Defects in transforming growth factor-β signaling cooperate with a Ras oncogene to cause rapid aneuploidy and malignant transformation of mouse keratinocytes

Genetic inactivation of the transforming growth factor-β (TGF-β) signaling pathway can accelerate tumor progression in the mouse epidermal model of multistage carcinogenesis. By using an in vitro model of keratinocyte transformation that parallels in vivo malignant conversion to squamous cell carcinoma, we show that v-rasHa transduced primary TGF-β1−/− keratinocytes and keratinocytes expressing a TGF-β type II dominant-negative receptor transgene have significantly higher frequencies of spontaneous transformation than control genotypes. Malignant transformation in the TGF-β1−/− keratinocytes is preceded by aneuploidy and accumulation of chromosomal aberrations. Similarly, transient inactivation of TGF-β signaling with a type II dominant-negative receptor adenovirus causes rapid changes in ploidy. Exogenous TGF-β1 can suppress aneuploidy, chromosome breaks, and malignant transformation of the TGF-β1−/− keratinocytes at concentrations that do not significantly arrest cell proliferation. These results point to genomic instability as a mechanism by which defects in TGF-β signaling could accelerate tumor progression in mouse multistage carcinogenesis.

Plakoglobin deficiency protects keratinocytes from apoptosis

The armadillo family protein plakoglobin (Pg) is a well-characterized component of anchoring junctions, where it functions to mediate cell-cell adhesion and maintain epithelial tissue integrity. Although its closest homolog beta-catenin acts in the Wnt signaling pathway to dictate cell fate and promote proliferation and survival, the role of Pg in these processes is not well understood. Here, we investigate how Pg affects the survival of mouse keratinocytes by challenging both Pg-null cells and their heterozygote counterparts with apoptotic stimuli. Our results indicate that Pg deletion protects keratinocytes from apoptosis, with null cells exhibiting delayed mitochondrial cytochrome c release and activation of caspase-3. Pg-null keratinocytes also exhibit increased messenger RNA and protein levels of the anti-apoptotic molecule Bcl-X(L) compared to heterozygote controls. Importantly, reintroduction of Pg into the null cells shifts their phenotype towards that of the Pg+/- keratinocytes, providing further evidence that Pg plays a direct role in regulating cell survival. Taken together, our results suggest that in addition to its adhesive role in epithelia, Pg may also function in contrast to the pro-survival tendencies of beta-catenin, to potentiate death in cells damaged by apoptotic stimuli, perhaps limiting the potential for the propagation of mutations and cellular transformation.Journal of Investigative Dermatology advance online publication, 16 November 2006; doi:10.1038/sj.jid.5700615.

Plakoglobin-dependent disruption of the desmosomal plaque in pemphigus vulgaris

We recently reported that the pathogenesis of pemphigus vulgaris (PV), an autoimmune blistering skin disorder, is driven by the accumulation of c-Myc secondary to abrogation of plakoglobin (PG)-mediated transcriptional c-Myc suppression. PG knock-out mouse keratinocytes express high levels of c-Myc and resemble PVIgG-treated wild-type keratinocytes in most respects. However, they fail to accumulate nuclear c-Myc and loose intercellular adhesion in response to PVIgG-treatment like wild-type keratinocytes. This suggested that PG is also required for propagation of the PVIgG-induced events between augmented c-Myc expression and acantholysis. Here, we addressed this possibility by comparing PVIgG-induced changes in the desmosomal organization between wild-type and PG knock-out keratinocytes. We found that either bivalent PVIgG or monovalent PV-Fab (known to trigger blister formation in vivo) disrupt the linear organization of all major desmosomal components along cell borders in wild-type keratinocytes, simultaneously with a reduction in intercellular adhesive strength. In contrast, PV-Fab failed to affect PG knock-out keratinocytes while PVIgG cross-linked their desmosomal cadherins without significantly affecting desmoplakin. These results identify PG as a principle effector of the PVIgG-induced signals downstream of c-Myc that disrupt the desmosomal plaque at the plasma membrane.

Modulation of cytochrome P450 enzyme activity and PAH -induced skin carcinogenesis by naturally occurring coumarins

The present study was designed to determine the potential anticarcinogenic activity of naturally occurring coumarins and their mechanism of action. The results indicated that several naturally occurring coumarins including bergamottin, coriandrin, imperatorin, isopimpinellin, and ostruthin, to which humans are routinely exposed in the diet, were effective inhibitors and/or inactivators of CYP1A1-mediated ethoxyresorufin-O-dealkylase (EROD) or CYP2B1-mediated pentoxyresorufin-O-dealkylase (PROD) in mouse liver microsomes. In addition, bergamottin and corandrin were also found to be inhibitors of purified human P450 1A1 in vitro. Further studies with coriandrin revealed that this compound was a mechanism-based inactivator of P450 1A1 and covalently bound to the P450 1A1 apoprotein. In cultured mouse keratinocytes, bergamottin and coriandrin effectively inhibited the B(a) P metabolism and significantly decreased covalent binding of B(a) P and DMBA to keratinocyte DNA and anti-diol-epoxide-DNA adducts derived from both B(a) P and DMBA in keratinocytes. The data from in vivo experiments showed that bergamottin and coriandrin were potent inhibitors of covalent binding of B (a) P to epidermal DNA and the formation of (+) anti BPDE-DNA adduct, whereas imperatorin and isopimpinellin were more potent inhibitors of covalent binding of DMBA to epidermal DNA. The ability of coumarins to inhibit covalent binding of B (a) P to DNA in mouse epidermis was positively correlated with their inhibitory effect P450 1A1 in vitro, while the inhibitory effect of coumarins on covalent binding of DMBA to epidermal DNA was positively correlated with their inhibitory effects on P450 2B1 and negatively to their inhibitory activity toward P450 1A1. The data from tumor experiments indicated that bergamottin, ostruthin, and coriandrin inhibited tumor initiation by B (a) P in a two-stage carcinogenesis protocol. Bergamottin was most effective in this regard and produced a dose dependent inhibition of papilloma formation in these experiments. In addition, imperatorin was an effective inhibitor of skin tumorigenesis induced by DMBA in SENCAR mouse skin using both a two-stage and a complete carcinogenesis protocol. At dose levels higher than those effective against DMBA, imperatorin also inhibited tumor initiation by B (a) P. The results to date demonstrate that several naturally occurring coumarins possess the ability to block tumor initiation and tumorigenesis by PAHs such as B (a) P and DMBA through inhibition of the P450s involved in the metabolic activation of these hydrocarbons. A working model for the involvement of specific P450s in the metabolic activation of these two PAHs was proposed. ^

Ultraviolet radiation modulates the immune system through the platelet -activating factor receptor

Ultraviolet radiation plays a critical role in the induction of non-melanoma skin cancer. UV radiation is also immune suppressive. Moreover, UV-induced systemic immune suppression is a major risk factor for skin cancer induction. Previous work had shown that UV exposure in vivo activates a cytokine cascade involving PGE2, IL-4, and IL-10 that induces immune suppression. However, the earliest molecular events that occur immediately after UV-exposure, especially those upstream of PGE2, were not well defined. To determine the initial events and mediators that lead to immune suppression after a pathological dose of UV, mouse keratinocytes were analyzed after sunlamp irradiation. It is known that UV-irradiated keratinocytes secrete the phospholipid mediator of inflammation, platelet-activating factor (PAF). Since PAF stimulates the production of immunomodulatory compounds, including PGE2, the hypothesis that UV-induced PAF activates cytokine production and initiates UV-induced immune suppression was tested. Both UV and PAF activated the transcription of cyclooxygenase (COX)-2 and IL-10 reporter gene constructs. A PAF receptor antagonist blocked UV-induced IL, 10 and COX-2 transcription. PAF mimicked the effects of UV in vivo and suppressed delayed-type hypersensitivity (DTH), and immune suppression was blocked when UV-irradiated mice were injected with a PAF receptor antagonist. This work shows that UV generates PAF-like oxidized lipids, that signal through the PAF receptor, activate cytokine transcription, and induce systemic immune suppression. ^

A role for the epithelial-cell-specific tyrosine kinase Sik during keratinocyte differentiation

Sik, the mouse homologue of the breast tumor kinase Brk, is expressed in differentiating cells of the gastrointestinal tract and skin. We examined expression and activity of Sik in primary mouse keratinocytes and a mouse embryonic keratinocyte cell line (EMK). Calcium-induced differentiation of these cells has been shown to be accompanied by the activation of tyrosine kinases and rapid phosphorylation of a 65-kDa GTPase-activating protein (GAP)-associated protein (GAP-A.p65). We demonstrate that Sik is activated within 2 min after calcium addition in primary keratinocytes and EMK cells. In EMK cells, Sik binds GAP-A.p65, and this interaction is mediated by the Sik Src homology 2 domain. Although Sik directly complexes with GAP-A.p65, overexpression of wild-type or kinase defective Sik in EMK cells does not lead to detectable changes in GAP-A.p65 phosphorylation. These data suggest that Sik is not responsible for phosphorylation of GAP-A.p65. GAP-A.p65 may act as an adapter protein, bringing Sik into proximity of an unidentified substrate. Overexpression of Sik in EMK cells results in increased expression of filaggrin during differentiation, supporting a role for Sik in differentiation.

Overexpression of the c-Myc oncoprotein blocks the growth-inhibitory response but is required for the mitogenic effects of transforming growth factor beta 1.

One of the more intriguing aspects of transforming growth factor beta 1 (TGF beta 1) is its ability to function as both a mitogenic factor for certain mesenchymal cells and a potent growth inhibitor of lymphoid, endothelial, and epithelial cells. Data are presented indicating that c-myc may play a pivotal role in both the mitogenic and antiproliferative actions of TGF beta 1. In agreement with previous studies using C3H/10T1/2 fibroblasts constitutively expressing an exogenous c-myc cDNA, we show that AKR-2B fibroblasts expressing a chimeric estrogen-inducible form of c-myc (mycER) are able to form colonies in soft agar in the presence of TGF beta 1 only when c-myc is activated by hormone. Whereas these findings support a synergistic role for c-myc in mitogenic responses to TGF beta 1, we also find that c-myc can antagonize the growth-inhibitory response to TGF beta 1. Mouse keratinocytes (BALB/MK), which are normally growth-arrested by TGF beta 1, are rendered insensitive to the growth-inhibitory effects of TGF beta 1 upon mycER activation. This ability of mycER activation to block TGF beta 1-induced growth arrest was found to occur only when the fusion protein was induced with hormone in the early part of G1. Addition of estradiol late in G1 had no suppressive effect on TGF beta 1-induced growth inhibition.

Regulation of MAPK activation, AP-1 transcription factor expression and keratinocyte differentiation in wounded fetal skin

Fetal epithelium retains the ability to re-epithelialize a wound in organotypic culture in a manner not dependent on the presence of underlying dermal substrata. This capacity is lost late in the third trimester of gestation or after embryonic day 17 (E-17) in the rat such that embryonic day 19 (E-19) wounds do not re-epithelialize. Moreover, wounds created in E-17 fetuses in utero heal in a regenerative, scar-free fashion. To investigate the molecular events regulating re-epithelialization in fetal skin, the wound-induced expression profile and tissue localization of activator protein 1 (AP-1) transcription factors c-Fos and c-Jun was characterised in E-17 and E-19 skin using organotypic fetal cultures. The involvement of mitogen-activated protein kinase (MAPK) signaling in mediating wound-induced transcription factor expression and wound re-epithelialization was assessed, with the effect of wounding on the expression of keratinocyte differentiation markers determined. Our results show that expression of AP-1 transcription factors was induced immediately by wounding and localized predominantly to the epidermis in E-17 and E-19 skin. c-fos and c-jun induction was transient in E-17 skin with MAPK-dependent c-fos expression necessary for the re-epithelialization of an excisional wound in organotypic culture. In E-19 skin, AP-11 expression persisted beyond 12 h post-wounding, and marked upregulation of the keratinocyte differentiation markers keratin 10 and loricrin was observed. No such changes in the expression of keratin 10 or loricrin occurred in E-17 skin. These findings indicate that re-epithelialization in fetal skin is regulated by wound-induced AP-1 transcription factor expression via MAPK and the differentiation status of keratinocytes.

Multiphoton high-resolution 3D imaging of Langerhans cells and keratinocytes in the mouse skin model adopted for epidermal powdered immunization

Langerhans cells (LCs) can be targeted with DNA-coated gold micro-projectiles ("Gene Gun") to induce potent cellular and humoral immune responses. It is likely that the relative volumetric distribution of LCs and keratinocytes within the epidermis impacts on the efficacy of Gene Gun immunization protocols. This study quantified the three-dimensional (3D) distribution of LCs and keratinocytes in the mouse skin model with a near-infrared multiphoton laser-scanning microscope (NIR-MPLSM). Stratum corneum (SC) and viable epidermal thickness measured with MPLSM was found in close agreement with conventional histology. LCs were located in the vertical plane at a mean depth of 14.9 mum, less than 3 mum above the dermo-epidermal boundary and with a normal histogram distribution. This likely corresponds to the fact that LCs reside in the suprabasal layer (stratum germinativum). The nuclear volume of keratinocytes was found to be approximately 1.4 times larger than that of resident LCs (88.6 mum3). Importantly, the ratio of LCs to keratinocytes in mouse ear skin (1:15) is more than three times higher than that reported for human breast skin (1:53). Accordingly, cross-presentation may be more significant in clinical Gene Gun applications than in pre-clinical mouse studies. These interspecies differences should be considered in pre-clinical trials using mouse models.

Participation of adult mouse bone marrow cells in reconstitution of skin

Results of recent studies have indicated that bone marrow cells can differentiate into various cells of ectodermal, mesodermal, and endodermal origins when transplanted into the body. However, the problems associated with those experiments such as the long latent period, rareness of the event, and difficulty in controlling the processes have hampered detailed mechanistic studies. In the present study, we examined the potency of mouse bone marrow cells to differentiate into cells comprising skin tissues using a skin reconstitution assay. Bone marrow cells from adult green fluorescent protein (GFP)-transgenic mice were transplanted in a mixture of embryonic mouse skin cells (17.5 days post-coitus) onto skin defects made on the backs of nude mice. Within 3 weeks, fully differentiated skin with hair was reconstituted. GFP-positive cells were found in the epidermis, hair follicles, sebaceous glands, and dermis. The localization and morphology of the cells, results of immunohistochemistry, and results of specific staining confirmed that the bone marrow cells had differentiated into epidermal keratinocytes, sebaceous gland cells, follicular epithelial cells, dendritic cells, and endothelial cells under the present conditions. These results indicate that this system is suitable for molecular and cellular mechanistic studies on differentiation of stem cells to various epidermal and dermal cells.

EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer.

The Notch1 gene has an important role in mammalian cell-fate decision and tumorigenesis. Upstream control mechanisms for transcription of this gene are still poorly understood. In a chemical genetics screen for small molecule activators of Notch signalling, we identified epidermal growth factor receptor (EGFR) as a key negative regulator of Notch1 gene expression in primary human keratinocytes, intact epidermis and skin squamous cell carcinomas (SCCs). The underlying mechanism for negative control of the Notch1 gene in human cells, as well as in a mouse model of EGFR-dependent skin carcinogenesis, involves transcriptional suppression of p53 by the EGFR effector c-Jun. Suppression of Notch signalling in cancer cells counteracts the differentiation-inducing effects of EGFR inhibitors while, at the same time, synergizing with these compounds in induction of apoptosis. Thus, our data reveal a key role of EGFR signalling in the negative regulation of Notch1 gene transcription, of potential relevance for combinatory approaches for cancer therapy.

Short- and long-term cultivation of embryonic and neonatal murine keratinocytes

Studies using cultured cells allow one to dissect complex cellular mechanisms in greater detail than when studying living organisms alone. However, before cultured cells can deliver meaningful results they must accurately represent the in vivo situation. Over the last three to four decades considerable effort has been devoted to the development of culture media which improve in vitro growth and modeling accuracy. In contrast to earlier large-scale, non-specific screening of factors, in recent years the development of such media has relied increasingly on a deeper understanding of the cell's biology and the selection of growth factors to specifically activate known biological processes. These new media now enable equal or better cell isolation and growth, using significantly simpler and less labor-intensive methodologies. Here we describe a simple method to isolate and cultivate epidermal keratinocytes from embryonic or neonatal skin on uncoated plastic using a medium specifically designed to retain epidermal keratinocyte progenitors in an undifferentiated state for improved isolation and proliferation and an alternative medium to support terminal differentiation.

Effect of thiazolidinedione, a class of anti-diabetic drugs, on the mouse skin tumorigenesis

Thiazolidinediones (TZDs), a novel class of anti-diabetic drugs, have been known as ligands of peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor that belongs to the nuclear receptor superfamily. These synthetic compounds improve insulin sensitivity in patients with type II diabetes likely through activating PAPRγ. Interestingly, they were also shown to inhibit cell growth and proliferation in a wide variety of tumor cell lines. The aim of this study is to assess the potential use of TZDs in the prevention of carcinogenesis using mouse skin as a model. ^ We found that troglitazone, one of TZD drugs, strongly inhibited cultured mouse skin keratinocyte proliferation as demonstrated by [3H]thymidine incorporation assay. It also induced a cell cycle G1 phase arrest and inhibited expression of cell cycle proteins, including cyclin D1, cdk2 and cdk4. Further experiments showed that PPARγ expression in keratinocytes was surprisingly undetectable in vitro or in vivo. Consistent with this, no endogenous PPARγ function in keratinocytes was found, suggesting that the inhibition of troglitazone on keratinocyte proliferation and cell cycle was PPARγ-independent. We further found that troglitazone inhibited insulin/insulin growth factor I (IGF-1) mitogenic signaling, which may explains, at least partly, its inhibitory effect on keratinocyte proliferation. We showed that troglitazone rapidly inhibited IGF-1 induced phosphorylation of p70S6K by mammalian target of rapamycin (mTOR). However, troglitazone did not directly inhibit mTOR kinase activity as shown by in vitro kinase assay. The inhibition of p70S6K is likely to be the result of strong activation of AMP activated protein kinase (AMPK) by TZDs. Stable expression of a dominant negative AMPK in keratinocytes blocked the inhibitory effect of troglitazone on IGF-1 induced phosphorylation of p70S6K. ^ Finally, we found that dietary TZDs inhibited by up to 73% mouse skin tumor development promoted by elevated IGF-1 signaling in BK5-IGF-1 transgenic mice, while they had no or little effect on skin tumor development promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) or ultraviolet (UV). Since IGF-1 signaling is frequently found to be elevated in patients with insulin resistance and in many human tumors, our data suggest that TZDs may provide tumor preventive benefit particularly to these patients. ^

Significance of signal transducer and activator of transcription 3 during multistage mouse skin carcinogenesis

Signal transducer and activator of transcription 3 (Stat3) is a signaling molecule that transduces signal from cell surface receptors, itself translocates into the nucleus, binds to consensus promoter sequences and activates gene transcription. Here, we showed that Stat3 is constitutively activated in both premalignant tumors (papillomas) and squamous cell carcinomas of mouse skin that is induced by topical treatment with an initiator 7,12-dimethylbenz[a]anthracene (DMBA) followed by a tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Additional data demonstrated that epidermal growth factor signaling contributes to the activation of Stat3 in this model. Using mice where Stat3 function is abrogated in keratinocytes via the Cre-LoxP system (K5Cre.Stat3 flox/flox), we demonstrated that Stat3 is required for de novo carcinogenesis since Stat3 deficiency leads to a complete abrogation of skin tumor development induced by DMBA and TPA. We subsequently showed that Stat3 plays a role in both the initiation and promotion stages of carcinogenesis. During initiation, Stat3 functions as an anti-apoptotic molecule for maintaining the survival of DNA-damaged keratinocyte stem cells. During promotion, Stat3 functions as a critical regulator for G1 to S phase cell cycle progression to confer selective clonal expansion of initiated cells into papillomas. On the other hand, using transgenic mice over-expressing a constitutively dimerized form of Stat3 (Stat3C) in keratinocytes (K5.Stat3C), we revealed a role for Stat3 in tumor progression. After treatment with DMBA and TPA, K5.Stat3C transgenic mice developed skin tumors with a shorter latency when 100% bypassed the premalignant stage and became carcinoma in situ. Histological and immunohistochemical analysis revealed these tumors as highly vascularized and poorly differentiated. More strikingly, these tumors exhibited invasion into surrounding mesenchymal tissue, some of which metastasized into lung. The tumor-mesenchymal front was characterized by partial loss of E-cadherin and elevation of vimentin, markers characterizing epithelial-mesenchymal transition. On the other hand, inhibition of Stat3 via a decoy oligonucleotide led to a significant reduction of tumor size in approximately 50% of all papillomas tested. In conclusion, we demonstrated that Stat3 plays a critical in all three stages (initiation, promotion and progression) of skin carcinogenesis, and it may potentially become a good target for cancer prevention and anti-cancer therapy. ^