Signaling In Keratinocytes Research Articles

Regulation of dioxin receptor function by different beta-carboline alkaloids

The dioxin receptor, also known as arylhydrocarbon receptor (AhR), is a ligand-activated transcription factor that mediates the toxicity of dioxins and related environmental contaminants. In addition, there is a growing list of natural compounds, mainly plant polyphenols that can modulate AhR function and downstream signaling with quite unknown consequences for cellular function. We investigate the potential of four different beta-carboline alkaloids to stimulate AhR signaling in human hepatoma cells and keratinocytes. Three test substances, namely rutaecarpine, annomontine and xestomanzamine A, increase AhR-driven reporter gene activity as well as expression of two AhR target genes in a dose-dependent and time-dependent manner. Additionally, the three test alkaloids stimulate cytochrome P450 (CYP) 1 enzyme activity without showing any antagonistic effects regarding benzo(a)pyrene-stimulated CYP1 activation. The AhR-activating property of the beta-carbolines is completely abrogated in AhR-deficient cells providing evidence that rutaecarpine, annomontine and xestomanzamine A are natural stimulators of the human AhR. The toxicological relevance of beta-carboline-mediated AhR activation is discussed.

RasGRP1 Is Essential for Ras Activation by the Tumor Promoter 12-O-Tetradecanoylphorbol-13-acetate in Epidermal Keratinocytes

RasGRP1 is a guanine nucleotide exchange factor for Ras that binds with high affinity to diacylglycerol analogs like the phorbol esters. Recently, we demonstrated a role for RasGRP1 in skin carcinogenesis and suggested its participation in the action of tumor-promoting phorbol esters like 12-O-tetradecanoylphorbol-13-acetate (TPA) on Ras pathways in epidermal cells. Given the importance of Ras in carcinogenesis, we sought to discern whether RasGRP1 was a critical pathway in Ras activation, using a RasGRP1 knockout (KO) mouse model to examine the response of keratinocytes to TPA. In contrast to the effect seen in wild type keratinocytes, Ras(GTP) levels were barely detected in RasGRP1 KO cells even after 60 min of exposure to phorbol esters. The lack of response was rescued by enforced expression of RasGRP1. Furthermore, small hairpin RNA-induced silencing of RasGRP1 abrogated the effect of TPA on Ras. Analysis of Ras isoforms showed that both H-Ras and N-Ras depended on RasGRP1 for activation by TPA, whereas activation of K-Ras could not be detected. Although RasGRP1 was dispensable for ERK activation in response to TPA, JNK activation was reduced in the KO keratinocytes. Notably, TPA-induced phosphorylation of JNK2, but not JNK1, was reduced by RasGRP1 depletion. These data identify RasGRP1 as a critical molecule in the activation of Ras by TPA in primary mouse keratinocytes and suggest JNK2 as one of the relevant downstream targets. Given the role of TPA as a skin tumor promoter, our findings provide additional support for a role for RasGRP1 in skin carcinogenesis.

Abstract LB-112: The human papillomavirus type 16 E5 protein represses X-box binding protein 1 and cyclooxygenase-2 stress signaling in keratinocytes

Abstract Human papillomavirus (HPV) is the causative agent of 99% of cervical cancers, around 60% of which are caused by the high-risk type 16. While two of its three oncoproteins, E6 and E7, have been well characterized, the biological functions of the E5 protein remain elusive. Recent studies have suggested that E5 enhances HPV-induced oncogenesis, although in isolation it is not tumorigenic. In this study, we analyzed the gene expression changes induced by the endoplasmic reticulum (ER) resident HPV-16 E5 protein in primary human keratinocytes. Using real-time RT-PCR, we identified the downregulation of spliced X-box binding protein 1 (XBP-1), a key player in the ER-stress response, as a biochemical marker of E5 expression. The transmembrane kinase receptor responsible for the cleavage of XBP-1, ER-to-nucleus signaling 1 protein (IRE1α/ERN1), was also downregulated in E5-transduced cells. Furthermore, a microarray analysis of E5-transduced keratinocytes revealed the downregulation of another member of the ER-stress response pathway, prostaglandin G/H synthase and cyclooxygenase (COX-2/PTGS2). In addition, levels of COX-2 and XBP-1 were measured in keratinocytes expressing either low-risk HPV-6b E5 or a C-terminal 16E5 mutant containing a two amino acid mutation. The latter mutant had the histidine and alanine residues (conserved in the high-risk HPVs) replaced with tyrosine and isoleucine (conserved in the low-risk HPVs). Importantly, neither 6bE5 nor the H77YA78I 16E5 mutant was able to affect levels of these ER-stress response genes, suggesting that the ability to induce such changes in gene expression is specific to high-risk 16E5. Since preliminary data showed that expression of the E6 and E7 proteins in keratinocytes greatly induced XBP-1 splicing, we hypothesized that 16E5 might play a protective role against the host response to viral infection. In agreement with this postulate, we found that cells co-expressing E6, E7 and E5 had lower levels of COX-2 expression than cells expressing E6 and E7 alone. Interestingly, the levels of COX-2 in E6/E7-transduced cells were also supressed when compared to vector alone, suggesting that multiple pathways of COX-2 repression may exist. The mechanism by which E5 downregulates COX-2 is currently unknown, but it can be overcome by treatment with thapsigargin or tunicamycin, which initiate ER stress via calcium fluxes and abnormal protein glycosylation respectively. It is therefore unlikely that E5 is specifically tempering these pathways. Our data suggest a potential role for E5 in repressing the cellular ER-stress response following HPV infection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-112.

Abstract 1026: A non-apoptotic function for Mcl-1 as an activator of canonical Wnt signaling in keratinocytes

Abstract Mcl-1 is an anti-apoptotic Bcl-2 family member whose rapid down-regulation is required for UV apoptosis. We recently showed that not only is Mcl-1 a major human keratinocyte (KC) survival protein, but it is required for proper KC differentiation in organotypic culture, implicating a non-apoptotic function for Mcl-1 involved in regulating epidermal morphogenesis. To further explore the function of Mcl-1 in vivo, we generated and analyzed transgenic mice expressing human Mcl-1 under control of the keratin 5 (K5) promoter. K5-Mcl-1 transgenic mice had >2-fold increase in epidermal thickness (p<0.001), increased basal KC BrdU incorporation (∼2-fold) and altered expression of KC differentiation markers. Several K5-Mcl-1 mice developed benign follicular cysts. Since both proliferation and hair follicle morphogenesis are features of increased Wnt/β-catenin signaling, we examined components of the Wnt/β-catenin pathway. Epidermises from K5-Mcl-1 mice had elevated steady state β-catenin, P-GSK3β (Ser9, Ser21) and P-Akt (Ser473), consistent with Wnt pathway activation. To assess Wnt pathway in more detail, we over-expressed Mcl-1 in human KCs by retroviral transduction. Over-expression of Mcl-1, but not Bcl-2 or Bcl-xL, also induced KC proliferation (3.3-fold). Mcl-1 increased the levels of total and active β-catenin, c-Myc, P-GSK-3β, and P-Akt, as well as inducing TCF-4-dependent luciferase reporter activity (p<0.05) in keratinocytes. Q-RT-PCR analysis of Wnt pathway genes identified Disheveled-1, Frizzled-1 and Cyclin D3 as the most highly up-regulated genes common to both Mcl-1 transduced human KCs and K5-Mcl-1 mice. Mcl-1 truncation mutants encoding the N-terminal 208 and 103 amino acids, but lacking the BH domains, were generated and over expressed in KCs. Both Mcl-1 mutants induced 3H-thymidine incorporation (3.4- and 2.9-fold respectively), indicating that the mitogenic activity of Mcl-1 resides in its N-terminal region. Since β-catenin signaling is activated in cutaneous SCC and involved in stem cell maintenance, this non-apoptotic function of Mcl-1 represents a novel and potentially oncogenic activity in addition to its well characterized anti-apoptotic function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1026.

Abstract 3975: CLIC4 induces a Smad7 variant to enhance TGF-β signaling in keratinocytes

Abstract CLIC4 (Chloride intracellular channel 4) is a member of a family of intracellular chloride channel proteins that are ubiquitously expressed in multiple tissue types. It is a dimorphic protein that exists in both soluble and membrane-bound forms. CLIC4 is multifunctional, traffics between the cytoplasm and nucleus, and participates in cell cycle control and differentiation in keratinocytes and other cell types. CLIC4 expression is lost in multiple human epithelial cancers. We have recently shown that nuclear CLIC4 enhances transforming growth factor beta (TGF-β) signaling through stabilization of phospho-Smad2/3. The TGF-β signaling pathway is a major growth regulator of multiple cell types, and its regulation is altered in many human cancers suggesting that CLIC4 could participate in altering TGF-β regulation in tumor cells. Here we show that in addition to stabilizing phospho-Smads 2/3, CLIC4 alters Smad7, a feedback inhibitor of the TGF-β pathway. Elevated levels of CLIC4 in primary keratinocytes lead to the expression of a truncated form of Smad7 (Smad7Δ), which is detectable as a PCR product. The Smad7Δ variant contains a missing region of 94bp in exon 4 of Smad7 and appears to be an alternately spliced form. Absence of these 94bp leads to a frameshift mutation and an early protein truncation causing the Smad7Δ variant to lack the MH2 domain of Smad7, the domain responsible for inhibition of TGF-β signaling. While wild-type Smad7 expression inhibits TGF-β signaling, exogenously expressed Smad7Δ variant does not inhibit TGF-β signaling, based upon analysis of TGF-β dependent proliferation, reporter assays and phosphorylation of Smad proteins. Together, these data suggest that CLIC4 regulates TGF-β signaling by multiple mechanisms. In light of this, CLIC4 may serve as a target for modifying TGF-β function in cancer and other conditions where this pathway is involved in pathological changes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3975.

Regulation of keratinocyte signaling and function via changes in epidermal growth factor presentation

Motivated by the need for bioactive materials that can accelerate dermal wound healing, this work describes the responses of keratinocytes to covalently immobilized epidermal growth factor (EGF) and how differences in the physical presentation of this growth factor affect cell function. Specifically, human keratinocytes were cultured with EGF delivered in soluble form, immobilized in a homogeneous pattern or immobilized in a gradient pattern, followed by analysis of cellular signaling, proliferation and migration. By changing the manner in which EGF was presented, keratinocyte behavior was dramatically altered. Keratinocytes responded to immobilized EGF patterns with high EGF receptor (EGFR) but low ERK1/2 and Akt phosphorylation, accompanied by low proliferation, high migratory activity and coordinated cell alignment. In contrast, keratinocytes treated with soluble EGF experienced lower EGFR but higher ERK1/2 and Akt phosphorylation and displayed a highly proliferative, rather than migratory, phenotype. Keratinocytes also responded to differences in immobilized EGF patterns, as migration was fastest upon immobilized gradients of EGF. A better understanding the interaction of cells with soluble vs. immobilized growth factors can help elucidate native healing events and achieve greater control over cell function, which may be useful in the development of wound repair treatments for many types of tissues.

β-catenin Activity in the Dermal Papilla Regulates Morphogenesis and Regeneration of Hair

The activity of keratinocytes in the hair follicle is regulated by signals from a specialized mesenchymal niche, the dermal papilla (DP). Here, mice expressing cre recombinase in the DP were developed to probe the interaction between follicular keratinocytes and the DP in vivo. Inactivation of the beta-catenin gene within DP of fully developed hair follicles results in dramatically reduced proliferation of the progenitors and their progeny that generate the hair shaft, and, subsequently, premature induction of the destructive phase of the hair cycle. It also prevents regeneration of the cycling follicle from stem cells. Gene expression analysis reveals that beta-catenin activity in the DP regulates signaling pathways, including FGF and IGF, that can mediate the DP's inductive effects. This study reveals a signaling loop that employs Wnt/beta-catenin signaling in both epithelial progenitor cells and their mesenchymal niche to govern and coordinate the interactions between these compartments to guide hair morphogenesis.

Toll-like receptor-2 and interleukin-1 signaling in keratinocytes during Staphylococcus aureus infections (46.9)

Abstract Staphylococcus aureus is a significant human pathogen. Subcutaneous infections in mice trigger neutrophil recruitment in an IL-1β, but not IL-1α, dependent manner. Since S. aureus can colonize the skin we explored the role keratinocytes play as first responders to bacterial challenges. In mice, topical application of IL-1, S. aureus or the bacterial cell wall components lipoteichoic acid (LTA) and peptidoglycan (PGN) triggered neutrophil recruitment to the skin surface. In human tissue culture models IL-1α and IL-1β increased mRNA and protein production of the neutrophil chemotactic CXCL1, CXCL2 and IL-8. S. aureus, LTA and PGN induced similar expression profiles in a TLR2 dependent manner. The S. aureus activated chemokine production was preceded by significant IL-1α and IL-1β secretion. Expression of IL-1α was significantly higher than that of IL-1β. IL-1RI neutralization experiments revealed that S. aureus derived LTA and PGN induced chemokine expression requires IL-1RI signaling. Surprisingly, we further found that LTA and PGN initiated chemokine secretion is dependent upon endocrine IL-1α, but not IL-1β, signaling. Our data demonstrate that the innate immune response of keratinocytes is regulated differently than those of subcutaneous tissues. A multifaceted IL-1 scheme involving IL-1α in the skin and IL-1β in underlying tissues may represent a fail-safe system, which protects the host against genetic variation and immune evasion mechanisms developed by pathogens.

COP1 Contributes to UVB-Induced Signaling in Human Keratinocytes

UVB irradiation has been shown to trigger a broad range of changes in gene expression in human skin; however, factors governing these events are still not well understood. In this study, we show that human constitutive photomorphogenic protein-1 (huCOP1), an E3 ligase, contributes to the orchestration of UVB response of keratinocytes. Accordingly, our data show that (i) huCOP1 protein is expressed both in the nucleus and in the cytoplasm of cultured keratinocytes, (ii) UVB reduces the levels of the huCOP1 mRNA and protein, and (iii) induces changes in the subcellular localization of huCOP1. Finally, we show that gene-specific silencing of huCOP1 induces the accumulation of the tumor suppressor p53 protein, which is further increased after UVB irradiation.

Processing of CD109 by furin and its role in the regulation of TGF-β signaling

CD109 is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, whose expression is upregulated in squamous cell carcinomas of the lung, esophagus, uterus and oral cavity. CD109 negatively regulates transforming growth factor (TGF)-beta signaling in keratinocytes by directly modulating receptor activity. In this study, we further characterized CD109 regulation of TGF-beta signaling and cell proliferation. We found that CD109 is produced as a 205 kDa glycoprotein, which is then processed in the Golgi apparatus into 180 kDa and 25 kDa proteins by furin (furinase). 180 kDa CD109 associated with GPI-anchored 25 kDa CD109 on the cell surface and was also secreted into the culture medium. To investigate whether furinase cleavage of CD109 is necessary for its biological activity, we mutated arginine 1273 in the CD109 furinase cleavage motif (amino acid 1270-RRRR-1273) to serine (R1273S). Interestingly, CD109 R1273S neither significantly impaired TGF-beta signaling nor affected TGF-beta-mediated suppression of cell growth, although it was expressed on the cell surface as a 205 kDa protein. Consistent with this finding, the 180 kDa and 25 kDa CD109 complex, but not CD109 R1273S, associated with the type I TGF-beta receptor. These findings indicate that processing of CD109 into 180 kDa and 25 kDa proteins by furin, followed by complex formation with the type I TGF-beta receptor is required for the regulation of TGF-beta signaling in cancer cells and keratinocytes.

Langerhans cell (LC) proliferation mediates neonatal development, homeostasis, and inflammation-associated expansion of the epidermal LC network

Most tissues develop from stem cells and precursors that undergo differentiation as their proliferative potential decreases. Mature differentiated cells rarely proliferate and are replaced at the end of their life by new cells derived from precursors. Langerhans cells (LCs) of the epidermis, although of myeloid origin, were shown to renew in tissues independently from the bone marrow, suggesting the existence of a dermal or epidermal progenitor. We investigated the mechanisms involved in LC development and homeostasis. We observed that a single wave of LC precursors was recruited in the epidermis of mice around embryonic day 18 and acquired a dendritic morphology, major histocompatibility complex II, CD11c, and langerin expression immediately after birth. Langerin+ cells then undergo a massive burst of proliferation between postnatal day 2 (P2) and P7, expanding their numbers by 10–20-fold. After the first week of life, we observed low-level proliferation of langerin+ cells within the epidermis. However, in a mouse model of atopic dermatitis (AD), a keratinocyte signal triggered increased epidermal LC proliferation. Similar findings were observed in epidermis from human patients with AD. Therefore, proliferation of differentiated resident cells represents an alternative pathway for development in the newborn, homeostasis, and expansion in adults of selected myeloid cell populations such as LCs. This mechanism may be relevant in locations where leukocyte trafficking is limited.

Constitutive Expression of MC1R in HaCaT Keratinocytes Inhibits Basal and UVB‐induced TNF‐α Production

Alpha-melanocyte stimulating hormone (alpha-MSH) binds to melanocortin-1 receptor (MC1R) on melanocytes to stimulate pigmentation and modulate various cutaneous inflammatory responses. MC1R expression is not restricted to melanocytic cells and may be induced in keratinocytes after UVB exposure. We hypothesized that MC1R signaling in keratinocytes, wherein basal conditions are barely expressed, may modulate mediators of inflammation, such as nuclear factor-kappa B (NF-kappaB) and tumor necrosis factor-alpha (TNF-alpha). Therefore, we generated HaCaT cells that stably express human MC1R or the Arg151Cys (R151C) nonfunctional variant. We demonstrate that: (1) the constitutive activity of MC1R results in elevated intracellular cAMP level, reduced NF-kappaB activity and decreased TNF-alpha transcription; (2) binding of alpha-MSH to MC1R and the subsequent increase in cAMP production do not inhibit TNFalpha-mediated NF-kappaB activation; (3) MC1R signaling is sufficient to strongly inhibit UVB-induced TNF-alpha expression and this inhibitory effect is further enhanced by alpha-MSH stimulation. Our findings suggest that the constitutive activity of the G-protein-coupled MC1R in keratinocytes may contribute to the modulation of inflammatory events and immune response induced by UV light.

Apurinic/Apyrimidinic Endonuclease 1 Is a Key Modulator of Keratinocyte Inflammatory Responses

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) functions in both DNA repair and redox signaling, making it an attractive emerging therapeutic target. However, the role of APE1 in cutaneous inflammatory responses is largely unknown. In this study, we report that APE1 is a key upstream regulator in TLR2-dependent keratinocyte inflammatory responses. We found that nuclear expression of APE1 in epidermal layers was markedly up-regulated in psoriatic skin. APE1 was essential for the transcriptional activation and nuclear translocation of hypoxia-inducible factor-1alpha and NF-kappaB, both of which are crucial for inflammatory signaling in keratinocytes. Moreover, APE1 played a crucial role in the expression of TLR2-mediated inflammatory mediators, including TNF-alpha, CXCL8, and LL-37, in HaCaT cells and human primary keratinocytes. Silencing of APE1 attenuated cyclin D1/cyclin-dependent kinase 4 expression and phosphorylation of ERK1/2 and Akt, thereby affecting keratinocyte proliferation. Importantly, TLR2-induced generation of reactive oxygen species contributed to the nuclear translocation and expression of APE1, suggesting an autoregulatory circuit in which the subcellular localization of APE1 is associated with the production of APE1 per se through reactive oxygen species-dependent signaling. Taken together, these findings establish a role for APE1 as a master regulator of TLR2-dependent inflammatory responses in human keratinocytes.

Bcl-3 Acts as an Innate Immune Modulator by Controlling Antimicrobial Responses in Keratinocytes

Innate immune responses involve the production of antimicrobial peptides (AMPs), chemokines, and cytokines. We report here the identification of B-cell leukemia (Bcl)-3 as a modulator of innate immune signaling in keratinocytes. In this study, it is shown that Bcl-3 is inducible by the Th2 cytokines IL-4 and IL-13 and is overexpressed in lesional skin of atopic dermatitis (AD) patients. Bcl-3 was shown to be important to cutaneous innate immune responses as silencing of Bcl-3 by small-interfering RNA (siRNA) reversed the downregulatory effect of IL-4 on the HBD3 expression. Bcl-3 silencing enhanced vitamin D3 (1,25D3)-induced gene expression of cathelicidin AMP in keratinocytes, suggesting a negative regulatory function on cathelicidin transcription. Furthermore, 1,25D3 suppressed Bcl-3 expression in vitro and in vivo. This study identified Bcl-3 as an important modulator of cutaneous innate immune responses and its possible therapeutic role in AD.

Hydrogen sulfide impairs keratinocyte cell growth and adhesion inhibiting mitogen-activated protein kinase signaling

The effects of exogenous hydrogen sulfide (H2S) on normal skin-derived immortalized human keratinocytes have been investigated in detail. We show in vitro that exogenous hydrogen sulfide reduces clonal growth, cell proliferation and cell adhesion of human keratinocytes. H2S, in fact, decreases the frequency of the putative keratinocyte stem cell subpopulation in culture, consequently affecting clonal growth, and impairs cell proliferation and adhesion of mature cells. As a mechanistic explanation of these effects, we show at the molecular level that (i) H2S reduces the Raf/MAPK kinase/ERK signaling pathway; (ii) the reduced adhesion of sulfur-treated cells is associated to the downregulation of the expression of β4, α2 and α6 integrins that are necessary to promote cell adhesion as well as anti-apoptotic and proliferative signaling in normal keratinocytes. One specific interest of the effects of sulfurs on keratinocytes derives from the potential applications of the results, as sulfur is able to penetrate the skin and a sulfur-rich balneotherapy has been known for long to be effective in the treatment of psoriasis. Thus, the relevance of our findings to the pathophysiology of psoriasis was tested in vivo by treating psoriatic lesions with sulfurs at a concentration comparable to that most commonly found in sulfurous natural springs. In agreement with the in vitro observations, the immunohistochemical analysis of patient biopsies showed a specific downregulation of ERK activation levels, the key molecular event in the sulfur-induced effects on keratinocytes.

Immune evasion during varicella zoster virus infection of keratinocytes

T cells are sensitized during varicella-zoster virus (VZV) infection and are important for control of viral spread and reactivation. In this report, we show that human keratinocytes infected with VZV inhibited upregulation of major histocompatibility complex (MHC) class I, MHC class II and intercellular adhesion molecule-1 after interferon (IFN)-gamma treatment. The ability of keratinocytes to upregulate MHC class I in response to IFN-alpha, tumour necrosis factor (TNF)-alpha and Toll-like receptor (TLR)-3 ligand was also diminished upon VZV infection. VZV-infected keratinocytes treated with IFN-gamma had significantly reduced capacity to stimulate antigen-specific T cells compared with uninfected cells. Interference with IFN-alpha, TNF-alpha, IFN-gamma and TLR-3 signalling in keratinocytes by VZV may contribute to immune evasion of the adaptive immune response.

Activation of fibroblast growth factor receptor 3 and oncogene-induced senescence in skin tumours

The activation of oncogenes is an important step in tumorigenesis, and recently, oncogene-induced senescence (OIS) was proposed as a critical barrier against malignant transformation in normal primary cells. The aim of this study was to examine the activation of fibroblast growth factor receptor 3 (FGFR3) as an oncogene product and OIS in human skin tumours. We investigated the activation of FGFR3 and OIS by mutation and immunohistochemical analysis in skin tumours, including seborrhoeic keratosis, actinic keratosis (AK), Bowen's disease (BD), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Activated point mutations of FGFR3 were identified in four of 22 cases (18%) of seborrhoeic keratosis, but no mutation was detected in the other skin tumours. Twenty-seven of 31 cases (87%) of seborrhoeic keratosis showed moderately to strongly positive expression of the FGFR3 protein, but almost all the other skin tumours were negative. On the other hand, almost all the seborrhoeic keratoses showed negative immunoreactivity for antiphoshohistone H2AX (gamma-H2AX) as a marker of OIS, but 17 of 22 cases (77%) of AK were moderately to strongly positive. Immunoreactivity for gamma-H2AX was significantly greater in AK than in seborrhoeic keratosis, BD, BCC and SCC. The activation of FGFR3 might be a common feature in the tumorigenesis in seborrhoeic keratosis, although the activation does not induce a typical oncogenic signal in keratinocytes. In addition, OIS due to some oncogenic signals rather than activation of FGFR3 might be involved in the early skin carcinogenesis related to chronic ultraviolet radiation exposure.

Inhibition of NF-κB signaling interferes with phorbol ester-induced growth arrest of keratinocytes in a TNFR1-independent manner

A skin-specific block in NF-κB signaling leads to hyperproliferation of the keratinocytes, inflammation, and spontaneous development of squamous cell carcinoma (SCC). Here we show that an inhibition of NF-κB signaling in keratinocytes, via the expression of the super-repressor/ degradation-resistant form of the IκBα protein (IκBαDN), interferes with the growth arrest induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). The IκBαDN cells are able to overcome the TPA-induced cell cycle block. Although SCC development as well as hyperproliferation due to IκBαDN expression in keratinocytes is known to require TNFR1 signaling, the effect of IκBαDN on phorbol ester signaling is downstream/independent of TNFR1. These data thus identify an interaction between IκBαDN and the tumor promoter TPA in the growth regulation of keratinocytes. The proposed mechanism is also likely to be significant in the process of cancer development due to NF-κB inhibition.

Cholesterol and Lipid Rafts as Regulators of Signaling Through the EGF Receptor in Keratinocytes

The EGF receptor (HER1/EGFR) and its cognate ligands are of considerable interest in epidermal biology and pathology. HER1/EGFR is expressed throughout the entire epidermis and its signaling has been shown involved in the control of keratinocyte proliferation, adhesion, and differentiation. The signaling of HER1/EGFR can be modulated by the receptor's immediate environment, particularly by its localization or not in lipid rafts. These membrane domains are nowadays described as heterogeneous dynamic signaling platforms where lipid molecules, cholesterol especially, are highly ordered and where transmembrane proteins encounter a specialized environment. Cholesterol depletion has been used in order to disorganize lipid rafts in keratinocytes and induces activation of HER1/EGFR (without ligand addition), as well as downstream signaling. The activation of p38 MAPK obtained after cholesterol depletion is responsible for the induction of involucrin expression while keratin expression is suppressed. However in keratinocytes recovering from cholesterol depletion the activation of p38 MAPK induces a strong expression of the heparin-binding EGF-like growth factor, a ligand of HER1/EGFR. Lipid rafts could thus be membrane platforms that regulate cell signaling in keratinocytes in normal, but also pathological conditions.

Arsenite Suppresses Notch1 Signaling in Human Keratinocytes

Arsenic is a well-known human skin carcinogen whose mechanism of action remains to be elucidated. In this work using cultured human epidermal cells, arsenite suppressed accumulation of the transcriptionally active intracellular domain of Notch1. The cells responded to an active peptide from the Notch1 ligand, Jagged1, with increased levels of differentiation marker mRNAs and decreased colony-forming ability. Arsenite suppressed Jagged1 effects and expression of Jagged1 mRNA as well. Moreover, exposure of the cells to a gamma-secretase inhibitor prevented Notch1 processing, decreased cell size and differentiation marker expression, and increased proliferative potential, all effects that occur with arsenite treatment. Thus, arsenite action in suppressing keratinocyte differentiation while maintaining germinative capability could be due to inhibition of Notch1 signaling subsequent to ligand binding. This work also revealed that such arsenite action depends upon epidermal growth factor receptor kinase activity. These findings may help to explain how arsenite, by decreasing generation of the tumor suppressor Notch1, contributes to skin carcinogenesis.