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1.
J Cell Biol ; 151(2): 401-12, 2000 Oct 16.
Article in English | MEDLINE | ID: mdl-11038186

ABSTRACT

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.VS) exhibited erythrokeratoderma with an epidermal barrier dysfunction. 4 d after birth, high-expressing transgenic animals showed a generalized scaling of the skin, as well as a constricting band encircling the tail and, by day 7, a thickening of the footpads. Histologically, ML. VS transgenic mice also showed retention of nuclei in the stratum corneum, a characteristic feature of VS and PSEK. Immunofluorescence and immunoelectron microscopy showed the mutant loricrin protein in the nucleus and cytoplasm of epidermal keratinocytes, but did not detect the protein in the cornified cell envelope. Transfection experiments indicated that the COOH-terminal domain of the mutant loricrin contains a nuclear localization signal. To determine whether the ML.VS phenotype resulted from dominant-negative interference of the transgene with endogenous loricrin, we mated the ML.VS transgenics with loricrin knockout mice. A severe phenotype was observed in mice that lacked expression of wild-type loricrin. Since loricrin knockout mice are largely asymptomatic (Koch, P.K., P. A. de Viragh, E. Scharer, D. Bundman, M.A. Longley, J. Bickenbach, Y. Kawachi, Y. Suga, Z. Zhou, M. Huber, et al., J. Cell Biol. 151:389-400, this issue), this phenotype may be attributed to expression of the mutant form of loricrin. Thus, deposition of the mutant protein in the nucleus appears to interfere with late stages of epidermal differentiation, resulting in a VS-like phenotype.


Subject(s)
Deafness/etiology , Keratosis/etiology , Membrane Proteins/genetics , Amino Acid Sequence , Animals , Cell Compartmentation , Cell Membrane/chemistry , Deafness/genetics , Frameshift Mutation , Keratosis/genetics , Mice , Mice, Mutant Strains , Mice, Transgenic , Molecular Sequence Data , Nuclear Localization Signals , Phenotype , Protein Transport , Skin/pathology , Skin Physiological Phenomena/genetics , Syndrome
2.
J Cell Biol ; 151(2): 389-400, 2000 Oct 16.
Article in English | MEDLINE | ID: mdl-11038185

ABSTRACT

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. We have generated mice that are deficient for this protein. These mice showed a delay in the formation of the skin barrier in embryonic development. At birth, homozygous mutant mice weighed less than control littermates and showed skin abnormalities, such as congenital erythroderma with a shiny, translucent skin. Tape stripping experiments suggested that the stratum corneum stability was reduced in newborn Lor(-/-) mice compared with wild-type controls. Isolated mutant CEs were more easily fragmented by sonication in vitro, indicating a greater susceptibility to mechanical stress. Nevertheless, we did not detect impaired epidermal barrier function in these mice. Surprisingly, the skin phenotype disappeared 4-5 d after birth. At least one of the compensatory mechanisms preventing a more severe skin phenotype in newborn Lor(-/-) mice is an increase in the expression of other CE components, such as SPRRP2D and SPRRP2H, members of the family of "small proline rich proteins", and repetin, a member of the "fused gene" subgroup of the S100 gene family.


Subject(s)
Epidermis/physiology , Membrane Proteins/genetics , Skin Physiological Phenomena/genetics , Adaptation, Biological , Amino Acid Sequence , Animals , Biomechanical Phenomena , Cell Membrane , Cloning, Molecular , Cornified Envelope Proline-Rich Proteins , Intermediate Filament Proteins/biosynthesis , Membrane Proteins/deficiency , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Permeability , S100 Proteins/biosynthesis , Up-Regulation
3.
Mol Cell Biol ; 20(14): 5248-55, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10866680

ABSTRACT

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. Upon wounding, MK6a was induced in the outer ORS and the interfollicular epidermis including the basal cell layer of MK6a(+/+) mice, whereas MK6b induction in MK6a(-/-) mice was restricted to the suprabasal layers of the epidermis. After superficial wounding of the epidermis by tape stripping, MK6a(-/-) mice showed a delay in reepithelialization from the hair follicle. However, the healing of full-thickness skin wounds was not impaired in MK6a(-/-) animals. Migration and proliferation of MK6a(-/-) keratinocytes were not impaired in vitro. Furthermore, the migrating and the proliferating keratinocytes of full-thickness wounds in MK6a(-/-) animals expressed neither MK6a nor MK6b. These data indicate that MK6a does not play a major role in keratinocyte proliferation or migration but point to a role in the activation of follicular keratinocytes after wounding. This study represents the first report of a keratin null mutation that results in a wound healing defect.


Subject(s)
Keratins/genetics , Wound Healing/physiology , Animals , Cell Division , Cell Movement/genetics , Cells, Cultured , Epidermis/metabolism , Epithelial Cells/pathology , Gene Deletion , Genetic Engineering , Hair Follicle/metabolism , Hair Follicle/pathology , Keratinocytes/cytology , Keratinocytes/pathology , Keratins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Recombination, Genetic , Stem Cells , Wound Healing/genetics
4.
Differentiation ; 65(2): 97-112, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10550543

ABSTRACT

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. Surviving Delta2B-P animals showed transgene expression only in isolated epidermal cells and not in all cells of the ORS, but nevertheless developed severe alopecia. Expression of two different C-terminal mutant transgenes also caused alopecia while a third C-terminal mutant had no phenotypic conse- quences. Electron microscopy revealed that Delta2B-P expression resulted in the collapse of keratin filaments, while destruction of hair follicles in the two phenotypic C-terminal mutant lines occurred in the absence of filament abnormalities. The latter finding indicates that the innermost ORS cells are uniquely sensitive to expression of even slightly altered K6 proteins, suggesting that mutations affecting an HK6 isoform expressed in this cell layer could result in alopecia in humans as well.


Subject(s)
Epidermis/metabolism , Genes, Dominant , Hair Follicle/metabolism , Keratins/genetics , Transgenes , Age of Onset , Alopecia/genetics , Amino Acid Sequence , Animals , Animals, Newborn , Epidermis/pathology , Epidermis/ultrastructure , Gene Expression , Hair Follicle/pathology , Hair Follicle/ultrastructure , Keratins/biosynthesis , Keratins/ultrastructure , Mice , Mice, Inbred BALB C , Mice, Transgenic , Microscopy, Electron , Molecular Sequence Data , Mutagenesis, Site-Directed , Phenotype , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Skin Diseases/genetics , Skin Diseases/pathology , Time Factors
5.
Differentiation ; 65(2): 119-30, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10550545

ABSTRACT

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. Here we report on the characterization of two isoforms of mouse K6 that are encoded by separate genes. The two genes (denoted K6a and K6b) are linked, have the same orientation and are actively transcribed. Sequence analysis revealed, that although they encode almost identical products, they have distinctly different regulatory regions, suggesting that the two K6 genes would be differentially expressed. In an attempt to define the expression characteristics of the K6 isoforms, we produced transgenic mice with each gene after modifying the C-terminal sequences to enable detection of the transgenic proteins with specific antibodies. The constitutive expression of the K6a transgene paralleled that of the endogenous genes in all K6 expressing tissues, except in the tongue. The K6b transgene was also expressed in these tissues but, in contrast to K6a, was only expressed in suprabasal cells. Both K6 transgenes were also induced in the interfollicular epidermis in response to phorbol esters, with K6a induced in all layers of the treated epidermis, while K6b was expressed only in suprabasal cells. These studies suggest that the K6 isoforms have overlapping yet distinct expression profiles.


Subject(s)
Epidermis/metabolism , Hair Follicle/metabolism , Hindlimb/metabolism , Keratins/biosynthesis , Keratins/genetics , Tongue/metabolism , Animals , Blotting, Western , Fluorescent Antibody Technique , Gene Expression Regulation , Genetic Linkage , Mice , Mice, Inbred BALB C , Mice, Transgenic , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic
6.
Differentiation ; 64(4): 225-35, 1999 May.
Article in English | MEDLINE | ID: mdl-10365440

ABSTRACT

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. The additional sequences limit expression of a reporter construct to differentiated keratinocytes in culture. The expression of the 6.5-kb and 14-kb loricrin constructs were also analyzed in transgenic mice. Significantly, loricrin was found in all layers of the epidermis of the 6.5-kb transgenics, including basal and spinous cells. The expression of the 14-kb clone was indistinguishable from that of the endogenous gene, confirming that the additional sequences contain negative regulatory elements that restrict loricrin expression to the granular layer in vivo. In addition, we show the AP-1 element localized in the loricrin proximal promoter is necessary but not sufficient for expression of the loricrin gene in vivo in transgenic mice. Finally, to gain further insight into how AP-1 family members regulate expression of the loricrin gene, we co-transfected the loricrin reporter constructs with expression plasmids for various fos and jun family members and demonstrated that c-Fos/Jun-B heterodimers could mimic the differentiation-specific induction of loricrin.


Subject(s)
Gene Expression Regulation , Keratinocytes/cytology , Membrane Proteins/genetics , Animals , Animals, Newborn , Cell Differentiation , Cells, Cultured , Chloramphenicol O-Acetyltransferase/genetics , Cloning, Molecular , Dimerization , Genes, Reporter , Genomic Library , Keratinocytes/physiology , Mice , Mice, Transgenic , Mutagenesis, Site-Directed , Promoter Regions, Genetic , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Recombinant Proteins/biosynthesis , Regulatory Sequences, Nucleic Acid , Restriction Mapping , Skin/cytology , Skin/metabolism , Transcription Factor AP-1/metabolism , Transfection , beta-Galactosidase/genetics
7.
Oncogene ; 16(7): 853-63, 1998 Feb 19.
Article in English | MEDLINE | ID: mdl-9484776

ABSTRACT

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. In order to evaluate the significance cell death deregulation during skin carcinogenesis, we generated a transgenic mouse model (HK1.bcl-2) using the human keratin 1 promoter to target the expression of a human bcl-2 minigene to the epidermis. Transgenic HK1.bcl-2 protein was expressed at high levels specifically in the epidermis extending from the stratum basale through the stratum granulosum. The epidermis of HK1.bcl-2 mice exhibited multifocal hyperplasia without associated hyperkeratosis and aberrant expression of keratin 6. The rate of proliferation was similar in HK1.bcl-2 and control epidermis although suprabasal BrdUrd incorporating cells were present only in HK1.bcl-2 skin. Keratinocytes from the HK1.bcl-2 mice were significantly more resistant to cell death induction by U.V.-B, DMBA, and TPA, compared to control keratinocytes. Furthermore, papillomas developed at a significantly greater frequency and shorter latency in the HK1.bcl-2 mice compared to control littermates following initiation with DMBA and promotion with TPA. Together these results support a role for bcl-2 in the pathogenesis of NMSC.


Subject(s)
Apoptosis , Genes, bcl-2 , Keratinocytes/cytology , Keratins/genetics , Mice, Transgenic , Skin Neoplasms/genetics , 9,10-Dimethyl-1,2-benzanthracene/pharmacology , Animals , Cell Division , DNA Fragmentation , Gene Expression Regulation, Developmental , Humans , Mice , Promoter Regions, Genetic , Skin/cytology , Skin/pathology , Skin Neoplasms/pathology , Tetradecanoylphorbol Acetate/pharmacology , Ultraviolet Rays
8.
Proc Natl Acad Sci U S A ; 94(6): 2386-91, 1997 Mar 18.
Article in English | MEDLINE | ID: mdl-9122204

ABSTRACT

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 2.5-fold increase in the labeling index over controls, with labeled nuclei occurring in both basal and suprabasal cells of transgenic epidermis. In heterozygotes, this skin phenotype gradually diminished, and by 10-14 days after birth the transgenic mice were indistinguishable from their normal siblings. However, when F1 mice were mated to homozygosity, perinatal lethality occurred due to the severe hyperkeratotic phenotype, which restricted movement. Cultured primary keratinocytes from delta beta RII mice also exhibited an increased rate of growth in comparison with nontransgenic controls, and were resistant to TGF-beta-induced growth inhibition. These data document the role of the type II TGF-beta receptor in mediating TGF-beta-induced growth inhibition of the epidermis in vivo and in maintenance of epidermal homeostasis.


Subject(s)
Epidermis/physiology , Keratinocytes/cytology , Keratinocytes/physiology , Receptors, Transforming Growth Factor beta/biosynthesis , Skin Aging , Skin Physiological Phenomena , Transforming Growth Factor beta/pharmacology , Amino Acid Sequence , Animals , Animals, Newborn , Cell Division/drug effects , Cells, Cultured , Epidermis/drug effects , Epidermis/pathology , Epitopes/chemistry , Female , Genes, myc , Humans , Hypertrophy , Keratinocytes/drug effects , Keratosis/genetics , Male , Mice , Mice, Inbred ICR , Mice, Inbred Strains , Mice, Transgenic , Mitotic Index , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins c-myc/biosynthesis , Receptor, Transforming Growth Factor-beta Type II , Skin/pathology
9.
Differentiation ; 61(2): 129-39, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8983179

ABSTRACT

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. Patients with EHK first exhibit erythroderma with severe blistering, which later is replaced by thick patches of scaly skin. To assess the effect of a mutated K1 gene on skin biology and to produce an animal model for EHK, we removed 60 residues from the 2B segment of HK1 and observed the effects of its expression in the epidermis of transgenic mice. Phenotypes of the resultant mice closely resembled those observed in the human disease, first with epidermal blisters, then later with hyperkeratotic lesions. In neonatal mice homozygous for the transgene, the skin was thicker, with an increased labeling index, and the spinous cells showed a collapse of the keratin filament network around the nuclei, suggesting that a critical concentration of the mutant HK1, over the endogenous MK1, was required to disrupt the structural integrity of the spinous cells. Additionally, footpad epithelium, which is devoid of hair follicles, showed blistering in the spinous layer, suggesting that hair follicles can stabilize or protect the epidermis from trauma. Blisters were not evident in adult mice, but instead they showed a thick, scaly hyperkeratotic skin with increased mitosis, resulting in an increased number of corneocytes and granular cells. Irregularly shaped keratohyalin granules were also observed. To date, this is the only transgenic model to show the typical morphology found in the adult form of EHK.


Subject(s)
Hyperkeratosis, Epidermolytic/genetics , Keratins/genetics , Mice, Transgenic/genetics , Age Factors , Animals , Animals, Newborn , Disease Models, Animal , Epidermis/metabolism , Epithelium/pathology , Female , Humans , Hyperplasia/genetics , Keratins/biosynthesis , Keratins/ultrastructure , Male , Mice , Mice, Inbred ICR , Mice, Inbred Strains , Mutation , Phenotype , Skin/pathology , Transgenes
10.
J Invest Dermatol ; 106(6): 1163-8, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8752651

ABSTRACT

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. In addition, human A375-SM melanoma cells exhibited a greater sensitivity in vitro to the cytotoxic effects of transduction with tk-adv and treatment with GCV, which was mediated by a strong bystander effect. In vivo, intratumoral injection of relatively large human melanomas (160 mm3) with 1.2 X 109 pfu of tk-adv, followed by intraperitoneal GCV treatment (60 mg/kg twice daily) over 4 days, typically resulted in a 50% reduction in melanoma growth rate compared to mock or untreated controls. Moreover, histometrical analysis employing a rigorous computerized imaging system revealed that the residual viable tumor area in the tk-adv/GCV-treated group was only one-fifth that of solvent controls. These data show that adv is a highly efficient in vivo gene delivery system to treat experimental human melanomas. In comparison to a previous murine melanoma study, human melanomas appeared to exhibit a greater sensitivity to this cytotoxic treatment in vivo, which may hold significant promise for development of effective gene therapy modalities to treat melanoma in humans.


Subject(s)
Adenoviridae , Gene Transfer Techniques , Genetic Vectors , Melanoma/therapy , Simplexvirus/enzymology , Thymidine Kinase/genetics , Adenoviridae/genetics , Animals , Cell Division/drug effects , Ganciclovir/therapeutic use , Genes, Reporter , Humans , Injections, Intraperitoneal , Melanoma/drug therapy , Melanoma/pathology , Mice , Mice, Nude , Transduction, Genetic , Tumor Cells, Cultured , beta-Galactosidase/genetics
11.
J Biol Chem ; 270(18): 10792-9, 1995 May 05.
Article in English | MEDLINE | ID: mdl-7738016

ABSTRACT

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. High expression levels were observed in both undifferentiated as well as differentiating cells. Transgenic mice bearing a similar construct, but with beta-galactosidase as the reporter gene, corroborated these in vitro findings and showed tissue- and cell type-specific, but not differentiation-specific expression. Deletion analysis of the promoter region determined that sequences up to -60 base pairs from the start of transcription could be removed without significant loss of promoter activity. Within these proximal 60 base pairs is an evolutionarily conserved AP-1 element that is recognized by both purified c-Jun and AP-1 factors from keratinocytes in vitro. Mutation of this AP-1 site abolished the activity of the loricrin promoter. These studies show that elements directing expression of the loricrin gene to the stratified squamous epithelia are contained within a 6.5-kilobase genomic fragment, and those elements required to restrict expression to differentiated keratinocytes lie outside this region.


Subject(s)
Genes , Membrane Proteins/genetics , Promoter Regions, Genetic , Transcription Factor AP-1/metabolism , Animals , Base Sequence , Binding Sites , Cell Differentiation , DNA Primers/chemistry , Gene Expression Regulation , Keratinocytes/physiology , Mice , Molecular Sequence Data , Sequence Alignment , Sequence Homology, Nucleic Acid , Transcription, Genetic
12.
J Invest Dermatol ; 104(3): 313-7, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7860993

ABSTRACT

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). In vivo, melanomas established from subcutaneous injection of 4 x 10(5) B16 cells were injected after 14 d with 1 x 10(10) ADV/RSV-tk viral particles. Subsequent treatment for 6 d with GCV resulted in an inhibition of melanoma growth, with an approximately 40-50% reduction in melanoma volume in comparison to controls in repeated experiments. These data demonstrate that adenovirus-mediated gene transfer can function as an efficient delivery system to reduce established tumor burden in vivo. This result may hold significant promise for the development of effective in situ gene therapy for melanoma in humans.


Subject(s)
Adenoviridae/genetics , Genetic Therapy , Genetic Vectors , Melanoma, Experimental/prevention & control , Simplexvirus/genetics , Thymidine Kinase/genetics , Animals , Ganciclovir/therapeutic use , Gene Transfer Techniques , Melanoma, Experimental/genetics , Mice , Transduction, Genetic , Tumor Cells, Cultured
13.
J Invest Dermatol ; 104(3): 405-10, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7861010

ABSTRACT

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. Interestingly, both loricrin and profilaggrin were initially expressed focally in areas corresponding to more advanced morphologic stages of maturation. Because the cornified envelope is a composite structure consisting of both protein and lipid components, we also monitored the appearance of lipid lamellar granules during epidermal development. These granules were first evident at E16 and the extrusion of lipids from the granules into the intercellular space occurred at E17, prior to the cross linking of loricrin into the cell envelope. Our results document that loricrin is expressed and accumulates at the cell periphery subsequent to the extrusion of lipids, but prior to processing of profilaggrin. We suggest that the sequential regulation of these events is critical for formation of epidermal barrier function during development.


Subject(s)
Membrane Proteins/genetics , Animals , Base Sequence , Cell Differentiation , Epidermis/growth & development , Female , Fluorescent Antibody Technique , Lipids/physiology , Male , Mice , Mice, Inbred ICR , Molecular Sequence Data , Polymerase Chain Reaction/methods , RNA, Messenger/analysis , Skin/growth & development
14.
Genes Dev ; 9(3): 317-29, 1995 Feb 01.
Article in English | MEDLINE | ID: mdl-7867929

ABSTRACT

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. Histological changes in the epidermis were subtle with the phenotypic stratum corneum appearing slightly thinner and more loosely packed than in controls. Electron microscopic studies revealed that lipid multilamellar structures were not present between cells in the stratum corneum of phenotypic mice. When assayed for transepidermal water loss, phenotypic skin lost water at a rate three times faster than controls, suggesting that neonatal lethality resulted from loss of epidermal barrier function. The absence of a functional lipid barrier in transgenic mice first became evident at E17 when lipids were extruded initially into the intercellular space. We have identified a potential pathway linking inhibition of retinoid signaling with disruption of the lipid barrier that involves peroxisome proliferator-activated receptors. This study documents the role of the retinoid signaling pathway in formation and maintenance of a functional epidermis and provides the first evidence that this is mediated in part by modulation of lipid metabolism.


Subject(s)
Lipid Metabolism , Mice, Transgenic/metabolism , Receptors, Retinoic Acid/genetics , Skin/metabolism , Animals , Base Sequence , Cells, Cultured , Keratinocytes/metabolism , Mice , Molecular Sequence Data , Mutation , Receptors, Retinoic Acid/biosynthesis , Skin/pathology , Skin/ultrastructure , Water/metabolism
15.
Genomics ; 23(2): 450-6, 1994 Sep 15.
Article in English | MEDLINE | ID: mdl-7835895

ABSTRACT

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. The gene has a simple structure consisting of a single intron of 1091 bp within the 5' noncoding sequence and an uninterrupted open reading frame. Using PCR analyses of DNAs isolated from mouse x Chinese hamster somatic cell hybrids, we have mapped both the loricrin and the profilaggrin genes to chromosome 3. Genetic linkage analysis has shown that mouse loricin and profilaggrin lie within 1.5 +/- 1.1 centimorgans of each other. We have further shown that both genes map in the vicinity of the flaky tail (ft) and soft coat (soc) loci. These mouse mutants exhibit a number of changes in their integument, suggesting that abnormalities in these genes may contribute to the mutant phenotype.


Subject(s)
Genetic Linkage , Intermediate Filament Proteins/genetics , Membrane Proteins/genetics , Protein Precursors/genetics , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Cloning, Molecular , Cricetinae , DNA, Complementary/genetics , Filaggrin Proteins , Humans , Hybrid Cells , Introns , Mice , Mice, Inbred BALB C , Mice, Mutant Strains , Molecular Sequence Data , Mutation , Open Reading Frames , Phenotype , Polymerase Chain Reaction , Rats
16.
J Dermatol Sci ; 7 Suppl: S164-9, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7528048

ABSTRACT

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. In order to test this we have produced transgenic mice that express a mutant K6 gene. These mice develop a progressive scarring alopecia at about 6 months of age. Later, the denuded areas developed a keratosis which was prone to infection. Ultrastructural analysis suggests that hair loss is due to the destruction of the outer root sheath. We believe that these mice are models of another keratin disorder.


Subject(s)
Alopecia/genetics , Keratins/genetics , Alopecia/classification , Alopecia/pathology , Animals , Disease Models, Animal , Epidermis/metabolism , Epidermis/pathology , Humans , Keratinocytes/metabolism , Keratinocytes/pathology , Keratins/chemistry , Keratins/metabolism , Mice , Mice, Transgenic , Molecular Structure , Multigene Family , Mutation
17.
Cell Growth Differ ; 5(6): 667-75, 1994 Jun.
Article in English | MEDLINE | ID: mdl-7522035

ABSTRACT

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. The long latency in lesion appearance may reflect the low level of intact E6 transcripts and the requirement for additional genetic or epigenetic events before production of an overt lesion. In agreement with this proposal, spontaneous papillomas developed that expressed an activated rasHa oncogene (codon 61, A-->T; codon 13, G-->T). All lesions expressed keratin genes K1, K6, and K13 in a fashion characteristic of hyperproliferative or benign tumors with no evidence of malignant conversion. Our results demonstrate that the mouse epidermis represents a relevant in vivo model system to analyze the interaction between HPV and cellular genes in neoplasia.


Subject(s)
DNA-Binding Proteins , Mice, Transgenic , Oncogene Proteins, Viral/genetics , Papilloma/genetics , Papillomaviridae/genetics , Animals , Base Sequence , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/biosynthesis , DNA Probes, HPV , Epidermis/pathology , Genes, ras , Keratins/genetics , Mice , Molecular Sequence Data , Papilloma/pathology
18.
Cell Growth Differ ; 4(12): 1071-82, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8117621

ABSTRACT

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. Juvenile transgenic mouse epidermis was uniformly hyperkeratotic, but this pattern was generally less pronounced in adult transgenic mice unless they expressed high levels of the HK1.TGF-alpha transgene. Spontaneous, squamous papillomas occurred at sites of wounding in adult mice expressing high levels of HK1.TGF-alpha; however, most were prone to regression. Immunoreactive TGF-alpha was 2-6 times higher in the epidermis of these HK1.TGF-alpha lines. Immunoreactive epidermal growth factor receptor had a normal pattern of expression in nonphenotypic adult epidermis, but a marked reduction in the receptor population was detected in hyperplastic newborn epidermis and phenotypic adult epidermis. Autoradiographic localization of 125I-epidermal growth factor showed a similar pattern of distribution, suggesting that the sites of increased TGF-alpha expression induced epidermal growth factor receptor down-regulation. These data demonstrate the in vivo effect of deregulated TGF-alpha expression on epidermal proliferation and differentiation and suggest a potential role for TGF-alpha in carcinogenesis and other hyperproliferative epidermal disorders.


Subject(s)
Epidermis/metabolism , Transforming Growth Factor alpha/biosynthesis , Animals , Base Sequence , Cell Division/genetics , Epidermal Growth Factor/metabolism , Epidermis/pathology , ErbB Receptors/metabolism , Hyperplasia/genetics , Hyperplasia/metabolism , Keratosis/genetics , Keratosis/metabolism , Mice , Mice, Transgenic , Molecular Sequence Data , Papilloma/genetics , Papilloma/metabolism , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Transforming Growth Factor alpha/genetics
19.
Oncogene ; 8(8): 2145-57, 1993 Aug.
Article in English | MEDLINE | ID: mdl-7687760

ABSTRACT

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. This indicates that a second promoting event, such as wounding or friction, is required to elicit these pathological changes. Highly keratotic benign ear lesions and benign squamous papillomas appeared after long latency at sites of phenotypic epidermis. These data suggest that v-fos may be interfering with c-fos function in normal keratinocyte differentiation, but by itself is insufficient to elicit overt benign lesions.


Subject(s)
Epidermis/pathology , Genes, fos , Keratosis/genetics , Skin Neoplasms/genetics , Alopecia/etiology , Animals , Base Sequence , Cell Differentiation , Gene Expression Regulation, Neoplastic , Hyperplasia , Keratins/analysis , Mice , Mice, Transgenic , Molecular Sequence Data , Oncogene Proteins v-fos/analysis , Proto-Oncogene Proteins c-fos/analysis
20.
Proc Natl Acad Sci U S A ; 90(11): 5237-41, 1993 Jun 01.
Article in English | MEDLINE | ID: mdl-7685120

ABSTRACT

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. The skin was very shiny and tautly stretched. These animals were rigid and appeared to be restricted in their ability to move and breathe; death occurred within 24 hr. Histologically, the most prominent features of the skin were a compact orthohyperkeratosis and a reduction in the number of hair follicles. Pulse-labeling studies with 5-bromodeoxyuridine demonstrated a marked reduction in the number of replicating cells in the epidermis and hair follicles. Thus, the macro- and microscopic appearance of these mice, as well as their neonatal lethality, most likely result from inhibition of normal skin development and suppression of epithelial cell proliferation by the overexpression of transforming growth factor beta 1.


Subject(s)
Skin/growth & development , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/physiology , Amino Acid Sequence , Animals , Animals, Newborn , Cysteine , DNA/genetics , Gene Expression , Immunohistochemistry , Introns , Mice , Mice, Transgenic , Microscopy, Electron , Mutagenesis, Site-Directed , RNA/genetics , RNA/isolation & purification , Reference Values , Serine , Skin/pathology , Skin/ultrastructure , Swine , Transforming Growth Factor beta/analysis
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