A bichromatic fluorescent reporter for cell-based screens of alternative splicing.

Alternative splicing is the primary source of proteome complexity in metazoans and its regulation shapes the proteome in response to shifting physiological requirements. We developed a bichromatic splicing reporter that uses a peculiar feature of some fluorescent protein coding regions to express two different fluorescent proteins from a single alternative splicing event. The mutually exclusive expression of different fluorescent proteins from a single reporter provides a uniquely sensitive approach for high-throughput screening and analysis of cell-specific splicing events in mixed cell cultures and tissues of transgenic animals.

Transgenic mice expressing CUG-BP1 reproduce splicing mis-regulation observed in myotonic dystrophy.

Myotonic dystrophy type I (DM1) is an RNA-mediated disease caused by a non-coding CTG repeat expansion. A key feature of the RNA-mediated pathogenesis model for DM is the disrupted splicing of specific pre-mRNA targets. A link has been established between splicing regulation by CUG-BP1, a member of the CELF family of proteins, and DM1 pathogenesis.

Redundant pathways for negative feedback regulation of bile acid production.

The orphan nuclear hormone receptor SHP has been proposed to have a key role in the negative feedback regulation of bile acid production. Consistent with this, mice lacking the SHP gene exhibit mild defects in bile acid homeostasis and fail to repress cholesterol 7-alpha-hydroxylase expression in response to a specific agonist for the bile acid receptor FXR. However, this repression is retained in SHP null mice fed bile acids, demonstrating the existence of compensatory repression pathways of bile acid signaling.

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.

Transgenic mice expressing a mutant form of loricrin reveal the molecular basis of the skin diseases, Vohwinkel syndrome and progressive symmetric erythrokeratoderma.

Mutations in the cornified cell envelope protein loricrin have been reported recently in some patients with Vohwinkel syndrome (VS) and progressive symmetric erythrokeratoderma (PSEK). To establish a causative relationship between loricrin mutations and these diseases, we have generated transgenic mice expressing a COOH-terminal truncated form of loricrin that is similar to the protein expressed in VS and PSEK patients. At birth, transgenic mice (ML.

Lessons from loricrin-deficient mice: compensatory mechanisms maintaining skin barrier function in the absence of a major cornified envelope protein.

The epidermal cornified cell envelope (CE) is a complex protein-lipid composite that replaces the plasma membrane of terminally differentiated keratinocytes. This lamellar structure is essential for the barrier function of the skin and has the ability to prevent the loss of water and ions and to protect from environmental hazards. The major protein of the epidermal CE is loricrin, contributing approximately 70% by mass.

Delayed wound healing in keratin 6a knockout mice.

Keratin 6 (K6) expression in the epidermis has two components: constitutive expression in the innermost layer of the outer root sheath (ORS) of hair follicles and inducible expression in the interfollicular epidermis in response to stressful stimuli such as wounding. Mice express two K6 isoforms, MK6a and MK6b. To gain insight into the functional significance of these isoforms, we generated MK6a-deficient mice through mouse embryonic stem cell technology.

The mouse keratin 6 isoforms are differentially expressed in the hair follicle, footpad, tongue and activated epidermis.

Keratin 6 (K6) is expressed constitutively in a variety of internal stratified epithelia as well as in palmoplantar epidermis and in specialized cells of the hair follicle. K6 expression can also be induced by hyperproliferative conditions as in wound healing or by conditions that perturb normal keratinocyte function. The functional significance of the expression of K6 on keratinocyte biology under these disparate conditions is not known.

Expression of MK6a dominant-negative and C-terminal mutant transgenes in mice has distinct phenotypic consequences in the epidermis and hair follicle.

Mouse keratin 6a (MK6a) is constitutively expressed in a single cell layer of the outer root sheath (ORS) of hair follicles, but its synthesis can be induced in interfollicular epidermis including the basal cell layer in response to perturbing stimuli. A basally inducible human K6 (HK6) isoform has not been described, and it is not clear which of the known HK6 isoforms is expressed in the ORS. In this study we show that expression of a dominant-negative MK6a construct (Delta2B-P) in the interfollicular epidermis caused severe blistering and neonatal lethality, suggesting that mutations in a yet to be identified basally expressed HK6 isoform might result in a severe blistering phenotype.

Characterization of loricrin regulation in vitro and in transgenic mice.

We have previously shown that the promoter of a 6.5 kb mouse loricrin clone contains a functional AP-1 element and directs tissue-specific, but not differentiation-specific, expression. We now report the isolation of a 14-kb genomic clone containing an additional 7 kb of genomic sequence.

Human keratin-1.bcl-2 transgenic mice aberrantly express keratin 6, exhibit reduced sensitivity to keratinocyte cell death induction, and are susceptible to skin tumor formation.

Nonmelanoma skin cancers (NMSC) are among the most common malignancies in the world. Typically, these neoplasms grow slowly and are comparatively indolent in their clinical behavior. The most frequent molecular alterations implicated in the pathogenesis of these neoplasms involve genes known to be regulators of cell death including p53, Ha-ras and bcl-2.

Expression of a dominant-negative type II transforming growth factor beta (TGF-beta) receptor in the epidermis of transgenic mice blocks TGF-beta-mediated growth inhibition.

To determine whether a functional type II receptor of transforming growth factor beta (TGF-beta) is required to mediate the growth inhibitory effect of TGF-beta on the skin in vivo, we have generated transgenic mice that overexpress a dominant negative-type II TGF-beta receptor (delta beta RII) in the epidermis. The delta beta RII mice exhibited a thickened and wrinkled skin, and histologically the epidermis was markedly hyperplastic and hyperkeratotic. In vivo labeling with BrdUrd showed a 2.

A transgenic mouse model that recapitulates the clinical features of both neonatal and adult forms of the skin disease epidermolytic hyperkeratosis.

Keratins are the major structural proteins of keratinocytes, which are the most abundant cell type in the mammalian epidermis. Mutations in epidermal keratin genes have been shown to cause severe blistering skin abnormalities. One such disease, epidermolytic hyperkeratosis (EHK), also known as bullous congenital ichthyosiform erythroderma, occurs as a result of mutations in highly conserved regions of keratins K1 and K10.

Adenoviral-mediated herpes simplex virus-thymidine kinase gene transfer in vivo for treatment of experimental human melanoma.

To assess the efficacy of an in vivo adenoviral-mediated cytotoxic gene therapy, human melanomas were established in nude mice and transduced with herpes simplex virus-thymidine kinase (tk) followed by treatment with ganciclovir (GCV). In initial experiments, adenovirus (adv) containing the beta-galactosidase reporter gene was employed to determine melanoma cell infectivity in vitro. In comparison to murine melanoma cell lines B16 and K1735-M2, human A375-SM cells exhibited up to a 10-fold greater susceptibility to adenoviral transduction, similar to the degree of infectivity found for human epidermal HaCaT cells.

The proximal promoter of the mouse loricrin gene contains a functional AP-1 element and directs keratinocyte-specific but not differentiation-specific expression.

Loricrin gene expression is limited to terminally differentiating keratinocytes of stratified squamous epithelia. To define the regulatory elements that mediate the expression of the loricrin gene, we replaced the loricrin coding sequences from a 6.5-kilobase genomic fragment with the chloramphenicol acetyltransferase gene and transfected this construct into cultured mouse keratinocytes.

Loricrin expression is coordinated with other epidermal proteins and the appearance of lipid lamellar granules in development.

In mouse, epidermal development proceeds from a single basal cell layer covered by a specialized single cell layer called the periderm at E14 to a fully differentiated stratified squamous epithelium at E18. To determine when loricrin, a major cell envelope component, is expressed during development, we examined fetal skin from mice of gestational ages E13 through E19 and compared the temporal pattern of loricrin expression with that of other differentiation markers. We found that loricrin mRNA and protein were expressed by E16, following the expression of keratins K1 and K10 and preceding the expression of profilaggrin.

Inhibition of melanoma growth by adenoviral-mediated HSV thymidine kinase gene transfer in vivo.

To assess the potential of an in vivo, adenovirus-mediated gene therapy approach for the treatment of malignant melanoma, the efficacy of adenovirus-mediated herpes simplex virus thymidine kinase gene (HSV-Ek) transfer and administration of ganciclovir (GCV) was investigated using a nude mouse model. Initially, B16 murine melanoma cells were efficiently transduced in vitro by a recombinant replication-defective adenovirus containing the HSV-tk gene (ADV/RSVtk), and rendered sensitive to cell killing by 10 micrograms/ml GCV. A significant "bystander effect" was observed at low multiplicity of infection in comparison of cell killing to control B16 transduction by adenovirus containing the beta-galactosidase gene (ADV/RSV-beta-gal).

Targeting expression of a dominant-negative retinoic acid receptor mutant in the epidermis of transgenic mice results in loss of barrier function.

To study the effects of retinoic acid on the skin in vivo, we have subverted the activity of endogenous receptors by targeting expression of a dominant negative mutant of retinoic acid receptor alpha (RAR alpha) to the epidermis of transgenic mice. At birth, mice expressing the mutant RAR alpha transgene exhibited a marked phenotype of a red, shiny skin that was somewhat sticky to touch. Severely affected neonates died within 24 hr.

Characterization of the mouse loricrin gene: linkage with profilaggrin and the flaky tail and soft coat mutant loci on chromosome 3.

Loricrin is the major component of a specialized structure, termed the cornified cell envelope, that is formed beneath the plasma membrane of stratified squamous epithelial cells and is coexpressed with profilaggrin in terminally differentiating epidermal keratinocytes. Full-length cDNAs for both mouse and human loricrin have been cloned and characterized, as has the human gene. Here we report the isolation and characterization of the mouse loricrin gene.

Genetic disorders of keratin: are scarring alopecias a sub-set?

Recent advances have challenged the prevailing view that keratins are merely passive bystanders of keratinocyte biology. With the exciting discovery that three autosomal dominant genetic skin disorders, epidermolysis bullosa simplex (EBS), epidermolytic hyperkeratosis (EHK) and palmoplantar keratoderma (PPK), are in fact disorders of keratins comes the realization that the integrity of the keratin filament network is crucial to the structural integrity of the skin. Since it has been recently established that mutations in keratins K5/K14, K1/K10 and K9 are causative for these keratinocyte disorders, it is very likely that mutations in K6 or in its obligate partner, K16 will result in disease.

Transgenic mice expressing targeted HPV-18 E6 and E7 oncogenes in the epidermis develop verrucous lesions and spontaneous, rasHa-activated papillomas.

In order to create a transgenic model for human papilloma virus (HPV)-associated carcinogenesis, we have used the regulatory elements of a human keratin K1 (HK1) gene to target the expression of the E6 and E7 oncogenes of HPV-18 exclusively to the epidermis. All murine expressors were viable and lived normal lifetimes; older mice (> 1 year) possessed numerous small lesions with a verrucous (wart-like) histotype. Analysis of newborn epidermis and lesions revealed that the HPV-18 E6/E7 genes were being expressed with a predominance of the E6*/E7 transcript over the full length E6/E7 message.

Targeted overexpression of transforming growth factor alpha in the epidermis of transgenic mice elicits hyperplasia, hyperkeratosis, and spontaneous, squamous papillomas.

To assess the effects of transforming growth factor alpha (TGF-alpha) on mammalian skin in vivo, we have targeted its expression to the epidermis of transgenic mice using a vector based on the human K1 (HK1) gene. Neonatal mice expressing the HK1.TGF-alpha transgene were often smaller than normal littermates and had precocious eyelid opening and wrinkled, scaly skin with diffuse alopecia.

Hyperplasia, hyperkeratosis and benign tumor production in transgenic mice by a targeted v-fos oncogene suggest a role for fos in epidermal differentiation and neoplasia.

A vector, derived from the human K1 keratin gene, has been employed to target v-fos expression exclusively in the epidermis of transgenic mice. Adult transgenic mice expressors (3-4 months) displayed hyperplasia and hyperkeratosis, initially in wounded (tagged) ears, which later became bilateral. This phenotype appeared at other epidermal sites, most notably in the axilla and inguinal areas.

Inhibition of skin development by overexpression of transforming growth factor beta 1 in the epidermis of transgenic mice.

To assess the effect of transforming growth factor beta 1 on the skin in vivo, we have targeted its expression to the epidermis of transgenic mice. To ensure that active TGF-beta 1 was expressed, we used a porcine TGF-beta 1 cDNA with mutations of Cys-223-->Ser and Cys-225-->Ser, which allow constitutive activation. Mice expressing the mutant transforming growth factor beta 1 transgene exhibited a marked phenotype at birth.

Induction of epidermal hyperplasia, hyperkeratosis, and papillomas in transgenic mice by a targeted v-Ha-ras oncogene.

The regulatory elements of the human keratin K1 gene have been used to target expression of the v-Ha-ras oncogene exclusively in the epidermis of transgenic mice. We developed 12 transgenic mouse lines that express the HK1.ras transgene, producing epidermal hyperplasia in neonates and hyperkeratosis in juveniles.