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Dennis R Roop

Publications and source records attributed to Dennis R Roop.

36 records · Page 2Linked to original sources
PubMed2004

Development of transgenic mice that inducibly express an active form of c-Src in the epidermis.

Recent work from our laboratory has shown that elevated src kinase activity enhances tumor promotion, malignant progression, and metastasis during multistage skin carcinogenesis. In this study, we have generated "gene-switch" src(530) transgenic mice to further analyze the role of this nonreceptor tyrosine kinase in multistage carcinogenesis. Target transgenic mice that have an activated form of the human c-src (src(530)) gene fused with GAL4 binding sites upstream of the thymidine kinase (TK) promoter were generated. Two lines of epidermis-specific transactivator mice were used that targeted the expression of GLVPc or GLp65 transactivators, fusion molecules containing a truncated progesterone receptor with a GAl4-DNA binding domain, with either a mouse loricrin (ML) or human keratin 14 (HK14) promoter, respectively. The transactivator mice (ML.GLVPc or HK14.GLp65) and the target mice (TK.src(530)) were mated to generate bitransgenic mice, and src(530) transgene expression was induced by topical application of RU486 (mifepristone, a progesterone receptor antagonist). In both ML.GLVPc/TK.src(530) and HK14.GLp65/TK.src(530) bitransgenic mice, histological analysis revealed that only the bitransgenic mice had marked epidermal hyperplasia and hyperkeratosis after treatment with RU486. Neither the nontransgenic mice nor the mice hemizygous for either the transactivator transgene or the target transgene alone showed any response to treatment with RU486. In addition, no differences were observed in the skin of the bitransgenic mice versus nontransgenic littermates without treatment of RU486. Interestingly, in HK14.GLp65/TK.src(530) bitransgenic mice, squamous cell carcinomas (SCCs) arose along the periphery of the area of the punch biopsies in 25% of the bitransgenic mice several weeks after taking the biopsy and subsequent to RU486 treatment. Collectively, the data support a role of c-src activation in epidermal hyperproliferation. Furthermore, the data support the conclusion that src activation can substitute for an initiating event in the presence of a tumor promoting stimulus (i.e., wounding). Finally, inducible src(530) transgenic mice provide a new tool for dissecting the role of src activation in multistage carcinogenesis by allowing temporal control of the expression of this oncogene.

Animals↗
PubMed2004

The role of p63 in development and differentiation of the epidermis.

Expression of p63, a transcription factor that is transcribed into six isoforms, is required for proper development of stratified epithelia, such as the epidermis. In the absence of p63, epithelia remain single-layered. The molecular role of p63 in development and differentiation of stratified epithelia, however, remains controversial. Based on recent studies, we now believe that p63 has a dual role and is essential for development as well as maintenance of the epidermis. During embryogenesis, p63 may be the molecular switch required for initiation of epithelial stratification. This is based on our recent data demonstrating that ectopic expression of a p63 isoform in single-layered epithelia results in the induction of a stratification program. Furthermore, in the mature epidermis, p63 may maintain the proliferative potential of basal keratinocytes. This is suggested by the observation that p63 is primarily expressed in the basal compartment of the epidermis, that p63 expression induces hyperproliferation, and that its expression needs to be downregulated for terminal differentiation to take place. In this review, we discuss recent evidence supporting this dual role for p63 and place it in the context of our increasing knowledge of epidermal development and differentiation.

Animals↗
PubMed2004

Genetic pathways required for epidermal morphogenesis.

The epidermis is composed of keratinocytes which undergo a highly reproducible terminal differentiation program resulting in the formation of a protective barrier, which is established during embryogenesis. Significant progress has recently been made in understanding the genetic pathways associated with the earliest event characteristic of epidermal morphogenesis, commitment to stratification. This process depends on the expression of p63, a transcription factor which is transcribed into isoforms that contain (TA) or lack (AN) a transactivation domain. In the absence of p63 expression, epithelia remain single-layered, while ectopic TAp63alpha expression in single-layered epithelia initiates stratification. Later events during epidermal morphogenesis require withdrawal from the cell cycle and commitment to terminal differentiation. Some of the genetic pathways underlying these events are beginning to be elucidated, however, the exact molecular events remain to be determined. In this review, we summarize the involvement of several signaling pathways in different stages of epidermal morphogenesis.

Animals↗
PubMed2004

Yin-yang 1 negatively regulates the differentiation-specific transcription of mouse loricrin gene in undifferentiated keratinocytes.

Loricrin is a major component of the epidermal cornified cell envelope, and is expressed only in terminally differentiated keratinocytes. This cell differentiation-specific expression pattern suggests specific suppression of loricrin gene expression in undifferentiated keratinocytes as well as its activation in differentiated keratinocytes. We identified a negative regulatory sequence element in the first intron of the mouse loricrin gene involved in suppression of loricrin gene expression in undifferentiated keratinocytes. A database search indicated that this sequence contained the putative inverted Yin-Yang 1 (YY1)-binding motif. Constructs with point mutations in the putative YY1-binding motif showed increased reporter activity, indicating that YY1 negatively regulates loricrin gene transcription. Co-transfection experiments using a YY1 expression vector revealed that YY1 represses loricrin promoter activity. Western blotting and immunohistochemical analyses indicated that YY1 is more abundant in undifferentiated than in differentiated keratinocytes. These findings suggest that YY1 contributes to specific loricrin gene expression in differentiated keratinocytes by suppression of its transcription in undifferentiated keratinocytes. Furthermore, we demonstrated that forced expression of YY1 in differentiated keratinocytes results in specific downregulation of expression of other early and late differentiation markers.

Animals↗
PubMed2004

Genes involved in stem cell fate decisions and commitment to differentiation play a role in skin disease.

Multipotent stem cells residing in the bulge region of the hair follicle give rise to cells of different fates including those forming hair follicles, interfollicular epidermis, and associated glands. Stem cell fate determination is regulated by genes involved in both proliferation and differentiation, which are tightly regulated processes. Understanding the molecular mechanisms by which proliferation and differentiation are regulated will provide useful insight into treating human diseases caused by the deregulation of these processes. Two genes involved in regulating proliferation and differentiation are c-Myc and p63, both of which have been found to be deregulated/mutated in several human diseases. Accelerating proliferation leads to neoplastic human diseases and deregulated c-Myc has been implicated in a variety of cancers. Evidence indicates that c-Myc also diverts stem cells to an epidermal and sebaceous gland fate at the expense of the hair follicle fate. Therefore, deregulation of c-Myc has the potential to not only accelerate tumorigenesis, but also influence skin tumor phenotype. In addition, the inhibition of differentiation may also predispose to the development of skin cancer. Recent evidence suggests that the transcription factor p63, is not only responsible for the initiation of an epithelial stratification program during development, but also the maintenance of the proliferative potential of basal keratinocytes in mature epidermis. Mutations in the p63 gene have been shown to cause ectodermal dysplasias and deregulated expression of p63 has been observed in squamous cell carcinomas. In this review, we will discuss recent data implicating a role for both c-Myc and p63 in human skin diseases.

Animals↗
PubMed2004

c-Myc and epidermal stem cell fate determination.

The epidermis is sustained by a multipotent stem cell population that gives rise to cells of different fates including those forming hair follicles, interfollicular epidermis and associated glands such as sebaceous glands. The most intriguing evidence comes from using a variety of mouse models with which studies have found the WNT pathway to be involved in regulating stem cell fate decisions. These mouse models have been used to analyze different members of the WNT pathway in the epidermis and altogether suggest that different levels of beta-catenin correlate with the adoption of different epidermal stem cell fates. Recent data specifically analyzing c-Myc, a downstream target of the WNT pathway, has found that c-Myc can divert epidermal stem cells to a sebaceous gland fate at the expense of hair follicles. Also, newly emerging data using gene expression profiling techniques have uncovered a more direct role of c-Myc in stem cell fate determination.

Animals↗
PubMed2004

p63 and epithelial appendage development.

Abstract Epithelial appendages share a common developmental program that relies on extensive interactions between epithelia and adjacent mesenchyme. The transcription factor p63 has a critical role in epithelial appendage development in both vertebrates and non-vertebrates. Both mice and zebrafish lacking p63 expression fail to develop epithelial appendages and other structures that develop as a result of epithelial-mesenchymal interactions. Furthermore, dominantly inherited mutations in p63 are the cause of a subset of human ectodermal dysplasias, which are characterized by developmental abnormalities in epithelia and epithelial appendages. While the importance of p63 for epithelial appendage development is evident, the molecular mechanisms by which p63 functions are largely unknown. In this review, we will discuss the current knowledge of the developmental role of p63 and the implications for epithelial appendage development.

Animals↗
PubMed2004

Inducible mouse models for inherited skin diseases: implications for skin gene therapy.

Stem cells are crucial for the formation and maintenance of tissues and organs. To understand the role of stem cells in the pathogenesis of mosaic skin disorders, we generated inducible mouse models for two autosomal dominant keratin disorders, epidermolytic hyperkeratosis (EHK) and epidermolysis bullosa simplex (EBS), that enable activation of the respective mutation in epidermal stem cells in a spatially and temporally controlled manner using a ligand-inducible Cre recombinase. Whereas mosaic forms have been reported for EHK, which is caused by mutations in the suprabasal keratins K1 or K10, this has never been reported for EBS, which is due to mutations in the basal keratins K5 or K14. When we induced the phenotype in these models by topical application of the inducer, we found phenotypic areas in the EHK model that persisted for the life of the mouse. On the contrary, the induced blisters in the EBS model healed within a few weeks by migration of surrounding non-phenotypic stem cells into the wound bed. Our results indicate that lack of selective pressure against certain mutations in epidermal stem cells could explain why mosaic forms exist for EHK, but not for EBS. These findings have important implications for the development of new strategies for somatic gene therapy of dominant genodermatoses, and we are currently using these inducible mouse models to test gene therapy approaches.

Animals↗
PubMed2003

Transcriptional regulation of bcl-2 mediated by the sonic hedgehog signaling pathway through gli-1.

Basal cell carcinomas (BCCs) express high levels of the antiapoptotic proto-oncogene, bcl-2, and we have shown that bcl-2 contributes to the malignant phenotype in a transgenic mouse model. The basis of bcl-2 transcriptional regulation in keratinocytes is unknown. The sonic hedgehog (SHH) signaling pathway is frequently altered in BCCs. Mediators of shh signaling include the downstream transactivator, gli-1, and transrepressor, gli-3. Seven candidate gli binding sites were identified in the bcl-2 promoter. Cotransfection of increasing amounts of gli-1 in keratinoycytes resulted in a corresponding dose-dependent increase in bcl-2 promoter luciferase activity. Gli-1 was also able to up-regulate endogenous bcl-2. Gli-3 cotransfection resulted in no significant changes in bcl-2 promoter activity compared with control. Gli-3 has been demonstrated to be proteolytically processed into an N-terminal repressive form that can inhibit downstream transactivation by gli-1. Gli-3 mutants possessing only the N-terminal region or the C-terminal region were made and used in luciferase assays. The N terminus of gli-3 inhibited gli-1 transactivation of the bcl-2 promoter. Gel shift analysis and luciferase assays demonstrated that gli binding site 4 (-428 to -420), is important for gli transcriptional regulation. Skin samples from transgenic mice expressing an RU486 gli-1 transgene exhibited significantly higher levels of endogenous bcl-2 protein in epidermal keratinocytes as assessed by immunoblotting and immunohistochemistry. Together, these findings provide consistent evidence that gli proteins can transcriptionally regulate the bcl-2 promoter and that gli-3 can inhibit transactivation by gli-1. These studies further suggest that one consequence of the deregulation of shh signaling in BCC is the up-regulation of bcl-2.

3T3 Cells↗
PubMed2003

Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation.

Transcription factor Nrf2 (encoded by Nfe2l2) regulates a battery of detoxifying and antioxidant genes, and Keap1 represses Nrf2 function. When we ablated Keap1, Keap1-deficient mice died postnatally, probably from malnutrition resulting from hyperkeratosis in the esophagus and forestomach. Nrf2 activity affects the expression levels of several squamous epithelial genes. Biochemical data show that, without Keap1, Nrf2 constitutively accumulates in the nucleus to stimulate transcription of cytoprotective genes. Breeding to Nrf2-deficient mice reversed the phenotypic Keap1 deficiencies. These experiments show that Keap1 acts upstream of Nrf2 in the cellular response to oxidative and xenobiotic stress.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Disruption of dihydronicotinamide riboside:quinone oxidoreductase 2 (NQO2) leads to myeloid hyperplasia of bone marrow and decreased sensitivity to menadione toxicity.

Dihydronicotinamide riboside (NRH):quinone oxidoreductase 2 (NQO2) is a flavoenzyme that catalyzes the reductive metabolism of quinones. To examine the in vivo role of NQO2, NQO2-null (NQO2-/-) mice were generated using targeted gene disruption. Mice lacking NQO2 gene expression showed no detectable developmental abnormalities and were indistinguishable from wild-type (NQO2+/+) mice. However, NQO2-null mice exhibited myeloid hyperplasia of the bone marrow and increased neutrophils, basophils, eosinophils, and platelets in the peripheral blood. Decreased apoptosis of bone marrow cells and circulating granulocytes contributed to myeloid hyperplasia and hyperactivity of bone marrow in NQO2-null mice. The hematological changes in NQO2-/- mice were specifically associated with loss of the NQO2 gene because histological analysis of various tissues including spleen, thymus, blood cultures, and urine analysis demonstrated no sign of infection. NQO2-null mice also demonstrated decreased toxicity when exposed to menadione or menadione with NRH. These results establish a role for NQO2 in protection against myelogenous hyperplasia and in metabolic activation of menadione, leading to hepatic toxicity. The NQO2-null mice are a model for NQO2 deficiency in humans and can be used to determine the role of this enzyme in sensitivities to toxicity and carcinogenesis.

Animals↗
PubMed2002

The epidermis as a bioreactor: topically regulated cutaneous delivery into the circulation.

Previous studies have documented that the skin can be used as a bioreactor to produce proteins for systemic release to treat diseases. A gene-switch system has been developed that allows regulated expression of therapeutic genes. To determine whether this system could be used in the skin, we developed a transgenic mouse model in which expression of a therapeutic gene could be topically induced in epidermal keratinocytes. After a single induction, high levels of the therapeutic protein, human growth hormone (hGH), were released from keratinocytes into the circulation. The serum levels of hGH were dependent on the amount of inducer applied, and repeated induction resulted in increased weight gain by transgenic versus control mice. Furthermore, physiological levels of hGH were detected in the serum of nude mice after topical induction of small transgenic skin grafts. These results clearly demonstrate the feasibility of using the gene-switch system to regulate the delivery of therapeutic proteins into the circulation via genetically modified keratinocytes.

Animals↗
PubMed2002

Quasi-normal cornified cell envelopes in loricrin knockout mice imply the existence of a loricrin backup system.

The cornified cell envelope, a lipoprotein layer that assembles at the surface of terminally differentiated keratinocytes, is a resilient structure on account of covalent crosslinking of its constituent proteins, principally loricrin, which accounts for up to 60%-80% of total protein. Despite the importance of the cell envelope as a protective barrier, knocking out the loricrin gene in mice results in only mild syndromes. We have investigated the epidermis and forestomach epithelium of these mice by electron microscopy. In both tissues, corneocytes have normal-looking cell envelopes, despite the absence of loricrin, which was confirmed by immunolabeling, and the absence of the distinctive loricrin-containing keratohyalin granules (L-granules). Isolated cell envelopes were normal in thickness (approximately 15 nm) and mass per unit area (approximately 7.3 kDa per nm2); however, metal shadowing revealed an altered substructure on their cytoplasmic surface. Their amino acid compositions indicate altered protein compositions. Analysis of these data implies that the epidermal cell envelopes have elevated levels of the small proline-rich proteins, and cell envelopes of both kinds contain other protein(s) that, like loricrin, are rich in glycine and serine. These observations imply that, in the absence of loricrin, the mechanisms that govern cell envelope assembly function normally but employ different building-blocks.

Amino Acids↗
PubMed2002

Epidermal differentiation: transgenic/knockout mouse models reveal genes involved in stem cell fate decisions and commitment to differentiation.

Epidermal development and differentiation are similar processes and therefore the study of one is likely to provide insight into the other. The signaling cascades required for epidermal differentiation are largely unknown. Recent evidence, however, has implicated two proteins, p63 and c-Myc, in different stages of epidermal development and differentiation. p63 was shown to be required for embryonic epidermal development. Mice lacking p63 do not develop stratified epithelia and appendages suggesting a role for p63 in the commitment to squamous epithelial lineages. Subsequent stem cell fate decisions are required to form the different structures of stratified epithelia including hair follicles, sebaceous glands, and epidermis. Several genes of the Wnt signaling pathway have been implicated in this process, including c-Myc, a downstream target of the Wnt pathway. Interestingly, targeted overexpression of c-Myc in the basal layer of the epidermis results in an increase in sebaceous gland size and number at the expense of hair follicles. This suggests that c-Myc promotes differentiation of epidermal stem cells into sebaceous glands. In this review, we discuss transgenic/knockout mouse models that have provided evidence linking c-Myc and p63 to different stages of epidermal development and differentiation.

Animals↗