FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1972145289> ?p ?o ?g. }

• W1972145289 endingPage "2779" @default.
• W1972145289 startingPage "2771" @default.
• W1972145289 abstract "We recently demonstrated that mice lacking the epidermal glucocorticoid (GC) receptor (GR) (GR epidermal knockout (GREKO) mice) have developmental defects and sensitivity to epidermal challenge in adulthood. We examined the susceptibility of GREKO mice to skin chemical carcinogenesis. GREKO mice treated with a low dose of 12-dimethylbenz(a) anthracene (DMBA) followed by phorbol 12-myristate 13-acetate (PMA) promotion exhibited earlier papilloma formation with higher incidence and multiplicity relative to control littermates (CO). Augmented proliferation and inflammation and defective differentiation of GREKO keratinocytes contributed to the phenotype, likely through increased AKT and STAT3 (signal transducer and activator of transcription 3) activities. GREKO tumors exhibited signs of early malignization, including delocalized expression of laminin A, dermal invasion of keratin 5 (K5)–positive cells, K13 expression, and focal loss of E-cadherin. Cultured GREKO keratinocytes were spindle like, with loss of E-cadherin and upregulation of smooth muscle actin (SMA) and Snail, suggesting partial epithelial–mesenchymal transition. A high DMBA dose followed by PMA promotion generated sebaceous adenomas and melanocytic foci in GREKO and CO. Importantly, the number, growth kinetics, and extent of both tumor types increased in GREKO mice, suggesting that in addition to regulating tumorigenesis from epidermal lineages, GR in keratinocytes is important for cross-talk with other skin cells. Altogether, our data reinforce the importance of GR in the pathogenesis of skin cancer. We recently demonstrated that mice lacking the epidermal glucocorticoid (GC) receptor (GR) (GR epidermal knockout (GREKO) mice) have developmental defects and sensitivity to epidermal challenge in adulthood. We examined the susceptibility of GREKO mice to skin chemical carcinogenesis. GREKO mice treated with a low dose of 12-dimethylbenz(a) anthracene (DMBA) followed by phorbol 12-myristate 13-acetate (PMA) promotion exhibited earlier papilloma formation with higher incidence and multiplicity relative to control littermates (CO). Augmented proliferation and inflammation and defective differentiation of GREKO keratinocytes contributed to the phenotype, likely through increased AKT and STAT3 (signal transducer and activator of transcription 3) activities. GREKO tumors exhibited signs of early malignization, including delocalized expression of laminin A, dermal invasion of keratin 5 (K5)–positive cells, K13 expression, and focal loss of E-cadherin. Cultured GREKO keratinocytes were spindle like, with loss of E-cadherin and upregulation of smooth muscle actin (SMA) and Snail, suggesting partial epithelial–mesenchymal transition. A high DMBA dose followed by PMA promotion generated sebaceous adenomas and melanocytic foci in GREKO and CO. Importantly, the number, growth kinetics, and extent of both tumor types increased in GREKO mice, suggesting that in addition to regulating tumorigenesis from epidermal lineages, GR in keratinocytes is important for cross-talk with other skin cells. Altogether, our data reinforce the importance of GR in the pathogenesis of skin cancer. control littermate 12-dimethylbenz(a) anthracene extracellular signal–regulated kinase glucocorticoid glucocorticoid receptor GR epidermal knockout keratin melanocytic foci phorbol 12-myristate 13-acetate smooth muscle actin signal transducer and activator of transcription 3 Glucocorticoid (GC) derivatives are the most widely used therapeutic agents for treating numerous cutaneous diseases (Schmuth et al., 2007Schmuth M. Watson R.E. Deplewski D. et al.Nuclear hormone receptors in human skin.Horm Metab Res. 2007; 39: 96-105Crossref PubMed Scopus (47) Google Scholar). In addition, GCs are routinely used in cancer therapy as coadjuvants to reduce side effects of chemotherapeutic agents, and their use as immunosuppressors is standardized in clinical practice (De Bosscher and Haegeman, 2009De Bosscher K. Haegeman G. Minireview: latest perspectives on antiinflammatory actions of glucocorticoids.Mol Endocrinol. 2009; 23: 281-291Crossref PubMed Scopus (213) Google Scholar; Barnes, 2011Barnes P.J. Glucocorticosteroids: current and future directions.Br J Pharmacol. 2011; 163: 29-43Crossref PubMed Scopus (377) Google Scholar). It is thus crucial to understand the mechanisms by which these compounds influence the development and progression of skin tumors in order to improve the design of combined therapies to combat cancer. Both endogenous and synthetic GCs exert their actions through the ubiquitous intracellular GC receptor (GR), a member of the steroid nuclear receptor superfamily of ligand-dependent transcription factors (De Bosscher and Haegeman, 2009De Bosscher K. Haegeman G. Minireview: latest perspectives on antiinflammatory actions of glucocorticoids.Mol Endocrinol. 2009; 23: 281-291Crossref PubMed Scopus (213) Google Scholar; Revollo and Cidlowski, 2009Revollo J.R. Cidlowski J.A. Mechanisms generating diversity in glucocorticoid receptor signaling.Ann NY Acad Sci. 2009; 1179: 167-178Crossref PubMed Scopus (163) Google Scholar; Nicolaides et al., 2010Nicolaides N.C. Galata Z. Kino T. et al.The human glucocorticoid receptor: molecular basis of biologic function.Steroids. 2010; 75: 1-12Crossref PubMed Scopus (316) Google Scholar). GR is required for postnatal survival and has a crucial role in skin pathophysiology, as demonstrated by the generation and characterization of genetically modified mice with both gain and loss of function of GR (Cole et al., 1995Cole T.J. Blendy A.P. Monaghan K.A. et al.Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation.Genes Dev. 1995; 9: 1608-1621Crossref PubMed Scopus (784) Google Scholar; Tronche et al., 1999Tronche F. Kellendonk C. Kretz O. et al.Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety.Nat Genet. 1999; 23: 99-103Crossref PubMed Scopus (1435) Google Scholar; Tuckermann et al., 1999Tuckermann J.P. Reichardt H.M. Arribas R. et al.The DNA-binding-independent function of the glucocorticoid receptor mediates repression of AP-1-dependent genes in skin.J Cell Biol. 1999; 147: 1365-1370Crossref PubMed Scopus (167) Google Scholar; Pérez, 2011Pérez P. Glucocorticoid receptors, epidermal homeostasis and hair follicle differentiation.Dermatoendocrinol. 2011; 3: 1-9Crossref PubMed Scopus (30) Google Scholar). We previously reported that transgenic mice with keratinocyte-specific GR overexpression were highly resistant to developing skin papillomas, with fewer papillomas that appeared with slower kinetics relative to control mice (Budunova et al., 2003Budunova I.V. Kowalczyky D. Perez P. et al.Glucocorticoid receptor functions as a potent suppressor of mouse skin carcinogenesis.Oncogene. 2003; 22: 3279-3287Crossref PubMed Scopus (29) Google Scholar). However, whether mice lacking GR in the epidermis would be more susceptible to tumorigenesis was not known. We recently demonstrated that the selective inactivation of GR in mouse epidermis (GR epidermal knockout or (GREKO) mice) led to abnormal skin development and exacerbated susceptibility to various epidermal challenges in the adult age (Sevilla et al., 2013Sevilla L.M. Latorre V. Sanchis A. et al.Epidermal inactivation of the glucocorticoid receptor triggers skin barrier defects and cutaneous inflammation.J Invest Dermatol. 2013; 133: 361-370Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In fact, acute phorbol 12-myristate 13-acetate (PMA) treatment of GREKO skin resulted in exaggerated epidermal keratinocyte hyperproliferation and increased immune infiltrates. With these antecedents, we examined the susceptibility of GREKO mice to skin tumor formation and development using two different protocols with distinct doses of initiating and promoting agents (Abel et al., 2009Abel E.L. Angel J.M. Kiguchi K. et al.Multi-stage chemical carcinogenesis in mouse skin: Fundamentals and applications.Nat Protoc. 2009; 4: 1350-1362Crossref PubMed Scopus (383) Google Scholar). The use of either low or high 12-dimethylbenz(a) anthracene (DMBA) dosage followed by distinct dosage of PMA produced either skin papillomas or sebaceous tumors and dermal melanocytic foci (MFs), respectively, in the mouse strain used in this study. GREKO mice exhibited earlier kinetics of skin tumor growth, higher tumor incidence and multiplicity, as well as increased tumor size, as compared with control littermates (CO). Altogether, our data reinforce the importance of GR in the pathogenesis of skin cancer. We examined the susceptibility of GREKO mice to skin tumor formation and development by using different dosage and kinetics of the two-stage DMBA/PMA carcinogenesis protocol. It is relevant to highlight that different mouse strains require different concentrations of DMBA and PMA for effective tumor formation in this protocol (Abel et al., 2009Abel E.L. Angel J.M. Kiguchi K. et al.Multi-stage chemical carcinogenesis in mouse skin: Fundamentals and applications.Nat Protoc. 2009; 4: 1350-1362Crossref PubMed Scopus (383) Google Scholar). In a first set of experiments, the dorsal skin of age-matched GREKO and CO mice was topically treated with a low dose of DMBA (23nmol), followed by twice-weekly doses of PMA (10μg). As expected, DMBA initiation caused mutation of H-ras in codon 61 (A–T) in papillomas from both genotypes (Supplementary Figure S1a online; Abel et al., 2009Abel E.L. Angel J.M. Kiguchi K. et al.Multi-stage chemical carcinogenesis in mouse skin: Fundamentals and applications.Nat Protoc. 2009; 4: 1350-1362Crossref PubMed Scopus (383) Google Scholar). Papillomas developed in GREKO mice at 8 weeks after the start of promotion and thereafter continued to increase in both number and size (Figure 1a and b and Supplementary Figure S2 online). In CO mice, papillomas were not detected until 10 weeks, and only 75% of animals developed tumors relative to 100% of GREKO mice (Supplementary Figure S2b online). We observed an increase in both the average number of tumors per mouse and the total number of tumors developed by GREKO mice relative to their CO counterparts, with statistical significance from 10 weeks onward (Figure 1a and Supplementary Figure S2b online). The mean number of papillomas <2mm was 3-fold increased, whereas the average number of tumors 2>mm was 5-fold higher in GREKO relative to CO mice (Figure 1b). We did not observe a statistical bias toward the susceptibility to tumor formation by gender within a given genotype (Supplementary Figure S2a online). Download .pdf (1.42 MB) Help with pdf files Supplementary Information All CO tumors generated by this protocol were well-differentiated papillomas, the epidermal–dermal border was well defined, and tumor differentiation was histologically evident by the presence of the so-called keratin pearls (Figure 1c–e). GREKO skin tumors were also papillomas, but they showed several features of poor differentiation including a diffuse epidermal–dermal border and frequent signs of keratinocyte atypia (Figure 1d′ and e′; discontinuous lines and arrows). Moreover, the expression of laminin A was delocalized in GREKO tumors instead of being confined to the basement membrane of the epidermis, as in CO papillomas (Figure 1f and f′). These features were unique to GREKO tumors, as adjacent skin showed similar epidermal hyperplasia and basement membrane deposition of laminin as CO (Supplementary Figure S3 online). In 40% of GREKO tumors assessed, and none of CO, keratinocytes invaded the underlying dermis, as shown by K5 immunostaining (Supplementary Figure S1g online, arrowheads). In another set of experiments, a high DMBA dose (390nmol) followed by three-times-weekly topical application of PMA (6μg) for 31 weeks generated sebaceous adenomas, as well as MFs in GREKO and CO littermates (Figure 2). The generation of sebaceous tumors as well as the accumulation of dermal melanocytes, which were visualized as nevi, have been reported in other studies using similar high doses of DMBA in different mouse strains (Rice and Anderson, 1986Rice J.M. Anderson L.M. Sebaceous adenomas with associated epidermal hyperplasia and papilloma formation as a major type of tumor induced in mouse skin by high doses of carcinogens.Cancer Lett. 1986; 33: 295-306Abstract Full Text PDF PubMed Scopus (10) Google Scholar; Husain et al., 1991Husain Z. Pathak M.A. Flotte T. et al.Role of ultraviolet radiation in the induction of melanocytic tumors in hairless mice following 7,12- dimethylbenz(a)anthracene application and ultraviolet irradiation.Cancer Res. 1991; 51: 4964-4970PubMed Google Scholar; Indra et al., 2007Indra A.K. Castaneda E. Antal M.C. et al.Malignant transformation of DMBA/TPA-induced papillomas and nevi in the skin of mice selectively lacking retinoid-X-receptor alpha in epidermal keratinocytes.J Invest Dermatol. 2007; 127: 1250-1260Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In both GREKO and CO mice, sebaceous tumors contained the characteristic A-to-T mutation in codon 61 of the H-ras gene (Supplementary Figure S1a online). GREKO mice showed early kinetics of sebaceous tumor appearance relative to CO littermates (13 vs. 19 weeks), as well as an elevated total tumor number (5- to 7-fold at late stages of tumor promotion; Figure 2a); however, the histology of GREKO sebaceous tumors was indistinguishable to that of controls (Figure 2b and b′). The sebaceous glands of GREKO mice are also targeted by the Krt5-driven recombinase, and thus GR is inactivated in sebocytes (Supplementary Figure S1b online). It has been reported that low levels of β-catenin and activator protein-1 signaling can favor sebaceous instead of epidermal tumor formation (Gerdes et al., 2006Gerdes M.J. Myakishev M. Frost N.A. et al.Activator protein-1 activity regulates epithelial tumor cell identity.Cancer Res. 2006; 66: 7578-7588Crossref PubMed Scopus (61) Google Scholar; Niemann et al., 2007Niemann C. Owens D.M. Schettina P. et al.Dual role of inactivating Lef1 mutations in epidermis: tumor promotion and specification of tumor type.Cancer Res. 2007; 67: 2916-2921Crossref PubMed Scopus (60) Google Scholar). We observed a decrease in the levels of β-catenin and p-c-Jun in sebaceous tumors relative to the epidermis of GREKO and CO mice; however, there were no clear differences between genotypes (Supplementary Figure S4 online). Although we cannot rule out the contribution of other signaling pathways to the development of sebaceous adenomas in our experiment, it is likely that the doses and kinetics of DMBA/PMA treatments together with the genetic background are responsible for the generation of sebaceous rather than epidermal tumors. With regard to the MFs, their total number, kinetics of growth, and extent were markedly increased in mice lacking epidermal GR (Figure 2c, d, and d′). We detected a 3-fold increase of these foci that appeared on average 3 weeks earlier in GREKO relative to CO mice (Figure 2c). In addition, the MFs in GREKO mice consistently invaded the deep dermis (Figure 2). As K5-Cre is not expressed in melanocytes (Supplementary Figure S1b online), these data highlight a role for keratinocytic GR in the regulation of other cell types that populate the skin. Our data are consistent with previous results describing that GCs can inhibit melanoma growth in mice, and are also compatible with reports indicating that GC-based therapy was protective against melanoma incidence in a Mediterranean population (Landi et al., 2001Landi M.T. Baccarelli A. Calista D. et al.Glucocorticoid use and melanoma risk.Int J Cancer. 2001; 94: 302-303Crossref PubMed Scopus (7) Google Scholar). Importantly, there were similarities in the response of GREKO mice to chemical carcinogenesis protocols and that of mice lacking skin expression of other nuclear hormone receptors including retinoid X receptor α, peroxisome proliferator–activated receptor γ, and vitamin D receptor (Indra et al., 2007Indra A.K. Castaneda E. Antal M.C. et al.Malignant transformation of DMBA/TPA-induced papillomas and nevi in the skin of mice selectively lacking retinoid-X-receptor alpha in epidermal keratinocytes.J Invest Dermatol. 2007; 127: 1250-1260Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). This is likely due to the production of soluble factors such as En1, Fgf2, and Scf by keratinocytes, which contributes to melanocyte activation and migration (Wang et al., 2011Wang Z. Coleman D.J. Bajaj G. et al.RXRa ablation in epidermal keratinocytes enhances UVR-induced DNA damage, apoptosis, and proliferation of keratinocytes and melanocytes.J Invest Dermatol. 2011; 131: 177-187Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Analogous to these hormone receptors, the increased skin tumor formation and progression of GREKO mice suggests a role for GR as a tumor suppressor in both nonmelanoma and melanoma skin cancers, as well as in the cross-talk between keratinocytes and melanocytes. Consistent with the growth inhibitory role of GR in keratinocytes, the epidermal-specific loss of GR caused an increase in basal keratinocyte proliferation of ∼30% in transgenic versus CO tumors (Figure 3a, a′ and d). In addition, we observed increased suprabasal (white arrows) keratinocyte proliferation in GREKO tumors, whereas the majority of proliferation was confined to the basal layer (black arrows) in CO. It is well known that CycD1 is a key target for activated ras in the mouse skin carcinogenesis protocol and that it is an indicator of the proliferative response (Robles et al., 1998Robles A.I. Rodriguez-Puebla M.L. Glick A.B. et al.Reduced skin tumor development in cyclin D1-deficient mice highlights the oncogenic ras pathway in vivo.Genes Dev. 1998; 12: 2469-2474Crossref PubMed Scopus (203) Google Scholar). We detected augmented CycD1 expression in keratinocytes throughout all epidermal layers (Figure 3b and b′), as well as strong upregulation of Ccnd1 mRNA levels in GREKO tumors (data not shown), which is consistent with the GR-mediated inhibition of this gene in mouse epidermis (Surjit et al., 2011Surjit M. Ganti K.P. Mukherji A. et al.Widespread negative response elements mediate direct repression by agonist-liganded glucocorticoid receptor.Cell. 2011; 145: 224-241Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). GR exerts anti-inflammatory effects in skin and, accordingly, the lack of GR in keratinocytes resulted in marked immune infiltrates in the stroma (Figure 3e′, black arrows) as well as within the epidermal layers of GREKO tumors (Figure 3e′, white arrows). These histological changes correlated with the increased expression of the proinflammatory cytokine IL-6 in the GREKO tumor epithelia relative to CO (Figure 3f and f′). As Il6 and other cytokines are transcriptional targets of GR repression, we examined the relative mRNA levels of Il6, Tnfa, and Il1b in the skin of GREKO and CO mice adjacent to the tumors (Figure 3h). We detected statistically significant upregulation of all cytokines (7-fold for Il6 and 2- to 3-fold for Tnfa and Il1b) in GREKO skin compared with CO (Figure 3h), thus demonstrating that the lack of GR augments cutaneous inflammation during carcinogenesis that may contribute to the development and progression of skin tumors. Well-differentiated benign papillomas express normal markers of the epidermal basal (K5) and suprabasal (K10, loricrin) layers, but not those of internal stratified epithelia such as K13 (Abel et al., 2009Abel E.L. Angel J.M. Kiguchi K. et al.Multi-stage chemical carcinogenesis in mouse skin: Fundamentals and applications.Nat Protoc. 2009; 4: 1350-1362Crossref PubMed Scopus (383) Google Scholar). Assessment of the expression of these keratins is routinely used to classify papillomas into low, intermediate, or high risk of malignant conversion. The immunohistochemical analysis of CO skin tumors showed the expression of K5 throughout all layers, as well as suprabasal K10 and loricrin restricted to the most differentiated outer layers (Figure 4a–c). We did not detect any CO papilloma with K13 expression (Figure 4d). In contrast, GREKO tumors showed a reduction in K10-positive layers and focal loss of loricrin (Figure 4b′ and c′, arrowheads), with 30% of the tumors staining positive for K13 (Figure 4d′). Moreover, we detected reduced expression of the epithelial marker E-cadherin, which mediates cell–cell adhesion, in regions of GREKO but not in CO tumors (Figure 4e and e′). Immunoblotting of protein extracts from papillomas for E-cadherin failed to show a statistically significant difference between genotypes, perhaps owing to the focal nature of the decreased expression in GREKO tumors (not shown). Our data show that augmented proliferation and inflammation, as well as defective differentiation of epidermal keratinocytes lacking GR, contributed to the accelerated onset and development of skin tumors of GREKO mice. Overall, these features indicate that the lack of epidermal GR triggers early stages of malignant conversion in GREKO mouse skin tumors. Previous reports have demonstrated that several signaling pathways including AKT, extracellular signal–regulated kinase (ERK), and signal transducer and activator of transcription 3 (STAT3) have a critical role throughout the process of epithelial carcinogenesis in mouse skin (Segrelles et al., 2002Segrelles C. Ruiz S. Pérez P. et al.Functional roles of Akt signaling in mouse skin carcinogenesis.Oncogene. 2002; 21: 53-64Crossref PubMed Scopus (159) Google Scholar; Kataokay et al., 2008Kataokay K. Kimy D.J. Carbajal S. et al.Stage-specific disruption of Stat3 demonstrates a direct requirement during both the initiation and promotion stages of mouse skin tumorigenesis.Carcinogenesis. 2008; 6: 1108-1114Crossref Scopus (57) Google Scholar). We have also shown that AKT and ERK activities were constitutively increased in the epidermis of mice with either total or keratinocyte-specific inactivation of GR (Bayo et al., 2008Bayo P. Sanchis A. Bravo A. et al.Glucocorticoid receptor is required for skin barrier competence.Endocrinology. 2008; 149: 1377-1388Crossref PubMed Scopus (43) Google Scholar; Sevilla et al., 2013Sevilla L.M. Latorre V. Sanchis A. et al.Epidermal inactivation of the glucocorticoid receptor triggers skin barrier defects and cutaneous inflammation.J Invest Dermatol. 2013; 133: 361-370Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Moreover, growth kinetics and malignancy of skin tumors induced by the injection of AKT-transfected mouse keratinocytes was greatly reduced by GR coexpression owing to a nongenomic interaction of GR and p85α/phosphatidylinositol-3-kinase (Leis et al., 2004Leis H. Page A. Ramírez A. et al.Glucocorticoid receptor counteracts tumorigenic activity of Akt in skin through interference with the phosphatidylinositol-3-kinase (PI3K) signalling pathway.Mol Endocrinol. 2004; 18: 303-311Crossref PubMed Scopus (58) Google Scholar). Accordingly, we examined whether the increased sensitivity of GREKO mice to develop epidermal tumors correlated with the increased activity of these signaling pathways. Phosphorylated (p)-ERK is normally restricted to the granular layer of the epidermis (Figure 4f, white arrows); however, increased levels of p-ERK were detected in all epidermal layers in GREKO tumors (Figure 4f′, white and black arrows). p-AKT was detected in tumors from both genotypes, although the levels and the number of positive cells were increased in GREKO relative to CO mice (Figure 4g and g′). STAT3 was localized in some nuclei of CO epidermal tumors (Figure 4h), whereas virtually all nuclei of GREKO tumors were STAT3 positive (Figure 4h′), consistent with the critical role of this transcription factor in multistage epithelial carcinogenesis (Kataokay et al., 2008Kataokay K. Kimy D.J. Carbajal S. et al.Stage-specific disruption of Stat3 demonstrates a direct requirement during both the initiation and promotion stages of mouse skin tumorigenesis.Carcinogenesis. 2008; 6: 1108-1114Crossref Scopus (57) Google Scholar). Quantification of immunoblotting experiments showed that the ratios of both p-AKT/AKT and p-STAT3/STAT3, but not p-ERK/ERK, were increased with statistical significance in GREKO relative to CO papillomas (Figure 4i and j). The inability to observe changes in p-ERK levels may be due to dilution effects, as stromal cells are also present in the protein extracts. These results suggest that the lack of epidermal GR not only accelerates the growth of epidermal tumors but also increases their malignancy, likely owing, at least in part, to the increased activation of the AKT and STAT3 pathways. Given that GREKO mouse skin tumors featured signs of malignant conversion (Figures 1 and 4), we further analyzed the morphology and cell–cell interactions of GREKO versus CO cultured keratinocyte cell lines (Sevilla et al., 2013Sevilla L.M. Latorre V. Sanchis A. et al.Epidermal inactivation of the glucocorticoid receptor triggers skin barrier defects and cutaneous inflammation.J Invest Dermatol. 2013; 133: 361-370Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). At confluency, GREKO keratinocytes featured marked morphological alterations, showing spindle-like phenotype suggestive of a partial EMT (Figure 5a and a′; Moreno-Bueno et al., 2009Moreno-Bueno G. Peinado H. Molina P. et al.The morphological and molecular features of the epithelial-to-mesenchymal transition.Nat Protoc. 2009; 4: 1591-1613Crossref PubMed Scopus (170) Google Scholar). This was confirmed by immunofluorescence staining for E-cadherin and smooth muscle actin (SMA), as epithelial and mesenchymal expression markers, respectively. Whereas confluent CO keratinocytes exposed to 1.2mM calcium showed E-cadherin-mediated cell–cell contacts, GREKO keratinocytes showed decreased cell–cell contacts with almost undetectable E-cadherin expression levels (Figure 5b and b′). Consistent with the absence of E-cadherin, GREKO keratinocytes did not form mature intercellular junctions shown by the reduced accumulation of β-catenin and desmoplakin at sites of cell–cell contact (Figure 5c, c′). Importantly, the expression of SMA was greatly increased in GREKO keratinocytes, although these cells still retained K5 expression (Figure 5e and e′). These results were further confirmed by immunoblotting (Figure 5g and h). Downregulation of E-cadherin is mediated by members of the Snail family of proteins through binding to an E-box located in its promoter (Moreno-Bueno et al., 2009Moreno-Bueno G. Peinado H. Molina P. et al.The morphological and molecular features of the epithelial-to-mesenchymal transition.Nat Protoc. 2009; 4: 1591-1613Crossref PubMed Scopus (170) Google Scholar). Indeed, we detected a statistically significant increase in Snail but not Slug/Snai2 levels in GREKO relative to CO keratinocytes (Figure 5g and h). These data are consistent with the observation that Dex-activated GR abolished Snail binding to the E-cadherin promoter in lung epithelial cells, thus contributing to EMT suppression (Zhang et al., 2010Zhang L. Lei W. Wang X. et al.Glucocorticoid induces mesenchymal-to-epithelial transition and inhibits TGF-β1-induced epithelial-to-mesenchymal transition and cell migration.FEBS Lett. 2010; 584: 4646-4654Crossref PubMed Scopus (44) Google Scholar). The observed changes in GREKO cultured keratinocytes were independent of media composition (Supplementary Figure S5 online). We evaluated the actin cytoskeleton by phalloidin staining and observed a reduction in cortical actin and an increase in stress fibers in GREKO versus CO cells (Figure 5f and f′), another hallmark of EMT (Moreno-Bueno et al., 2009Moreno-Bueno G. Peinado H. Molina P. et al.The morphological and molecular features of the epithelial-to-mesenchymal transition.Nat Protoc. 2009; 4: 1591-1613Crossref PubMed Scopus (170) Google Scholar). As cytoskeleton reorganization could be linked to altered cell motility, we evaluated the migration of GREKO versus CO keratinocytes in a scratch wound assay but did not detect statistically significant changes in wound closure (not shown). This might be related to the lack of Slug upregulation in GREKO keratinocytes, which is essential for re-epithelialization of healing wounds and cannot be substituted by Snail (Sou et al., 2010Sou P.W. Delica N.C. Hallidaya G.M. et al.Snail transcription factors in keratinocytes: enough to make your skin crawl.Int J Biochem Cell Biol. 2010; 42: 1940-1944Crossref PubMed Scopus (17) Google Scholar). Our in vitro results were consistent with the in vivo findings showing that the lack of epidermal GR increases the malignancy of epidermal tumors (Figures 1, 4, and 5). However, immunoblotting of protein extracts from tumors of both genotypes did not show statistically significant changes in E-cadherin or SMA (not shown), likely owing to the fact that in vivo EMT is restricted to a subset of tumor cells. There are contradictory reports regarding the role of GR in EMT, and cell-type specificity seems to be key for the final outcome of GC treatments. Recent studies have shown negative regulation of GCs in the process of EMT, which is vital for tumor invasion and metastasis (Godoy et al., 2010Godoy P. Lakkapamu S. Schug M. et al.Dexamethasone-dependent versus -independent markers of epithelial to mesenchymal transition in primary hepatocytes.Biol Chem. 2010; 391: 73-83Crossref PubMed Google Scholar; Zhang et al., 2010Zhang L. Lei W. Wang X. et al.Glucocorticoid induces mesenchymal-to-epithelial transition and inhibits TG" @default.
• W1972145289 created "2016-06-24" @default.
• W1972145289 creator A5026925465 @default.
• W1972145289 creator A5031976046 @default.
• W1972145289 creator A5049079881 @default.
• W1972145289 creator A5051076709 @default.
• W1972145289 date "2013-12-01" @default.
• W1972145289 modified "2023-10-16" @default.
• W1972145289 title "Selective Ablation of Glucocorticoid Receptor in Mouse Keratinocytes Increases Susceptibility to Skin Tumorigenesis" @default.
• W1972145289 cites W1605768991 @default.
• W1972145289 cites W1761323037 @default.
• W1972145289 cites W1977445011 @default.
• W1972145289 cites W1990968397 @default.
• W1972145289 cites W1991739587 @default.
• W1972145289 cites W1994410887 @default.
• W1972145289 cites W2000333623 @default.
• W1972145289 cites W2005436781 @default.
• W1972145289 cites W2006066882 @default.
• W1972145289 cites W2008654807 @default.
• W1972145289 cites W2020459118 @default.
• W1972145289 cites W2020896282 @default.
• W1972145289 cites W2054731876 @default.
• W1972145289 cites W2059793858 @default.
• W1972145289 cites W2065367870 @default.
• W1972145289 cites W2068246413 @default.
• W1972145289 cites W2068568330 @default.
• W1972145289 cites W2069787603 @default.
• W1972145289 cites W2083951559 @default.
• W1972145289 cites W2085023568 @default.
• W1972145289 cites W2085944780 @default.
• W1972145289 cites W2089195710 @default.
• W1972145289 cites W2092900546 @default.
• W1972145289 cites W2100521322 @default.
• W1972145289 cites W2107570915 @default.
• W1972145289 cites W2114765375 @default.
• W1972145289 cites W2115646733 @default.
• W1972145289 cites W2122181371 @default.
• W1972145289 cites W2137615558 @default.
• W1972145289 cites W2149450040 @default.
• W1972145289 cites W2153086661 @default.
• W1972145289 cites W2169796269 @default.
• W1972145289 cites W4254512484 @default.
• W1972145289 cites W2005523988 @default.
• W1972145289 doi "https://doi.org/10.1038/jid.2013.255" @default.
• W1972145289 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23756710" @default.
• W1972145289 hasPublicationYear "2013" @default.
• W1972145289 type Work @default.
• W1972145289 sameAs 1972145289 @default.
• W1972145289 citedByCount "17" @default.
• W1972145289 countsByYear W19721452892013 @default.
• W1972145289 countsByYear W19721452892015 @default.
• W1972145289 countsByYear W19721452892016 @default.
• W1972145289 countsByYear W19721452892017 @default.
• W1972145289 countsByYear W19721452892018 @default.
• W1972145289 countsByYear W19721452892019 @default.
• W1972145289 countsByYear W19721452892020 @default.
• W1972145289 countsByYear W19721452892021 @default.
• W1972145289 countsByYear W19721452892022 @default.
• W1972145289 crossrefType "journal-article" @default.
• W1972145289 hasAuthorship W1972145289A5026925465 @default.
• W1972145289 hasAuthorship W1972145289A5031976046 @default.
• W1972145289 hasAuthorship W1972145289A5049079881 @default.
• W1972145289 hasAuthorship W1972145289A5051076709 @default.
• W1972145289 hasBestOaLocation W19721452891 @default.
• W1972145289 hasConcept C104317684 @default.
• W1972145289 hasConcept C126322002 @default.
• W1972145289 hasConcept C134018914 @default.
• W1972145289 hasConcept C170493617 @default.
• W1972145289 hasConcept C2778902805 @default.
• W1972145289 hasConcept C2780841215 @default.
• W1972145289 hasConcept C502942594 @default.
• W1972145289 hasConcept C54355233 @default.
• W1972145289 hasConcept C555283112 @default.
• W1972145289 hasConcept C59493245 @default.
• W1972145289 hasConcept C71924100 @default.
• W1972145289 hasConcept C86803240 @default.
• W1972145289 hasConcept C95444343 @default.
• W1972145289 hasConceptScore W1972145289C104317684 @default.
• W1972145289 hasConceptScore W1972145289C126322002 @default.
• W1972145289 hasConceptScore W1972145289C134018914 @default.
• W1972145289 hasConceptScore W1972145289C170493617 @default.
• W1972145289 hasConceptScore W1972145289C2778902805 @default.
• W1972145289 hasConceptScore W1972145289C2780841215 @default.
• W1972145289 hasConceptScore W1972145289C502942594 @default.
• W1972145289 hasConceptScore W1972145289C54355233 @default.
• W1972145289 hasConceptScore W1972145289C555283112 @default.
• W1972145289 hasConceptScore W1972145289C59493245 @default.
• W1972145289 hasConceptScore W1972145289C71924100 @default.
• W1972145289 hasConceptScore W1972145289C86803240 @default.
• W1972145289 hasConceptScore W1972145289C95444343 @default.
• W1972145289 hasIssue "12" @default.
• W1972145289 hasLocation W19721452891 @default.
• W1972145289 hasLocation W19721452892 @default.
• W1972145289 hasLocation W19721452893 @default.
• W1972145289 hasOpenAccess W1972145289 @default.
• W1972145289 hasPrimaryLocation W19721452891 @default.
• W1972145289 hasRelatedWork W2013774132 @default.
• W1972145289 hasRelatedWork W2023035210 @default.

• next