by small interfering RNA (siRNA)-mediated gene silencing blocked the Ca(2+)(o)-induced association of Fyn with E-cadherin and suppressed the Ca(2+)(o)-induced tyrosine phosphorylation of β-, γ-, and p120-catenin and formation of intercellular adherens junctions. Rho A silencing also decreased the Ca(2+)(o)-stimulated expression of terminal differentiation markers. Elevating the Ca(2+)(o) level induced interactions among CaR, Rho A, E-cadherin, and the scaffolding protein filamin A at the cell membrane. Inactivation of CaR expression by adenoviral expression of a CaR antisense complementary DNA inhibited Ca(2+)(o)-induced activation of endogenous Rho. Ca(2+)(o) activation of Rho required a direct interaction between CaR and filamin A. Interference of CaR-filamin interaction inhibited Ca(2+)(o)-induced Rho activation and the formation of cell-cell junctions. These results indicate that Rho is a downstream mediator of CaR in the regulation of Ca(2+)(o)-induced E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation.

Newly formed E‐cadherin contacts do not activate Cdc42 or induce filopodia protrusion in human keratinocytes1

The appropriate regulation of cell-cell adhesion is an important event in the homoeostasis of different cell types. In epithelial cells, tight adhesion mediated by E-cadherin receptors is essential for the differentiation and functionality of epithelial sheets. Upon assembly of cadherin-mediated cell-cell contacts, it is well established that the small GTPases Rho and Rac are activated and are necessary for junction stability. However, the role of the small GTPase Cdc42 in cadherin adhesion is less clear. Cdc42 can be activated by E-cadherin in a breast tumour cell line, but the requirement for Cdc42 function for new junction assembly or maintenance has been contradictory. Cdc42 participation in cell-cell contacts has been inferred from the presence of filopodia, the typical F-actin structure induced by Cdc42 activation, as cells approach each other to establish cell-cell contacts. Yet, under these conditions, the contribution of migration to filopodia protrusion cannot be excluded and the results are difficult to interpret. In the present study, we set out to address (a) whether Cdc42 is activated by new E-cadherin cell-cell contacts when junction assembly occurs without prior migration and (b) whether Cdc42 function is necessary for cadherin stability. We found that junction formation in confluent keratinocytes or upon E-cadherin clustering decreased Cdc42-GTP levels. In the absence of serum- and migration-induced Cdc42 activation, we demonstrated that cell-cell contacts do not induce filopodia or require Cdc42 function to assemble. We conclude that Cdc42 does not participate in the early events that initiate stable cadherin adhesion in keratinocytes. Yet, it is feasible that Cdc42 may be activated at later time points or by other receptors. Cdc42 can then participate in additional functions during polarization, such as Golgi re-positioning or basolateral trafficking.

Induction of hyper-adhesion attenuates autoimmune-induced keratinocyte cell–cell detachment and processing of adhesion molecules via mechanisms that involve PKC

In confluent keratinocyte monolayers, desmosomal adhesion gradually becomes calcium-independent and this is associated with an increase in the strength of intercellular adhesion (hyper-adhesion). In this study, we investigated the functional and molecular significance of hyper-adhesion in a system challenged by autoimmune sera from patients with Pemphigus Vulgaris (PV), a disease primarily targeting desmosomal adhesion. The results show that keratinocytes with calcium-independent desmosomes are resistant to disruption of intercellular contacts (acantholysis) in experimental PV. Furthermore, both the desmosomal cadherins desmoglein (Dsg) 1 and Dsg3 and the adherens junction protein E-cadherin were decreased in confluent keratinocytes at Day 1, but not in hyper-adhesive cells (Day 6) after incubation with PV serum. Pharmacological induction of the hyper-adhesive state with the PKC inhibitor Go6976 reduced both the acantholysis rate and the processing of cell adhesion molecules induced by PV serum. When the establishment of the hyper-adhesive state was prevented by cell adhesion recognition (CAR) peptides that perturbed desmosomal interactions, Go6976 could still partially attenuate PV acantholysis. Taken together, these data demonstrate that keratinocyte hyper-adhesion decreases the morphological, functional and biochemical dys-cohesive effects of PV serum via mechanisms that involve, at least in part, the function of PKC. This suggests that reinforcing keratinocyte adhesion may be a promising way to inhibit the effects of this most debilitating disorder.

The transcriptional repressor RIP140 is a cell-cycle regulated gene which controls E2F1 activity and cell proliferation

E2F and DP family members are established regulators of the cell cycle. In this study, we examined their activity/expression during keratinocyte growth arrest. Treating human epidermal keratinocytes with the growth inhibitors TPA or IFN-γ or allowing the cells to reach confluence resulted in 90% inhibition of DNA synthesis, whereas a keratinocyte-derived squamous carcinoma cell line (SCC25) was resistant to growth inhibitors. Gel shift analysis of keratinocytes using an E2F response element indicated that growth arrest was associated with a decrease in all E2F binding complexes. This indicates that growth inhibition is not due to negative regulation by pocket proteins. Conversely, gel shift analysis of growth inhibitor-resistant SCC25 cells showed no decrease in E2F binding. If deregulated E2F expression/activity is involved in tumor development, then the deliberate deregulation of E2F activity may make keratinocytes resistant to growth inhibitors in much the same way as the SCC cells. The HPVI6 E7 protein is known to activate E2F. Retroviral infection of keratinocytes with E7-expressing constructs resulted in growth inhibitor resistance, whereas infection with E6 constructs did not. E2F is a heterodimeric complex consisting of E2F family members (1–5) and DP proteins (1–3). Examination of the expression levels for E2F genes and other genes associated with the cell cycle indicated that E2F1 was profoundly decreased in growth-arrested keratinocytes (90%), whereas E2F3, E2F5, and DP1 were not. E2F2 and E2F4 were increased in IFN-γ-treated keratinocytes but not in TPA-treated or confluent keratinocytes. In contrast, SCC2S cells did not undergo growth arrest and did not downregulate E2F1 mRNA expression in response to growth inhibitors.

AKT1 Provides an Essential Survival Signal Required for Differentiation and Stratification of Primary Human Keratinocytes

Keratinocyte differentiation and stratification are complex processes involving multiple signaling pathways, which convert a basal proliferative cell into an inviable rigid squame. Loss of attachment to the basement membrane triggers keratinocyte differentiation, while in other epithelial cells, detachment from the extracellular matrix leads to rapid programmed cell death or anoikis. The potential role of AKT in providing a survival signal necessary for stratification and differentiation of primary human keratinocytes was investigated. AKT activity increased during keratinocyte differentiation and was attributed to the specific activation of AKT1 and AKT2. Targeted reduction of AKT1 expression, but not AKT2, by RNA interference resulted in an abnormal epidermis in organotypic skin cultures with a thin parabasal region and a pronounced but disorganized cornified layer. This abnormal stratification was due to significant cell death in the suprabasal layers and was alleviated by caspase inhibition. Normal expression patterns of both early and late markers of keratinocyte differentiation were also disrupted, producing a poorly developed stratum corneum.

Defining the transcriptome of accelerated and replicatively senescent keratinocytes reveals links to differentiation, interferon signaling, and Notch related pathways

Epidermal keratinocytes (KCs) undergo highly orchestrated morphological and molecular changes during transition from proliferative compartment into growth arrested early and late differentiation layers, prior to dying in outermost cornified layers of normal skin. Creation of stratum corneum is vital to barrier function protecting against infection. Transcriptional events in KCs regulating complex processes of differentiation and host defense required to maintain constant epidermal thickness and resistance to infection in either young or aged skin are largely unknown. Furthermore, as terminal differentiation is characterized by irreversible loss of replicative potential culminating in dead layers at the skin surface, this process may be viewed as a form of senescence. However, a complete transcriptional profile of senescent (SN) human KCs has not been previously defined to permit delineation of molecular boundaries involving differentiation and senescence. To fill this void, we utilized global transcriptional analysis of KCs maintained in vitro as either cultures of proliferating (PR) cells, early and late confluent (LC) (accelerated senescence) cultures, or KCs undergoing replicative senescence. Global gene expression profiling revealed early confluent (EC) KCs were somewhat similar to PR KCs, while prominent differences were evident when compared to LC KCs; which were also distinct from replicatively SN KCs. While confluent KCs have in common several genes regulating differentiation with replicatively SN KCs, the latter cells expressed elevated levels of genes involved in interferon signaling and inflammatory pathways. These results provide new insights into cell autonomous transcriptional-based programs operative within KCs contributing to replicative senescence, with partial sharing of genes involved in differentiation. In addition, regulation of KC senescence may involve participation of interferon signaling pathways derived from the important role of KCs in protecting skin from infection. Integrating all of the transcriptional data revealed a key role for Notch receptor mediated signaling in the confluency induced differentiation phenotype using this model system.

A simple in vitro model for investigating epithelial/mesenchymal interactions: keratinocyte inhibition of fibroblast proliferation and fibronectin synthesis

Hypertrophic scarring and graft contracture are major causes of morbidity after burn injuries. It is well established that application of a split-thickness skin graft reduces scarring and contraction, and cultured epithelial autografts have a similar effect. To investigate the influence of keratinocytes on fibroblast proliferation and fibronectin synthesis, we used an in vitro separated co-culture model in which epithelial sheets were cultured above fibroblast monolayers without physical contact. We also investigated the response of fibroblasts to keratinocyte-conditioned medium (KCM) obtained from confluent and subconfluent keratinocyte monolayers. Both cultured epithelial sheets, composed of adherent fully confluent keratinocytes, and their conditioned medium, reduced fibroblast proliferation. However, KCM from subconfluent keratinocytes stimulated fibroblast proliferation at low concentrations while inhibiting it at higher concentrations, indicating that keratinocytes can produce both mitogenic and growth-inhibiting factors for fibroblasts. KCM, but not epithelial sheet co-culture, also inhibited fibroblast fibronectin synthesis. This indicates regulation of fibroblast phenotype by soluble factors released by the keratinocyte and also suggests that there is a dialogue between keratinocytes and fibroblasts with respect to fibronectin production. We conclude that this separated co-culture model is a simple way to study epithelial/mesenchymal communication particularly with respect to the role of the fibroblast in wound healing.