Angustias Page

Altered skin development and impaired proliferative and inflammatory responses in transgenic mice overexpressing the glucocorticoid receptor

Glucocorticoids (GCs) are potent inhibitors of epidermal proliferation and effective anti‐inflammatory compounds, which make them the drug of choice for a wide range of inflammatory and hyperproliferative skin disorders. GC action is mediated via the glucocorticoid receptor (GR). To study the role of GR in skin development and the molecular mechanisms underlying its action, we generated transgenic mice overexpressing GR in epidermis and other stratified epithelia, under the control of the keratin K5 promoter. Newborn mice show altered skin development, manifested as variable‐sized skin lesions that range from epidermal hypoplasia and underdeveloped dysplastic hair follicles to a complete absence of this tissue. In the most affected individuals, skin was absent at the cranial and umbilical regions, and the vibrissae and eyebrows appear scarce, short, and curly. In addition, as a consequence of transgene expression in other ectodermally derived epithelia, K5‐GR mice exhibited further abnormalities that strikingly resemble the clinical findings in patients with ectodermal dysplasia, which includes aplasia cutis congenita. In adult transgenic skin, topical application of the tumor promoter TPA did not elicit hyperplasia or transcriptional induction of several proinflammatory cytokines. This anti‐inflammatory role of GR was due at least in part to interference with NF‐κB, leading to a strong reduction in the κB‐binding activity without altering the transcriptional levels of the inhibitor IκBα.

A cutaneous gene therapy approach to human leptin deficiencies: correction of the murine ob/ob phenotype using leptin-targeted keratinocyte grafts

Leptin deficiency produces a phenotype of obesity, diabetes, and infertility in the ob/ob mouse. In humans, leptin deficiency occurs in some cases of congenital obesity and in lipodystrophic disorders characterized by reduced adipose tissue and insulin resistance. Cutaneous gene therapy is considered an attractive potential method to correct circulating protein deficiencies, since gene‐transferred human keratinocytes can produce and secrete gene products with systemic action. However, no studies showing correction of a systemic defect have been reported. We report the successful correction of leptin deficiency using cutaneous gene therapy in the ob/ob mouse model. As a feasibility approach, skin explants from transgenic mice overexpressing leptin were grafted on immunodeficient ob/ob mice. One month later, recipient mice reached body weight values of lean animals. Other biochemical and clinical parameters were also normalized. In a second human gene therapy approach, a retroviral vector encoding both leptin and EGFP cDNAs was used to transduce HK and, epithelial grafts enriched in high leptin‐producing HK were transplanted to immunosuppressed ob/ob mice. HK‐derived leptin induced body weight reduction after a drop in blood glucose and food intake. Leptin replacement through genetically engineered HK grafts provides a valuable therapeutic alternative for permanent treatment of human leptin deficiency conditions.—Larcher, F., Del Rio, M., Serrano, F., Segovia, J. C., Ramírez, A., Meana, A., Page, A., Abad, J. L., González, M. A., Bueren, J., Bernad, A., Jorcano, J. L. A cutaneous gene therapy approach to human leptin deficiencies: correction of the murine ob/ob phenotype using leptin‐tar‐geted keratinocyte grafts. FASEB J. 15, 1529–1538 (2001)

Tumor formation in mice with conditional inactivation of Brca1 in epithelial tissues.

The BRCA1 tumor-suppressor protein has been implicated in the regulation of transcription, DNA repair, proliferation, and apoptosis. BRCA1 is expressed in many proliferative tissues and this is at least in part due to E2F-dependent transcriptional control. In this study, inactivation of a conditional murine Brca1 allele was achieved in a variety of epithelial tissues via expression of the Cre recombinase under the control of a keratin 5 (K5) promoter. The K5 Cre:Brca1 conditional knockout mice exhibited modest epidermal hyperproliferation, increased apoptosis, and were predisposed to developing tumors in the skin, the inner ear canal, and the oral epithelium after 1 year of age. Overexpression of the E2F1 transcription factor in K5 Cre:Brca1 conditional knockout mice dramatically accelerated tumor development. In addition, Brca1 heterozygous female mice that had elevated E2F1 expression developed tumors of the reproductive tract at high incidence. These findings demonstrate that in mice Brca1 functions as a tumor suppressor in other epithelial tissues in addition to the mammary gland. Moreover, inactivation of Brca1 is shown to cooperate with deregulation of the Rb-E2F1 pathway to promote tumorigenesis.

A Transposon-Based Analysis of Gene Mutations Related to Skin Cancer Development

Nonmelanoma skin cancer (NMSC) is by far the most frequent type of cancer in humans. NMSC includes several types of malignancies with different clinical outcomes, the most frequent being basal and squamous cell carcinomas. We have used the Sleeping Beauty transposon/transposase system to identify somatic mutations associated with NMSC. Transgenic mice bearing multiple copies of a mutagenic Sleeping Beauty transposon T2Onc2 and expressing the SB11 transposase under the transcriptional control of regulatory elements from the keratin K5 promoter were treated with TPA, either in wild-type or Ha-ras mutated backgrounds. After several weeks of treatment, mice with transposition developed more malignant tumors with decreased latency compared with control mice. Transposon/transposase animals also developed basal cell carcinomas. Genetic analysis of the transposon integration sites in the tumors identified several genes recurrently mutated in different tumor samples, which may represent novel candidate cancer genes. We observed alterations in the expression levels of some of these genes in human tumors. Our results show that inactivating mutations in Notch1 and Nsd1, among others, may have an important role in skin carcinogenesis.

An inactivating CYLD mutation promotes skin tumor progression by conferring enhanced proliferative, survival and angiogenic properties to epidermal cancer cells

In this study, we demonstrate that the expression in tumorigenic epidermal cells of a catalytically inactive form of CYLD (CYLDC/S) that mimics the identified mutations of cyld in human tumors and competes with the endogenous CYLD results in enhanced cell proliferation and inhibition of apoptosis; it also stimulates cell migration and induces the expression of angiogenic factors, including vascular endothelial growth factor-A. Altogether, these characteristics indicate an increased oncogenicity of the tumorigenic epidermal CYLDC/S mutant cells in vitro. Moreover, we show the increase in malignancy of epidermal squamous cell carcinomas that express the CYLDC/S transgene in an in vivo xenograft model. Tumors carrying the mutated CYLDC/S exhibit a fast growth, are poorly differentiated and present a robust angiogenesis. CYLDC/S tumors are also characterized by their elevated proliferation rate and decreased apoptosis. In contrast with previous studies showing the development of benign tumors by mutations in the CYLD gene, here we provide evidence that the occurrence of mutations in the CYLD gene in tumorigenic epidermal cells (carrying previous mutations) increases the aggressiveness of carcinomas, mainly through enhancement of the expression of angiogenic factors, having therefore a key role in epidermal cancer malignancy.

IKKβ Leads to an Inflammatory Skin Disease Resembling Interface Dermatitis

IKKbeta is a subunit of the IkappaB kinase (IKK) complex required for NF-kappaB activation in response to pro-inflammatory signals. NF-kappaB regulates the expression of many genes involved in inflammation, immunity, and apoptosis, and also controls cell proliferation and differentiation in different tissues; however, its function in skin physiopathology remains controversial. In this study we report the alterations caused by increased IKKbeta activity in skin basal cells of transgenic mice. These animals suffered chronic inflammation with abundant macrophages and other CD45(+) infiltrating cells in the skin, which resulted in epidermal basal cell injury and degeneration of hair follicles. They showed histological features characteristic of interface dermatitis (ID). This phenotype is accompanied by an increased production of inflammatory cytokines by transgenic keratinocytes. Accordingly, transcriptome studies show upregulation of genes associated with inflammatory responses. The inflammatory phenotype observed as a consequence of IKKbeta overexpression is independent of T and B lymphocytes, as it also arises in mice lacking these cell types. In summary, our data indicate the importance of IKKbeta in the development of ID and in the homeostasis of stratified epithelia. Our results also support the idea that IKKbeta might be a valid therapeutic target for the treatment of skin inflammatory diseases.

IKKα enhances human keratinocyte differentiation and determines the histological variant of epidermal squamous cell carcinomas

Squamous cell carcinomas (SCCs) of the skin display different clinical features according to their epithelial differentiation grade and histological variant. Understanding the causes of these divergences might increase the curability of SCCs. Therefore, it is important to study the mechanisms of differentiation in keratinocytes. IKK (IκB kinase) α is an important protein for epidermal morphogenesis, although the pathways through which it exerts its function are unknown and controversy exists about its role in cancer development. We show that enhanced IKKα expression increases both early and terminal differentiation of human keratinocytes through an E-cadherin-dependent mechanism. Increased expression of IKKα in mouse tumorigenic epidermal cells leads to changes in the differentiation pattern of the resulting SCCs, originating a distinct histological variant that resembles the human acantholytic SCC (ASCC) variant. Although human ASCCs have an aggressive clinical course and high risk of metastasis, nothing is known about their etiology. We show that human ASCCs, as observed in the counterpart IKKα murine tumors, express high levels of both IKKα and E-cadherin, with absence of keratins K1 and K10, usually co-expressed with IKKα and E-cadherin. The tight correlation between the properties of both murine and human ASCC variants strongly suggests that IKKα is responsible for the development of this human SCC variant.

Role of phosphorylated p50-NF-?B in the ultraviolet response of mouse skin

The skin constitutes a primary target for stimuli such as ultraviolet (UV) radiation and tumor promoters, leading to both inflammatory and altered proliferative responses. Since the nuclear factor κB (NF‐κB) family of transcription factors plays a major role in these biological processes, we sought to elucidate its expression in newborn mouse skin upon UV and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) exposures. We have identified the nuclear NF‐κB binding activity in mouse skin as composed of p50/p65 heterodimers and p50 homodimers by supershift assays using different NF‐κB–containing sequences. After UV exposure, but not TPA treatment, we detected increased NF‐κB binding activity that correlated with a decrease of IκBα protein levels, although it was not accompanied by p50 or p65 translocation. Immunostaining of newborn mouse sections confirmed that p50 was predominantly localized in the cytoplasm of epidermal basal cells before and after UV treatment. By immunoblotting, we found distinct phosphorylated forms of p50 in cytoplasmic extracts, while only a hyperphosphorylated form was detected in nuclear extracts. In vitro dephosphorylation of skin extracts dramatically reduced the affinity of p50‐containing dimers for DNA. Our data suggest that the NF‐κB response of mouse skin to UV exposure, contrary to most stimuli in other tissues, implies additional mechanisms other than translocation, such as p50 phosphorylation. Mol. Carcinog. 27:272–279, 2000.

Increased IKKα Expression in the Basal Layer of the Epidermis of Transgenic Mice Enhances the Malignant Potential of Skin Tumors

Non-melanoma skin cancer is the most frequent type of cancer in humans. In this study we demonstrate that elevated IKKα expression in murine epidermis increases the malignancy potential of skin tumors. We describe the generation of transgenic mice overexpressing IKKα in the basal, proliferative layer of the epidermis and in the outer root sheath of hair follicles. The epidermis of K5-IKKα transgenic animals shows several alterations such as hyperproliferation, mislocalized expression of integrin-α6 and downregulation of the tumor suppressor maspin. Treatment of the back skin of mice with the mitogenic agent 12-O-tetradecanoylphorbol-13-acetate causes in transgenic mice the appearance of different preneoplastic changes such as epidermal atypia with loss of cell polarity and altered epidermal tissue architecture, while in wild type littermates this treatment only leads to the development of benign epidermal hyperplasia. Moreover, in skin carcinogenesis assays, transgenic mice carrying active Ha-ras (K5-IKKα-Tg.AC mice) develop invasive tumors, instead of the benign papillomas arising in wild type-Tg-AC mice also bearing an active Ha-ras. Therefore we provide evidence for a tumor promoter role of IKKα in skin cancer, similarly to what occurs in other neoplasias, including hepatocarcinomas and breast, prostate and colorectal cancer. The altered expression of cyclin D1, maspin and integrin-α6 in skin of transgenic mice provides, at least in part, the molecular bases for the increased malignant potential found in the K5-IKKα skin tumors.

IKKβ Overexpression Leads to Pathologic Lesions in Stratified Epithelia and Exocrine Glands and to Tumoral Transformation of Oral Epithelia

Alterations in nuclear factor kappaB (NFκB) signaling have been related with several diseases and importantly also with cancer. Different animal models with increased or diminished NFκB signaling have shown that NFκB subunits and their regulators are relevant to the pathophysiology of different organs and tissues. In particular, both the deletion of the regulatory subunit β of the kinase of the inhibitor of NFκB (IKKβ) and its overexpression in epidermis lead to the development of skin inflammatory diseases not associated with tumoral lesions. In this work, we have studied the consequences of IKKβ overexpression in other organs and tissues. We found that elevated IKKβ levels led to altered development and functionality of exocrine glands (i.e., mammary glands) in transgenic female mice. In oral epithelia, increased IKKβ expression produced lichenoid inflammation with abundant granulocytes, macrophages, and B cells, among other inflammatory cells. This inflammatory phenotype was associated with high incidence of tumoral lesions in oral epithelia, contrary to what was found in skin. Moreover, IKKβ also increased the malignant progression of both spontaneous and experimentally induced oral tumors. These results highlight the importance of IKKβ in epithelial and glandular homeostasis as well as in oral tumorigenesis and open the possibility that IKKβ activity might be implicated in the development of oral cancer in humans. Mol Cancer Res; 9(10); 1329–38. ©2011 AACR.