Biomedical subjects

José L Jorcano

Publications and source records attributed to José L Jorcano.

15 recordsLinked to original sources

PubMed2005

Targeted overexpression of leptin to keratinocytes in transgenic mice results in lack of skin phenotype but induction of early leptin resistance.

The epidermis has a great potential as a bioreactor to produce proteins with systemic action. However, the consequences of ectopic epidermal protein overexpression need to be carefully addressed to avoid both local and systemic adverse effects. Thus, the long-term effects of leptin on skin physiology have not been studied, and the metabolic consequences of sustained keratinocyte-derived leptin overexpression are unknown. Herein we describe that very high serum leptin levels can be achieved from a cutaneous source in transgenic mice in which leptin cDNA overexpression was driven by the keratin K5 gene regulatory sequences. Histopathological analysis including the study of skin differentiation and proliferation markers in these transgenic mice revealed that keratinocyte-derived leptin overexpression appears not to have any impact on cutaneous homeostasis. Although young K5-leptin transgenic mice showed remarkable thinness and high glucose metabolism as shown in other leptin transgenic mouse models, a marked leptin insensitivity become apparent as early as 3-4 months of age as demonstrated by increased weight gain and insulin resistance development. Other signs of leptin/insulin resistance included increased bone mass, organomegaly, and wound healing impairment. In addition, to provide evidence for the lack of untoward effects of leptin on epidermis, this transgenic mouse helps us to establish the safe ranges of keratinocyte-derived leptin overexpression and may be useful as a model to study leptin resistance.

Adipose Tissue↗

PubMed2005

Ectoderm-targeted overexpression of the glucocorticoid receptor induces hypohidrotic ectodermal dysplasia.

Hypohidrotic ectodermal dysplasia is a human syndrome defined by maldevelopment of one or more ectodermal-derived tissues, including the epidermis and cutaneous appendices, teeth, and exocrine glands. The molecular bases of this pathology converge in a dysfunction of the transcription factor nuclear factor of the kappa-enhancer in B cells (NF-kappaB), which is essential to epithelial homeostasis and development. A number of mouse models bearing disruptions in NF-kappaB signaling have been reported to manifest defects in ectodermal derivatives. In ectoderm-targeted transgenic mice overexpressing the glucocorticoid receptor (GR) [keratin 5 (K5)-GR mice], the NF-kappaB activity is greatly decreased due to functional antagonism between GR and NF-kappaB. Here, we report that K5-GR mice exhibit multiple epithelial defects in hair follicle, tooth, and palate development. Additionally, these mice lack Meibomian glands and display underdeveloped sweat and preputial glands. These phenotypic features appear to be mediated specifically by ligand-activated GR because the synthetic analog dexamethasone induced similar defects in epithelial morphogenesis, including odontogenesis, in wild-type mice. We have focused on tooth development in K5-GR mice and found that an inhibitor of steroid synthesis partially reversed the abnormal phenotype. Immunostaining revealed reduced expression of the inhibitor of kappaB kinase subunits, IKKalpha and IKKgamma, and diminished p65 protein levels in K5-GR embryonic tooth, resulting in a significantly reduced kappaB-binding activity. Remarkably, altered NF-kappaB activity elicited by GR overexpression correlated with a dramatic decrease in the protein levels of DeltaNp63 in tooth epithelia without affecting Akt, BMP4, or Foxo3a. Given that many of the 170 clinically distinct ectodermal dysplasia syndromes still remain without cognate genes, deciphering the molecular mechanisms of this mouse model with epithelial NF-kappaB and p63 dysfunction may provide important clues to understanding the basis of other ectodermal dysplasia syndromes.

Abnormalities, Multiple↗

PubMed2004

Epidermal abnormalities and increased malignancy of skin tumors in human epidermal keratin 8-expressing transgenic mice.

Keratins K8 and K18 are the major components of the intermediate filament cytoskeleton of simple epithelia. Increased levels of these keratins have been associated with invasive growth and progression to malignancy in different types of human and murine epithelial tumors (including skin tumors), and even in tumors from nonepithelial origin. However, it has not yet clarified whether K8/K18 expression in tumors is cause or consequence of malignancy. Given the increasing incidence of epidermal cancer in humans (40% of all tumors diagnosed), we generated a mouse model to examine the role of simple epithelium keratins in the establishment and progression of human skin cancer. Transgenic mice expressing human K8 in the epidermis showed severe epidermal and hair follicle dysplasia with concomitant alteration in epidermal differentiation markers. The severity of the skin phenotype of these transgenic mice increases with age, leading to areas of preneoplastic transformation. Skin carcinogenesis assays showed a dramatic increase in the progression of papillomas toward malignancy in transgenic animals. These results support the idea that K8 alters the epidermal cell differentiation, favors the neoplastic transformation of cells, and is ultimately responsible of the invasive behavior of transformed epidermal cells leading of conversion of benign to malignant tumors.

Aging↗

PubMed2003

Glucocorticoid Receptor Counteracts Tumorigenic Activity of Akt in Skin through Interference with the Phosphatidylinositol 3-Kinase Signaling Pathway.

The skin-targeted overexpression of the glucocorticoid receptor (GR) in transgenic mice dramatically impairs the inflammatory responses to tumor promoter agents and suppresses skin tumor development. The antiinflammatory, rapid effects of corticosteroids are partially exerted through interference of GR with the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in several tissues, a highly relevant pathway in the mouse skin tumor progression process. In this work, we aimed to elucidate whether a cross-talk mechanism between GR and PI3K/Akt occurred in intact skin as well as the biological relevance of this interaction during skin tumorigenesis. We report that, in transgenic mice overexpressing the receptor, GR physically associated with p85 alpha/PI3K in skin, resulting in decreased Akt and I kappa B kinase activity. GR activation by dexamethasone in normal mouse skin also decreased Akt activity within minutes, whereas cotreatment with the GR antagonist RU486 abolished dexamethasone action. Indeed, GR exerted a nongenomic action because keratinocyte transfection with a transcriptionally defective receptor mutant still decreased PI3K and Akt activity. Moreover, GR coexpression greatly reduced the accelerated growth of malignant tumors and increased Akt activity induced by Akt-transfected keratinocytes, as shown by in vivo tumorigenic assays. Overall, our data strongly indicate that GR/PI3K-Akt cross-talk constitutes a major mechanism underlying the antitumor effect of glucocorticoids in skin.

Administration, Topical↗

PubMed2003

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.

Animals↗

PubMed2003

Glucocorticoid receptor functions as a potent suppressor of mouse skin carcinogenesis.

Glucocorticoids are effective inhibitors of epidermal proliferation and skin tumorigenesis. Glucocorticoids affect cellular functions via glucocorticoid receptor (GR), a well-known transcription factor. Recently, we generated skin-targeted transgenic mice overexpressing GR under control of the keratin5 promoter (K5-GR mice). To test the hypothesis that GR plays a role as a tumor suppressor in skin, we bred K5-GR transgenic mice with Tg.AC transgenic mice, which express v-Ha-ras oncogene in the skin, and compared the susceptibility of F1 offspring to TPA-induced skin carcinogenesis. GR overexpression in the epidermis dramatically inhibited skin tumor development. In K5-GR/ras+ double transgenic mice papillomas developed later and the average number of tumors per animal was 15% (in males) and 40% (in females) of the number seen in wild type (w.t./ras+) littermates. In addition, the papillomas in w.t./ras+ animals were eight to nine times larger. GR overexpression resulted in a decrease in keratinocyte proliferation combined with a modest increase in apoptosis and differentiation of keratinocytes in K5-GR/ras+ papillomas. Our data clearly indicate that interference of GR transgenic protein with nuclear factor kappa B (NF-kappaB) transcription factor had resulted in NF-kappaB blockage in K5-GR/ras+ tumors. We discuss the role of NF-kappaB blockage in tumor-suppressor effect of GR.

Animals↗

PubMed2003

Impaired NF-kappa B activation and increased production of tumor necrosis factor alpha in transgenic mice expressing keratin K10 in the basal layer of the epidermis.

Both the diversity and the precisely regulated tissue- and differentiation-specific expression patterns of keratins suggest that these proteins have specific functions in epithelia besides their well known maintenance of cell integrity. In the search for these specific functions, our previous results have demonstrated that the expression of K10, a keratin expressed in postmitotic suprabasal cells of the epidermis, prevents cell proliferation through the inhibition of Akt kinase activity. Given the roles of Akt in NF-kappa B signaling and the importance of these processes in the epidermis, a study was made into the possible alterations of the NF-kappa B pathway in transgenic mice expressing K10 in the proliferative basal layer. It was found that the inhibition of Akt, mediated by K10 expression, leads to impaired NF-kappa B activity. This appears to occur through the decreased expression of IKK beta and IKK gamma. Remarkably, increased production of tumor necrosis factor alpha and concomitant JNK activation was observed in the epidermis of these transgenic mice. These results confirm that keratin K10 functions in vivo include the control of many aspects of epithelial physiology, which affect the cells not only in a cell autonomous manner but also influence tissue homeostasis.

Animals↗

PubMed2003

Inhibition of tumor angiogenesis by cannabinoids.

Cannabinoids, the active components of marijuana and their derivatives, induce tumor regression in rodents (8). However, the mechanism of cannabinoid antitumoral action in vivo is as yet unknown. Here we show that local administration of a nonpsychoactive cannabinoid to mice inhibits angiogenesis of malignant gliomas as determined by immunohistochemical analyses and vascular permeability assays. In vitro and in vivo experiments show that at least two mechanisms may be involved in this cannabinoid action: the direct inhibition of vascular endothelial cell migration and survival as well as the decrease of the expression of proangiogenic factors (vascular endothelial growth factor and angiopoietin-2) and matrix metalloproteinase-2 in the tumors. Inhibition of tumor angiogenesis may allow new strategies for the design of cannabinoid-based antitumoral therapies.

Angiogenesis Inducing Agents↗

PubMed2003

Modulation of the angiogenesis response through Ha-ras control, placenta growth factor, and angiopoietin expression in mouse skin carcinogenesis.

Tumor angiogenesis is governed by a complex balance of positive and negative angiogenic factors. Development of chemically-induced mouse skin tumors appears to be highly dependent on an early burst of neovascularization. We have previously shown that Ha-ras-driven vascular endothelial growth factor (VEGF) expression plays a pivotal role in this process. However, the status of other critical positive and negative angiogenic factors throughout skin tumorigenesis has not been studied to the same extent. In the present study, we show that another VEGF family member, placenta growth factor (PlGF), was highly upregulated at all tumor stages in a ras-dependent manner. The study of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), ligands of receptor tyrosine kinase 2 (Tie-2), showed that while stroma-derived Ang-2 was increased, epidermal Ang-1 expression was completely abolished at early papilloma formation. Studies using epidermal tumor cell lines suggest that the disappearance of Ang-1 also depends on ras activation, extending the plethora of events controlled by this oncogene in mouse skin carcinogenesis. Our results indicated that tumor development occurred in a strong angiogenesis-prone scenario in which PlGF and Ang-2 acted cooperatively with VEGF, whereas the negative or stabilizing effect of Ang-1 was abrogated. A time-course sequence of expression of angiogenic factors expressed throughout tumor growth, as well as the identification of key signaling molecules triggering the angiogenic response, may contribute to the development and testing of antiangiogenic therapeutic strategies with this in vivo tumor model.

Angiogenesis Inducing Agents↗

PubMed2003

Inhibition of skin tumor growth and angiogenesis in vivo by activation of cannabinoid receptors.

Nonmelanoma skin cancer is one of the most common malignancies in humans. Different therapeutic strategies for the treatment of these tumors are currently being investigated. Given the growth-inhibiting effects of cannabinoids on gliomas and the wide tissue distribution of the two subtypes of cannabinoid receptors (CB(1) and CB(2)), we studied the potential utility of these compounds in anti-skin tumor therapy. Here we show that the CB(1) and the CB(2) receptor are expressed in normal skin and skin tumors of mice and humans. In cell culture experiments pharmacological activation of cannabinoid receptors induced the apoptotic death of tumorigenic epidermal cells, whereas the viability of nontransformed epidermal cells remained unaffected. Local administration of the mixed CB(1)/CB(2) agonist WIN-55,212-2 or the selective CB(2) agonist JWH-133 induced a considerable growth inhibition of malignant tumors generated by inoculation of epidermal tumor cells into nude mice. Cannabinoid-treated tumors showed an increased number of apoptotic cells. This was accompanied by impairment of tumor vascularization, as determined by altered blood vessel morphology and decreased expression of proangiogenic factors (VEGF, placental growth factor, and angiopoietin 2). Abrogation of EGF-R function was also observed in cannabinoid-treated tumors. These results support a new therapeutic approach for the treatment of skin tumors.

Angiogenesis Inducing Agents↗

PubMed2003

Disruption of eyelid and cornea development by targeted overexpression of the glucocorticoid receptor.

Glucocorticoid hormones act through the glucocorticoid receptor (GR) and they affect almost all physiological systems in the organism. We have previously reported that transgenic mice overexpressing GR under the control of the keratin k5 promoter (K5-GR mice) display severe phenotypic alterations in the epidermis and other ectoderm derivatives (Perez et al., 2001). In this work, we aimed to characterize the pathological consequences of GR targeted overexpression in the eyelid and cornea at late developmental stages. Despite glucocorticoids being widely prescribed as a topical treatment in ophthalmology, their potential role during ocular development in the embryo is not well understood. As shown by scanning electron microscopy analysis as well as by our histopathological and immunohistochemical data, long-term and newborn transgenic embryos showed unfused eyelids, along with proptosis of the globe and exposure of the anterior surface. In addition, epithelial defects were evident at the cornea. Our results indicate that GR overexpression affected the proliferation rate of targeted epithelia of the cornea and eyelid, thus demonstrating that GR was responsible for the arrest of epithelial proliferation of the developing eyelid edges, as well as for their destruction. We conclude that constitutive targeted overexpression of GR in the eyelid and corneal epithelium dramatically impairs ocular function in these transgenic mice.

Animals↗

PubMed2002

Severe abnormalities in the oral mucosa induced by suprabasal expression of epidermal keratin K10 in transgenic mice.

Previous studies have demonstrated that keratin K10 plays an important role in mediating cell signaling processes, since the ectopic expression of this keratin induces cell cycle arrest in proliferating cells in vitro and in vivo. However, apart from its well known function of providing epithelial cells with resilience to mechanical trauma, little is known about its possible roles in nondividing cells. To investigate what these might be, transgenic mice were generated in which the expression of K10 was driven by bovine K6beta gene control elements (bK6(beta)hK10). The transgenic mice displayed severe abnormalities in the tongue and palate but not in other K6-expressing cells such as those of the esophagus, nails, and hair follicles. The lesions in the tongue and palate included the cytolysis of epithelial suprabasal cells associated with an acute inflammatory response and lymphocyte infiltration. The alterations in the oral mucosa caused the death of transgenic pups soon after birth, probably because suckling was impaired. These anomalies, together with others found in the teeth, are reminiscent of the lesions observed in some patients with pachyonychia congenita, an inherited epithelial fragility associated with mutations in keratins K6 and K16. Although no epithelial fragility was observed in the bK6(beta)hK10 oral epithelia of the experimental mice, necrotic processes were seen. Collectively, these data show that the carefully regulated tissue- and differentiation-specific patterns displayed by the keratin genes have dramatic consequences on the biological behavior of epithelial cells and that changes in the specific composition of the keratin intermediate filament cytoskeleton can affect their physiology, in particular those of the oral mucosa.

Animals↗

PubMed2002

The expression of keratin k10 in the basal layer of the epidermis inhibits cell proliferation and prevents skin tumorigenesis.

Forced expression of K10, a keratin normally expressed in postmitotic, terminally differentiating epidermal keratinocytes, inhibits the progression of the cell cycle in cultured cells (Paramio, J. M., Casanova, M. Ll., Segrelles, C., Mittnacht, S., Lane, E. B., and Jorcano, J. L. (1999) Mol. Cell. Biol. 19, 3086-3094). This process requires a functional retinoblastoma (pRb) gene product and is mediated by K10-induced inhibition of Akt and PKCzeta, two signaling intermediates belonging to the phosphoinositide (PI) 3-kinase signal transduction pathway (Paramio, J. M., Segrelles, C., Ruiz, S., and Jorcano, J. L. (2001) Mol. Cell. Biol. 21, 7449-7459). Extending earlier in vitro studies to the in vivo situation, this work analyzes the alterations found in transgenic mice that ectopically express K10 in the proliferative basal cells of the epidermis. Increased expression of K10 led to a hypoplastic and hyperkeratotic epidermis due to a dramatic decrease in skin keratinocyte proliferation in association with the inhibition of Akt and PKCzeta activities. The inhibition of cell proliferation and Akt and PKCzeta activities was also observed although to a minor extent in low hK10-expressing mice. These animals displayed no overt epidermal phenotype nor overexpression of K10. In these non-phenotypic mice, ectopic K10 expression also resulted in decreased skin tumorigenesis. Collectively, these data demonstrate that keratin K10 in vivo functions include the control of epithelial proliferation in skin epidermis.

Animals↗

PubMed2002

Analysis of the ultraviolet B response in primary human keratinocytes using oligonucleotide microarrays.

UV radiation is the most important environmental skin aggressor, causing cancer and other problems. This paper reports the use of oligonucleotide microarray technology to determine changes in gene expression in human keratinocytes after UVB treatment. Examination of the effects of different doses at different times after irradiation gave a global picture of the keratinocyte response to this type of insult. Five hundred thirty-nine regulated transcripts were found and organized into nine different clusters depending on behavior patterns. Classification of these genes into 23 functional categories revealed that several biological processes are globally affected by UVB. In addition to confirming a majority up-regulation of the transcripts related to the UV-specific inflammatory and stress responses, significant increases were seen in the expression of genes involved in basal transcription, splicing, and translation as well as in the proteasome-mediated degradation category. On the other hand, those transcripts belonging to the metabolism and adhesion categories were strongly downregulated. These results demonstrate the complexity of the transcriptional profile of the UVB response, describe several cellular processes previously not known to be affected by UV irradiation, and serve as a basis for the global characterization of UV-regulated genes and pathways.

3T3 Cells↗

PubMed2002

Beyond structure: do intermediate filaments modulate cell signalling?

Intermediate filament (IF) proteins form the largest family of cytoskeletal proteins in mammalian cells. The function of these proteins has long been thought to be only structural. However, this single function does not explain their diverse tissue- and differentiation-specific expression patterns. Evidence is now emerging that IF also act as an important framework for the modulation and control of essential cell processes, in particular, signal transduction events. Here, we review the most recent developments in this growing and exciting new field.

Animals↗