Angel Ramírez

Increased epidermal tumors and increased skin wound healing in transgenic mice overexpressing the catalytic subunit of telomerase, mTERT, in basal keratinocytes

Telomerase transgenics are an important tool to assess the role of telomerase in cancer, as well as to evaluate the potential use of telomerase for gene therapy of age‐associated diseases. Here, we have targeted the expression of the catalytic component of mouse telomerase, mTERT, to basal keratinocytes using the bovine keratin 5 promoter. These telomerase‐transgenic mice are viable and show histologically normal stratified epithelia with high levels of telomerase activity and normal telomere length. Interestingly, the epidermis of these mice is highly responsive to the mitogenic effects of phorbol esters, and it is more susceptible than that of wild‐type littermates to the development skin tumors upon chemical carcinogenesis. The epidermis of telomerase‐transgenic mice also shows an increased wound‐healing rate compared with wild‐type littermates. These results suggest that, contrary to the general assumption, telomerase actively promotes proliferation in cells that have sufficiently long telomeres and unravel potential risks of gene therapy for age‐associated diseases based on telomerase upregulation.

Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes

β1 integrins are ubiquitously expressed receptors that mediate cell–cell and cell–extracellular matrix interactions. To analyze the function of β1 integrin in skin we generated mice with a keratinocyte‐restricted deletion of the β1 integrin gene using the cre–loxP system. Mutant mice developed severe hair loss due to a reduced proliferation of hair matrix cells and severe hair follicle abnormalities. Eventually, the malformed hair follicles were removed by infiltrating macrophages. The epidermis of the back skin became hyperthickened, the basal keratinocytes showed reduced expression of α6β4 integrin, and the number of hemidesmosomes decreased. Basement membrane components were atypically deposited and, at least in the case of laminin‐5, improperly processed, leading to disruption of the basement membrane and blister formation at the dermal–epidermal junction. In contrast, the integrity of the basement membrane surrounding the β1‐deficient hair follicle was not affected. Finally, the dermis became fibrotic. These results demonstrate an important role of β1 integrins in hair follicle morphogenesis, in the processing of basement membrane components, in the maintenance of some, but not all basement membranes, in keratinocyte differentiaton and proliferation, and in the formation and/or maintenance of hemidesmosomes.

Hedgehog Signaling Regulates Sebaceous Gland Development

Epithelial progenitor cells in skin give rise to multiple lineages, comprising the hair follicle, an associated sebaceous gland, and overlying epidermis; however, the signals that regulate sebocyte development are poorly understood. We tested the potential involvement of the Hedgehog pathway in sebaceous gland development using transgenes designed to either block or stimulate Hedgehog signaling in cutaneous keratinocytes in vivo. Whereas inhibition of the Hedgehog pathway selectively suppressed sebocyte development, Hedgehog pathway activation led to a striking increase both in size and number of sebaceous glands. Remarkably, ectopic Hedgehog signaling also triggered the formation of sebaceous glands from footpad epidermis, in regions normally devoid of hair follicles and associated structures. These ectopic sebaceous glands expressed molecular markers of sebocyte differentiation and were functional, secreting their contents directly onto the skins surface instead of into a hair canal. The Hedgehog pathway thus plays a key role in sebocyte cell fate decisions and is a potential target for treatment of skin disorders linked to abnormal sebaceous gland function, such as acne.

Endogenous Myc controls mammalian epidermal cell size, hyperproliferation, endoreplication and stem cell amplification

The transcription factor Myc (c-Myc) plays an important role in cell growth and cell death, yet its physiological function remains unclear. Ectopic activation of Myc has been recently suggested to regulate cell mass, and Drosophila dmyc controls cellular growth and size independently of cell division. By contrast, it has been proposed that in mammals Myc controls cell division and cell number. To gain insights into this debate we have specifically knocked out Myc in epidermis. Myc epidermal knockout mice are viable and their keratinocytes continue to cycle, but they display severe skin defects. The skin is tight and fragile, tears off in areas of mechanical friction and displays impaired wound healing. Steady-state epidermis is thinner, with loss of the proliferative compartment and premature differentiation. Remarkably, keratinocyte cell size, growth and endoreplication are reduced, and stem cell amplification is compromised. The results provide new and direct evidence for a role for endogenous Myc in cellular growth that is required for hyperproliferative cycles and tissue homeostasis.

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)

Exocrine pancreatic disorders in transsgenic mice expressing human keratin 8

Keratins K8 and K18 are the major components of the intermediate-filament cytoskeleton of simple epithelia. Increased levels of these keratins have been correlated with various tumor cell characteristics, including progression to malignancy, invasive behavior, and drug sensitivity, although a role for K8/K18 in tumorigenesis has not yet been demonstrated. To examine the function of these keratins, we generated mice expressing the human K8 (hk8) gene, which leads to a moderate keratin-content increase in their simple epithelia. These mice displayed progressive exocrine pancreas alterations, including dysplasia and loss of acinar architecture, redifferentiation of acinar to ductal cells, inflammation, fibrosis, and substitution of exocrine by adipose tissue, as well as increased cell proliferation and apoptosis. Histological changes were not observed in other simple epithelia, such as the liver. Electron microscopy showed that transgenic acinar cells have keratins organized in abundant filament bundles dispersed throughout the cytoplasm, in contrast to control acinar cells, which have scarce and apically concentrated filaments. The phenotype found was very similar to that reported for transgenic mice expressing a dominant-negative mutant TGF-beta type II receptor (TGFbetaRII mice). We show that these TGFbetaRII mutant mice also have elevated K8/K18 levels. These results indicate that simple epithelial keratins play a relevant role in the regulation of exocrine pancreas homeostasis and support the idea that disruption of mechanisms that normally regulate keratin expression in vivo could be related to inflammatory and neoplastic pancreatic disorders.

Conditional ablation of C/EBPβ demonstrates its keratinocyte-specific requirement for cell survival and mouse skin tumorigenesis

The CCAAT/enhancer binding protein β (C/EBPβ) is implicated in the regulation of many different molecular and physiological processes. Mice with a germline deletion of C/EBPβ (C/EBPβ−/−) display phenotypes in a multitude of cell types and organ systems, including skin where C/EBPβ−/− mice exhibit increased apoptosis in epidermal keratinocytes in response to carcinogen treatment and are completely resistant to carcinogen-induced skin tumorigenesis. To determine the contribution of systemic versus cell autonomous functions of C/EBPβ to specific phenotypes, mice with a conditional ‘floxed’ C/EBPβ null allele were generated. Epidermal-specific deletion of C/EBPβ was achieved by Cre recombinase expression from a keratin 5 (K5) promoter. Similar to C/EBPβ−/− mice, K5-Cre;C/EBPβfl/fl mice were completely refractory to 7,12 dimethylbenz[a]anthracene (DMBA)-induced skin tumorigenesis and these mice displayed increased DMBA-induced apoptosis in epidermal keratinocytes compared to wild-type mice. In contrast, mice lacking the related gene, C/EBPδ, were not resistant to DMBA-induced skin tumorigenesis, indicating a unique role of C/EBPβ in skin tumor development. Our findings demonstrate that C/EBPβ exerts an essential, keratinocyte-intrinsic role in cell survival in response to carcinogen treatment and the elimination of C/EBPβ in keratinocytes is sufficient to confer complete resistance of the skin to chemical carcinogenesis.

Chronic lymphocytic leukaemia with 17p deletion: a retrospective analysis of prognostic factors and therapy results.

Patients with chronic lymphocytic leukaemia (CLL) whose tumour cells harbour a 17p deletion (17p‐) are universally considered to have a poor prognosis. The deletion can be detected at diagnosis or during the evolution of the disease, particularly in patients who have received chemotherapy. We sought to evaluate the natural history of patients with 17p‐ CLL, identify predictive factors within this prognostic subgroup, and evaluate the results of different therapeutic approaches. Data from 294 patients with 17p‐ CLL followed up at 20 different institutions was retrospectively collected and analysed. Median age was 68 (range 27–98) years at the time of fluorescence in situ hybridization analysis. After 17p‐ documentation, 52% received treatment, achieving an overall response rate of 50%. Median overall survival was 41 months, and was significantly shorter in patients with elevated beta2‐microglobulin concentration (P < 0·001), B symptoms (P = 0·016), higher percentage of cells with deletion (P < 0·001), and acquired deletions (P = 0·012). These findings suggest that patients with 17p‐ CLL have a variable prognosis that can be refined using simple clinical and laboratory features, including 17p‐ clone size, beta2‐microglobulin concentration, presence of B symptoms and type of deletion (de novo versus acquired).

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.